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A Book on Plant Breeding and Genetics by Jiban Shrestha, Scientist, Nepal Agricultural Research Council, National Maize Research Program, Rampur, Chitwan, Nepal

A Book on Plant Breeding and Genetics by Jiban Shrestha, Scientist, Nepal Agricultural Research Council, National Maize Research Program, Rampur, Chitwan, Nepal



A Book on Plant Breeding and Genetics by Jiban Shrestha, Scientist, Nepal Agricultural Research Council, National Maize Research Program, Rampur, Chitwan, Nepal

A Book on Plant Breeding and Genetics by Jiban Shrestha, Scientist, Nepal Agricultural Research Council, National Maize Research Program, Rampur, Chitwan, Nepal



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    A Book on Plant Breeding and Genetics by Jiban Shrestha, Scientist, Nepal Agricultural Research Council, National Maize Research Program, Rampur, Chitwan, Nepal A Book on Plant Breeding and Genetics by Jiban Shrestha, Scientist, Nepal Agricultural Research Council, National Maize Research Program, Rampur, Chitwan, Nepal Document Transcript

    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. PLANT BREEDING AND GENETICS Compiled and edited by: Jiban Shrestha Nepal Agricultural Research Council National Maize Research Program, Rampur, Chitwan, Nepal January, 2014
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. PLANT BREEDING AND GENETICS Compiled and edited by: Jiban Shrestha Scientist (Plant Breeding and Genetics) Nepal Agricultural research Council National Maize Research Program, Rampur, Chitwan, Nepal January, 2014
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1. General 1.1Organizations involved for crop improvement in Nepal 1.2 International institutes for crop improvement 1.3 Relationship of national and international research institutes in crop improvement, 1.4 Intellectual Property Rights (IPRs) in relation to improved varieties and plant genetic resources (Breeders’ and Farmers’ Rights), 1.5 National Seed Act (2045) and Seed Regulations 1.6 Objectives and activities of plant breeding, 1.7 Domestication, plant introduction and acclimatization 1.1. ORGANIZATIONS INVOLVED FOR CROP IMPROVEMENT IN NEPAL 1. National Rice Research Program (NRRP): Hardinath, Dhanusa 2. National Wheat Research Program (NWRP): Bh airahawa, Rupendehi 3. National Maize Research Program (NMRP) Rampur, Chitwan 3. National Grain Legumes Research Program ( NGLRP) Rampur, Chitwan 4. National Oilseeds Research Program (NORP): Nawalpur, sarlahi 5. National Sugarcane Research Program (NSRP): Jitpur, Bara 6 . National Jute Research Program (NJRP): Itahari, Sunsari 7. Cotton Development Program : Khajura, Nepaljung 8. Coordinated Maize Program (CMO): Khumaltar, Lalitpur. 9. Institute of Agriculture and Animal Science (IAAS): Rampur, Chitwan ACTIVITIES of organization involved for crop improvement in Nepal.: 1.Varietal improvement Development of high-yielding and disease-resistant crop varieties Development of crop varieties suitable for different agro-ecological domains. 2. Resource management: Development of improved crop production technologies (Irrigation and fertilizer management, time and method of crop establishment, weed management etc.) suitable for different agro climatic conditions Identification of farmers' problems through on-site inspection of farmers' fields and solving them through adaptive research 3. Research on crop protection Detection of major diseases and insects on crop, estimation of their damage, identification and development of insects and disease resistant crop varieties and disease management techniques
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 4. Outreach research activities On-farm verification of station developed technologies through farmers' field testing and mini -kit distribution of recently released and pre-release wheat varieties and other technologies under farmers' situations. Provide farmers with technical knowledge through different media. 5. Source Seed production Produce breeder seeds and provide them to different farms /stations, seed companies for foundation seed production and supervise them Assist farmers in seed multiplication program through technical advice 1.2.INTERNATIONAL INSTITUTES FOR CROP IMPROVEMENT 1. IRRI ( International Rice Research Institute): Los Banos, Philippines 2. CIMMYT ( International Centre for Maize and Wheat Improvement): el Baton, Mexico 3. CIAT ( International Centre for Tropical Agriculture): Palmira, Colombia 4. IITA ( International Institute of Tropical Agriculture): Ibadan, Nigeria 5. WARDA ( West African Rice Development Association): Monrovia, Liberia 6. CIP ( International Centre for Potato): Lima, Peru 7. ICRISAT ( International Crops Research Institute for the Semi-Arid Tropics) : Hyderabad, India 8. IBPGR (International Board for Plant Genetic Resources) : Rome, Italy. 9. ICARDA (International Centre for Agricultural Research in Dry Areas): Aleppo, Syria MANDATES of International Institutes for crop Improvements: 1. Genetic improvement of crops; develop high yielding and disease resistant lines suited to various environments 2. Collection and conservation of germplasm of concerned crops and their relatives 3. Conduct research on farming sy stems for an efficient use of available resources 4. Determine appropriate technology suitable for needs and resources of the region 5. Extension activities to popularize the new technology so that the cultivars is able to adopt them INSTITUTES INVOLVED IN PGR ACTIVITIES A. Nepal Agriculture Research Council 1. Division of Agriculture Botany, Khumaltar, Lalitpur - leading role in conservation of crop genetic resources 2. National Rice Research Program, Parwanipur 3. National Wheat Research Program, Bhairahawa 4. National Maize Research Program, Rampur
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 5. National Oilseed Research Program, Nawalpur 6. National Hill crops Research Program, Kabre 7. National Grain Legume Research Program, Rampur 8. National Sugarcane Research Program, Jitpur 9. National Tobacco Research Program, Belachapi 10.National Potato Research Program, Khumaltar 11.Horticulture Research Station, Pokhara/ Tarhara/ Dhankuta/ Nepalganj B. Department of Agriculture Development 1. Division of Horticulture, Kirtipur, Kathmandu 2. Division of vegetable, Khumaltar, Lalitpur 3. Horticulture Farm, Daman/Marpha/Solukhumbu/Sarlahi/Sindhuli/Dolkha/ Panchkhal/Godawari/Kirtipur/Trisuli/Mustang/Palpa/Jumla/Dailekh/Baitadi/ Humla/Chitwan C. Department of Plant Resources 1. National Herbarium and Plant laboratory, Godawari, Lalitpur - leading role in conservation of medicinal plants 2. Herbal conservatory, Hetauda/ Daman/ Tistung, Makwanpur D. Forest Research and Survey Center, Babar Mahal, Kathmandu E. Department of Wildlife Resources and National Parks - Wildlife Reserves: Koshi-Tappu Wildlife Reserves Parsa Wildlife Reserves Shivapuri Wildlife Reserves Dhorpatan Wildlife Reserves Royal Shuklaphant Wildlife Reserves 1.3. RELATIIONSHIP OF NATIONAL AND INTERNATIONAL RESEARCH INSTITUTES IN CROP IMPROVEMENT NARC and IAAS achieve financial grants from international donor agencies for conducting researches on crops. The CIMMYT international and the IRRI has been collaraborating actively with NARC and IAAS in wheat and rice improvement programs. And germplasm exchange. International Agriculture Research Centers have played a definitive role in boosting up the national crop production and productivity. Elite lines/varieties generated by IRRI, CIMMYT, ICRISAT, ICARDA, and CIP. have been extensively tested under different agro-ecological zones of Nepal. High yielding varieties with resistance to biotic/abiotic stresses have been selected and
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. released for general cultivation. The collaborative activities between national programs and IARCs have been effective in exchange of germplasm, testing of advanced breeding lines/varieties, providing on-job training, visits/exchange of scientists, conservation of germplasm and supply of research reports, newsletters, special bulletins and books. CGIAR Centers have generously supported national programmes since last three decades. However, the level of support for training is declining. Visits/exchange of scientists and on-job training of research workers should be enhanced. These centers should play a leading role in supporting the developing nations for use of recent advances in biotechnology and their application in crop improvement. IBPGR/IPGRI has been successful in creating awareness of PGR conservation, use and management among national scientists and policy makers. It has supported the national programme by providing on-job training, supplying newsletters/ descriptors/ special bulletins/books and cosponsoring national workshops on PGR. IPGRI should continue to support in establishing national PGR system, formulating national policy on PGR, upgrading technical capabilities and attracting donor in establishing national facilities for conservation. NEPAL AGRICULTURAL RESEARCH COUNCIL (NARC) Background NARC was established in 1991 as an autonomous organization under ‗Nepal Agicultural Council Act-1991‘ with the prospect of having an efficient, effective and dynamic agricultural research system in the country to uplift the socioeconomic level of people through the increase of productivity by optimal use of available means and resources without depletion in environment. Nepal Agricultural Research Council (NARC) was established in 1991 under the Nepal. Agricultural Research Council Act, 2048 (1991). It is operated under two tier decision making bodies: a 16-member NARC Council chaired by Minister for Agriculture and Cooperatives and a 8-member Executive Board chaired by Executive Director of NARC. NARC is an autonomous national apex body responsible for overall agriculture research in the country. It is mandated to conduct research and study in different aspects of agriculture for increasing agricultural productivity and production by generating appropriate agrotechnologies suitable to various agro-ecological zones (AEZs) for the country's diversified commodities like cereal crops, grain legumes, oilseeds, cash/industrial crops, horticultural crops, livestock (bovine, swine, avian, goat, sheep) and fisheries. As stated in the Preamble of NARC Act, 1991, the mission of NARC is to conduct high level studies and research on problems of the agriculture sector and to find out measures of solutions of the problems and thereby uplift the quality of life of general public. So, the main mission of NARC is to develop and provide appropriate technologies to the farmers, extension agents, agro-entrepreneurs and other clients/ stakeholders in order to convert the agriculture into a viable/ dynamic system and thereby to improve the standard of living of Nepalese people. Nepal Agricultural Research Council (NARC), which came into being in 1991, was set up with the goal of promoting research work in the field of agriculture. The new organisation was started
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. with the institutional facilities of the then National Agricultural Research and Services Centre (NARSC) which had been serving in Nepal as a prime agricultural research organization in Nepal since 1985. The organisation functioned under the Department of Agriculture, Ministry of Agriculture. NARC had been established as an autonomous organization under "Nepal Agricultural Research Council Act - 1991". The goal behind its establishment was to provide an efficient, effective and dynamic agriculture research system in the kingdom of Nepal to give a boost to the economic level of the people involved in agriculture. Objectives To conduct high level studies and researches on various aspects of agriculture To identify the existing problems in agriculture and find out measures to solve To assist His Majesty's Government in the formulation of agricultural policies and strategies Functions and Responsibilities Conduct high level research work on various fields of agriculture required in the line with the national agricultural policies, Prioritize studies and researches to be conducted, Provide research and consultancy services to its clients, Coordinate, monitor and evaluate the agriculture research activities in Nepal, Document research activities Activities 1. Implement research programs, by itself or in collaboration with other institutions, on: Research areas of NARC are followings;I. Cereals and Cash Crops(Rice, Maize, Wheat, Potato, Sugarcane, Jute etc.) II.Horticulture III. Livestock and Animal Health IV. Fisheries V.Pasture and Fodder VI. Agro-forestry/Farm-forestry VII. Soil and Irrigation Management VIII.Botany and Bio-technology IX. Entomology, Plant Pathology and Plant Protection X.Farming Systems XI. Agri Extension XII. Agri Economic and Marketing XIII. Food Science
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. XIV.Agri Ecology and Environment XV.Socio Economics Xvi. Other fields related to Agriculture 2. Implement research with a focus on generation of agricultural technology suitable for various agro-climates of Nepal. 3. Provide client-oriented contract out research services to the farmers, agriextensionists and agro-entrepreneurs. 4. Conduct research works to increase long-term-agricultural productivity without depleting the environment. Financial Resources  Grants from His Majesty's Government  Grants from national and international donor agencies and governments  Funds obtained from research or consultancy services Organizational Setup 1. Council 2. Executive Board Executive Board NARC, at present, is governed by fourteen-member Executive Board, chaired by Honorable Minister for Agriculture. The Board comprises of  Minister for Agriculture -Chairperson  Member, National Planning Commission -Member  Secretary, Ministry of Agriculture -Member  Director General,  Department of Agriculture Development (DOAD) -Member  Joint Secretary, Ministry of Finance -Member  Dean,  Institute of Agriculture and Animal Science (IAAS) -Member  Dean, Institute of Forestry -Member  Representatives from Agricultural Scientists (Two) -Members  Representatives from farmers (Two) -Members  Representatives from Agri-Entrepreneurs (Two) -Members  Executive Director, NARC -Member-Secretary 3. NARC Headquarter (HQ): It has 3 components 3.1. NARI and NASRI, Disciplinary Divisions 3.2. Cross cutting Divisions/Units 3.3. Regional Directorates: Agriculture Research Stations 3.4. Commodity Programs NARC Organization: NARC has a two-tire body: the council and the executive board. The council is the apex body for policy level work on agricultural research. The sixteen-member council is chaired by the Minister for Agricultrue and Cooperatives. Executive Director acts as the member-
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. secretary of the council. The eight-member executive board, chaired by the executive director of NARC implements and executes research program approved by the council. One of the program directors acts as member secretary of the board. Mandates In pursuance of these objectives the following mandates have been provisioned: 1) To conduct and/or let to conduct agricultural research, 2) To determine priorities in studies and research related to agriculture, 3) To provide consultant services and research support services to agricultural research, 4) To coordinate, supervise, monitor, review and evaluate research activities related to agriculture in the country, 5) To maintain/document up-to-date records of agricultural research, and 6) To conduct and /or let to conduct other necessary activities related to agricultural research. Executive Director is the administrative head of the organization, who is responsible for all the activities of the institution. There are five line directors to help in the responsibility of the Executive Director. Director for Crops and Horticulture Research technically handles 11 national crops related research programs of rice, maize, wheat, grain legumes, oilseeds, hillcrops, sugarcane, citrus, potato, ginger and jute. Research and studies on these crops are also conducted in the multidisciplinary research stations and divisions as well. Director for Livestock and Fisheries Research handles 3 livestock commodity programs on Bovine, Swine and Avian, Sheep and Goat, and Fisheries. Director for Planning and Coordination handles the planning process of research programs and coordinates for resource management and development. The following Divisions function under the Director for Planning and Coordination: Planning, Outreach Research, Socioeconomics & Agricultural Policy Research, Monitoring & Evaluation, Training & Scholarship, and Communication, Publication & Documentation. All the research projects are monitored and evaluated by the chiefs of Monitoring & Evaluation, disciplinary divisions, Regional Directors, Commodity Coordinators and senior scientists. Directors for Finance and Administration handle the financial and administrative management of NARC respectively. In order to implement different research projects and activities, there are different organizational entities under NARC: Two institutes (National Agricultural Research Institute and National Animal Science Research Institute) are located at Khumaltar and separate wings of 14 National Commodity Research Programs (NCRPs), 4 Regional Agricultural Research Stations (RARSs), 14 Agricultural Research Stations (ARSs), 20 Technical Disciplinary Divisions (DDs) and units. National Agricultural Research Institute (NARI) deals mainly with the agronomical and horticultural crops research and related activities. It includes seven related disciplinary divisions: Agronomy, Agriculture Botany, Soil Science, Plant Pathology, Entomology, Agricultural Engineering, and Horticulture Research. Director of NARI has the responsibility to administer their overall activities.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. National Animal Science Research Institute (NASRI) deals mainly with the livestock and fisheries research. It includes five related disciplinary divisions such as Animal Breeding, Animal Nutrition, Animal Health, Pasture and Fodder Research and Fisheries Research. Its overa ll activities are administered by the Director for NASRI. Organizations for supporting Projects and Budget to NARC. The main international donor agencies/ financial institutions that supported NARC were: Japan International Cooperation Agency (JICA) Japan through KR-II (Fisheries) and KR-II (others), Department for International Development (DFID) UK through Hill Agriculture Research Project (HARP), World Bank (IDA) through Agricultural Research & Extension Project (AREP), Swiss Agency for Development and Cooperation (SDC) through Hill Maize Research Project (HMRP), IFAD through Hills Leasehold Forestry and Fodder Development Project and others. The agricultural research activities of crop horticulture, animal science and fisheries were accomplished, with the joint efforts of commodity programs, disciplinary divisions, Regional/ Agricultural Research Stations of NARC in collaboration with other partners. On-farm/ outreach research including farmers‘ acceptance tests (FATs) has been conducted by the RARSs, ARSs, commodity programs and some divisions as regular activities. Research programs are initiated with bottom-up planning through village level workshops followed by station/ regional level discussions and finally approved by the NARC Council. Joint meetings are also held among NARC, DOA and DLS and other allied agencies at different levels to identify problems, prioritize them and determine research agenda. Some Facts About Narc, Nepal 1. NARC was established in 1991. 2. Two institutions: 2 (NARI and NASRI) 3. National Commodity Research Program:14 4. Regional Agricultural Research Stations-5 5. Disciplinary divisions: 13 (Crop Science-9, Animal Science-5) 6. Agricultural Research Stations: 11 7. Units: 4 (Food-research, agriculture environment, post-harvest and biotechnology) NARC Highlights: 1) Varieties released. High yielding, disease and insect resistant/ tolerant crop varieties released for different agro-ecological zones are: Khumal 8, Loktantra, Mithila, Ram, Barkhe 3004 and Pokhareli Jethobudho of Rice; Deuti and Shitala of Maize; Pragati and Unnati of Rapeseed; Kalyan and Prateekchha of Greengram/Moongbean and Baidehi and Rajarshi of Groundnut. Many pipeline varieties of different crops to be released are identified . Sagun variety of lentil is developed 2) Rice genotypes were analyzed for physical and physicochemical characteristics, milling recovery, protein and ash content for development of fine and aromatic rice varieties.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 3) Community-based seed production (CBSP) program found useful for the supply of source seeds to the users clients locally NATIONAL COMMODITY RESEARCH PROGRAMS OF NARC: 1) National Rice Research Program 2) National Wheat Research Program 3) National Maize Research Program 4) National Grain Legumes Research Program 5) National Oilseeds Research Program 6) National Sugarcane Research Program 7) National Citrus Research Program 8) National Potato Research Program 9) National Jute Research Program 10) National Ginger Research Program 11) Hill Crops Research Program 12) National Bovine Research Program 13) National Sheep and Goat Research Program 14) National Swine and Avian Research Program REGIONAL DIRECTORATES OF NARC: Regional Research Stations 1) Regional Agricultural Research Station (Far Western Development Region) 2) Regional Agricultural Research Station (Mid Western Development Region) 3) Regional Agricultural Research Station (Western Development Region) 4) Regional Agricultural Research Station (Central Development Region) 5) Regional Agricultural Research Station (Eastern Development Region) AGRICULT URE RESEARCH STAT IONS: Regional Agricultural Research Station (Mid Western Development Region), Nepalgunj 1) Agricultural Research Station, Surkhet 2) Agricultural Research Station, Jumla 3) Agricultural Research Station, Dailekh Regional Agricultural Research Station (Western Development Region), Lumle 4) Agricultural Research Station (Fish) Begnas, Pokhara 5) Agricultural Research Station (Horticulture) Malepatan, Pokhara 6) Agricultural Research Station (Goat) Bandipur, Tanahun Regional Agricultural Research Station (Central Development Region), Parwanipur 7) Agricultural Research Station (Pasture) Dhunche Rasuwa 8) Agricultural Research Station (Fish) Trisuli, Nuwakot 9) Agricultural Research Station (Agriculture Implements) Ranighat, Parsa 10) Agricultural Research Station, Belachapi, Dhanusa Regional Agricultural Research Station (Eastern Development Region), Tarahara
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 11) Agricultural Research Station, Pakhribas, Dhankuta CROSS CUTTING DIVISIONS/UNITS Name of Division: o Communication, Publication and Documentation o Socio Economics and Agricultural Research Policy o Outreach Research  Name of Unit : a. Food_Research b. Bio-Technology c. Agri-Environment d. National Agriculture Genetic Resources Centre (Genebank) DISCIPLINARY DIVISIO NS: A) Crop Science: 1) Agronomy 2) Plant Pathology 3) Entomology 4) Soil Science 5) Agri-Engineering 6) Horticulture 7) Agri-Botany 8) Commercial Crops 9) Seed Science & Technology B) Animal Science 10) Animal Nutrition 11) Animal Breeding 12) Fishery Research 13) Animal Health Research 14) Pasture and Forage Research 1.4 INTELLECTUAL PROPERTY RIGHTS (IPRS) IN RELATION TO IMPROVED VARIETIES AND PLANT GENETIC RESOURCES (BREEDERS’ AND FARMERS’ RIGHTS) INTELLECTUAL PROPERTY RIGHT (IPR) in relation to improved varieties Intellectual property rights are exclusive rights provided by a nation to a creator over the use of his/her creation for a certain period of time. The major forms of intellectual property rights are Trade Secrete, Patent, Copy Right and Plant Variety Protection. The major problems with intellectual properties are that they can be copied, imitated or reproduced, which minimizes the returns to the original inventor. IPRs are considered important for following reasons
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1. 2. 3. 4. 5. 6. 7. To control trade distortions and barriers in national and international trade To enhance creativity by rewarding inventiveness To develop affordable products for consumer‘s satisfaction To generate additional funds by attracting private sector to invest in research and development To allow free and fair competition in trade sectors To facilitate transfer of technology for clients To protect consumers from counterfeit goods and services. PLANT GENETIC RESOURCES (BREEDERS’ AND FARMERS’ RIGHTS) PLANT BREEDER’S RIGHT (PBR) Plant breeder‘s rights are the rights granted by the government to a plant breeder, originator or owner of a variety to exclude others from producing or commercializing the propagating material of that variety for fixed period of time. (15-20 years). The requirements of PBRs are novelty, distinctiveness, uniformity and stability of varieties. The main considerations for development of PBR system are; 1) it allows breeders to benefit from varieties developed by them 2) private sector is encouraged to invest in plant breeding and seed industry. 3) Development of a new plant variety. Benefits from PBR: 1) it encourages private companies to invest in plant breeding activities. 2) It enables access to varieties developed in other countries 3) Increased competition among various organizations engaged in plant breeding is beneficial to both farmers and nations 4) The opportunities to breeders of obtaining profits from varieties developed by them. Disadvantages of PBR: 1) PBR will encourage monopolies in genetic material for specific traits. 2) It suppresses free exchange of gentic material and may encourage unhealthy practices 3) The holder of PBR-title may produce less seed than demand in order to increase prices for achieving more profit 4) PBR may result in increased cost of seed. FARMER’S RIGHT Farmers rights are rights arising from the past, present and future contributions of farmers in conserving, improving and making available plant genetic resources particularly those in the context of origin and diversity. The basic rights of farmers are:  right to own seed, plant seed, sell seed and exchange seed
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal.  legal status in respect of their agro-biological resources and knowledge base  recognition of an indigenous knowledge base as the property of a local community  Benefits accruing from the use of their resources in terms of royalty. 1.5 NATIONAL SEED ACT (2045) AND SEED REGULATIONS Seed Act was enacted in Nepal in 2045 (1988). It is intended to protect the farmers against the risk of sowing seed of inferior quality. The purpose of the act is to regulate the various aspects of seed industry such as seed production, processing, seed quality and seed certification, imports, exports and marketing of seeds. The act could be regarded as an expression of all the concerned agencies of seed sub sector for seed quality and government‘s commitment for the same. Some of highlights of the seed Act are as follows: 1) The preamble of the act indicates the use of high quality seed for increased crop production along with the economic interest and convince of the public 2) The act has provided the definitions of Seed, Crop, Notified Seed, Agricultural Work, Seed Board, Seed Testing Laboratory, Seed Container, Plant Breeder, Seed Species, Seed Variety and Seed Labelling 3) It has prescribed the formation, functions duties and responsibilities of National Seed Board and its Sub-Committees 4) The establishment of Seed Certification Office and Central Seed Testing Laboratory as well as their duties and authorities have been prescribed. 5) Notification of seed varieties and kinds and minimum germination and purity level are specified 6) In seed marketing restriction on the sale and distribution of Notified Seeds and Prohibition to sell seeds treated with toxic chemicals are specifically mentioned 7) For the export and import of seeds, provisions are made for the approval from authorized body. 8) The appointment of seed Inspector and Analyst, their duties, functions and authorities are also prescribed 9) Finally, the act includes section on Penalty and Punishment, Prosecution by Government of Nepal, Investigation and filling of the cases, Power to hear the case, Protection of Action in Good Faith and authorization to formulate the rules and regulations for the purpose of implementing the objectives of the act. However, the act was not yet implemented due to lack of rules and regulations. 1.6 OBJECTIVES AND ACTIVITIES OF PLANT BREEDING Objectives of Plant Breeding:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1. to produce higher crop yields by developing more efficient genotypes. E.g. hybrid varieties of maize, bajra etc. 2. to improve quality of plant produce which determines its suitability for various uses e.g. to improve cooking quality in rice, malting quality in barley, keeping quality in vegetable, protein content in cereals and legumes 3. to develop disease and insect resistant varieties which not only increase production but also stabilize it 4. to change in maturity duration in order to develop early or late maturity crop varieties 5. to modify agronomic characteristics such as plant height, tillering, branching, erect or trailing habit etc. 6. to develop photoinsensitive and thermoinsensitive wheat and photoinsensitive rice varieties 7. to improve synchronous maturity and non-shattering characteristics e.g. in legumes 8. to develop varieties with determinate growth in crops like mung, pigeonpea, cotton etc. 9. to develop winter hardiness and to remove dormancy of crop seeds 10. to develop varieties for new seasons 11. to eliminate toxic substances in crops in order to increase nutritive value 12. to develop varieties for rainfed areas and for saline soils for increase crop production. Activities of Plant Breeding: 1) Creation of variation: Genetic variation can be created by domestication, germplasm collection, plant introduction, hybridization, polyploidy, somaclonal variation and genetic engineering 2) Selection: The identification and isolation of plants having desirable combinations of characters and growing their progeny is called selection. Selection is necessarily based on phenotype. Various breeding methods have been designed to increase efficacy of selection. Selection finally yields an improved lines or population. 3) Evaluation: Newly selected lines/population are tested for yield and other traits and performance is compared with existing best varieties (checks). Evaluation is step wise process, ordinary conducted at several locations for three or more years. 4) Multiplication: This step concerns with the large scale production of certified seed of the released and notified varieties. Seed production is usually done by seed production agencies in a stepwise manner and seed is certified by a seed certification agencies.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 5) Distribution: Certified seed is ultimately sold to the farmers who use it for commercial crop production. This activity alone makes it possible to reap the economic benefits from above activities. For an efficient crop improvement programmes, the above activities have to be properly coordinated and efficiently geared to maximize outputs from a programmes. UNDESIRABLE EFFECTS OF CROP BREEDING OR CROP IMPROVEMENT: 1) Genetic erosion: crop breeding activities lead to reduction in variability, this reduction is produced by replacement of heterozygous local varieties by few homozygous improved varieties and use of similar related varieties parents in breeding programmes. 2) Narrow genetic base: The improved varieties of crop species has increased similar to each other due to commonness of one or more parents in their ancestry. This has led to narrow genetic base of these varieties. The narrow genetic base has created genetic vulnerability i.e. susceptibility to a disease, insect pest etc. 3) Increased susceptibility to minor diseases: the breeding activities for resistance to major disease and insects has resulted increased susceptibility to minor diseases. 1.7 DOMESTICATION, PLANT INTRODUCTION AND ACCLIMATIZATION DOMESTICATION The process of bringing a wild species under human management is referred to as domestication. There is bound to be some selection during domestication. This is likely to give rise to better types than wild ones. Domestication continues till today and is likely to continue for some time in future. The domestication may be regarded as a method , in fact, the most basic method, of plant breeding; all other breeding methods become applicable to a plant species only after it has been successfully domesticated. The results/effects of plant domestication include: (changes in plant species under domestication) Elimination of dormancy of crop seeds Higher germination rates and more uniform timing of germination Increased size of reproductive organs Reduced complexity of reproductive organs Reduction of toxicity (humans select against self defense mechanisms) Increase in economic yields of crops. Change in life cycle (normally from perennial to annual for seed crops, and from annual to biennial for vegetable crops) Promotion of asexual reproduction
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Shift in sex form of species Modification of plant type. Decreases in variability within a variety PLANT INTRODUCTION Farmers rights are rights arising from the past, present and future contributions of farmers in conserving, improving and making available plant genetic resources particularly those in the context of origin and diversity. The basic rights of farmers are: Plant introduction consists of taking a genotype or a group of genotypes of plants into a new area or region where they were not being grown before. Types of plant introduction 1.Primary Introduction: When the introduced variety is well adapted to the new environment, it is released for commercial cultivating without any alteration in the original genotype; this constitutes primary introduction. It is less common, particularly in countries having well organized crop improvement programmes. 2.Secondary introduction: The introduced variety may be subject t o selection to isolate a superior variety . Alternatively it may be hybridized with local varieties to transfer one or few characters from this variety to the local ones. Such introduction constitutes secondary introduction. It is much common than primary introduction. Purpose of plant introduction: 1. to obtain an entirely new crop species 2. to serve as new varieties 3. to be used in crop improvement programmes 4. to save a crop form disease or pest 5. for scientific studies 6. used for aesthetic value 7. for germplasm collection. Procedure for Plant Introduction: Introduction of germplasm consists of the follwing steps: 1) procurement of germplasm: The germplasm may be obtained from foreign countries as gifts from institution/individuals, purchased, collected from plant exploration. Plant
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. propagules like seeds, tubers, runners, suckers, stolens, bulbs, rooted cuttings, seedlings etc.may be imported from foreign countries. 2) Quarantine : it means to keep materials in isolation to prevent the spread of diseases present in them to other materials.The contaminated materials are fumigated and heavily contaminated samples are destroyed. Quarantine check is done at quarantine check post. 3) Cataloguing: during the cataloguing of germplasm, each germplasm accession is given an accession number such as IC (indigenous collection), EC (exotic collection) or IW (indigenous wild). Information on species and varieties names, place of origin, adaptation and on its various features is also recorded. 4) Evaluation: it consists of assessment of the germplasm accessions for their various features or traits of some known or potential use in breeding programmes. Germplasm accessions are evaluated for morphological, physiological, biochemical, plant pathological, entomological etc features. The charactgers assessed must be related to the need of breeders and other users. 5) Multiplication: the germplasm accessions requested by breeders/researchers are multiplied. 6) Distribution: The germplasm are distributed to plant breeders or researchers. ACCLIMATISATION Acclimatization is the reversible process by which an individual becomes adapted to a change in the environment often involving temperature, moisture, food, often relating to seasonal climate changes. The process that leads to the adaptation of a variety, line or population to a new environment is known as acclimatization. Acclimatization is brought about by a faster multiplication of those genotypes (present in the original population) that are better adapted to the new environment Factors affecting extent of acclimatization: 1. mode of pollination 2. the magnitude of genetic variability present in the original population 3. the duration of life cycle of the crop 4. mutation 5. nature and intensity of environmental stress. 2. Plant Genetic Resources 2.1 Biodiversity and agrobiodiversity, 2.2 Evolution of major crop species, 2.3 Centers of origin 2.4 Germplasm collection and exchange, 2.5 Plant exploration and conservation 2.6 Evaluation and utilization of plant genetic resources,
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2.7 Agrobiodiversity policy in Nepal, 2.8 Status of plant genetic resources in Nepal. 2.1. BIODIVERSITY AND AGROBIODIVERSITY Biological diversity or biodiversity embraces the variability within and between all living organism from all sources like micro-organisms, plants, animals and the ecological system in which they inhabitat. Biodiversity is the totality of genes, species and ecosystem in a region. It refers to variety of life on earth. Biodiversity (Biological diversity) is the variety and variability among living organisms and ecological complexes in which they occur and encompasses ecosystem or community diversity, species diversity and genetic diversity. a) Ecosystem diversity: it includes variety of habitats that occurs within a region b) Species diversity: it is variety and abundance of different types of organisms which inhibit an area c) Genetic diversity: it is combination of different genes found within a population of a single species. Biodiversity or Biological diversity embraces the variability within and between all living organisms from all sources like microorganisms, plants and animals and the ecological systems which they inhabit. It starts with genes and manifests itself as organisms, populations, species and communities, which give life to ecosystems, landscapes and ultimately the biosphere. In other words, it encompasses the total number, variety and variability of life forms, levels and combinations existing within the living world. It is not the sum of all ecosystems, species and genetic material. Rather it includes diversity within species, between species and of ecosystems. According to the definition of the 1992 UN conference on Environment and Development Convention, biodiversity includes all of its manifestations. Therefore, in addition to terrestrial biodiversity, it also covers marine and other aquatic biodiversity as well. As such biodiversity means the richness and variety of living things in the world as a whole or in any location within it. Biodiversity consists of three main fundamental and hierarchical categories. They are: Ecosystem diversity, Species diversity and genetic diversity. Ecosystem Diversity: An ecosystem comprises a dynamic complex of plants, animals and microorganisms communities and their non-living environment, which interact as a functional unit. Non living components include sunlight, air, water, minerals and nutrients. Ecosystem can be small and ephemeral, for example, water filled tree holes or rotting logs on a forest floor or large and long lived like forest s or lakes. Thus, ecosystem commonly exists within ecosystems. Biologists are often concerned with small-scale ecosystems, but for conservation purposes larger units (such as particular forests,
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. grass lands) are generally used. Ecosystem diversity refers to the variety and frequency of distinct ecosystems including the variety of habitats, biotic communities and their change in composition and structure over time and ecological processes in the biosphere. Species Diversity Species is defined as a population of organisms whose members are able to interbred freely under natural conditions. A species represents a group of organisms, which has evolved distinct inheritable features and occupies a unique geographical area. Species usually do not freely interbreed with other species. Species Diversity is used to describe the frequency and variety of species in the globe has been estimated to range from 5 to 30 million, out of which approximately 1.7 million living species of all kinds of organisms have been described to date. The World Conservation and Monitoring Centre suggests that there are many different ways to describe species diversity. Species richness is the total number of species within a geographical area. It is expressed as an enumeration of the species occurring within a particular sample area, and is one often used to measure species diversity. Measures of species richness are the basis for the observation that diversity increases with decreasing latitude on earth, for example, tropical areas are richer in species than temperate areas. Species evenness is also used to measure species diversity that is expressed as relationship of species to each other. This includes relative abundance of species in various categories. It is also known as taxonomic diversity. For example, an island of two species of birds and one species of lizard has greater taxonomic diversity than an island with three species of birds but no lizards. Species dominance is expressed as the most abundant species as dominant. The country presents a great diversity of flora and fauna that are found in the dense tropical monsoon forest of the Terai to deciduous and coniferous forests of the sub tropical and temperate regions, and finally to the sub alpine and alpine pastures and snow converted Himalayan picks. Why is Biodiversity Important? The diversity of life enriches the quality of our lives in ways that are not easy to quantify. Biodiversity is intrinsically valuable and is important for our emotional, psychological, and spiritual well-being. Some consider that it is an important human responsibility to be stewards for the rest of the world's living organisms. Diversity breeds diversity. Having a diverse array of living organisms allows other organisms to take advantage of the resources provided. For example, trees provide habitat and nutrients for birds, insects, other plants and animals, fungi, and microbes. Humans have always depended on the Earth's biodiversity for food, shelter, and health. Biological resources that provide goods for human use include: food-species that are hunted, fished, and gathered, as well as those cultivated for agriculture, forestry, and aquaculture;
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. shelter and warmth-timber and other forest products and fibers such as wool and cotton; medicines-both traditional medicines and those synthesized from biological resources and processes. Biodiversity also supplies indirect services to humans which are often taken for granted. These include drinkable water, clean air, and fertile soils. The loss of populations, species, or groups of species from an ecosystem can upset its normal function and disrupt these ecological services. Recent declines in honeybee populations may result in a loss of pollination services for fruit crops and flowers Biodiversity provides medical models for research into solving human health problems. For example, researchers are looking at how seals, whales, and penguins use oxygen during deep water dives for clues to treat people who suffer strokes, shock, and lung disease. The Earth's biodiversity contributes to the productivity of natural and agricultural systems. Insects, bats, birds, and other animals serve as pollinators. Parasites and predators can act as natural pest controls. Various organisms are responsible for recycling organic materials and maintaining the productivity of soil. Genetic diversity is also important in terms of evolution. The loss of individuals, populations, and species decreases the variety of genes-the material needed for species and populations to adapt to changing conditions or for new species to evolve. Importance of biodiversity: 1.Biodiversity and its ecological processes sustain our lives and lives of other species with which we share the planet 2.Biodiversity provides the raw materials we need 3.Biodiversity provides economic benefit through attracting visitors. Factors affecting biodiversity: Rainfall: diversity increases as the rainfall is increased winter snowfall: diversity increases as the winter snowfall is increased Latitude: diversity increases as the latitude is increased. Altitude: Vegetation is decreased as the altitude is increased. AGROBIODIVERSITY Definition Of Agrobiodiversity The variety and variability of animals, plants and micro-organisms that are used directly or indirectly for food and agriculture, including crops, livestock, forestry and fisheries. It comprises the diversity of genetic resources (varieties, breeds) and species used for food, fodder, fibre, fuel and pharmaceuticals. It also includes the diversity of non-harvested species that support production (soil micro-organisms, predators, pollinators), and those in the wider environment
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. that support agro-ecosystems (agricultural, pastoral, forest and aquatic) as well as the diversity of the agro-ecosystems. Distinctive Features of Agrobiodiversity There are several distinctive features of agrobiodiversity, compared to other components of biodiversity: 1. Agrobiodiversity is actively managed by male and female farmers; 2. many components of agrobiodiversity would not survive without this human interference; local knowledge and culture are integral parts of agrobiodiversity management; 3. many economically important agricultural systems are based on ‗alien‘ crop or livestock species introduced from elsewhere (for example, horticultural production systems or Friesian cows in Africa). This creates a high degree of interdependence between countries for the genetic resources on which our food systems are based; 4. as regards crop diversity, diversity within species is at least as important as diversity between species; 5. because of the degree of human management, conservation of agrobiodiversity in production systems is inherently linked to sustainable use - preservation through establishing protected areas is less relevant; and 6. in industrial-type agricultural systems, much crop diversity is now held ex situ in gene banks or breeders‘ materials rather than on-farm. The Role of Agro biodiversity 1. Increase productivity, food security, and economic returns 2. Reduce the pressure of agriculture on fragile areas, forests and endangered species 3 . Make farming systems more stable, robust, and sustainable 4. Contribute to sound pest and disease management Conserve soil and increase natural soil fertility and health 5. Contribute to sustainable intensification 6. Diversify products and income opportunities 7. Reduce or spread risks to individuals and nations 8. Help maximize effective use of resources and the environment 9. Reduce dependency on external inputs 10. Improve human nutrition and provide sources of medicines and vitamins, and 11. Conserve ecosystem structure and stability of species diversity. Genetic Diversity A. Wheat 1. Origin Centres of origin are Mediterranean (bread wheat), and Ethiopian (durums). He species of Triticum are classified into three ploidy levels; diploid (2n=2x=14), tetraploid (2n=4x=28) and hexaploid (2n=6x=42). The hexaploid bread wheat contain three different sets of genome AA, BB, DD.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Diploid species might have originated from the same common parent species, which are now differentiated into several genomes. Evidences indicate that tetraploid species T. turgidum (AABB), the cultivated emmer and durum wheat evolved from an alloploid T. monococcum (AA) and an unknown and possibly extinct species containing BB genomes. Bread wheat T. aestivum (AABBDD) originated as an alloploid combining T. turgidum and the diploid species T. tauschi (DD). 2. Genetic diversity for adaptation of wheat Produced in diverse climatic conditions. Best adapted in cool climates while least in warm and moist climates. Inherent variations for tolerance to heat, drought, cold, diseases, insect pests affect adaptation to a particular climate. Important species of wheat. Species name chromosome genome Number formula Triticum monococcum Unknown species T. dichasians T. tauschii T. turgidum T. durum T. dicoccum T. timopheevii T. aestivum T. spelta T. compactum T. sphaerococcum T. macha T. vavilovi 14 14 14 14 28 28 28 28 42 42 42 42 42 42 common name domestication AA einkorn BB CC DD AABB solid stem wheat AABB durum AABB emmer AAGG AABBDD bread AABBDD Spelt AABBDD club AABBDD AABBDD AABBDD cultivated wild wild cultivated cultivated cultivated wild cultivated cultivated cultivated wild wild wild B. Maize 1. Origin Southern Mexico and central American regions are considered as its centre of origin. Corm now seems to be generally accepted it originated from teosinte ( Zea mays L. spp. Paviglumis, or Zea mays L. spp. Mexicana), the nearest known relative of corn.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2. Genetic diversity Maize (Zea mays) belongs to tribe Maydeae of family gramineae. It is the only cultivated species in the genus Zea. It is considered to have originated from Teosinte. Maize is diploid with 20 chromosomes. Produced in diverse climatic conditions. Best adapted in warm climates while least in cool climates. Inherent variations for tolerance to heat, drought, cold, diseases, insect pests affect adaptation to a particular climate. Classification 1. Dent corn (Zea mays var. indentata Sturt.) 2. Flint corn (Z. mays var. indurata Sturt.) 3. Flour corn (Z. mays var. amylacea Sturt.) 4. Pod corn (Z. mays var. tunicata Sturt.) 5. Pop corn (Z. mays var. everta Sturt.) 6. Sweet corn (Z. mays var. saccharata Sturt.) 7. Waxy corn (Z. mays var. carabina Sturt.) C. Paddy 1. Origin Oryza sativa L., the principal cultivated species of rice, is believed to have been domesticated nearly 10,000 years ago in an area that includes northeastern India, bangladesh, Burma, Thailand, Laos, Vietnam, and southern China. Rice spread from its area of primary diversity throughout southeast asia and adjacent islands of the Pacific region. The only other cultivated species of rice, O. glaberrima Steud., is indigenous to the upper valley of the Niger river in west Africa. Genetic diversity in rice: The genus Oryza contains 20 species with a basic chromosome number of 12. The genus includes both diploid and tetraploid species with 6 genomes groups, A, B, C, D, E and F. The cultivated species O. sativa (2n=2x=24) has AA genome formula. O. glaberrima (2n=2x=24) does not pair with Oryza sativa and has AgAg genome formula. Six of the species of Oryza are annuals and the remainders are perennial. Species name chromosome genome Number formula domestication Oryza sativa Oryza glaberrima Oryza nivara Oryza rufipogon Zizania palustris L. 24 24 24 24 cultivated cultivated wild wild wild AA AgAg AA AA
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. There is great genetic diversity in rice. Natural mutants have occurred with a rather high frequency, enabling O sativa to adapt to a wide range of agroclimates. The 18 wild species, and wild relatives in related genera, provide a rich spectrum of useful genes for the breeder. Genetic diversity for adaptation of rice Produced in diverse climatic conditions. Adapted in tropical climates to temperateclimates. Inherent variations for tolerance to stress, diseases, insect pests affect adaptation to a particular climate. The species O. sativa has evolved into 3 types or ecographic races, generally characterised as follows: Indica: the tropical type, sensitive to low temperature and photoperiod, typically with tall plants, weak stems, long and droopy leaves, slender grains that shatter easily and remain dormant for long period, and the source for dried cooked rice. Japonica or Sinika: the temperate type, typically with short leaves and stems, moderate tillering, resistant to low temperature, short rounded grains with low amylose content, sticky when cooked. Javanica: tall with thick stems and broad stiff leaves, low tillering, long panicles, resistant to shattering, and large bold grains. D. Legumes (Soybean) 1. Origin The soybean was domesticated in northeastern China about 2500 BC. From the area of its origin, the soybean spread to southern China, Korea, Japan and other countries in southeastern Asia. The soybean was introduced intermittently into the United States in the late 1700s. 2. Genetic diversity Taxonomically, the soybean is classified in the legume family, Leguminoseae, subfamily Papilionoideae, tribe Phaseoleae, genus Glycine. The genus Glycine contains three subgenera: Soja and Glycine. a. Subgenus Glycine Twelve species under subgenus Glycine, all wide perennial. Ten species are diploid. Two are mixtures of diploid and tetraploid plants. Diploid (2n=2x=40), Tetraploid (2n=4x=80); both are found. b. Subgenus Bracteata Glycine wightii, perennial vine with limited cultivation. 2n = 22, 44. c. Subgenus Soja Glycine max: 2n = 2x = 40; cultivated, annual Clycine soja: 2n = 2x = 40; wild, annual vine.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. E. Jute 1. Origin Chorcorus capsularis: Southeast Asia (Indo-Burma) Chorcorus olitorius: Africa 2. Genetic diversity Family: Tiliaceae Genus: Chorcorus Chorcorus genus contains 40 species and only 2 species are cultivated for fiber. They are: Chorcorus capsularis and Chorcorus olitorius. Both cultivated species are diploid (2n = 2x = 14). Several wild species are tetraploid (2n = 2x = 28). F. Cotton 1. Origin Diploid species containing the D genome originated in the western Hemispere and are referred to as New World species. Diploid species with the A, B, E and F genome are African or Asian in origin and are referred to as old world species. Tetraploid cotton contains AADD genomes are referred as New World tetraploid. 2. Genetic Diversity Genus: Gossypium Thirty diploid (2n = 2x = 26) species. Four tetraploid (2n =4x =52) species. Six genomes: A, B, C, D, E and F. Species of cotton Species 2n genome geographic origin cultivation G. herbaceum G. arboreum G. hirsutum G. barbadense G. anomalum G. sturtianum G. stocksii G. longicalyx G. thurberi 26 26 52 52 26 26 26 26 26 A A AD AD B C E F D Africa India America America Africa Australia Indo-Arabia Africa America cultivated cultivation cultivated cultivated wild wild wild wild wild
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. G. armourianum G. tomentosum G. caicoense 26 52 52 D AD AD America Hawai Brazil wild wild wild G. Sugarcane 1. Origin Cultivated sugarcane had two geographic centres of origin, New Guinea and the northern India Burma-China region. The large barrelled, tropical species, Saccharum officinarum, probably originated from the wild species S. robustum, in the New Guinea region. 2. Genetic Diversity Family: Gramineae Genus: Saccharum Species of Sugarcane Species Saccharum officinarum S. sinense S. barberi S. spontaneum S. robustum Sclerostachya fusca Narenga porphyrocoma Erianthus maximus Chromosome No. 2n = 80 2n = 116, 118 2n = 82 – 124 2n = 40 – 128 2n = 60 – 148 2n = 30 2n = 30 2n = 60 – 100 Cultivation cultivated cultivated cultivated wild wild wild wild wild H. Potato 1. Origin The cultivated potato is generally believed to have originated in the Andes region from central Peru to central Bolivia. Chile may be a secondary centre of origin of Solanum tuberosum, where evolutionary changes occurred similar to those which later took place under selection in Europe. 2. Genetic Diversity Family: Solanaceae Genus: Solanum The genus Solanum contains approximately 2000 species, including over 150 tuber bearing species which form a polyploid series from diploids (2x) to hexaploids (6x) with 75 % of them being diploid. The cultivated potato belongs to the species Solanum tuberosum is considered to be an autotetraploid with a genomic formula (2n = 4x = 48). Evidence have found that Andean region of Peru and Bolivia the native cultivated diploid species, Solanum stenotomum, believed to be the first
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. domesticated potato species and a progenitor of the tetraploid tuberosum species. Solanum phujure (2n = 2x = 24) is one of the wild species of potato. I. Oilseeds crop (Brassica spp.) 1. Origin 2. Genetic Diversity Species of Brassica. Species Chromosome No. Brassica nigra 16 Brassica campestris 20 Brassica juncea 36 Brassica napus 38 Common name Genome black sarson rape rai, mustard rape BB AA AABB AACC Genetic erosion The gradual loss of variability from cultivated species, and their wild forms and wild relatives is called genetic erosion. The variability lead arisen in nature over an extremely long period of time. Therefore, if allowed to be lost, it would be impossible to create it again during a short period. The chief cause of genetic erosion is human since varieties are created by human using the natural genetic diversity and the natural diversity is destroying gradually. The main causes of genetic erosion are as given below: Replacement of genetically variable land races by the improved, genetically uniform pureline or hybrid varieties. Improved crop management practices have virtually eliminated the weedy forms of many crops. Increasing human needs have extended farming and grazing into forests the habitats of most wild species. This has led to the extinction of many wild relatives of crops. Developmental activities like hydroelectric projects, roads, industrial areas, railways, buildings etc have also disturbed the wild habitat. Sometimes, introduction (deliberarate or accidental) of a weedy species may result in the invasion of wild habitats by this species and lead to the elimination of the native wild relatives of crop plants. 2.2 EVOLUTION OF MAJOR CROP SPECIES Selection by nature and man is responsible for evolution of crops. selection is effective in altering a species only when genetic variability exists in population of that species. The patterns of evolution of various crops are broadly classified according to mode of origin of genetic variation for evolution of that species. These patterns are following;
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1) Mendalian variation: Many crops have evolved through variation generated by gene mutation, and by hybridization between different genotypes within the same species followed by recombination. Ultimately all variability in any species originates from gene mutations. Mutations may be macromutation and micromutation. A single macromutation has led to differentiation of modern maize (Zea mays) plant from grassy pod corn. Similarly, cabbage, cauliflower, broccoli and Brussel‘s sprouts have originated from a common wild species. Mutation tends to be accumulated in a population. Several important crops have evolved through mendalian variation like barley, rice beans, peas, tomato, bajra, jowar, linseed etc. 2) Interspecific hybridization: It refers to crossing of two different species of plants. The resulting F1 is generally more vigorous than parents. But segregation in F2 and later generations produces a vast range of genotypes. Interspecific hybridization has led to the development of several strawberry varieties. Many varieties in pear, plum, cherries, grapes and ornamentals like roses, lilies, etc. are developed through interspecific hybridization. 3) Polyploidy: Generally autopolyploidy leads to increased vigor, larger flowers and fruits etc. over diploid forms. Many varieties of ornamental plants are autopolyploids. The commercial banana is autoptriploid (3x). Triploid varieties are developed in apples, watermelons, sugarbeet etc. Potato (Solanum tuberosum) is autotetraploid. Autopolyploid crops are sweet potato (6x), oat (4x), alfalfa (4x). Allopolyploidy is more important in crop evolution. Allopolyploidy results from chromosome doubling of interspecific F1 hybrids. Crops like wheat, tobacco, cotton, sugarcane, rai, rapeseed are allopolyploid. Common bread wheat (Triticum aestivum) is allohexaploid, while cotton and tobacco are allotetraploids. 2.3 CENTERS OF ORIGIN In 1926, N.I Vavilov proposed that crop plants evolved from wild species in the areas showing great diversity and termed them as primary centres of origin. In some areas, certain crop species show considerable diversity of forms although they did not originate there; such areas are known as secondary centrea of origin of these species. Eight main centres of origin proposed by N.I. Vavilov are following; 1.) The China centre of origin: It consists of mountainous regions of central and western china and neighbouring lowlands A) Primary centre of origin: soybean, radish, buckwheat, brinjal , pear, peach, plum, apricot, Chinese cabbage, onion, cucumber,litchi, walnut B) Secondary centre of origin: maize, rajma, cowpea, turnip and seas am 2.) The Hindustan centre of origin:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. This centre consists of Burma, Assam, Java, Borneo, Sumatra, Malaya, Archipelago and Philippines A.) Primary centre of origin: rice, arhar, gram, cowpea, mung, brinjal, cucumber, yams, Indian lettuce, Indian radish, noble cane (sugarcane), black pepper, indigo, mango, orange, sour lime, coconut, banana, turmeric etc. 3.) The Central Asia Centre of origin: Includes Northwest India (Punjab, Northwest Frontier Provinces and Kashmir), Afghanistan, Tadjikistan, Uzbekistan, and western Tian-Shan. A.) Primary centre of origin: wheat, pea, broad bean, mung, linseed, sesame, cotton, radish, safflower, carrot, onion, garlic, spinach, apricot, pear, almond, grape, apple B..) Secondary centre of origin: rye 4.) The Asia Minor Centre of Origin: It includes the interior of Asia minor, the whole of Transcaucasia, Iran and highlands of Turkmenistan a. Primary centre of origin: rye, alfalfa, carrot, cabbage, oat, lettuce, fig, pomegranate, apple, almond, chestnut b. Secondary centre of origin: rape, black mustard, leaf mustard, turnip 5.) The Mediterranean centre of origin: It Includes all Mediterranean regions like Labnan, Jordan etc. A,) Primary centre of origin: durum wheat, emmer wheat, barley, lentil, pea, broadbean, lupins, chick pea, clovers, vetch, onion, garlic, beet, lettuce, asparagus, pepper mint 6.) The Abyssinian centre of origin: it includes Ethiopia and hill country of Eritrea. A.) primary centre of origin: barley, jowar, bajra, gram, lentil, pea, linseed, safflower, sesame, castor, coffee, onion, okra B.) Secondary centre of origin: broad bean 7.) The Central American centre of origin: It Includes southern sections of Mexico, Guatemala, Honduras and Costa Rica. A.) Primary centre of origin: Maize, rajma, lima bean, melon , pumpkin, sweet potato, cotton, chillies, papaya, guava, avocado 8.) The south American Centre of origin: Includes the high mountainous regions of peru, Bolivia, Ecuador, Columbia, part of Chile and Brazil and Whole of Paraguay.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. A.) Primary centre of origin: potato, maize, lima bean, peanut, pineapple, pumpkin, tomatoes, guava, tobacco, rubber and cassava Law of Homologous series in variation: This law states that characters found in one species also occur in other related species. Genus secale duplicates the variation found in genus Triticum. What is importance of centre of origin to plant breeder? Plant breeder can improve the crop variety in aspect of yield, height, resistance to insect and diseases and edaphic and climatic stress. To cross the plant a great diversity in qual ity is required or high variation is desirable. The centre of origin denotes they having large vars of wild type and great diversity of quality and quantity. Therefore it is necessary to have knowledge of centre of origin of plant to plant breeder. In addition to these, varieties in centre of origin develops gene combination through mutation and natural selection which are suitable and more efficient to that place. 2.4 GERMPLASM COLLECTION AND EXCHANGE GERMPLASM COLLECTION: A germplasm collection of a crop species consists of a large number of lines, varieties and related wild species of the crop. Such collection is called gene banks. The major concerns in gene banks relate to following; a) agroecological coverage of collections, b) the number of distinct accessions and c) the proportion of viable accessions. In Nepal gene bank is going to be established in four places namely Tarahara, Parwanipur, Lumle and Khajura. The process of obtaining the various germplasm accessions for a germplasm collection is known as collection of germplasm. This can be done in two ways: 1. Exploration: Explorations are trips for collection of various forms of crop plants and their related species. They are primary source of all the germplasm present in various germpalsm collection. The explorations are done to collect germplasm needed by breeder and to collect variability in crops and their relatives for their conservation. 2. Procurement from other agencies: Germplasm can be obtained form other agencies concerned with germplasm conservation, from research institutions, individuals or companies. Generally this involves an import of the germplasm GERMPLASM EXCHANGE:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Import and export of germplasm is called germplasm exchange. Germplasm exchange is an important aspect of this binational collaboration, The germplasm exchange has played an important role in the development of a strong and diversified agriculture. The introduction of new sources of germplasm from different plant species is regular process and essential aspect of crop improvement programs. Developing countries receive germplasm of different crops for testing in their local conditions. Similarly various institutions of developed countries collect land races of different crops and conserve for breeding purpose. Today, exchange programmes of seed materials is continued throughout the world. Due to movement of plants, plant materials and seeds from country to country, several diseases, insects and weeds have been introduced along with those materials into countries where they never existed. Therefore, to control these harrmful biotic factors, germplasm should be subjected to quarantine. In Nepal, there are six quarantine check post, one at Tribhuvan Internaitonal Airport, KTM, and Central Office of plant Quarantine in NARC, Khumaltar. The exchange of seeds among farmers is the most common practice for the dissemination of varieties. Procedure of Germplasm Exchange: The germplasm exchange procedure is following: 1. Import: Germplasm import is the major activity in plant introduction. Only healthy, viable and clean seed material (free from soil, pests, pathogens and weeds) are imported from foreign countries. The imported materials are subjected to quarantine checks. They must not be treated by sender with fungicides or insecticides. Imported samples must be accompanied by a phytosanitary certificate from sending scientists or institution. Contaminated entries may be fumigated to control insect or pathogens but samples that are heavily contaminated should be destroyed during quarantine. The process of quarantine is done ar quarantine check posts which are situated at boarder area of Nepal and also at Tribhuvan International Airport, KTM. 2. Export: The supply of seed and other materials to collaborating scientists/organisations abroad is done as per requests for seed/planting material received from them. Before the dispatch of the seed/planting materials, they should be checked from quarantine angle and phytosanitary certificates are issued to them. Only the healthy seed material (free from diseases, pests, weeds, soil clods, plant debris, etc.) should be sent to requesting countries. No seed dressing with insecticides or fungicides be given while dispatching the seed to the foreign countries. The seeds are usually forwarded by registered air parcel post, while perishable plant materials are despatched through air freight. 2.5 PLANT EXPLORATION AND CONSERVATION
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. PLANT EXPLORATION Plant Exploration is the search for new, undiscovered plants. Explorations are trips for collection of various forms of crop plants and their related species. They are primary source of all the germplasm present in various germpalsm collection. The explorations are done to collect germplasm needed by breeder and to collect variability in crops and their rel atives for their conservation. The principal objective of most plant exploration expeditions is to provide germplasm resources for existing breeding programs, biotechnology, and conservation. Objectives of plant exploration: 1) collection of gemplasm needed by breeders 2) collection of the variability remaining in the crop plants and their relatives for its conservation. Areas of exploration: The areas to be covered by explorations are usually the centres of origin of concerned crops. In addition, collections should be made from the peripheral regions of species distribution, and even in areas where it was introduced in comparatively recent times. Merits of Plant Exploration 1) it is the source of virtually all genetic diversity stored in gene banks 2) it is the only means of collecting and conseving the threatened genetic diversity 3) it provides access to materials of special interest, e.g. new genes (= alleles), new species etc. Limitations of Plant Exploration: 1) it is tedius, time taking and expensive. 2) It poses various hardships to the collectors, e.g. in boarding, transportation, etc. especially in remote areas 3) There may even be a threat to life, specially from wild animals. CONSERVATION Gene Bank: Gene Bank, term applied to a facility where plant genes are stored, usually in the form of seeds but also as whole plants, pollen, and cell cultures. Gene banks provide a broad range of plant genes from which breeders can develop new plant varieties with desired characteristics such as higher yield and better resistance to disease and weather. To this end, breeders should have as large a variety of plant genes as possible. The need for such gene banks increases as agricultural
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. lands impinge further upon undeveloped areas, with subsequent loss of species and strains of wild plants. Germplasm Conservation -.The sum total of all the genes present in a crop and its related species constitutes its germplasm. It is ordinarily represented by a collection of various strains and species. Germplasm provides the raw materials (= genes), which the breeder uses to develop commercial crop varieties. Therefore, germplasm is the basic indispensable ingredient of all breeding programmes, and a great emphasis is placed on collection, evaluation and conservation of germplasm. Conventionally, germplasm is conserved as seeds stored at ambient temperature, low temperature or ultralow temperature. Conservation of crop germplasm diversity involves the establishment of in situ and ex situ genebanks The major activities for ex situ genebanks include assembling, conserving, characterizing and providing easy access to germplasm for scientists. More than six million accessions are currently assembled in over 1300 genebanks worldwide. The following approaches of germplasm conservation may be applied: (i) freeze preservation, (ii) slow growth cultures, (iii) desiccated somatic embryos/artificial seeds, and (iv) DNA clones. The germplasm has to be maintained in such a state that there is minimum risk for its loss and that either it can be planted directly in the field or it can be prepared for planting with relative ease; this is called germplasm conservation.Germplasm can be connserved either 1) in situ or 2) ex situ. 1). In situ germplasm conservation: In-situ germplasm conservation means "on-site conservation". Conservation of germplasm in its natural habitat or in the area where it grows naturally is known as in situ germplasm conservation. This is achieved by protecting this area from human interference; such an area is often called natural park, biosphere reserve or gene. Merits of gene sanctuaries: 1) it is not only conserve the existing genetic diversity present in the population,it also allows evolution to continue. 2) The risks associated with ex situ conservation are not operative. Demerits of gene sanctuaries: 1) they are easiest to demarcate, difficult to establish and very difficult to maintain. 2) These can not conserve the variability found in crop plants, for which ex situ conservation is the only answer.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2). Ex situ germplasm conservation: Ex-situ conservation means literally, "off-site conservation". Conservation of germplasm away from its natural habitat is called ex situ germplasm conservation. It can be achieved by following ways: a) seed gene banks b) plant or field gene banks c) shoot-tip gene banks d) cell and organ gene banks e) DNA gene banks. Importance of germplasm conservation: 1) germplasm may be directly release as a variety. 2) it may be subjected to selection for developing a new variety. 3) it may be used as parent in hybridization programmes. GENE BANKS Large collection of germplasm representing materials from various parts of the world is called gene bank. Plant genetic resources genebanks store, maintain and reproduce living samples of the world's huge diversity of crop varieties and their wild relatives. They ensure that the varieties and landraces of the crops and their wild relatives that underpin our food supply are both secure in the long term and available for use by farmers, plant breeders and researchers. What do genebanks do? Genebanks conserve genetic resources. The most fundamental activity in a genebank is to treat a new sample in a way that will prolong its viability as long as possible while ensuring its quality. The samples (or accessions as they are called) are monitored to ensure that they are not losing viability. A cornerstone of genebank operations is the reproduction-called regeneration-of its plant material. Plant samples must periodically be grown out, regenerated, and new seed harvested because, even under the best of conservation conditions, samples will eventually die.To conserve and regenerate genetic resources, genebanks first must collect genetic resources. But genebanks aren't built just to conserve genetic resources; they are intended to ensure that these resources are used, whether it is in farmers' fields, breeding programmes or in research institutions. This means making sure the collections are properly characterized and documented; and that the documentation is available to those who need it. The information syste ms used by genebanks are becoming increasingly important tools for researchers and breeders seeking data on the distribution of crops and their wild relatives. Finally, genebanks must be able to deliver healthy samples to the farmers, breeders and researchers. Major concerns in gene banks:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1)ecogenetic coverage of the collections 2) the number of distinct accessions 3) the proportion of viable accessions. TYPES OF GENE BANKS 1) Seed genebanks The most widely used technique for conserving plant genetic resources is seed banking. Seeds are dried to low moisture content and stored at subzero temperatures in cold stores or deep freezers. According to FAO, this accounts for 90 percent of the 6 million accessions conserved ex situ globally. However, this technique is only possible for species with seeds that can tolerate desiccation and low temperatures. Many species have seeds that cannot survive under such conditions. For species with so-called 'recalcitrant' seeds or species that are vegetatively propagated, such as roots tubers and aroids, different conservation techniques are used. 2) Community seed banks Seed banks don't have to be high-tech and managed by governments or businesses. In many developing countries, farmers rely on informal seed systems based on local growers retention of seed from previous harvests, storage, treatment and exchange of this seed within and between communities. The informal seed sector is typically based on indigenous structures for information flow and exchange of seed. Seed banks managed within this local seed system operate on a small scale at the community level with few resources. These community seed banks and the institutions that support them are extremely important in the preservation of local varieties and for agricultural production. Much could be gained from learning more about these seed banks and working with communities to improve them. In spite of this, informal seed banks have until now received little attention or support from the scientific community or the state. 3) Field genebanks The conservation of germplasm in field genebanks involves the collecting of materials and planting in the orchard or field in another location. Field genebanks have traditionally been used for perennial plants, including: species producing recalcitrant seeds; species producing little or no seeds; species that are preferably stored as clonal material; and species that have a long life cycle to generate breeding and/or planting material. Field genebanks are commonly used for such species as cocoa, rubber, coconut, coffee, sugarcane,
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. banana, tuber crops, tropical and temperate fruits, vegetatively propagated crops, such as wild onion and garlic, and forage grasses. 4) Shoot tip gene banks: In such gene banks, germplasm is conserved as slow growth cultures of shoot tips and nodal segments. Their regeneration consists of subculturing the cultures, which may be done every 6 months to 3 years. Through this gene bank conservation, genotypes of accessions can be conserved free from diseases and pests. 5) cells and organs gene banks (cryopreservation). In vitro storage and cryopreservation In vitro storage and cryopreservation are two technologies used in genebanks to conserve species with recalcitrant seeds or species that are vegetatively propagated. In vitro storage involves keeping plant tissues under strict sterile conditions in glass tubes and vessels. Cryopreservation involves storing the living tissues at ultra-low temperature, usually at -196C in liquid nitrogen, to guarantee long-term preservation of germplasm in genetically unaltered state. Research work on cryopreservation have led to the development of protocols for cryopreservation of no less than over 150 different plant species. A wide range of species can now be routinely cryopreserved: banana (Musa spp.), cassava (Manihot esculenta), bramble fruits (Rubus), pear Pyrus, vegetables in the Solanum family, coffee (Coffea arabica), oil palm (Elaeis guineensis) and tea (Camellia sinensis). In vitro storage is not always suitable for long-term conservation and requires advanced infrastructure and equipment along with highly trained staff - adding to the cost. Cryopreservation is a more promising technique for the long-term conservation of 'difficult' species. Cryopreservation (Gr. Kryo: frost) means preservation in frozen state. It is the methods of germplasm conservation where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as (typically) 77 K or −196 °C (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. However, when vitrification solutions are not used, the cells being preserved are often damaged due to freezing during the approach to low temperatures or warming to room temperature. Applications of cryopreservation: 1) conservation of genetic materials 2) freeze storage of cell cultures 3) maintenance of disease free stocks
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 4) cold acclimation and frost resistance 6) DNA banking The rapidly expanding study of DNA in so many areas of science has created an odd surplus: the DNA itself. Reasonably easy and inexpensive to store, with established techniques for almost infinite multiplication, the samples of DNA created in laboratories around the world have almost by accident become an important resource for future research. DNA resources can be maintained at 20C for short- and mid-term storage (up to 2 years), and at 70C or in liquid nitrogen for longer periods. DNA banking is not at present widespread. It is not viewed as a substitute for existing techniques for the conservation of genetic resources. However, DNA banks can complement conservation strategies that make use of ex situ and in situ conservation, and they can help to ensure the optimal use of plant and animal populations. For many species that are difficult to conserve by conventional means that are highly threatened in the wild, DNA storage may provide a way to conserve the genetic diversity of these species and their populations in the short term, until effective methods can be developed. REQUISITES FOR A GENE BANK: 1) the accession maintained in the gene bank should provide comprehensive and representative coverage of the germplasm of the concerned species. 2) It should have dependable and cost effective preservation facilities. 3) It should generate sufficient seed stocks for distribution and exchange. 4) It should publish/make available easily and freely accessible information on the accessions maintained in the bank 5) It should enjoy adequate financial and technical support 6) The staff of the gene bank should be technically capable and more particularly willing to perform their assigned duties. 2.6 EVALUATION AND UTILIZATION OF PLANT GENETIC RESOURCES EVALUATION OF PGR Evaluation goes deeper than characterization. It may require special biochemical techniques and usually include agronomic performance, yield and biotic and abiotic stresses, such as drought or pest. These traits are important to plant breeders and researchers in crop improvement. Such evaluation may also use DNA-based methods to analyze a plant's genetic diversity.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. The evaluation descriptors, although contributing to some extent to identifying an accession, are more interesting than characterization descriptors because of their value in crop improvement. In general, effective evaluation is possible when there is close institutional and personal interaction between curators and breeders or other crop improvement scientists, and where breeding objectives are reflected in evaluation programmes. Evaluation is primarily carried out by users, in multidisciplinary teams that include breeders, entomologists, pathologists and agronomists. The potential value of the germplasm depends on the efficiency of the techniques designed to differentiate among accessions. Because the farmers are the ultimate users of the product of any crop improvement programme and possess valuable traditional knowledge, due consideration must be given to involve farmers views and expectations at some point during any evaluation programme. Evaluation consists of assessment of plant genetic resources for their various features or traits of some known or potential use in breeding programmes. It is multi-disciplinary activity; generally germplasm accessions are evaluated for morphological, physiological, biochemical, plant pathological (i.e. disease resistance), entomological (i.e. insect resistance) and other fatures. Obviously, evaluation involves experts from different disciplines. The evaluation of germplasm helps to eliminate duplicates.It is the most critical step determining the utilization of a collection. A poorly assessed germplasm is extremely valuable for germplasm collections. In Nepal, evaluation and characterization of food crop species have been done by the commodity programs and PGRU staff using IBPGR/IPGRI descriptors. The evaluation and characterization data of barley, buckwheat, finger millets, amaranths and grain legumes have been published. Screening of rice germplasm against Pyricularia oryzae is being done at Khumaltar. Physical facilities are not available for the systematic evaluation of the germplasm for biochemical and genetic finger printing. Such studies could be undertaken in collaboration with international institutes. However, the facilities should be developed in the country itself. National program should play a leading role in collaborating with international institutes for characterization and evaluation at molecular level. Objectives of Germplasm Characterization/Evaluation The major objectives of germplasm characterization are: 1. Describe accessions, establish their diagnostic characteristics and identify duplicates; 2. Classify groups of accessions using sound criteria; 3. Identify accessions with desired agronomic traits and select entries for more precise evaluation; 4. Develop interrelationships between, or among traits and between geographic groups of cultivars; and 5. Estimate the extent of variation in the collection.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. UTILIZATION OF PGR (GERMPLASM UTILIZATION) PGR provides basic raw materials to plant breeders and these resources are valuable only if they are utilized for the betterment of mankind. In Nepal, plant breeders have successfully selected superior landraces and blended the landraces in crop breeding schemes as well. Utilization of plant genetic resources can be improved substantially when national priorities are directed towards strengthening national crop research programmes. The PGR or germplasm can be used in a breeding programmes in following ways: 1). it may be directly release as variety 2). it may be subjected to selection for developing a variety 3). it may be used a as parent in hybridization programmes. Utilization of germplasm collections having several thousand accessions is an extremely demanding task. In 1984, Frankel and Brown put forth the concept of core collection to facilitate germplasm utilization. Core collection is a selected and limited set (5–20% of the total collection) of accessions derived from an existing germplasm collection, chosen to represent the genetic spectrum in the whole collection (reserve collection) and including as much as possible of its diversity. Core collection consists of a set of the minimum number of accessions that together represent the genetic diversity of the concerned crop and its wild relatives. Thus a core collection is a subset of the total accessions of a crop; it provides the users most of genetic variability of the concerned crop in a set of workable number of accessions. Therefore, each accession in a core collection is, to some extent, representative of a number of accessions in the gene bank. The land races usually have poor agronomic features and can not be used directly in breeding programmes. Therefore, such accessions that have desirable traits are first crossed with modern varieties and breeding lines to improve their agronomic features. The lines derived from these crosses are usually deficient in certain desirable traits, but they can be used directly in breeding programmes, whereas the original accessions could not be used directly. This phase is often called prebreeding, germplasm enhancement or developing breeding, and is critical for the utilization of primitive, agronomically inferior germplasm. Inadequate number of plant breeders is the major constraint in PGR utilization. Germplasm conserved at ICRISAT genebank has become an important source of diversity available to researchers in both public and private sectors throughout the world.For example, between 1975 and 2007, ICRISAT genebank has distributed over 686,000 samples of its mandate crops and small millets to users in 144 countries .
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Impact of germplasm utilization Genetic resources provide basic material for selection and improvement through breeding to ensure food security needs of the world‘s rapidly rising population. The germplasm accessions can be directly used as superior varieties.. A a vast number of germplasm accessions have been used as building blocks for numerous varieties and hybrids that are cultivated in many parts of the world. Germplasm can contribute significantly towards food security. Germplasm can be shared through joint exploration with international institutes and friendly countries.This enhances good relationship with foreign countries. Introduced varieties can be tested and utilized in plant breeding activities. The elite varieties suitable to local environments can be released and recommended for general cultivation. PGR can provide food, cloths and shelter to the growing population since ancient times. Medicinal plants are being exploited to meet the national needs as Ayurvedic medicines and exported to foreign countries. Acacia catechu is being utilized for extracting Catechin. Paddy and wheat straw and Eulaliopsis binnata are being utilized in paper industries. Orchids and cut flowers are exported and can earn foreign currencies. USES OF PGR COLLECTION PGRS supplies national collections to commodity research programmes for utilization in crop improvement activities. Utilization of PGR in present day crop improvement programs would increase the crop production and productivity. 2.7 AGROBIODIVERSITY POLICY IN NEPAL Policy 1. Prioritize and implement programs on scientific studies, research, extension and other programs for conservation, maintenance and sustainable use of agro biodiversity. 2. Rights to authorize ownership of the agricultural genetic resources of Kingdom of Nepal shall remain with the His Majesty‘s Government, Ministry of Agriculture and Cooperative. 3. The ownership of local agricultural genetic resources shall remain with farmers, farming communities and the His Majesty‘s Government of Nepal for their roles in conservation, maintenance and sustainable use of genetic resources. Policy of Nepal 4. The ownership of traditional knowledge, skills and techniques shall remain with farming communities. 5. Agro biodiversity registration shall be initiated to prepare a document for which the ownership shall remain with the farming communities.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 6. Rights to grant prior informed consent (PIC) for accessing local agricultural genetic resources and traditional knowledge, skills and techniques for foreign institutions shall remain with the National Agro- biodiversity Conservation Committee of the Ministry of Agriculture and Cooperative, His Majesty‘s Government, Nepal. What is the good of the farmers‘ ownership if only the government, and NOT the farmers, has the right to grant (or not to grant) PIC? I can imaging farmers (and NGOs) not liking this part. 7. Prior informed consent (PIC) shall not be required for the scientific studies and research at the national level. What happens if the research turns out later to lead to commercialization? 8. Accessors/ users of genetic resources with the intention of export and commercialization shall apply to NABC for approval with the following details:a) Organization details b) Objective of Access (Research/Commercialization) c) Organizational commitment on the Convention on Biodiversity. d) Organizational policy on farmer‘s right, sharing of benefits and intellectual property rights. e) Organizational Policy on technology transfer. f) Other necessary detail 9. Institutions with prior informed consent for access to genetic resources and traditional knowledge shall undergo in the process of agreement for developing technology and commercialization of accessed genetic resources and knowledge. However, agreements shall not be allowed if it has an adverse effect on environment and biodiversity. 10. IPR for innovation on genetic resources and knowledge shall not be claimed inside or outside Nepal? without prior approval of the NABC.of Nepal 11. Traditional knowledge, skill and techniques on genetic resources / materials shall be protected as per the national IPR protection legislations. 12. Benefits arising from use, commercialization and IP rights of agricultural genetic resources and IKT shall be based on the agreements made with the NABC . 13. One window policy shall be adopted for the registration, ownership, access, use, commercialization and IPR etc of the genetic resources/ materials. 14. Traditional seed system shall be strengthened to protect farmers to-farmers seed exchanges and their access to a wide diversity materials for inclusion in their innovation/production systems. 15. The NABC shall approve and monitor scientific studies and research for i mport and innovation of GMO, LMOs and infectious organisms. 2.8 STATUS OF PLANT GENETIC RESOURCES IN NEPAL Nepal has a diverse environment ranging from warm subtropical in plains to temperate in mountain region of Himalayas. Diverse agro ecological condition and a long history of cultivation has resulted in evolution of a large number of landraces in major cultivated species. Some crops have originated in foot hills of Himalayas, regarded as secondary centre of origin of plant species. Nepal is rich in indigenous wild and landrace plant genetic resources for agronomic, horticultural, forestry, or medicinal uses, but much of this germplasm remains uncollected and
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. awaits economic development. Existence of wild rice species and related wild taxa found in Nepal. The genetic erosion is maximum in rice and wheat and intermediate in maize. The institutions namely Nepal Agricultural Research Council (NARC), Royal Nepal Academy of Science and Technology (RONAST), Ministry of Forest and Soil Conservation, Tribhuwan University, Non Government Organizations (NGOs): LI-BIRD, Donor Agencies etc. are involved in plant genetic resources conservation in Nepal. 3. Genetic Basis of Plant Breeding 3.1 Cell division and molecular biology, 3.2 Partitioning of genetic variance, 3.3 Hybridization and selfing, 3.4 Modes of reproduction and pollination, 3.5 Inbreeding depression and heterosis, 3.6 Mating systems, 3.7 Heritability, 3.8 Selection and response to selection, 3.9 Choice of breeding methods, 3.10 Genotype x environment interaction, 3.11 Combining abilities (GCA and SCA). 3.1 CELL DIVISION AND MOLECULAR BIOLOGY CELL DIVISION: Cell division occurs as either mitosis or meiosis: 1. MITOSIS: Is an essential process for increasing number of cell Allows accurate replacement of old cells by new cells and ensures that all of the somatic cells of an organism have same genetic composition Is only means of reproducing for asexual species. Stages of mitosis: prophase: chromosome thicken and shorten nuclear membrane disintegrates nucleolus disappears centrioles migrates to opposite pole of cells
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. spindle apparatus forms from centrioles towards late prophase, spindle fibres attach to kinetochores. Metaphase: Chromosomes separate themselves in equatorial plane of spindle Anaphase: Separation of sister chromatids with division of centrioles sepsichro Chromatids are pulled to opposite ends of the cells Centromeres are pulled towards the poles with arms trailing behind. Telophase: Cytokinesis, the division of cytoplasm Reversal of prophase: reform nuclear membrane, spindle apparatus disappears, chromosomes uncoil and nucleoli reform 2. MEIOSIS:  Occuring in reproductive cells which produces 4 haploid cells from a single diploid cells  Mechanism for the reproduction of chromosomal compliment prior to fertilization and zygotene formation  Allows a halving of chromosomal complement such that each gamete receives one member of each homologous pair  Includes 2 successive nuclear divisions. a) Meiosis I or reductional division b) Meiosis II or equational division: It is similar to mitosis. Stages of Meiosis I: 1. Prophase I: it includes 5 substages a) Leptonema: it is first stage - chromatin begins to condense b) Zygonema: it is second stage - homologous chromosomes are attracted to each other and pair up (synapse) - synaptonemal complex forms between homologues c) Pachynema: it is third stage - chromosomes continue to shorten and sister chromatids become obvious exchange of material between chromatids occurs - synaptonemal complex begins to disappear d) Diplonema: it is fourth stage: sister chromatids begin to separate except at chiasmata
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. e) Diakinesis: it is the fifth stage - chromosomes continue to shorten - chromosomes continue to repel each other and chiasmata move to ends of terad - spindles attach to centromers. 2. Metaphase I: -chromosomes align (separate) along the equatorial plane of cell 3. Anaphase I: - Homologous chromosomes are pulled apart by spindle fibres -centromeres do not divide, so that only homologous chromosomes are separated, not chromatids - results in a dyad (pair of sister chromatids) at each pole. 4. Telophase I: -Division of cytoplasm -reform nuclear membrane -spindle apparatus disappears -chromosomes uncoil Nucleoli reform Interphase: some cells may enter an interphase but no DNA synthesis will occur Stages of Meiosis II: (Equational division): Like mitosis 1. Prophase II 2. Metaphase II 3. Anaphase II 4. Telophase II DIFFERENCES BETWEEN MITOSIS AND MEIOSIS Mitosis 1) chromosome equally distributed into two daughter nuclei 2) occurs in somatic cells 3) whole process of division is completed in one sequence 4) prophase is shorter durational 5) no pairing occurs in chromosomes Meiosis 1) it gives rise 4 haploid nuclei having half number o f chromosome as compared to parent nucleus. 2) occurs in germ cells 3) it includes two successie division 4) prophase is long durational and is differentiated into five sub phases 5) chromosomes pair and form dyads sister
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 6) no crossing over and no chiasmata formation occurs 6) crossing over and chiasmata formation occur. 7) two cells are formed by this division. 7) four cells are formed by this division MOLECULAR BIOLOGY Molecular biology is a branch of biology that seeks to understand the molecular basis of life. In particular, it relates the structure of specific molecules of biological importance -such as protein, enzymes and nucleic acids DNA and RNA- to their functional roles in cells and organism. Molecular Biology, branch of biology that seeks to understand the molecular basis of life. In particular, it relates the structure of specific molecules of biological importance —such as proteins, enzymes, and the nucleic acids DNA and RNA—to their functional roles in cells and organisms. Structure of DNA: The following are the Watson and Crick conclusions regarding the structure of the DNA model; 1.two single stranded chains 2.purine are opposite a pyrimidine 3.chains held together by H-bonds: Guanine is paired with cytosine by three H-bonds Adenine is paired with thymine by two H-bonds 4.anti-parallel orientation of two chains 5.the molecule is stabilized by:  large number of H-bonds  Hydrophobic bonding between the stacked bases. 3.2 PARTITIONING OF GENETIC VARIANCE Variance due to genotypes of different plants or strains is called genetic variance. It is estimated from trials using homozygous lines arises from differences between homozygotes. Fisher in 1918 divided genotypic variance into 3 components; 1) Additive component: it is component arising from differences between two homozygotes for a gene, e.g. AA and aa 2) Dominance component: it is due to deviation of heterozygotes (Aa) phenotype from the average of phenotypic values of the two homozygotres (AA and aa). 3) Interaction or epistatic component: it results from an interaction between two or more genes. Genetic variance (Vg) is partitioned as following; Vg = Va + Vd + Vi Where, Va is additive genetic variance
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Vd is dominance genetic variance Vi is interaction variance. 3.3 HYBRIDIZATION AND SELFING The mating or crossing of two plants or lines of dissimilar genotypes is called hybridization. . In plants, mating is done by placing pollen grains from one genotype, called male parent, onto the stigma of flowers of the other genotype, called female parent. Hybridization is most common method of crop improvement. Objectives of hybridization: 1. to create genetic variation 2. transfer of one or few qualitative characters into a single variety from another varieties (combination breeding). 3. Improvement in one or more quantitative characters through trasgressive segregation (transgressive breeding). 4. use of F1 as a hybrid variety Types of hybridization: 1. Intervarietal hybridization (Intraspecific hybridization) It is crosses between parents belong to the same species. Types: a. Simple cross: two parents are crossed to produce F1 b. Complex cross: more than two parents are crossed to produce hybrids 2. Distant hybridization (Interspecific hybridization): it is crosses between different species of the same genus or of different genera. Procedure of Hybridization: 1) Choice of parents: At least one of the parents should be higher yielder, well adapted, popular or having desirable quality. 2) Evaluation of parents: If the performance of the parents in the area is not known, evaluation of parents is done. Selfing of parent is done to eliminate the undesirable characters and obtaining inbreeds. 3) Emasculation: It refers to removal of stamens or anthers or the killing of pollen grains of a flower without affecting in any way the female reproductive organs. It is done to prevent self fertilization in flower line/variety to be used as female parents. Methods like hand emasculation, suction
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. method, hot water treatment, alcohol treatment, cold treatment, genetic emasculation etc are used for emasculation. The emasculation should be done before anthers are mature and the stigma becomes receptive. 4) Bagging: Immediately after emasculation, flower or inflorescence are enclosed in suitable bags of appropriate size to prevent random cross-pollination. Bags are tied to base of inflorescence or stalk of the flower with wire, thread or pin. Remove bags 2-3 days after pollination. 5) Pollination: it consists of collecting pollen from freshly dehisced anthers of male parent and dusting this pollen onto the stigma of emasculated flowers. 6) Tagging: The emasculated flowers are tagged just after bagging. The information regarding date of emasculation, date of pollination, name of male and female parents, name of breeders should be recorded on tags. 7) Harvesting and storing the F1 seed: The crossed heads or pods should be harvested and threshed. The seeds should be dried and properly stored to protect from storage pests. Proper care should be taken to avoid mixture of other seeds. The seeds from each cross should be kept separately and preferably, the seeds should be kept along with the original tags. Difficulties in hybridization: A more serious problem of hybrid (F1) mortality due to lethal genes is encountered in several cross combinations of some crops. Hybrid necrosis occurs in F1 wheat plants. In mild case of necrosis, leaf tips are affected but in severe case of necrosis whole plant may die. SOMATIC HYBRIDIZATION Production of hybrid plants through fusion of protoplasts of two different plant species/varieties is called somatic hybridization and such hybrids are known as somatic hybids. Steps of somatic hybridization: 1) isolation of protoplast: it is done by treating cells/tissues with a suitable mixture of cell wall degrading enzymes (a mixture of pectinase, 0,1-1% and cellulose, 1-2 %). 2) Fusion of protoplast: the two protoplasts of desired stains/species are mixed in equal proportion, then subjected to high pH and high ca concentration or treated with 28-50 % polyethylene glycol. 3) Selection of hybrid cells: some visual markers e.g. pigmentation, of parental protoplasts is used for identification of hybrid cells under microscope; hybrid cells are then isolated and cultured.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 4) Regeneration of hybrid plants: once the hybrid calli are obtained, plants are induced to regenerate from them. SELFING The objective of selfing is to avoid cross –pollination and to ensure self pollination. The technique of selfing varies from one crop to the other depending upon their mode of reproduction. In the self pollinated crops, selfing is natural mode of reproduction, and to ensure selfing no operation is needed. But in case of often cross pollinated species, the flowers are generall y bagged to prevent cross pollination. In case of cross pollinated species with bisexual flowers or with both male and female flowers in a single inflorescence, bagging the entire inflorescence or sometimes the whole plant is adequate. The bags may be shaken daily to help pollen dissemination and pollination. In crops like maize, male and female inflorescences are bagged; pollen is collected in the tassel bag and dusted onto the silk of female inflorescence. Alternatively, the tassel may be cut and inclosed in the bag covering cob. The cut end of tassel may be kept in water contained in a small bottle to keep the tassel alive for a longer period of time. Selfing increases homozygosity with a corresponding decrease in heterozygosity. 3.4 MODES OF REPRODUCTION AND POLLINATION Modes of reproduction: 1) Asexual reproduction: It does not involve fusion of male and female gametes. Types: a) Vegetative reproduction: New plants may develop from vegetative parts of plant b).Apomixis: New plants may arise from embryos that develop without fertilization. 2) Sexual reproduction: It involves fusion of male and female gametes to form a zygote, which develops into an embryo. Modes of pollination: 1. Self pollination: Pollen from an anther may fall on to the stigma of the same flower Mechanism promoting self pollination are: a.cleistogamy: flowers do not open at all b. chasmogamy: flowers open but only after pollination takes place 2. Cross pollination: Transfer of pollen grains from flowers of one plant to stigmas of flowers of another plant is called cross pollination. Cross pollinatiton preserves and promotes heterozygosities. The mechanism promoting cross pollination are following: a.Dicliny or unisexuality: Condition in which flowers are either pistillate or staminate. b. Dichogamy: stamens and pistils of hermaphrodite flower mature at different time
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. c. Self incompatibility: failure of pollen from a flower to fertilize the same flower or other flowers on the same plant d. Male sterility: absence of functional pollen grains. Why modes of reproduction and pollination is important in plant breeding?. 1. mode of reproduction and pollination determine genetic constitution of a species. Cross pollinated crop species are highly heterozygous and show less vigour but self pollinated crop species are homozygous and not show inbreeding depression. 2) They determine the ease in pollination control. In the self pollinating crops, selfing occurs naturally, while in cross pollinated crop species, flowers have to be pollinated and protected from foreign pollen. 3) They determine stability of varieties after release. Self pollinated crop varieties are fairly stable in their genetic constitution. Farmers may plant same seed for several years to avoid off types but in cross pollinated crops, precaution taken to avoid contamination of foreign pollen. 3.5 INBREEDING DEPRESSION AND HETEROSIS INBREEDING DEPRESSION Inbreeding depression is reduction or loss in vigour and fertility as a result of inbreeding. The degree of inbreeding depression depends on plant species. a. High inbreeding depression: e.g. alfalfa, carrot b. Moderate inbreeding depression: e.g. maize, jowar, bajra c. Low inbreeding depression: e.g. onion, sunflower d. Lack of inbreeding depression: e.g. self pollinated species. Effects of Inbreeding: 1. appearance of lethal and sublethal alleles 2. reduction in vigour 3. reduction in reproductive ability 4. separation of the population into distinct lines 5. increases in homozygosity 6. reduction in yield. HETEROSIS (HYBRID VIGOUR) The term heterosis, also known as hybrid vigor or outbreeding enhancement, describes the increased strength of different characteristics in hybrids; the possibility to obtain a genetically superior individual by combining the virtues of its parents.. Heterosis is superiority of an F1 hybrid over both its parents in terms of yield or some other character. Heterosis can be classified
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. as: 1. Mutational heterosis: it results from dominance gene action 2. Balanced heterosis: it results from over dominance gene action. Manifestation of heterosis: 1. increased yield 2. increased reproductive ability 3. increase in size and general vigour 4. better quality 5. earlier flowering and maturity 6. greater resistance to disease and pests 7. greater adaptability 8. faster growth rate 9. increase in number of a plant part. 3.6 MATING SYSTEMS The scheme according to which individuals or lines are mated to produce sexual progeny is called mating system. Mating system is determinant for genetic differentiation of inter-population and intro-populations. As a bridge connecting two generations, mating system also determines the genotype distribution and population dynamic of offspring populations. There are following 5 basic mating schemes: 1) Random mating: Random mating is a system of mating in which an individual has equal chance of mating with every other individual of the same population. In this system of mating, each female gamete is equally likely to unite likely to unite with any male gamete and the rate of reproduction of each genotype is equal, i.e. there is no selection. Random mating is useful for production and maintenance of synthetic and composite varieties. 2) Genetic assortative mating: In this system of mating, the mating occurs between individuals that are more closely related by ancestry than in random mating. This is more commonly known as inbreeding. Genetic assertive mating is useful in development of inbreds. 3) Genetic Disassortative mating: Mating is done between individuals which are less closely related by ancestry. In this system of mating, totally unrelated individuals are mated. These individuals often belong to different populations. Examples of genetic disassortative mating are intervarietal and interspecific crosses. This mating reduces homozygosity and increases heterozygosity. 4) Phenotypic Assortative mating:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. In this system of mating, mating is carried out between individuals which are phenotypically more similar. This mating system is useful in the isolation of extreme phenotypes. It is used in some breeding scheme, e. g. in recurrent selection. 5) Phenotypic Disassortative mating: In this system of mating, mating is done between individuals which are phenotypically dissimilar belonging to the same population. This mating system is useful in making a population stable i. e. in maintaining variability. 3.7 HERITABILITY The ratio of genetic variance to the total variance i. e. phenotypic variance is known as heritability. Heritability is important in plant breeding because it helps to appreciate the proportion of variation that is due to genotypic (broad sense heritability) and additive ( narrow sense heritability ) effects. If h2 of a character is very high i.e. 0.8 or more, selection for such a character should be fairly easy. But if h2 is low i.e. less than 0.4, selection may be considerably difficult or virtually impractical due to the masking effect of environment on the genotypic effects. Types of heritability: 1.Broad sense heritability ( heritability of 0.8): The extent to which phenotypes are determined by their genotype is known as heritability in board sense. H2 bs = VG/VP 2. Narrow sense heritability (heritability of 0.3) : The ratio of additive component of variance to the total phenotypic variance is called narrow sense heritability H2 ns= VA/VP=VA/VG+VE=VA/VA+VD+VI+VE How does a heritability of 0.8 differ from 0.3: In heritabily of 0.8 (broad sense heritability), the proportion of variation is due to genetic effect. Here a relatively smaller contribution of environment to phenotypes and selection for the character is easy. But in case of heritability of 0.3 (narrow sense heritability), the proportion of variation is due to additive effects. Here masking effect of environment on genotypic effects and selection for the character is difficult or impossible. Methods of calculating heritability: 1. parent offspring regression method 2. Analysis of variance method 3. Six generation mean analysis method
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 3.8 SELECTION AND RESPONSE TO SELECTION SELECTION The differential reproduction rates of different genotypes is called selection. In theory, selection in a random mating population is able to 1. change the gene and genotype frequencies 2. produce new genotypes due to changed gene frequencies 3. cause a shift in mean in the direction of selection 4. change the variance of population to some extent. TYPES OF SELECTION Selection is of two types; a. Natural selection: it is due to natural forces like climate, soil, biological factors and other factors of environment. b. Artificial selection: it is carried out by man. On the basis of type of phenotypic classes, selection is of following types: 1) Directional selection: selection for individuals having extreme phenotype for a trait or group of traits. 2) Stabilizing selection : selection for individuals having intermediate phenotype. 3) Disruptive selection: selection of a different phenotypic optima in ecological niche so that population consists of two or more recognizable forms. RESPONSE TO SELECTION: Response to selection: The effects of selection in random mating populations is called response to selection. Types of response to selection: 1.Rapid gain followed by slow response: Selection produces rapid gains for some generations, which is followed by a period of slow gain under selection. This is typical of characters like plant height, resistance to diseases, days to flowering, color etc. 2.Slow progress for a long period: Selection produces slow progress for a large number of generations. The variability in the selected population after 50 generations of selection was comparable to that in the original population. The selection for high oil content in Burr‘s white maize is example of such response.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 3.Slow response for a short period: Selection for some characters shows slow gain for several generation, which ends in a plateau. The selection for low oil content in Burr ‘s white maize is example of such response. 4.Lack of response: Selection for some characters produces little or no gain. A typical example or this type of response is provided by selection for yield in maize. 5.Rapid gain-plateau-rapid gain response: Selection for some characters shows a period of rapid gain, followed by a plateau, which is followed by another period of rapid gain. The selection for increased abdominal bristle number in fruit fly is example of such kind of response. 3.9 CHOICE OF BREEDING METHODS The appropriate method of breeding would depends on how well the methods complements other aspects of breeding program. For the self pollinated crops, pureline selection, pedigree selection, bulk selection, back cross selection and single seed descent selection methods are used. The breeding methods like progeny selection (half sib, full sib, selfed progeny selection), recurrent selection (simple RS, reciprocal RS, RS for GCA, RS for SCA) are used for breeding cross pollinated crop species. Mass selection method is used for both self pollinated and cross pollinated crop species. For the transfer of disease and insect reisistance gene from one crop varieties to another crop varieties, generally back cross method of selection is used. 3.10. GENOTYPE X ENVIROMENT INTERACTION (G X E INTERACTION) Genotype X Environment interaction (GXE) is a common phenomenon in agricultural research.The association between the environment and the phenotypic expression of a genotype constitute the GXE interaction. The GXE interaction determines if a genotype is widely adapted for an entire range of environmental conditions or separate genotypes must be selected for different sub environments. When GXE interaction occurs, factors present in the environment (temperature, rainfall, etc.), as well as the genetic constitution of an individual (genotype), influence the phenotypic expression of a trait. The impact of an environmental factor on different genotypes may vary implying that the productivity of an animal or plant may also vary from one environment to the next. Breeding plans may focus on the GXE interaction to select the best genotype for a target population of environments. A basic principle indicated by the GXE interaction is that even if all plants were created equal (same genotypes), they will notnecessarily express their genetic potential in the same way when environmental conditions (drought, temperature, disease pressure, stress, etc.) vary. This important concept may require genetic engineering ofplants or animals specifically tailored to theirenvironmental conditions. Genotypes are normally tested over a widerange of diverse environments (e.g., locations, years, growing seasons) and agricultural experiments involving GXE interactions. G X E interaction is a measure of effects of different levels of an environment in the relative performance among genotype. G X E interaction describes the differential performance of genotypes/lines/varieties over environments. This interaction takes place because the different
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. genes have different genotypic values at different environment and different genes are active at different environments G X E interaction signifies that the relative performance of various genotypes is affected by environment. The performance of genotype A may be superior to that of genotype B in one environment, but in another environment it would be inferior to that of B. However if G X E interaction is absent, genotype A will be superior (or inferior) to B in all environment Example of G X E interaction , Mansuli variety of rice increases in yield with increasing in fertility status but Jaya remains constant Statistically G X E interactions occurs if performance of genotypes varies significantly across enviroments. (Fig.1). otherwise no G x E interaction occurs (Fig.2). The Presence of the GXE interaction indicates that the phenotypic expression of one genotype might be superior to another genotype in one environment but inferior in a different environment There are two types of G X E interaction: 1. Quantitative G X E interaction: response curves of genotyps over enviroments do not cross each other 2. Qulaitative G X E interaction: response curves of genotypes over enviroments cross each other. GEI is calculated as ўij = μ+Gi +Ej + (G X E)ij When relative level of performance of genotype are consistent over the levels of environment, GEI is said to be low or absent and when the ranking of performance among genotype changes significantly over levels of environment, the interaction is said to be large. AMMI Model The Additive Main effects and Multiplicated Interaction (AMMI) model combines regular analysis of variance for additive effects with principle component analysis for multiplicated
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. structure within interaction. It provides a powerful analytical tool to interpret large genotype X environment X replication tables without missing values. 3.11 COMBINING ABILITIES (GCA AND SCA) The ability of a strain to produce superior progeny upon hybridization with other strains is called combining ability. There are two types of combining abilities. General combining ability (GCA) Specific combining ability (SCA) 5. It is average performance of a strain in a series of crosses 6. It is due to additive genetic variance and additive X additive epistasis 7. It is estimated from half-sib families 8. It helps in the selection of suitable parents for hybridization 9. It has relationship with narrow sense heritability 1. It refers to the performance of specific cross in relation to GCA 2. It is due to dominance genetic variance and all the 3 types of epistasis 3. It is estimated from full sib- families 4. It helps in identification of superior cross combinations 5. It has relationship with heterosis 4. Biometrical Techniques in Plant Breeding 4.1 Assessment of variability, 4.2 Genetic diversity, 4.3 Correlation coefficient, correlation and path analysis, 4.4 Diallele cross analysis, 4.5 Experimental designs, 4.6 Analysis of co- variance, 4.7 Chi-square test, 4.8 Stability analysis, 4.9 Field Plot Technique 4.10. Regression and correlation analysis 4.11. Different computer statistical packages and their use in plant breeding. Biometry or biometrics is the science that deals with the application of statistical procedures to the study of biological problems. The various statistical procedures employed in biometical genetics are called biometrical techniques. Biometrical techniques are useful to the plant breeders in the following four different ways; 1) in the assessment of genetic variability present in a population (range, variance, standard deviation, coefficient of variation, standard error, D 2 statisctic, metroglyph analysis)
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2) in the selection of elite genotypes from mixed populations (correlation, path and discriminant function analyses) 3) in the choice of parents and breeding procedures ( diallel partial diallel, line x tester, generation means, triallel, quadriallel, biparental cross and triple testcross analyses) and 4) in determining the varietal adaptation (stability analysis). 4.1 ASSESSMENT OF VARIABILITY Following measures are used to assess the variability. 1.Range: it is difference between the lowest and the highest values present in the observations included in sample 2.Arithmetic Mean: it is computed by dividing the sum of all the observations in a sample by their number 3.Variance: it is expressed as the sum of squares of deviations of all observations of a sample from its mean divided by the degree of freedom (N-1). 4.Standard deviation: it is square root of variance. 5.Coefficient of variation: it is percent ratio of standard deviation of a sample to its mean 6.Standard error: it is estimated by dividing the estimate of standard deviation by the square root of the number of observations in sample. 4.2 GENETIC DIVERSITY The variability present among different genotypes of a species is called genetic diversity. Genetic diversity is a level of biodiversity that refers to the total number of genetic characteristics in the genetic makeup of a species. It is distinguished from genetic variability, which describes the tendency of genetic characteristic to vary. Genetic diversity arises either due to geographical separation or due to genetic barriers to crossability. Importance of genetic diversity: genetic diversity plays an important role in plant breeding because hybrids between lines of diverse origin generally display a greater heterosis than those between closely related strains. The magnitude of heterosis increased with genetic divergence in morphological traits and flowering time and also geographical origin of parents. For example, in maize, increased genetic difference between inbred lines resulted in a greater heterosis in their hybrids. The genetic diversity is assessed by following two biometric techniques. 1.D2 Statistic: It measures the forces of differentiation at two levels, namely, intracluster and intercluster levels and thus helps in the selection of genetically divergent parents for exploitation in hybridization programs. In addition to aiding in selection of divergent parents for hybridization, D2 statistic
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. measures the degree of diversification and determines the relative proportion of each component character to the total divergence. The genotypes grouped together are less divergent than the ones, which are placed in different clusters. The clusters which are separated by the greatest statistical distance, show the maximum divergence.D 2 statistic is used in assessing variability present in crops like maize, jowar, bajra, wheat, cotton etc. Following points should be taken into consideration while selecting parents on basis of D2 statistic; a) relative contribution of each character to the total divergence, b) the choice of clusters with the maximum statistical distance and c) selection of one or two genotypes from such clusters. 1. Metroglyph Analysis: It is a semigraphic method of studying variability in a large number of germplasm lines taken at a time. The analysis of variation is based on the mean values for different traits. To begin with, two characters exhibiting the highest variability are identified. One of the traits is used a the X-axis, while other is plotted on the Y-axis. For each line, the mean values X and Y are used to determine its position, which is marked by a small circle. Thus each line is represented by a small circle on the graph. For easy identification, exotic lines may be represented by solid circles, while indigenous lines may be denoted by open circles. The other characters for the different lines are represented by rays on the respective circles. The ray for each character occupies a definite position on the circle. The range of variation in a character is represented by the variation in the length of the corresponding ray on all the circles. For convenience, mean values for each trait are classified into 3 groups;i) low (index score, ii) medium (score 2) and iii) high (score 3). The length of each ray on circle, as a result is either short (low mean value), medium (medium mean value) or long (high mean value). A circle along with rays emanating from it is called a glyph. The worth of an individual line is assessed from sum of index scores for all characters represented in graph. The maximum and minimum scores that an individual line can get will be 3n and n, respectively, where n is the total number of character studied. 4.3 CORRELATION COEFFICIENT, CORRELATIN AND PATH ANALYSIS CORRELATION COEFFICIENT: The statistic that measures the relationship between two or more variables is called correlation coefficient. Types of correlation coefficient: i. Simple correlation: It is association between any two variables. It is estimated from both unreplicated as well replicated data using the following formulae:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. ii. Partial correlation: when the correlation between two variables, say X1 and X2 is estimated by taking into account the effect of a third variable, e. g. X3, it is called partial or net correlation iii. Multiple correlations: The estimate of the joint influence of two or more variables on a dependent variable is called multiple correlation. PATH ANALYSIS: In statistics, path analysis is used to describe the directed dependencies among a set of variables and can lead on to a type of multiple regression analysis.Path analysis is a standardized partial regression coefficient analysis, which splits the various correlation coefficients into the measures of direct and indirect effects of a set of independent variables on a dependent variable (usually yield). For e.g. in the black gram, grain yield (X5) is affected by number of primary branches (X1), secondary branches (X2), pods/plant (X3) and seeds/pod (X4). The path analysis shows whether the association of these characters with yield is due to their direct effects on yield, or it is a consequence of their indirect effects through some other traits. If the correlation between yield and a character is due to direct effect of character, it reflects a true relationship between them and selection can be practiced for such a character to improve yield. But if the correlation is mainly due to indirect effect of the character through another component trait, breeder has to select for the latter trait through which indirect effect is exerted. Path analysis is carried out us ing estimates of correlation coefficients. To begin with, all possible correlations among the dependent and independent variables are worked out. Path analysis is undertaken in following three steps; X 1 r1 2 r1 r1 X2 3 5 r2 5 r1 r3 4 r2 3 X3
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. X1 : primary branch X2 : secondary branch X3 : pods/plant X4 : seeds/pod Y: yield R: correlation coefficient For measurement of direct effects, a path diagram is constructed using estimates of simple correlation coefficients, with the help of path diagram, simultaneous equations are developed as following: r15 = P15 + r12 . P25 + r13 . P35 + r14 . P45 r25 = r12 . P15 + P25 + r23 . P35 + r24 . P45 r35 = r13 . P15 + r23 . P25 + P35 + r34 . P45 and r45 = r14 . P15 + r24 . P25 + r34 . P35 + P45 where, r12 , r13 , r14 etc. are estimates of simple correlation coefficients between variables X1 and X2 , X1 and X3 , X1 and X4 etc. respectively, and P 15 , P25 , P35 and P45 are estimates of direct effects of variable X 1 , X2 , X3 and X4 respectively, on dependent variable X5 (in this case yield). After putting values of the correlation coefficients in these equations, the values of P 15 , P25 etc are estimated by process of elimination. Indirect effects are computed by putting values of correlation coefficients and those of direct effects as described below. The indirect effects of primary branches (X 1 ) via the other traits are estimated as below; Via secondary branches (X 2 ) = r12 . P25 Via pods/plant (X 3 ) = r13 . P35 Via seeds/pod (X4 ) = r14 . P45
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. The indirect effects of other component traits e.g. pods/plant (X 3 ) and seeds/pod (X4 ) may be computed in similar fashion. Using the values of direct effects and correlation coefficients, the residual effect is estimated according to the following formula; the equation is based on the earlier example of black gram. 1 = P2R5 + P15 . r.15 + P25 .r25 + P35 .r35 + P45 . r45 where P2 R5 is square of residual effect. 4.4 DIALLELE CROSS ANALYSIS Diallele cross refers to all possible crosses among n lines, and the analysis based on such a set of crosses is known as diallele cross analysis. It has been extensively used in both self and cross pollinated species to understand the nature of gene action involved in expression of quantitative traits. Assumptions for diallele analysis: 1) normal diploid segregation 2) lack of maternal effects 3) absence of multiple alleles 4) homozygosity of parents 5) absence of linkage among genes affecting characters 6) lack of epistasis 7) random mating. There are two approaches for diallele analysis: 1) Hayman’s Graphical approach: It is based on the estimation of components of variation . Following six componets of variation are estimated; D = additive genetic variance. H1 = dominance variance. H2 = H1 {1 - (u-v)2 }, where u and v are proportions of positive and negative genes, respectively in parents. E = expected environmental component of variance F = mean of Fr over the arrays, where, Fr is covariance of additive and dominace effects in a single array. h 2 = dominance effect, as algebric sum over all the loci in heterozygous phase in all crosses 2) Griffing’s Numerical approach: it is based on the estimation of GCA ( general combining ability) and SCA (specific combining ability) variances and effects. In this approach, gene action is deduced through the estimates of GCA and SCA variances and effects.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. The GCA component is primarily a function of additive genetic variance. But if epistasis is present, GCA will include the additive x additive interaction component as well. On the other hand. The SCA variance is mainly a function of dominance variance, but it would include all the three types of epistatic interaction components , if epistasis is present. Appilcation of diallel analysis is used in both self and cross pollinated crop species to understand the nature of gene action involved in the expression of quantitative traits. Merits of diallel analysis: it provides a sensitive approach to large scale studies of quantitative characters. It yields reliable information on the components of variance, and on GCA and SCA variances and effects. Thus it helps in the selection of suitable parents for hybridization as well as in the choice of appropriate breeding program. Demerits of diallel analysis: this technique can test only a limited number of parents at a time. This is because for every increment of 1 in the number of parents, number of crosses increases by 2n – 2 in case of a diallel set including the reciprocals (where, n is the number of parents after the increment.). Partial dialle analysis: Number of sampled cross per parent or per array in all possible combination of a given sets of parents. K= (n+1-S) where, n= numbers of parents, S=number of sampled crosses and K=constant 2 4.5 EXPERIMENTAL DESIGNS Design of Experimental Techniques Commonly Used in Agricultural Research: Design of experiments deals with the study of methods for comparing the treatment, varieties, factors etc. under different experimental situations faced by agricultural research worker. The main objective of any experimental design is to provide the maximum amount of information relevant to the problem under the investigation. Experimental design provides maximum amount of information at minimum cost. There are three basic principles of experimental design. Replication Randomization Local Control Replication means repetition of the basic experiment. It is useful for more precise estimate of the mean effect of any factor and it is also useful for estimation of experimental error and determination of confidence interval. Randomization is the technique or device for eliminating the bias. Local Control: The purpose of the local control is to make the experimental design more efficient and reduce the experimental error.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Experimental Design and Design of Experiment: We are concerned with the analysis of data generated from an experiment. It is wise to take time and effort to organize the experiment properly to ensure that the right type of data and enough of it is available to answer the questions of interest as clearly and efficiently as possible. This process is called experimental design. We should also attempt to identify known or expected sources of variability in the experimental units since one of the main aims of a designed experiment is to reduce the effect of these sources of variability on the answers to questions of interest. That is, we design the experiment in order to improve the precision of our answers. Treatment: In experiments, a treatment is something that researchers administer to experimental units. For example, a rice field is divided into four, each part is 'treated' with different fertilizer to see which produces the most rice; a teacher practices different teaching methods on different groups in the class to see which methods yield the best results; a doctor treats a patient with different treatments to see which is most effective. Factor: A factor of an experiment is a controlled independent variable; a variable whose levels are set by the experimenter or researcher. A factor is a general type or category of treatments. Different treatments constitute different levels of a factor. For example, three different groups of farmers are subjected to different training methods. The farmers are the experimental units, the training methods, treatments; where the three types of training methods constitute three levels of the factor 'type of training'. Main Effect: This is the simple effect of a factor on a dependent variable. It is the effect of the factor alone averaged across the levels of other factors. Interaction: An interaction is the variation among the differences between means for different levels of one factor over different levels of the other factors. Blocking: This is the procedure by which experimental units are grouped into homogeneous clusters in an attempt to improve the comparison of treatments by randomly allocating the treatments within each cluster or 'block'. 1. Completely Randomized design (CRD): when the treatments are arranged randomly over whole set of experimental units, the design is called CRD. It is used when field is homogeneous. The structure of the experiment in a CRD is assumed to be such that the treatments are allocated to the experimental units completely at random. In the design of experiments, CRDs are for studying the effects of one primary factor without the need to take other irrelevant variables into account. The CRDs have one primary factor. The experiment compares the values of a response variable based on different levels of that primary factor. For CRDs, the levels of the primary factor are randomly assigned to the experimental units. It is the simplest design for researcher of agricultural sciences based on principle of randomization and replication. In this design of experiment, treatments are allotted randomly on experimental unit over the entire experimental material.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2. Randomized Complete Block Design (RCBD): When the treatments are assigned at random to a group of plots, the design is known as RCBD. It includes two principles ; formation of blocks and randomization of treatments. it is used when fertility gradient of field is in one direction, maximum treatments 15-20 The RCB is the standard design for agricultural experiments. The field is divided into units to account for any variation in the field. Treatments are then assigned at random to the subjects in the blocks-once in each block. In other words the RCBD is a design in which the subjects are matched according to a variable which the experimenter wishes to control. The subjects are put into groups (blocks) of the same size as the number of treatments. The members of each block are then randomly assigned to different treatment groups. 3. Latin Square Design (LSD): when the available experimental units are known to be subject to two major sources of variation which can be controlled by a double blocking of the units, the design is known as LSD. In LSD, number of treatments= number of replications and number of rows= number of columns= number of treatments. it is used when field having fertility gradient in two directions Latin Square Design: In randomized block design (RCBD) whole experiment is divided into homogeneous group and treatments are allocated randomly, but in the field of experimental area are not homogeneous. For example, land fertility varies in strips or high or low level fertility. In the design of experiments, Latin squares are a special case of row-column designs for two blocking factors: Many row-column designs are constructed by concatenating Latin squares. Layout of Design: In the Lattin Square Design, number of treatments (m) is equal to number of replications (n). ABDC BACD DCBA CDAB Two way elimination of variation as a result of cross grouping often results in small error mean sum of square. 4. Factorial Experimental Design: Experiments in which the effect of more than one factor are under investigation are known as factorial experiments. Thedesign adopted to carry out the experiment is termed as factorial experimental design. It is used when two or more factors are to be tested simultaneously with same direction. Factorial indicates the effect of several factors at different levels estimate the effects of each factors and also the interaction effect. In other words, a factorial design is used to evaluate two or more factors simultaneously. The treatments are combinations of levels of the factors. The main feature of factorial designs over one-factor at a time experiment is that they are more efficient and they allow interactions to be detected. In the simple example, if two fertilizers potash (K) and nitrogen (N) used and let p different levels of potash and K different levels of nitrogen and interaction NK. General form of factorial design experiment is Sn factorial design with n factors each at s level.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 5. Split-plot Design: The layout in which, each block is divided into number of plots known as main plots, equal to number of levels of the first factor and then main plot is subdivided into number of sub plots equal to the number of levels of second factor. The levels of the first factors are randomized in the main plots of each block and levels of second factor are randomized in subplots of each main plot. If there is a third factor also which is to be studied with still more precision as compared with the second factor, subplots will again further split into smaller ultimate plots to be assigned to level of third factor. Such type of design is called split plot design. It is used when two or more factors are to be tested simultaneously with different direction. Experimental Error: It is defined as plot to plot variation which are treated alike. Errors in field trails are of three types; 1) due to soil heterogeneity, 2) due to faulty technique and 3) random or chance error. Errors can be minimized or controlled by following ways Control of error: 1) Blocking or local control: putting the experimental units that are similar as possible together in the same groups (blocks) and by assigning all treatments into the block separately and independently 2) Proper plot techniques: treatment must be maintained uniformity for all plots in the experiment. 3) Proper choice of data analysis: proper choice of data analysis such as using covariance can reduce the variability among experimental units. Estimation of error: 1) Replication: to estimate the experimental error, a treatment has to be applied to at least twice i.e. two experimental units. Applying treatments to experimental units, which are homogeneous reduce experimental error 2) Randomization: Randomization ensures that each treatment well have an equal chance being assigned any experimental plots. So process of randomly allotting the treatments in experimental units prevent bias is called randomization. Boarder effect and intervarietal competition should be overcome by planting multiple rows and discarding boarder harvesting. The split-plot design is an experimental design that is applicable when a factorial treatment structure has two levels of experimental units. In the case of the split-plot design, two levels of randomization are applied to assign experimental units to treatments. The first level of andomization is applied to the whole plot and is used to assign experimental units to levels of treatment factor A. The whole plot is split into sub plots and the second level of randomization is used to assign the sub plot experimental units to levels of treatment factor B. Since the split-plot design has two levels of experimental units, the whole plot and sub-plot portions have separate experimental errors.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. The Strip-Plot Designs: In this design where there are only two factors, Factor A is applied to whole plots like the usual split-plot designs but factor B is also applied to strips which are actually a new set of whole plots orthogonal to the original plots used for factor A. Example of strip-plot design where both of the factors have three levels. Whole Plots A3 A1 A2 Strip Plot B1 A3B1 A1B1 A2B1 B3 A3B3 A1B3 A2B3 B2 A3B2 A1B2 A2B2 It is important to note that the split-block design has three sizes of experimental units where the units for effects of factor A and B are equal to whole plot of each factor and the experimental unit for interaction AB is a sub-plot which is the intersection of the two whole plots. ANOVA for Experimental design: 1) CRD: Source of variance tratment error Total df (t-1) t(r-1) (n-1) or (rt-1) 2) RCBD: Source of variance replication/block treatment df (r-1) (t-1) error Total (t-1) (r-1) (rt-1) Source of variance df rows column treatment error (r-1) (c-1) (t-1) (r-1) (c-1) (t-1) Total (rct-1) 3) LSD: 4) Factorial Design: RCBD with 2 factors A & B Source of variance df replication (r-1)
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. treatment (ab-1) Factor A B AxB error Total (a-1) (b-1) (a-1) (b-1) (r-1) (ab-1) (rab-1) 4.6 ANALYSIS OF CO-VARIANCE Analysis of covariance is a more sophisticated method of analysis of variance.It is a technique that sits between analysis of variance and regression analysis. It has a number of purposes but the two that are, perhaps, of most importance are: 1. to increase the precision of comparisons between groups by accounting to variation on important prognostic variables; 2. to "adjust" comparisons between groups for imbalances in important prognostic variables between these groups Analysis of covariance (ancova) is used when you have two measurement variables and two nominal variables. One of the nominal variables groups is the "hidden" nominal variable that groups the measurement observations into pairs, and the other nominal variable divides the regressions into two or more sets.The purpose of ancova to compare two or more linear regression lines. It is a way of comparing the Y variable among groups while statistically controlling for variation in Y caused by variation in the X variable. Analysis of covariance (ANCOVA) is a general linear model which blends ANOVA and regression. ANCOVA evaluates whether population means of a dependent variable are equal across levels of a categorical independent variable (IV), while statistically controlling for the effects of other continuous variables that are not of primary interest, known as covariates 4.7 CHI-SQUARE TEST It is the statistical test to determine if observed ratio differs significantly from an expected ratio or not. It is the statistical biometric test to test the agreement between hypothesis and observation. The chi-square statistic is calculated by finding the difference between each observed and theoretical (expected) frequency for each possible outcome, squaring them, dividing each b y the theoretical frequency, and taking the sum of the results. Pearson's chi-square is used to assess two types of comparison: tests of goodness of fit and tests of independence. A test of goodness of fit establishes whether or not an observed frequency distribution differs from a theoretical distribution. A test of independence assesses whether paired observations on two variables, expressed in a contingency table, are independent of each other. The formula of chi-square is following;
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Chi-square formula: X2 = Σ (Observed value – Expected value)2 (Expected value) The degree of freedom (df) = n-1, where n is the number of classes The chi-square statistic can then be used to calculate a p-value by comparing the value of the statistic to a chi-square distribution. If the calculated chi-square value is less than 0.05 value (0.05 probability level), we accept the hypothesis. If the calculated value is greater than the tabulated value, we reject hypothesis. A chi-squared test, also referred to as chi-square test or test, is any statistical hypothesis test in which the sampling distribution of the test statistic is a chi-squared distribution when the null hypothesis is true. Also considered a chi-squared test is a test in which this is asymptotically true, meaning that the sampling distribution (if the null hypothesis is true) can be made to approximate a chi-squared distribution as closely as desired by making the sample size large enough. Some examples of chi-squared tests where the chi-squared distribution is only approximately valid: Pearson's chi-squared test, also known as the chi-squared goodness-of-fit test or chisquared test for independence. When the chi-squared test is mentioned without any modifiers or without other precluding context, this test is usually meant (for an exact test used in place of , see Fisher's exact test). Yates's correction for continuity, also known as Yates' chi-squared test. Cochran–Mantel–Haenszel chi-squared test. McNemar's test, used in certain 2 × 2 tables with pairing Tukey's test of additivity The portmanteau test in time-series analysis, testing for the presence of autocorrelation Likelihood-ratio tests in general statistical modelling, for testing whether there is evidence of the need to move from a simple model to a more complicated one (where the simple model is nested within the complicated one). One case where the distribution of the test statistic is an exact chi-squared distribution is the test that the variance of a normally distributed population has a given value based on a sample variance. Such a test is uncommon in practice because values of variances to test against are seldom known exactly. Chi-squared test for variance in a normal population If a sample of size n is taken from a population having a normal distribution, then there is a result (see distribution of the sample variance) which allows a test to be made of whether the variance of the population has a pre-determined value. For example, a manufacturing process might have been in stable condition for a long period, allowing a value for the variance to be determined essentially without error. Suppose that a variant of the process is being tested, givi ng rise to a
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. small sample of n product items whose variation is to be tested. The test statistic T in this instance could be set to be the sum of squares about the sample mean, divided by the nominal value for the variance (i.e. the value to be tested as holding). Then T has a chi-squared distribution with n − 1 degrees of freedom. For example if the sample size is 21, the acceptance region for T for a significance level of 5% is the interval 9.59 to 34.17. Degrees of freedom Degrees of freedom can be described as the number of scores that are free to vary. For example, suppose you tossed three dice. The total score adds up to 12. If you rolled a 3 on the first die and a 5 on the second, then you know that the third die must be a 4 (otherwise, the total would not add up to 12). In this example, 2 die are free to vary while the third is not. Therefore, there are 2 degrees of freedom. In many situations, the degrees of freedom are equal to the number of observations minus one. Thus, if the sample size were 20, there would be 20 observations; the the degrees of freedom would be 20 minus 1 or 19. Chi-square critical value The chi-square critical value can be any number between zero and plus infinity. The chisquare calculator computes the probability that a chi-square statistic falls between 0 and the critical value. Suppose you randomly select a sample of 10 observations from a large population. In this example, the degrees of freedom (DF) would be 9, since DF = n - 1 = 10 - 1 = 9. Suppose you wanted to find the probability that a chi-square statistic falls between 0 and 13. In the chisquare calculator, you would enter 9 for degrees of freedom and 13 for the critical value. Then, after you click the Calculate button, the calculator would show the cumulative probability to be 0.84. Cumulative probability A cumulative probability is a sum of probabilities. The chi-square calculator computes a cumulative probability. Specifically, it computes the probability that a chi-square statistic falls between 0 and some critical value (CV). With respect to notation, the cumulative probability that a chi-square statistic falls between 0 and CV is indicated by P(Χ2 < CV). Chi-square statistic
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. A chi-square statistic is a statistic whose values are given by Χ2 = [ ( n - 1 ) * s2 ] / σ2 where σ is the standard deviation of the population, s is the standard deviation of the sample, and n is the sample size. The distribution of the chi-square statistic has n - 1 degrees of freedom. (For more on the chi-square statistic. Probability A probability is a number expressing the chances that a specific event will occur. This number can take on any value from 0 to 1. A probability of 0 means that there is zero chance that the event will occur; a probability of 1 means that the event is certain to occur. Numbers between 0 and 1 quantify the uncertainty associated with the event. For example, the probability of a coin flip resulting in Heads (rather than Tails) would be 0.50. Fifty percent of the time, the coin flip would result in Heads; and fifty percent of the time, it would result in Tails. 4.8 STABILITY ANALYSIS Analysis of stability is a biometrical method with great potential for characterization of the relative performance of a group of populations (varieties, hybrids, lines, clones, etc.) under different environmental conditions. With this methodology it is possible to de- sign the allocation of populations obtained in plant breeding programs. The statistical techniques used for assessing the agronomic stability of a set of cultivars or advanced lines may be following: 1) Analysis Of Variance: It is a systematic procedure for obtaining two or more estimates of variances and comparing them. The significant tests for combined analysis of variance are valid if error terms from different enviroments are homogeneous; this is determined by Bartlett‘s test. Analysis of variance for g genotypes evaluated at e locations in trials having r replications. Source of d.f. Mean squares F ratios variation Genotypes (G) Enviroments (E) Replicaiton within enviroments g-1 e-1 e (r-1) MS1 MS2 MS3 MS1/MS4 MS2/MS3 MS3/MS5 Genotype X environ ment (G X E) (e-1) (g-1) MS4 MS4/MS5 Error e (g-1) (r-1) MS5
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. A genotype‘s stability can be assessed as following; 1) by averaging of variance components determined from each pairwise combination involving genotype in question with every genotype 2) by determing the contribution of a genotype to G X E intercataon ; this is called ecovalence parameter 3) Using a modification of the ecovalance term to obtain unbiased estimate of G X E variance for every genotype. In the case of all the above measures, the larger the estimate or a genotype the poorer is its general agronomic stability or adaptation. All the three methods tend to rank genotypes in the same order, but only the last one permits statistical testing. 2) Regression analysis: The most commonly used methods for estimating stability of cultivars are based on regression analysis. In this approach, the slope of regression of individual genotypes yields against environmental mean yields, based on all genotypes in a trial, is determined. The regression analysis is done by following ways; Eberhart & Russel l (1966) model: In this model genotype x environment interaction of each variety are partitioned into two parts; a) slope of regression line b) deviations from regression line. In this model, b (regression coefficient) is considered as parameter of response and Source of variation d.f. Sum of squares Mean square Genotypes (G) Environments (E) + interaction (G x E) Enviroment (linear) G x E (linear) Pooled deviations Genotype 1 Genotype 2 . Genotype g. Pooled error g-1 g (e-1) 1 g-1 g (e-2) e-2 e-2 e-2 ge (r-1) Perkins and Jinks (1968) model: In this model, regression of genotypes X environments is obtained on the environme ntal index. The genotype X environment (G x E) sum of squares is further divided int two parts; a) sum of squares (SS) due to heterogeneity among regressions and b) SS due to the remainder. Source of variation d.f. Sum of squares Mean square Chi-squared Genotypes (G) Environments (E) g-1 e-1
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. GXE Heterogeneity among regressions Remainder Error (g-1) (e-1) g-1 (g-1) (e-2) ge (r-1) 3) Non Parametric Statistics: Analysis of ranks of genotypes across the environments represents commonly used non parametric methods. Two of these approaches are briefly described below; a.) in stratifies ranking, the proportion of locations where a genotype ranks in the top, middle or bottom third of the genotype included in the trials is determined. A genoty pe usually found in the top third of the genotypes across the sites can be considered as relatively well adapted. b.) Consistency of performance of genotypes is determined by plotting the average ranks across locations against the standard deviation of ranks for every genotype This allows the allocation of every genotype to one of the following 4 groups; a) consistently superior, b) inconsistently superior, c) consistently inferior, d) inconsistently inferior. Genotypes showing general adaptation are found in the first group, while those possessing specific adaptation will occur in the second group. Non parametric methods can be used for grouping environments and genotypes. Two environments, regardless of their yield level, may be considered alike for selecti on purposes if both rank genotypes similarly. 4) Multivariate Techniques: Multivariate Data Analysis refers to any statistical technique used to analyze data that arises from more than one variable. This technique is used for describing relationships amo ng locations and among genotypes, typically using yield data from genotype (G) x location (L) matrices generated by breeding programmes. The yield data are subjected to pattern analysis, which makes parallel use of classification and ordination techniques to present the maximum variation from G x L matrices in a few dimensions. Classification techniques like ‗Clustering’ assume discontinuities within data, while ordination techniques like principle component analysis assume a continuous distribution. Multivariate techniques are following types; i) Cluster Analysis In this method, a genotype is assigned to one group only. The purpose of cluster analysis is to reduce a large data set to meaningful subgroups of individuals or objects. The division is accomplished on the basis of similarity of the objects across a set of specified characteristics. Outliers are a problem with this technique, often caused by too many irrelevant variables. The sample should be representative of the population, and it is desirable to have uncorrelated factors. There are three main clustering methods: hierarchical, which is a treelike process appropriate for smaller data sets; nonhierarchical, which requires specification of the number of clusters a priori, and a combination of both. The results from a cluster analysis are usually displayed as a dendrogram or hierarchial tree.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Dendrogram analysis: The dendrogram is a graphical representation of the results of hierarchical cluster analysis . This is a tree-like plot where each step of hierarchical clustering is represented as a fusion of two branches of the tree into a single one. The branches represent clusters obtained on each step of hierarchical clustering. Dendrograms are often used in computational biology to illustrate the clustering of genes. ii) Factor Analysis Factor analysis is a statistical method used to describe variability among observed variables in terms of fewer unobserved variables called factors. In this method, entries are assigned to main groups on the basis of primary loads; it also accounts for affinities with other groups by secondary loads. When there are many variables in a research design, it is often helpful to reduce the variables to a smaller set of factors. This is an independence technique, in which there is no dependent variable. Rather, the researcher is looking for the underlying structure of the data matrix. There are two main factor analysis methods: common factor analysis, which extracts factors based on the variance shared by the factors, and principal component analysis, which extracts factors based on the total variance of the factors. Common factor analysis is used to look for the latent (underlying) factors, where as principal components analysis is used to find the fewest number of variables that explain the most variance. The first factor extracted explains the most variance. Typically, factors are extracted as long as the Eigen values are greater than 1.0 or the Screen test visually indicates how many factors to extract. The factor loadings are the correlations between the factor and the variables. Typically a factor loading of .4 or higher is required to attribute a specific variable to a factor. An orthogonal rotation assumes no correlation between the factors, whereas an oblique rotation is used when some relationship is believed to exist. 4.9. FIELD PLOT TECHNIQUE It is important for the crop scientists to have knowledge on field plot techniques, the study of size and shape of plot best suited for a particular type of experiment. It is of utmost importance to use the most efficient shape, size and arrangements of plots in a particular experiment for obtaining the reliable results. The precision of significance tests in field trial is largely controlled by size and shape of plots, which are further controlled by the size, and shape of area available for the particular trial, the nature of fertility or other variation. The problem was therefore selected to see a scientific basis for using plot size and shape within optimum limits. To cope with the problem of the research workers, it has become necessary to standardize a suitable plot size and shape for the experimental plot of major crops grown under different conditions, which will reduce the standard error of the experiments. Field-plot techniques deal with the various elements to properly plan an agricultural field experiment. The use of improper field-plot techniques may inflate experimental error and lead to erroneous inferences. Hence, to improve the quality as well as credibility of research results, there is a need to carry out research on field-plot techniques. Gomez and Gomez (1984) described that uniformity trial involves planting an experimental site with a single crop variety, applying cultural and management practices as uniformly as possible. All sources of variability, except that due to native soil differences, are kept constant. The planted area is sub divided into small units of the same size (generally referred to as basic
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. units) from which separate measurements of productivity, such as grain yield are made. The size of the basic unit is governed mostly by available resources. The smaller the basic unit, the more detailed is the measurement of soil heterogeneity. In field experiments, soil variability is one of the important external sources of variation. This variability may be random or systematic. Usually researchers use block experiments to minimize this source of variation. 4.10. REGRESSION AND CORRELATION ANALYSIS: REGRESSION Regression analysis is typically used to develop a regression line equation (y = a + bx) which assesses the linearity of a relationship (how well the relationship conforms to a line between an independent variable (X) and the dependent variable (Y)). By adding a squared or cubed term (y = a + bx + bx^2 + bx^3) regression can also be used to test if the relationship between two variables is nonlinear. This analysis involves identifying the relationship between a dependent variable and one or more independent variables. A model of the relationship is hypothesized, and estimates of the parameter values are used to develop an estimated regression equation. Various tests are then employed to determine if the model is satisfactory. If the model is deemed satisfactory, the estimated regression equation can be used to predict the value of the dependent variable given values for the independent variables. In statistics, regression analysis is a statistical process for estimating the relationships among variables. It includes many techniques for modeling and analyzing several variables, when the focus is on the relationship between a dependent variable and one or more independent variables. More specifically, regression analysis helps one understand how the typical value of the dependent variable (or 'Criterion Variable') changes when any one of the independent variables is varied, while the other independent variables are held fixed. Most commonly, regression analysis estimates the conditional expectation of the dependent variable given the independent variables – that is, the average value of the dependent variable when the independent variables are fixed. Less commonly, the focus is on a quantile, or other location parameter of the conditional distribution of the dependent variable given the independent variables. In all cases, the estimation target is a function of the independent variables called the regression function. In regression analysis, it is also of interest to characterize the variation of the dependent variable around the regression function which can be described by a probability distribution. Regression analysis is widely used for prediction and forecasting, where its use has substantial overlap with the field of machine learning. Regression analysis is also used to understand which among the independent variables are related to the dependent variable, and to explore the forms of these relationships. In restricted circumstances, regression analysis can be used to infer causal relationships between the independent and dependent variables. However this can lead to illusions or false relationships, so caution is advisable;[1] for example, correlation does not imply causation. Many techniques for carrying out regression analysis have been developed. Familiar methods such as linear regression and ordinary least squares regression are parametric, in that the regression function is defined in terms of a finite number of unknown parameters that are
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. estimated from the data. Nonparametric regression refers to techniques that allow the regression function to lie in a specified set of functions, which may be infinite-dimensional. The performance of regression analysis methods in practice depends on the form of the data generating process, and how it relates to the regression approach being used. Since the true form of the data-generating process is generally not known, regression analysis often depends to some extent on making assumptions about this process. These assumptions are sometimes testable if a sufficient quantity of data is available. Regression models for prediction are often useful even when the assumptions are moderately violated, although they may not perform optimally. However, in many applications, especially with small effects or questions of causality based on observational data, regression methods can give misleading results. Regression analysis implicitly assumes a linear cause and effect relationship between input variables and the output and then tests to see how good that assumption is. Regression fits the data to a line (for one input) or a surface (for multiple inputs). If the R -Squared value reported is high enough (the definition of "high enough" depends on the situation), the fit was good and the linear model is a good reflection of the relationship between the inputs and output. If the R Squared value is too low, the regression model is not an accurate reflection of the relationship between the inputs and outputs. CORRELATION Correlation is a measure describing the way two variables vary together. A correlation coefficient of +1.00 means two variables vary in the same way pefectly (positive correlation); a correlation coefficient of -1.00 means two variables vary perfectly in opposite directions (negative correlation). The correlation between heallth status and medical expenditures is negative: as health status goes down medical expenditures go up. A correlation coefficient of 0 means a change in one of the measures has no relationship with a change of the other variable. As noted by others, correlation evaluates the relationship between two interval or ratio level variables. Correlation and regression analysis are related in the sense that both deal with relationships among variables. The correlation coefficient is a measure of linear association between two variables. Values of the correlation coefficient are always between -1 and +1. A correlation coefficient of +1 indicates that two variables are perfectly related in a positive linear sense, a correlation coefficient of -1 indicates that two variables are perfectly related in a negative linear sense, and a correlation coefficient of 0 indicates that there is no linear relationship between the two variables. For simple linear regression, the sample correlation coefficient is the square root of the coefficient of determination, with the sign of the correlation coefficient being the same as the sign of b1, the coefficient of x1 in the estimated regression equation assumes no cause and effect relationship between inputs and outputs. In simplified terms, it simply asks the question "when this input is big, does my output tend to be bigger (a positive correlation) smaller (a negative correlation) or does the value of the input have no effect on the output (zero correlation). Correlation 4.11 DIFFERENT COMPUTER STATISTICAL PACKAGES AND THEIR USE IN PLANT BREEDING
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. The computer programmes for design and analysis of germplasm evaluation trials for the following software products: 1) Microsoft Excel 2) MSTAT 3) Agrobase 4) Genstat 5) S-PLUS 6) SAS 7) CycDesigN and 8) ASREML. The combination of Genstat, Agrobase and SAS could be used for the randomization of the trials and for the analysis. 5. Breeding self- pollinated crops by Introduction, Selection and Hybridization 5.1 Characteristics of self-pollinated crops 5.2 Methods of breeding self-pollinated crops - Mass selection method, - Pureline selection method, - Pedigree selection method, - Bulk selection method, - Back cross selection method, - Single seed descent method. 5. BREEDING SELF- POLLINATED CROPS BY INTRODUCTION, SELECTION AND HYBRIDIZATION 5.1. CHARACTERISTICS OF SELF-POLLINATED CROPS AND CROSS POLLINATE CROPS: S.N. Self pollinated crops Cross pollinated crops 1 2 Flowers are generally small, Flowers are generally large, showy, inconspicuous, colorless, colored, fragrant and with nector odourless and nectorless Both anthers and stigma mature Anthers and stigma mature at different simultaneously time 3 Pollination takes place under natural External agents condition pollination 4 Yield of plant falls with time is required Yield does not fall with an average for
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 5 Adaptability to changed environment is Plants are better adapted to changed poor enviroment The purpose of selection (breeding) in self pollinated crops is to isolate desirable plant types from a genetically variable population. 5.2.METHODS OF BREEDING SELF POLLINATED CROPS 1) MASS SELECTION This method of selection depends mainly on selection of plants according to their phenotype and performance. The seed from selected plants are bulked for the next generation. This method is used to improve the overall population by positive or negative mass selection. Mass selection is only applied to a limited degree in self fertilizing plants and is an effective method for the improvement of land races. This method of selection will only be effective for highly he ritable traits. one shortage of mass selection are the large influence that the environment has on the development, phenotype and performance of single plants. It is often unclear whether the phenotypically superior plants are also genotypically superior and strong environmental differences may lead to low selection efficacy (heritability). Application of mass selection: .1) improvement of desi or local varieties. 2) purification of existing pureline varieties. Merits of mass selection: 1) it retains considerable genetic variability in new variety. 2) often extensive and prolonged yield trials are not necessary. This reduces time and cost for developing new varieties. 3) no need of progeny testing. 4) varities developed through mass selection are likely to be more widely adapted than purelines. Demerits of mass selection: 1) it is not possible to know whether selected phenotype is superior in appearance is due to hereditary characters or due to environment. 2) varieties developed by mass selection are more difficult to identify than pure lines in seed certification programmes. 3) mass selection can not generate variability. It utilizes variability already present in a variety of in population. 4) not commonly used in improvement in self pollinated crops.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2) PURELINE SELECTION In pureline selection, a large number of plants are selected from a self pollinated crops and are harvested individually; individual plant progenies from them are evaluated and the best progeny is released as a pure line variety. A variety developed by this method will be more uniform than those developed by mass selection because all of the plants in such a variety will have the same genotype. The see d from selected plants are not added together but are kept apart and used to perform offspring tests. This is done to study the breeding behaviour of the selected plants. The high uniformity in stand and conditions performance has been stressed in the past, but the risk of highly specialized pathogens evolving is very high. More genetic variability could buffer the crop against such pathogens as well as stability of production under varied environmental. Application of pureline selections: a) improvement of local and old pureline varieties, b) selection for a new characteristic in a pure line c) selection in segregating generations from crosses d) pure line selection in introduced varieties to develop suitable varieties. Advantages of pureline selection: 1) it achieves the maximum possible improvement over original variety. 2) pureline varieties are extremely uniform since all the plants in variety have the same genotype. 3) due to its extreme uniformity, the variety is easily identified in seed certification programmes. 4) it is good method to exploit the variation in land races. Demerits of pure line selection: 1) varieties developed by pure line selection generally do not have wide ad aptation and stability in producitn. 2) the procedure of pureline selection requires more time, space and more expensive yield trials than mass selection. 3) the upper limit on improvement is set by genetic variation present in original population. 4) the breeder has to devote more time to pureline selection than to mass selection. This leaves less time for other breeding programmes. 3.PEDIGREE SELECTION: In pedigree selection, individual plants are selected in F2 generation and progenies of each selected plants are reselected in succeeding generation until genetic purity is obtained, a detail record of relationships between the selected plants and their progenies is maintained, each progeny in every generations can be traced back to the F2 plant from which it originated. Such a record of parent-offspring relationships is called pedigree. The pedigree record may be used as a basis for selection in a later generation.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Considerations while selecting plants in F2 in pedigree method: 1. selection of easily observable traits with moderate to high heritability is effective.e.g. seed color, presence of awn, disease resistanace etc. 2. selection for vigour is ineffective because vigour may be due to heterozygosity, G X E interaction and environment. 3. Selection for yield from F2 is not possible because yield is governed by polygenes and having low heritability. Application of pedigree selection: a) used for selection from segregating generations of crosses in self pollinated crops b) used to correct some specific weakness of an established varieties c) used in selection of new superior recombinant type Merits of pedigree selection: 1) plants and progenies with visible defects and weakness are eliminated at an early stage (in F2) in breeding programme; this saves resources and time of labour. 2) It takes less time than bulk method to develop new variety. 3) It is well suited for improvement of characters which can be easily identified and are simply inherited. 4) The breeder may often be able to obtain information about the inheritance of qualitative charcters from pedigree record. Demerits of pedigree selection: 1. valuable genotype may be lost in early segregation. 2. Maintenance of pedigree record takes valuable time. 3. Selection among and within a large number of progenies in every gene ration is laborious and time consuming 4. Selection for yield in F2 and F3 is ineffective because yield is governed by polygenes ad has low h2 4) BULK POPULATION SELECTION In the bulk method, F2 and subsequent generations are harvested in mass or as bulks to raise the next generation. At the end of bulking period, individual plants are selected and evaluated in a similar manner as in the pedigree method of breeding. The duration of bulking may vary from 6 -7 to 30 or more generations. With this method of selection the offspring from a crossing are planted at planting densities equal to commercial planting densities. During this period, which may include a number of generations, the level of homozygosity in the bulk population increases.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. This method is simple and cheap and involves less work than pedigree selection in the earlier generations. It is necessary to plant large populations to ensure that the best segregates are selected when selection starts. Segregating generations are subjected to another single plant selection step. Fewer records are kept during earlier generations than with pedigree selection. This type of selection is especially carried out with crops which are usually planted at high planting densities, e.g. small grain crops. Application of bulk selection: a) for isolation of homozygous lines with minimum effort and expenses b) waiting for the opportunity for selection c) to provide opportunity for natural selection to change population composition. Merits of bulk selection: 1) simple, convenient and inexpensive method. 2) No need of keeping pedigree records which saves time and labour. 3) Isolation of desirable types is easier. 4) Little work and attention is needed in F2 and subsequent generations. Demerits of bulk selection: 1. it takes a much longer time to develop a new variety. 2. it provides little opportunity for the breeder to exercise his skill or judgement in selection. 3. A large number of progenies have to be selected at the end of bulking method. 4. Information on inheritance of characters can not be obtained which is often available from progenies method. 5) BACK CROSS SELECTION: A cross between a hybrid (F1 or a segregating generation) and one of its parents is known as backcross. In backcross method, the hybrid and the progenies in the subsequent generations are repeatedly backcrossed to one of the parents of F1. As a result the genotype of backcross progeny becomes increasily similar to that of the parent to which the backcross is made. At the end of 6 -8 backcrosses, the progeny would be almost identical with the parent used for backcrossing. The backcross breeding method is often used to transfer recessive traits controlled by one or a few genes from one pure line to another. Requirements of a Backcross Programmes: 1) a suitable recurrent parent which lacks in one or two characteristics 2) a suitable donor parent which have characters to be transferred 3) characters to be transferred must have high heritability 4) a suitable no. of backcrosses should be made. Applications of Backcross Methods: 1) intervarietal transfer of simply inherited character. E.g. disease resistance, seed color
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2) 3) 4) 5) 6) 7) 8) intervarietal transfer of quantitative characters like earliness, plant ht. seed size etc. interspecific transfer of simply inherited characters Transfer of cytoplasm from one variety or species to another. Production of near isogenic lines. Germplasm conservation. Incorporation of male sterility and restorer genes for hybrid varieties production. Development of varieties with multiple disease resistance. Merits of backcross selection: 1) it is only method for interspecific gene transfer and for transfer of cytoplasm. 2) Much smaller population of plants are needed in backcross method than in case of pedigree method. 3) It is not necessary to test the variety developed by back cross method in extensive yield tests because the performance of recurrent parent is already known. 4) The backcross programme is not dependent upon the environment. Offseason nurseries and green houses can be used to grow 2-3 generations each year. This would drastically reduce the time required for developing new variety. 5) Adaptibility of developed new variety is not problem. Demerits of backcross method: 1) amount of crossing is more. This is often difficult, time consuming and costly. 2) Undesirable genes closely linked with genes being transferred may also be transmitted to new variety. 3) The new variety generally can not be superior to the recurrent parent, except for the characters that is transferred. 4) The characters of few or low h2 are hard to transfer. 5) By the time, backcross programme improves it, the recurrent parent may have been replaced by other varieties superior in yielding ability and other characters. 6) SINGLE SEED DESCENT METHOD: In this method, a single seed from each of one to two thousand F2 plants is bulked to raise the F3 generation. Similarly, in F3 and Subsequent generations, one random seed is selected from every plant present in the population and planted in bulk to raise the next generation. This procedure is followed till F5 or F6 when the plants would have become nearly homozygous. It is modification of bulk method. In this method, in F2 and subsequent generations only one seed is used from each plant in the population selected for advancing the next generations. For the practical reasons, a single pod (2-3 seeds) is taken from each plant but only one plant from pod is retained for advancing to the next generation. When desired level of inbreeding is attained, each progeny which traces back to a F2 plants, is maintained in bulk.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 6. Breeding Cross-pollinated crops 6.1 Mass selection method, 6.2 Progeny selection (Ear to row selection) 6.3 Recurrent selection method: 6.4 Types of varieties and their development, 6.5 Hybrid varieties development procedures and their seed production methods.   The aim of breeding for cross pollinated crops is to prevent inbreeding depression. The objective of selection (breeding for cross pollinated crops ) is to increase the frequency of desirable alleles in the population which result in increase the frequency of desirable gene combination or genotype. Methods of breeding cross pollinated crops: 6.1MASS SELECTION METHOD: It is the simplest, easiest and oldest method of selection where a number of plants are selected based on their phenotypic performance, and open pollinated seed from them is bulked to produce the next generation. Mass selection proved to be quite effective in maize improvement at the initial stages but its efficacy especially for improvement of yield, soon came under severe criticism that culminated in the refinement of the method of mass selection. The selection after pollination does not provide any control over the pollen parent as result of which effective selection is limited only to female parents.The heritability estimates are reduced by half, s ince only parents are used to harvest seed whereas the pollen source is not known after the cross pollination has taken place. Procedure of mass selection: First year: i) plants selected on basis of phenotype, ii) open pollinated seeds from plants harvested and bulked. Second year: i) grow the bulked seeds, ii) steps i) and ii) in first year may be repeated Third year: i) perform yield trail along with check varitety (standard checks) ii) superior may be released as var Fourth year: variety released , seed multiplication for distribution. Merits of mass selecton: 1. extremely simple breeding method 2. selection cycle is short 3. highly efficient in improving characters that are easily identified visually and have high h 2 e.g. plant height, ear size, maturity date etc. 4. efficient in improving yields of cross pollinated crops. Demerits of mass selection:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1. selection of superior phenotype may be poor basis for identification of superior genotype since phenotype caused by environment 2. the selected plants are pollinated by superior and inferior plants present in the population as the selected plants are allowed to open pollinate. This reduces effectiveness of selection 3. high intensities of selection reduce population size and as a result lead to some inbreeding. Stratified mass selection (Grid method of mass selection): the field, from which selection is to be done is divided into several small plots e.g. having 40-50 plants each equal number of superior plants are selected from each of small plots i.e. selection is done within the plots and not among the plots. The seed from all the selected plants as compared to raise the next generation. 6.2 PROGENY SELECTION (EAR TO ROW SELECTION): This method involves selection of a number of phenotypically desirable ears out of a populat ion grown in isolation. The seeds of harvested ears are not bulked but kept separate. A part of the seeds of each ear is grown in the next season to obtain information on progeny performance . Based on this progeny performance, the next cycle is taken from the remnant seeds of promising ears. Procedure of progeny selection: First year: i) plants are selected on the basis of phenotype, ii) open pollination is allowed and harvest seeds separately. Second year: i) grow individual plant progenies in a row, ii) select superior progenies and reject inferior ones. This selection is done first between progeny rows and then within the progenies, iii) open pollination is allowed and iv) seed harvested separetly Third year: same as in 2nd year. Fourth year: i) perform yield trials, ii) compare with standard check, iii) if superior , release as variety, seed multiplication. Merits of progeny selection: 1) selection is based on progeny test and not on the phenotypes of individual plants. 2) Selection scheme is relatively simple and easy 3) It is popular method than mass selection for maize breeders. Demerits of progeny selection: 1. there is no pollination and selection is based on the maternal parent only, this reduces efficiency of selection. 2. Many of the progeny selection schemes are complicated and involves considerable works. 3. Time requirement for selection is twice as much as that in case of mass selection. 4. Continued ear to row selection results in inbreeding and hence loss in vigour.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 3). Half sib selection: Half sibs are a plant or family of individual plants having either the same father or the same mother (i.e. common parent). Under the half-sib family scheme, plants used as males are selfed and also crossed to several females to produce half-sib families. The selected plants are composited from selfed seeds. In half sib test, no selfing is carried out and compositing is done from open pollinated seeds. . Selections are made based on progeny test performance instead of phenotypic appearance of the parental plants. Seed from selected half-sibs, which have been pollinated by random pollen from the population, is grown in unreplicated progeny rows for the purpose of selection. A part of the seed is planted to determine the yielding ability, or breeding value, for any character of each plant. The seed from the most productive rows or remnant seed from the outstanding half-sibs is bulked to complete one cycle of selection 1) Full-sib selection: Full-sibs refers to individuals having common parents but derived from different g ametes. Full sibs are family of individuals having both parents common and are derived from selfing or crossing of two plants from the same population. . . A number of full-sib families, each produced by making crosses between the two plants from the base population are evaluated in replicated trials. A part of each full -sib family is saved for recombination. Based on evaluation the remnant seed of selected full -sib families is used to recombine the best families. Steps of full sib selection: 1) First year: superior plants are selected based on phenotype. Perform the pairwise crosses and harvest seed separately. (The full sibs are developed by making crosses between phenotypically desirable and vigorous plants within the population.). 2) second year: Grow the individual plant progenies in rows, retain remnant seeds, select superior progenies and reject inferior ones, seeds of superior progenies can be bulked or the remnant seeds of parental source can be mixed to grow next generation. 3) Third year: grow the composited seeds in isolation condition, open pollination is allowed and seeds harvested as bulk. Reciprocal crosses are made to increase seeds. (selected families (80 -90) are planted ear to row in breeding nursery from remnant seeds. Crosses are made between best plants of selected families. The crosses form a new set of full sibs) 4) Fourth year: seed multiplication is done for distribution. 5) Selfed progeny selection:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. In this selection scheme, seeds for progeny testing are obtained by selfing the selected plants and not from the open pollination. This is known as S1-family selection. When the seeds for progeny test are obtained after two generations of selfing, the scheme is known as S2-family selection. The plants in the original base population are selfed to produce S1 progenies, which are evaluated in the next season in replicated multi-environmental trials to identify promising S1 families. The remnant S1 seed of such selected families is then recombined in the third season as a result o f which one cycle is completed in three seasons. Hence, the units of selection and recombination are S1 progenies. 6.3.Recurrent selection method: Recurrent selection is the method of plant breeding for quantitatively inherited traits by which the frequencies of favorable genes are increased in the population. The methodology is cyclic with each cycle having following 2 phases; a) selection of genotype that posses favorable genes b) crossing among the selected genotype The basic steps of recurrent selection are; a) isolation of one generation selfed lines b) testing of these lines for yield and other characters c) intercrossing the best selfed lines to produce new improved population. Recurrent selection is scheme of selection (on the basis of phenotype or progeny test), followed by intermating (in all combinations) of the selected plants or their selfed proegeny to produce the population for the next cycle of selection. More than one cycle of selection is practiced. This is breeding method for cross pollinated crops but not for self pollinated crops. Recurrent selection causes the gradual upgrading of frequencies of desired alleles in population. Selection of Recurrent Parents: 1) the recurrent parents be popular must 2) high yielding ability 3) desirable quality 4) high adaptability When recurrent selection is applicable?. 1. Recurrent selection is useful only where characters have high heritability because it is operated on phenotypic basis. 2. It is applicable for breeding in cross pollinated crops, not in self pollinated crops. Procedure/steps of simple recurrent selection: A) Original Selection cycle:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1) First year: a) several phenotypically superior plants selected, b) selected plants self pollinated, c) seeds harvested separately and d) seeds evaluated; superior seeds retained. 2) Second year: a) individual plant progenies planted, b) all possible intercrosses made, and c) equal amounts of seed from all intercrosses composited. B). First recurrent selection cycle: 1. Third year: a) compositeed intercross seed planted, b) as in all steps in first year. 2. Fourth year: a) individual plant progenies planted, b) all possible intercrosses made and c) equal amount of seed from all intercrosses composited. Difference between Simple recurrent selection and Reciprocal recurrent selection Simple recurrent selection (SRS) Reciprocal recurrent selection (RRS) 1) It is a procedure in which a number of phenotypically desirable plants are selected and self pollinated. At harvest, selection is again done for desirable plant and ear characters. Progenies from these are grown in next season and all possible crosses are made. At harvest, these crosses (equal amount of seed form each cross) are bulked for the next cycle of recurrent selection. In this scheme, progressively improved populations of two germplasm pools are not used reciprocally as tester. 1) it is a procedure in which progressively improved populations of two germplsm pools are used reciprocally as testers. The objective of RRS is to improve 2 different populations in their ability to combine well with each other. In this scheme, each of 2 population, say A and B, serve as tester for plants selected from population. ‗A‘ serves as the tester for plants selected from population B. Similarly a random sample of plants from population B serves as the tester for those selected from population A Difference between half sib family selection and full sib family selection Half sib family selection Full sib family selection 1. in this case, seeds for progeny testing are obtained by crossing the selected plants to a common tester (same father or same mother). 2) it is less efficicent method. The additive variance is smaller 1) in this case, seeds for progeny testing are obtained by mating the selected plants in pairs so that plants within progeny are full sibs. 2) it is more efficient method. The additive variance is larger
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Difference between recurrent selection for GCA and recurrent selection for SCA Recurrent selection for general combining ability Recurrent selection combining ability for specific 1) In this scheme, number of plants are selected on the 1) In this scheme, number of plants basis of phenotype and selfed (self pollinated) as well are selected on the basis of as crossed to a tester with broad genetic base like an phenotype and selfed (self open pollinated variety or synthetic variety. pollinated) as well as crossed to an inbred with narrow genetic base. 2) It is used to improve the yielding ability and 2) It is used to isolate from a agronomic characteristics of a population as well as used to accumulated genes for superior GCA. It is applied in developing synthetic variety. population such lines that will combine well with a given inbred. It is applied in developing hybrid varieties Hybrid varieties are first generation (F1) from crosses between two purelines, inbreds, open pollinated varieties, clones or other populations that are genetically dissimilar. 6.4.TYPES OF VARIETIES AND THEIR DEVELOPMENT 1.COMPOSITE VARIETIES: Varieties produced by open pollination among a number of outstanding strains usually not tested for combining ability with each other is called composite varieties. Procedure for production of composite varieties: 1) select a desired number of plants on the basis of phenotype 2) self pollination is allowed, harvest the seeds separately 3) individual plant progenies are grown and select a superior progeny and reject the inferior progeny 4) carry out selfing 5) after selfing, there is production of inbreds 6) select the outstanding line from the produced inbreds 7) mix the seeds of outstanding lines 8) then, grow the mixed seeds in next year in isolation condition 9) open pollination is allowed to plants grown in isolation condition, harvest the seeds in bulk That seeds harvested as bulk is known as composite seed 3. SYNTHETIC VARIETIES:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Synthetic variety refers to an improved commercial variety that has synthesized through open pollination of inbreds, strains or clones. It is especially suitable for forage crops where other conventional breeding technique are difficult to adopt. Procedure of producing synthetic variety: 1) Source population: Several thousand genetically different plants are collected. These may arise from farmer‘s field, bulk population, commercial varieties, plant introduction 2) Establishment of inbred lines Superior plants are selected from the source populations on the basis of phenotypes. Inbreds are produced through controlled pollination 3) Identification of superior inbreds: Superior inbreds are identified through evaluation for their performance and combining ab ility 4) Production of synthetic varieties: a) Syn 0 generation: 5-10 inbreds are planted in isolation and allowed open pollination. The open pollinated seeds are harvested and mixed to produce the next generation. b) Syn 1 generation: open pollinated seeds from syn 0 generation is grown in isolation to produce syn 1 generation. Open pollination is enforced. Seed multiplication is done. Seeds are harvested after syn 1 generation may either released as a new synthetic variety or advanced to syn 2 generation. c) Syn 2 generation: open pollinated seed from Syn 1 generation is grown in isolation and open pollination is enforced. The seed harvested in bulk after Syn 2 generation and released as a new synthetic variety to the farmers Merits of synthetic varieties: 1) less costly seeds compared to hybrids and requires less skill. 2) Heterosis is exploited. 3) They are good reservoir of genetic variability. 4) In variable enviroments, synthetic varities are likely to do better than hybrid varieties Demerits of synthetic varieties: 1) much work and resource needed to maintain the components of synthetic variety 2) extensive progeny testing is needed to identify superior parents for synthetic. 3) Synthetics can be produced and maintained only in cross pollinated crops.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 4.MULTILINE VARIETIES: Multiline varieties are mixtures of several purelines of similar height, flowering and maturity dates, seed color and agronomic characteristics, but having different genes for disease resistance. They are mixtures of isogenic lines with desirable agronomic characters including yield. Multiline varieties are produced or developed in order to break down of resistance to disease. Types of multiline varieties: 1) Clean multiline : if each of resitance gene in multiline is still effective to all races of pathogen, that multiline is called clean multiline 2) Dirty multilines: they consist of lines carrying gene for resistance that are not effective against all races of the pathognes. Each line carries a different single gene for resistance. Development of (clean) multiline varieties: A multiline variety is usually created by mixing the seeds of several lines that are similar in appearance but different genes for resistance to a given disease. There are two main steps in the development of multilines 1) Development of component lines : The productions of component lines of multiline variety involves following two steps: a) identification of several sources of distinct and preferably, known genes for resistance to the concerned disease. In order to avoid unnecessary duplication, test of allelic relationships among R genes should be a routine feature of the programme. b) The resistance genes are incorporated in an elite variety or line to produce as many nearisogenic lines as there are distinct R genes. This is done through a conventional backcross programme (5-6 backcrosses), a limited backcrossing (2-3 backcrossing followed by pedigree selection) or by making double or multiple crosses 2.) Evaluation of grouping of component lines: The component lines are first evaluated in multilocation trials for yielding ability, morphologic features, agronomic characteristics and produce quality. The evaluation is particularly important for such lines that are produced by 2-3 backcrosses or by using double or complex crosses. In addition, it is essential to screen the lines for resistanace to the known physiological races/pathotypes of the concerned pathogen both in the seedling and adult plant stages. Further, the component lines should have reasonable levels of resistance to other important diseases of the concerned crops. The selected lines are then evaluated for compatibility or nicking ability. Mixing of two genotypes may have a positive, neutral or negative effect on their performance Lines showing negative effects should be avoided, while those having positive effect should be preferred. The development of a multiline variety is a continuous process. The breeder must continuously search for new R gnes and develop component lines carrying them. As the race composition of the
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. pathogen population changes, the lines that have become susceptible are withdrawn and in the place, new resistant component lines are added. This process continues till the multiline become unattractive due to development of other superior varieties of the crops. Merits of multiline varieties: 1) All the lines are almost identical to the recurrent parent in agronomic characteristics, quality etc. 2) Only one or few lines of the mixture would become susceptible of pathogen in any one season. This would reduce the damage to the susceptible line as well. Merits of multiline varieties: 1) The farmers has to change the seed of multiline varieties every few years depending upon the change in the races of pathogen. 2) There is a possibility that a few race may attack all the lines of a multiline variety 3) Production and maintenance of multilines is a time taking job. 6.5 HYBRID VARIETIES DEVELOPMENT PROCEDURES AND THEIR SEED PRODUCTION METHODS.    development of inbred lines from genetically variable source population through continued inbreeding evaluation of inbreds for performance and combining abilities production of hybrid seeds by using cytoplasmic male sterility, genetic male sterility cytoplasmic-genetic male sterility, gametocide, manual emasculation and pollination, self incompatibility. Merits of hybrid varieties: 1) they produce higher yields than open pollinated or pureline varieties. 2) They can be produced both in self and cross pollinated crops. 3) They utilize heterosis Demerits of hybrid varieties: 1) Farmers have to use new hybrid seed every year, since they can not produce their own seed. 2) Hybrid seed production requires considerable technical skill. 3) High level of crop management is required. 4) Hybrid seed production is difficult where farm holdings are small. Why hybrid seed is more expensive?. Following factors increase price of hybrid seed;
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1) In most cases hybrid seed is produced on seed producing inbred line. Because the inbred lines are much lower yielding than open pollinated cultivars due to inbreeding depression, the seed yield per hectare in hybrid seed production is much lower. 2) Several extra activities have to be carried out to produce the hybrid seeds like detasseling in maize. Such extra activities increases hybrid seed price. How to solve this problem: 1) grow many rows of seed parent and as few of pollen parent as possible. 2) Making seed parent male sterile, extra activities can be reduced. 3) Low yield of hybrids can be solved by making three way and double crosses. METHODS OF HYBRID PRODUCTION: Hybrid production requires A-line, B-line and R-line. A-line is the female or seed producing parent line. It contains recessive non-restorer genes. It is a male sterile line. B-line is the maintainer line. It is used for maintaining a cytoplasmic male sterile line. It has the nuclear genes as found in the male sterile line. It contains normal cytoplasm. R-line is restorer or pollinator line. It is used for overcoming the effect of male sterile cytoplasm on male fertility. It prod uces functional male gametes even in the presence of the male sterile cytoplasm. The procedure in the production of hybrid seeds is accomplished in these steps; 1) introduction of a male sterile cytoplasm into A-line by back cross procedure 2) maintenance of the cytoplasmic male sterile line (A-line) and its fertile isogenic counterpart (B-line) 3) development of fertility restorer line (R-line) by introducing Rf genes into the pollinator line 4) crossing A-line and R-line to produce hybrid seeds. The methods of hybrid seed production are following; 1) Use of cytoplasmic-genetic male sterility: This system is the most widely used in hybrid seed production. It is commercially used in maize, onions, sunflower, cotton and sugarbeets. The system is based on a cytoplasm that produces male sterility and on a gene that restores fertility in the presence of male sterile cytoplasm. The fertiliy restorer gene, R is dominant and restores male fertility in the male sterile line. The use of this system in hybrid seed production is outlined below; Inbred 1 X inbred 2 (MS, rr) (Restorer, RR) Female male
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Single cross hybrid (MF, Rr) Inbred 1 X (MS, rr) Female inbred 2 Inbred 3 X (restorer, RR) ( Non-Restorer, rr) Male Single cross 1st (MF, rr) Female X female inbred 4 (restorer, RR) male single cross 2nd ( MF, RR) male Double cross hybrid (MF, Rr) 2) Use of cytoplasmic male sterility: This sterility is governed by a particular type of cytoplasm. The scheme for hybrid seed production is the same as that with cytoplasmic-genetic male sterility, except that male fertile line is non-restorer . The hybrid therefore is male sterile. It may be useful in crops where grain or seed is not the commercial product. Pollinator (R-line) Seed parent (A-line) maintainer (B-line) (F) RfRf-MF MF (s) rfrf-MS (F) rfrf
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. (F)RfRf Propagated B-line R-line (S) Rfrf F1 hybrid (S) rfrf (F) rfrf Propagated A line propagated Fig: production of single cross hybrid Line 1 (S) rfrf MS SC 1: X (S) rfrf MS Double cross Line 2 (F) rfrf MF Line 3 (S) rfrf MS X (S)Rfrf MF (hybrid) SC 2: + X Line 4 (F)RfRf MF (S) Rfrf MF (S) rfrf MS Fig: production of double cross hybrid 3) Use of genetic male sterility: Sterility is governed by a nuclear recessive gene (ms). The male sterile line (ms ms) is allowed to be cross pollinated with a male fertile line (Ms Ms) to yield a male fertile hybrid (Ms ms). It has been commercially exploited in pigeonpea. Inbred 1 X (MS, ms ms) inbred 2 (MF, Ms Ms)
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Female male Single cross hybrid (MF, Ms ms) Inbred 1 X inbred 2 (MS, ms ms) Female Inbred 3 X (MF, Ms Ms) ( MF, Ms ms) Male Single cross 1st (Ms, ms ms) Female X female inbred 4 (MF, Ms) male single cross 2nd ( MF, Ms Ms) male Double cross hybrid (MF, Ms ms) 4) Use of concept of self incompatibility: Two self-incompatible lines are planted in alternate rows; seed produced by both lines would be hybrid seed. Alternatively, a self compatible line may be interplanted with a self-incompatible line. In this case, the seed form self-incompatible line will be the hybrid seed, while that from the self compatible line will be a mixture of hybrid and selfed seed. Therefore, the seed from self incompatible line only is used as hybrid variety. 5) Manual Emasculaion and /or pollination: This method relies on manual emasculation and in many cases on manual polli nation. Early hybrid maize production is based on manual emasculation i. e. detasselling. It is used in hybrid seed production in tomatoes.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 6) Chemically-induced male sterility: Several chemicals induce male sterility when applied during specific developme natal stages of plants; such male sterility is known as chemically induced male sterility and chemical compounds are called chemical hybridizing agents (CHAs). The use of CHAs for rendering the pollen grains nonfunctional so that the treated lines is used as female parent in hybrid seed production is termed as chemical emasculation. Some of the CHAs are highly effective and are being used for commercial hybrid seed production in rice and wheat. Why hybrid seeds are not produced in cereals in Nepal?. For producing hybrid seeds; the technical skill is required for pollen control system. The inflorescence of cereals is spikelet in which flowers are small. It is difficult for hand emasculation and pollen transfer in cereals flowers. It is necessary for changing the seed every year in hybrid seed production and the cost of buying new hybrid seed is high. For the hybrid seed production, some what large farm area is needed. Hybrid varieties is less adapted as compared to local varieties. That is why farmer do not like to replace the local varieties by hybrid one. Most of Nepalese farmers are poor in their economic status, they have low land holding capacity and are mostly illiterate and ignorant about the technology. Therefore hybrid seeds are not produced in cereals in Nepal. OPERATIONS INVOLVED IN HYBRID PRODUCTION 1) Development of inbred lines: Inbred lines are developed form a genetically variable population (source population) through continued inbreeding or self pollination. Inbreds isolated from an open-pollinated variety, which may or may not have been subjected to population improvement , are known as first cycle inbreds. On the other hand, inbreds isolated from hybrid varieties are termed as second, third or fourth cycle inbred. Folllowing are steps for production of first cycle inbreds; a) Isolation of inbreds through inbreeding b) Development of inbreds from haploid plants c) Slection during inbreeding d) Early testing 2) Evaluation of inbreds: The most important operation in a hybrid programme is the identification of inbreds that would produce an outstanding hybrid suitable for commercial use. The modern practice of inbred evaluation may be divided into follwing; a) Phenotypic evaluation: it is done for elimination of weak and inferior inbreds b) Top cross test: it is done for selection of inbreds showing high GCA c) Single cross evaluation: it is done for identification of outstanding single or double cross. d) Prediction of the double cross performance from data on the performance of single crosses.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 3) Production of hybrids: The two requirements for commercial hybrid seed production are; a) easy emasculation of female parent and b) effective pollen dispersal from the male parent to ensure a satisfactory seed set in the female parent.The hybrid seed production can be done using following methods; a) use of cytoplasmic male sterility b) use of genetic male sterility c) use of cytoplasmic-genetic male sterility d) self incompatibility e) Manual emasculation and or pollination f) Chemically induced male sterility. 7. Special Techniques 7.1 Mutation breeding, 7.2 Polyploidy in plant breeding, 7.3 Apomixis, 7.4 Self incompatibility and its application in plant breeding, 7.5 Male sterility 7.6 Wide crossing and distant hybridization in plant breeding 7.7 Clonal breeding 7.8 Breeding for resistance to biotic stresses i.e. diseases and insects 7.9 Breeding for quality improvement, 7.10 Ideotype concept in crop improvement 7.1. MUTATION BREEDING Mutation is a sudden heritable change in a characteristic of an organism. The causes of mutation are; a change in allelic form of a gene, rearrangement of chromosomal materials, loss or duplication of chromosomes segment. Characteristics of mutation: 1) mutations are generally recessive. 2) Mutations are generally harmful to organisms 3) Mutations are random 4) Mutations are recurrent 5) Induced mutation show pleiotrophy. Types of mutation:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1. According to cell involvement: d) Somatic mutation e) Gamatic mutation 2. A/c to size and quality: a) Point mutation: it occurs in a very small segment of DNA molecule i.e. a single nucleo tide or nucleotide pair b) Gross mutation: it occurs in more than one nucleotide pairs 3. A/c to origin/mechanism of mutation: a) spontaneous mutation: it occurs by chance in natural population without any treatment by man at a low rate c) Induced mutation: it is artificially induced by a treatment with certain physical or chemical agents. It is important in plant breeding. Utilization of induced mutation: 1) direct release as variety, 2) used as parents in a hybridization programmes. Mutagenesis: treating a biological material with a mutagenic agents in order to induce mutation is called mutagenesis. Mutation breeding: The utilization of induced mutation for crop improvement. Mutagenic agents: Undiscriminating agents produce complex mixture mutations, chromosome structural change and non genic aberration A)Physical mutagens: 1) Ionising radiation: X- and gamma-rays are energetic enough that they produce reactive ions (charged atoms or molecules) when they react with biological molecules; thus they are referred to as ionizing radiation. This term also includes corpuscular radiation--streams of atomic and subatomic particles emitted by radioactive elements: these are of two types, alpha- and beta-particles [alpha are helium nuclei, 2 protons and 2 neutrons; beta are electrons]. The units now used for ionizing radiation of all types are rems (roentgen equivalent man): 1 rem of any ionizing radiation produces similar biological effects. The unit used previously was the rad (radiation absorbed dose). However, the effects of different types of radiation differ for one rad unit: one rad of alpha particles has a much greater damaging effect than one rad of gamma rays; alpha particles have a greater RBE (relative biological effectiveness) than gamma rays. The relationship between these units is that: # rads x RBE = # rems In addition to the energy type and total dose of radiation the dose rate should be considered: the same number of rems given in a brief, intense exposure (high dose rate) causes burns and skin damage versus a long-term weak exposure (low dose rate) which would only increase risk of mutation and cancer
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2) Non-ionising radiation: UV radiation is not ionizing but can react with DNA and other biological molecules and is also important as a mutagen. B). Chemical mutagens The first report of mutagenic action of a chemical was in 1942 by Charlotte Auerbach, who showed that nitrogen mustard (component of poisonous mustard gas used in World Wars I and II) could cause mutations in cells. Since that time, many other mutagenic chemicals have been identified and there is a huge industry and government bureaucracy dedicated to finding them in food additives, industrial wastes, etc. It is possible to distinguish chemical mutagens by their modes of action; some of these cause mutations by mechanisms similar to those which arise spontaneously while others are more like radiation (to be considered next) in their effects. 1. Base analogs These chemicals structurally resemble purines and pyrimidines and may be incorporated into DNA in place of the normal bases during DNA replication: bromouracil (BU)--artificially created compound extensively used in research. Resembles thymine (has Br atom instead of methyl group) and will be incorporated into DNA and pair with A like thymine. It has a higher likelihood for tautomerization to the enol form (BU*) aminopurine --adenine analog which can pair with T or (less well) with C; causes A:T to G:C or G:C to A:T transitions. Base analogs cause transitions, as do spontaneous tautomerization events. 2. Chemicals which alter structure and pairing properties of bases There are many such mutagens; some well-known examples are: nitrous acid--formed by digestion of nitrites (preservatives) in foods. It causes C to U, meC to T, and A to hypoxanthine deaminations. [See above for the consequences of the first two events; hypoxanthine in DNA pairs with C and causes transitions. Deamination by nitrous acid, like spontaneous deamination, causes transitions. nitrosoguanidine, methyl methanesulfonate, ethyl methanesulfonate--chemical mutagens that react with bases and add methyl or ethyl groups. Depending on the affected atom, the alkylated base may then degrade to yield a baseless site, which is mutagenic and recombinogenic, or mispair to result in mutations upon DNA replication. 3. Intercalating agents acridine orange, proflavin, ethidium bromide (used in labs as dyes and mutagens) All are flat, multiple ring molecules which interact with bases of DNA and insert between them. This insertion causes a "stretching" of the DNA duplex and the DNA polymerase is "fooled" into inserting an extra base opposite an intercalated molecule. The result is that intercalating agents cause frameshifts. 4. Agents altering DNA structure We are using this as a "catch-all" category which includes a variety of different kinds of agents. These may be: --large molecules which bind to bases in DNA and cause them to be noncoding--we refer to these as "bulky" lesions (eg. NAAAF)
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. --agents causing intra- and inter-strand crosslinks (eg. psoralens--found in some vegetables and used in treatments of some skin conditions) --chemicals causing DNA strand breaks (eg. peroxides) Applications/uses of mutation breeding: 1. Useful in improving specific characteristics of a well adapted high yielding variety im.spe.cha. 2. Used to improve various quantitative characters im.qua.cha. 3. F1 hybrids from intervarietal crosses may be treated with mutagens in order to increase genetic variability by inducing mautations in.ge.va. 4. possible to transfer desirable gene from wild to cultivated species. Tra.de.ge. wil.cul. Limitations of mutation breeding:  most of mutations are recessive. Detection of recessive mutation is imposible in clonal crops and is difficult in polyploidy species  desirable mutations are commonly associated with undesirable side effect due to other mutation, chromosomal aberration  mutation breeding programme requires long time, more labor and expenditure  the frequency of desirable mutation is very slow, about 0.1% of total mutation  breeder has to screen large population to select desirable mutation Gamma-garden: it is an area subjected to gamma irradiation. The purpose of a gamma garden is to irradiate whole plants during different stages of development and fo r varying durations. The first gamma-garden was built in Long Island near U.S.A. 7.2.POLYPLOIDY IN PLANT BREEDING Poidy : A.. Euploidy: It is the condition where the individuals contain multiples of basic chromosomal set (denoted by x) 1. Monoploidy (n) 2. Diploidy (2n) 3. Polyploidy (3n,4n,5n etc.): The organisms with multiple chromosomes having more than twice the basic number of chromosomes is called polyploidy. Polyploid is of two types; a) Autopolyploids: they are usually sterile b) Allopolyploids: they are commonly fertile.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. B. Aneuploidy: It is the condition where there is a difference of one or more chromosomes from the basic diploid number 1. Hypoploidy: a) Monosomy (2n-1) b) Nullisomy (2n-2) 2. Hyperploidy : a) Trisomy (2n+1) b) Tetrasomy (2n+2) Name Formula/symbol Somatic chromosome component Nullisomic Monosomic Double monosomic Trisomic Double trisomic Terasomic Monosomic-trisomic Euploid 2n-2 2n-1 2n-1-1 2n+1 2n+1+1 2n+2 2n-1+1 (AB) (AB) (ABC) (AB) (AB)(AC) (ABC) (ABC) (C) (ABC) (ABC) (A) (B) (ABC) (ABC) (A) (A) (ABC) (AB) (A) Monoploid Diploid Autoploid Autotriploid Auto tetraploid X 2x (ABC) (ABC) (ABC) 3x 4x (ABC) (ABC) (ABC) (ABC) (ABC) (ABC) (ABC) Aneuploid Alloploid Allotetraploid Allohexaploid 2x1+2x2 2x1+2x2+2x3 Role of polyploidy (Euploidy) in plant breeding: 1. used in propagating crops which are valued for vegetative organs 2. are important factors in the evolution of plant species. The genetic origin of common wheat, tobacco, cotton is an examples. 3. have evolutionary significance enabling plant breeders to cross cultivated spp with wild spp 4. are sources of genetic variabbiity in the form of multiple sets of chromosome or lacking one or more chromosomes 5. form a genetic linkage bridge in gene introgression Utilization of Autopolyploidy:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1. Auto triploids are highly sterile therefore used in production of seedless watermelon. Autotriploid is used in tea hybridization and drought tolerance 2. Auto tetraploids are useful in production of improved superior quality, overcoming self incompatibility, making distant crosses and used directly as variety. Limitations of Autopolyploidy: 1. Larger size of autopolyploids is generally accompanied with a higher water content 2. autopolyploids show high sterility accompanied with poor seed set 3. triploids cannot be maintained except through clonal propagation 4. polyploids are always characterized by a few or more undesirable features. E.g. poor strength of stem in grapes, irregular fruit size in watermelon 5. Fertility in autotetraploids can be increased by hybridization and selection at the tetraploid level. But due to the complex segregation in autotetraploids, progress under selection is slow. 6. Effects of autoplolyploidy can not be predicted. Utilization of Allopolyploidy: 1) act as bridging species in the transfer of characters from one species to another 2) In the production of new crop species 3) In widening the genetic base of existing allopolyploid crop species 4) used in identification of genetic origin of poplyploids. Limitation of Allopolyploids: 1) The effects of allopolyploidy can not be predicted 2) Newly synthesized allopolyploids have many defects e.g. low fertility, cytogenetic and genetic instability, other undesirable features. 3) the synthetic allopolyploids have to be improved through extensive breeding at the polyploidy level. This involves considerable time, labour and other resources 4) Only a small proportion of allopolyploids are promising; a vast majority of them are valueless for agricultural purposes. Role or importance or applications of Aneuploidy: 1. useful to see the effect or loss or gain of an entire chromosome or a chromosome arm on morphology and physiology of plant 2. useful in locating (detecting) a linkage group and a gene to a particular chromosome 3. useful in detection of homoeology between A, B, and D genomes of wheat 4. useful in the production of alien (foreign) substitution lines 5. useful in detection of translocated chromosomes. Limitations of Aneuploidy: 1. production, identification and maintenance of aneuploids requires elaborate cytogenetic analysis which is difficult, time consuming and requires considerable skill
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2. Maintenace of monosomic is difficult because of elimination of unpaired chromosomes 3. production and use of aneuploids as genetic study of alies substitution line requires cytogenetic analysis. 7.3. APOMIXIS Apomixis is an asexual mode of plant reproduction through seeds. A common feature of all apomicts is the autonomous development of embryos and the generation of progenies that are exact genetic replicas of the mother plant.. It refers to development of embryo or seeds without fertilization. It is development of seeds without completion of sexual process i. e. not due to fusion of male and female gametes. Plants producing only apomitic embryo is called obligate apomictics and those producing apomictic and sexual seedlings is called facultative apomictics. When embryos arise from haploid cells, apomixes is termed as nonrecurrent apomixes and when embryos arise from diploid cells, apomixes is called recurrent apomixes. Forms of Apomixis: 2. Parthenogenesis: it refers to development of embryos from embryo sac without pollination 3. Androgenesis: pollen grains induced in invitro to produce haploid embyos or planets is called androgenesis 4. Apogamy: it refers to development of embryo from synergids or antipodal cells without fertilization 5. Semigamy: in this case, male gamete enters into embyo sac. Male gamete and female gamete do not fuse nor it displace each otherbut both nuclei starts dividing and d evelop into plant 6. Adventitious embryony: In this case, embryos develop directly from vegetative cells of ovules such as nucellus, integument and chalaza and it does not involve production of embryo sac. Development of apomictic lines: 1) Gene transfer from wild species: genes controlling apomixes are transferred into a crop species from a related wild species through back cross method. 2) Induced mutation: apomictic lines are developed in sexually reproducing species by using induced or spontaneous mutations 3) isolation of apomictic recombinats from interspecific crosses. Significance of apomixes: The aims of studying apomixis are to unlock the diversity of apomictic plants and to make it feasible to transfer apomixis to agriculturally important genotypes, therefore conferring them the ability of cloning through seeds. "Apomixis is a natural way of cloning plants through seed. It offers plant breeders a unique system for developing new and distinctive cultivars in many species. "Apomixis will give scientists a potent tool to create hybrids that can produce generations of genetically identical
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. plants that retain their original hybrid genetics."Various forms of apomixis have been reported for over 300 species representing more than 35 families of plants. Applications of apomixes: 1) Fixation of heterosis 2) production of homozygous lines 3) development of phenotypically stable populations called vybrids. Advantages of apomixis: 1) obligate apomixes permits fixation of heterosis in hybrids. 2) Hybrid seed fields require minimal (3m) isolation to prevent mechnical mixtures Problems in utilization of apomixes: 1) it is complicated phenomena. 2) Estimation of level of apomixes is tedious and time consuming 3) Genetic basis of apomixes is not clear in most cases. 4) In case of facultative apomictics, the proportion of sexual progeny is affected by environmental factors like day length. 5) In the absence of morphological markers linked with apomictic development, maintenance of apomictic stocks becomes difficult. 7.4. SELF INCOMPATIBILITY AND ITS APPLICATION It is failure of pollen from a anther to fertilize the same or other flowers on the same plant. Self incompatibility is defined as ‗ prevention of fusion of fertile (or functional) male and female gametes after self pollination‘. It refers to inability of functional male and female gametes to effect fertilization in particular genotypic combination. Incase of self incompatibility, pollen grains fails to germinate on the stigma of flower. If some pollen grains do germinate, pollen tubes fails to enter stigma. Self-incompatibility (SI) is a general name for several genetic mechanisms in angiosperms, which prevent self-fertilization and thus encourage outcrossing. In plants with SI, when a pollen grain produced in a plant reaches a stigma of the same plant or another plant with a similar genotype, the process of pollen germination, pollen tube growth, ovule fertilization, and embryo development is halted at one of its stages, and consequently no seeds are produced. SI is one of the most important means to prevent selfing and promote the generation of new genotypes in plants, and it is considered as one of the causes for the spread and success of the angiosperms on the earth. Causes of self incompatibility: 1) pollen-stigma interaction: inability of pollen grains to penetrate on stigma. 2) Pollen tube- style interaction: growth of pollen tube is retarded within stigma. 3) Pollen tube-ovule interaction; embryo degenerates at the early stage of development. 4) Inability of pollen tube formation.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Consequences of self incompatibility: self incompatibility prevents self pollination and favors out crossing, cross pollination Elimination of self incompatibility: 1) by doubling the chromosome number 2) isolation of self-fertile mutants 3) self incompatibility alleles may be transferred from related species through backcross. Ways to overcome self incompatibility: 1) bud pollination: application of mature pollen to immature non receptive stigma 2) surgical removal of stigmatic surface. 3) maintaining high temperature to flowers 4) Treating with increased CO 2 concentration 5) Maintaining high humidity 6) Spraying of salt (Nacl) solution to flowers 7) Exposing flowers to gamma rays 8) Grafting of branches Types of self incompatibility: 1. Heteromorphic self incompatibility: it occurs because of flower morphology associated with differences in positioning and shape of flower parts. In this system, flowers of different incompatibility groups are different in morphology. Distyly: Pin flower: long style and short stamen Thrum flower: short style and long stamen. Pin and thrum flowers are produced on different plants. This characteristic is governed by single locus ‗s‘; Ss produces thrum while ss produces pin flowers. The gene governing self incompatibility reaction has two alleles S and s; allele S is dominant over s. 2. Homomorphic self incompatibility: it is not associated with morphological differences among flowers. The incompatibility reaction of pollen may be controlled by genotype of plant on which it is produced (sporophytic control) or by its own genotype (gametophytic control). it is due to genotype of plants. It is of two types: i. Sporophytic self-incompatibility (SSI) : It is governed by genotype of plant on which pollen is produced and not by genotype of pollen. In this system, S allele exhibits dominance, individual action (codominance or competition). This form of self-incompatibility has been studied intensively in members of the mustard family (Brassica), including turnips, rape, cabbage, broccoli, and cauliflower
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. ii. Gametophytic self-incompatibility (GSI): The incompatibility reaction of pollen is determined by its own genotype and not by genotype of the plant on which it is produced. The incompatibility reaction may be controlled by one or two genes; on this basis, gametophytic self incompatibility is two types; a) monofactorial gametophytic SI: SI reaction is controlled by a single gene designated by S, which usually has 50 or mote alleles. b) Difactorial gametophytic SI: SI reaction is controlled by two genes designated by S and Z, which are unlinked and exhibit multiple alleles. This is the more common type of SI, existing in the families: Solanaceae, Rosaceae, Plantaginaceae, Fabaceae, Onagraceae, Campanulaceae, Papaveraceae and Poaceae Applications of self incompatibility: 1) Used in hybrid seed production: 2) Gametophytic system is used for hybrid seed production of clover, trifolium 3) Sporophytic system is used for hybrid seed production of brassicas. Difference between Gametophytic self incompatibility and Sporophytic self incompatibility Gametophytic self incompatibility Sporophytic self incompatibility 1) this incompatibility reaction of pollen is determined by its own genotype and not by genotype of the plant on which it is produced 1) this incompatibility reaction of pollen is governed by genotype of plant on which the pollen is produced and not by genotype of pollen 2) dominance is absent 2) dominance is present on the male side but absent in female side 3) a homozygous system may not be 3) a homozygous system may be possible possible through crossing in a through crossing in sporophytic self gametophytic self incompatibility incompatibility. 7.5 MALE STERILITY Male sterility is defined as the failure of plants to produce functional anthers, pollen, or male gametes. It refers to the absence of functional pollen grains. Male sterility is the incapacity of flowering plants to produce or release functional gametes. Causes of male sterility 1) Enviromental factors: low temperature, low light intensity and climatic stress 2) presence of subvital or sub lethal genes 3) mutation 4) use of gametocides.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Manifestations of Male Sterility: 1) Absence or malformation of male organs (stamens) in bisexual plants or no male flowers in dioecious plants. 2) Failure to develop normal microsporogenous tissue- anther. 3) Abnormal microsporogenesis--deformed or inviable pollen. 4) Abnormal pollen maturation; inability to germinate on compatible stigmata. 5) Nondehiscent anthers but viable pollen,- sporophytic control. 6) Barriers other than incompatibility preventing pollen from reaching ovule. Applications of male sterility: 1. hybrid seed production: Genetic male sterility: hybrid seed production of arhar, castor Cytoplasmic male sterility: hybrid seed production in ornamentals Cytoplasmic genetic male sterility: hybrid seed production of maize, bajra, cotton, rice, sunflower etc. 2. Introduction of favorable gene in a population: the disease resistance gene in male can be crossed with female to develop disease resistant variety. Classification of Male Sterility : A. Phenotypic Male sterility: It is caused by extreme climatic condition and use of gametocides. It occurs in following forms: Absence, atrophy or malformation of male sex organs Lack of a normal anther sac or anther tissues Inability of pollen to mature or to be released from anther sacs Inability to develop normal microspores or pollen. B. Genotypic male sterility: It is of two types: a) True male sterility: it is of follwing types i) Structural male sterility: anomalies in male sex organs (or missing all together) ii) Pollen male sterility: it is of following types: 1) Cytoplasmic Male Sterility: it is determined by particular type of cytoplasm. CMS is the result of mutation in the mitrochondrial genome (mt DNA) which leads to mitochondrial disfunction. This results in
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. unfavorable nuclear mitochondrial interaction or incompatibility the ultimate consequence of which is male sterility. Steps in developing CMS lines: Introduction of male sterile cytoplasm into breeding stock Maintenance of CMS line (A-line) and its fertile isogenic counterpart (B-line) Development of fertility restorer line (R-line) by introducting Rf gene into pollinator line Production of hybrids by crossing A-line and R-line. 2) Genetic Male Sterility: it is caused by nuclear genes MS gene inhibiting normal development of anthers or an pollen. Generally this MS is governed by single recessive gene ms. When a male sterile plant (ms ms) is crossed with a male fertile (MS ms) one, the F1 (MS ms) is male fertile. Genetic MS is following types; o enviroment-insensitive GMS o environment-sensitive GMS 3) Cytoplasmic Genetic Male Sterility: This male sterility is caused by primarily by a particular type of cytoplasm but only in the absence of dominant sterility restorer gene. This is a case of cytoplasmic MS where a nuclear gene for resoring fertility in the male sterile line is known. The fertility restorer gene, R, is dominant and is found in certain strains of species or may be transferred form a related species. Limitations of cytoplasmic-Genetic male sterility: 1) undesirable effects of MS cytoplasm 2) unsatisfactory fertility restoration 3) unsatisfactory pollination 4) MS cytoplasm show a low frequency of spontaneous reversion 5) Cytoplasm may be contributed by sperm which lead to breakdown of male sterility mechanism. 6) MS sterility may breakdown under certain environmental condition.. b) Functional male sterility: viable pollen form, but barrier prevents fertilization (anther indehiscence, no exine formation, inability of pollen to migrate to stigma. 7.6 WIDE CROSSING AND DISTANT HYBRIDIZATION IN PLANT BREEDING Hybridization between individuals from different species, belonging to the same genus or to different genera, is termed as distant hybridization, and such crosses are known as distant crosses or wide crosses. The first authentic record of a distant hybridization for crop improvement is the production of a hybrid between carnation and sweetwillian byThomas Fairchild in 1717. Distant hybridization is used for transfer of specific characters like disease resistance to cultivated species. Many of genes for rust resistance in wheat have been transferred from related wild species.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Difficulties encountered in production of distant hybrids:  failure of zygote formation.  Failure of zygote development  Failure of F1 seedling development Sterility in distant hybrids: The sterility of distant hybrids may be caused by: a) Cytogenetic factors (cytogenetic basis of sterility): most of interspecific hybrids show reduced chromosome pairing cases, all the chromosomes may be present as univalents. The distribution of chromosomes in such cases is irregular, and it leads to the formation of unbalanced gametes resulting in partial to complete sterility. b) Genetic factors ( genetic basis of sterility): small structural changes in chromosomes may cause the sterility in distant hybrids. c) Cytoplasmic factors (cytoplasmic basis of sterility): in some interspecific crosses, sterility is produced by cytoplasm. Application of distant hybridization: 1) transfer of small chromosome segments: The genes for disease resistance, earliness and wider adaptation and genes affecting mode of reproduction, quality, yield etc are transferred from wild relative to cultivated species. 2) transfer of cytoplasm: cytoplasm are transferred in production of male sterile lines. 3) development of new crop species 4) Production of alien-addition lines and alien-substitution lines. Limitations of distant hybridization: 1) several distant combinations cannot be crossed 2) F1 hybrids from distant crosses generally exhibits sterility. 3) The production of new crop species through distant hybridization suffers from several problems like lower economic yields, poor agronomic characters, sterility etc. 4) In some cases, the F1 seeds of interspecific hybrids exhibit dormancy. 5) F1 hybrids from interspecific crosses fail to produce flowers. 6) In many distant hybridization, transfer of recessive traits and of quantitative characters is not feasible. 7) Transfer of undesirable linkages. Sexual polyploidization: it refers to the production of polyploidy progeny through sexual reprodcuiton; it occurs due to production of unreduced gametes. When unreduced gametes are produced by only one of the
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. parents, it is called unilateral sexual polyploidization and when unreduced gametes are produced by both of parents of a cross, it is called bilateral sexual polyploidization. 7.7 CLONAL BREEDING Clone: A clone is a group of plants produced from a single plant through asexual reproduction. Thus asexually propagated crops consists of a large number of clones and they are often known as clonal crops. All the members of a clone have same genotype as the parent plant. As a result they are identical with each other in genotype. Clonal Selection: The procedure of selection of asexually propagated crops is called clonal selection since the selected plants are used to produce new variety. The phenotypic value of a plant or clone is due to the effects of its genotype (G), the environment (E) and genotype x environment (G X E) interaction. Procedure of Clonal Selection: First year: Few to several hundred superior plants are selected Second year: 6) clones from the selected plants grown separately 7) desirable clones selected. Third year: 1) conduct priminary yield trial with standard checks 2) selection for quality, disease reistance etc. disease nurseries may be planted 3) few out standing clones selected. Fourth-sixth year: 1) multilocation yield trials with standard checks 2) best clone identified for release as a new variety Seven year: 1) the best clone released as a new variety 2) seed multiplication for distribution begins.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Merits of clonal selection: 1) useful in maintaining the purity of clones 2) it is the only method of selection applicable to clonal crop it avoids inbreeding depression and preserves the gene combinations present in the clones 3) clonal selection without any substantial modification can be combined with hybridization to generate variability necessary for selection. Demerits of clonal selection: 1) this selection method utilizes the natural variability already present in the population; it has not been devised to generate variability 2) sexual reproduction is prerequisite for the creation of variability through hybridizatrion. 7.8 BREEDING FOR RESISTANCE TO BIOTIC STRESSES (DISEASES AND INSECTS) A) Methods of breeding for disease resistance: Followings are methods of breeding for disease resistance; 6) Introduction: Resistant varieties may be introduced for cultivation in a new area. Introduction is quick but has certain limitations like introduced varieties may not perform well in a new area, may become susceptible to concerned disease in the new environment and may be susceptible to other races of the concerned diseases. 7) Selection: Selection of resistant plants from a commercial variety is the cheapest and quickest method of developing a resistant variety. e.g. Kufri red potato is a disease resistant selection form Darjeeling. 8) Mutation: The spontaneous as well as induced mutation is useful in breeding for disease resistance 9) Somaclonal variation: The disease resistant somaclonal variants can be obtained in following ways; b) plants regenerated from cultured cells or their progenies are subjected to disease test and resistant plants are isolated (screening) c) cultured cells are selected for resistance to toxin or culture filterate produced by the pathogen and plants are regenerated form selected cells. 10) Genetic Engineering: Genes expected to confer disease resistance are isolated and transferred into crops through genetic engineering.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 11) Hybridization: It is the most common method of breeding for disease resistance. It serves 2 purposes; i) transfer of disease resistance from an agronomically undesirable variety to a susceptible which is widely adapted and agronomically desirable (by backcrossing) ii) combining disease resistance and some other desirable characters of one variety with superior characteristics of another variety (by pedigree method). DIFFERENCE BETWEEN VERTICAL RESISTANCE AND HORIZONTAL RESISTANCE Vertical resistance{VR} Horizontal resistance{HR} 1) It is effective to some races of pathogen but not to others 2) A gene -for- gene relationship is involved in VR 1) It is effective against all races of pathogen 2) It is characterized by absence of differential interaction between host varieties and pathogenic races 3) It is determine by major genes and is 3) It is controlled by polygenes and pathotype -specificity pathotype-non specificity 4) Phenotypic expression is qualitative 4) Phenotypic expression is quantitaive 5) Selection and evaluation is relatively 5) Selection and evaluation is relatively easy difficult 6) Suitable host is annuals but not 6) Suitable host is both annuals and perennials perennials 7) Stage of expression is seedling to 7) Expression increases as plant matures maturity GENE-FOR-GENE RELATIONSHIP: The gene-for-gene relationship between a host and its pathogen was postulated by Flor in 1951. based on his work on linseed rust. According to gene-for-gene hypothesis ―A resistance gene ‗R‘ is only effective if the infecting pathogen carries the corresponding avirulence gene ‗A‘. It has been found that ― for every resistance gene present in the host, pathogen has gene for virulence‖. Susceptible reaction would result only when the pathogen is able to match all the resistance genes present in the host with appropriate virulence genes. If one or more resistance genes are not matched by the pathogen with the appropriate virulence genes, resistance reaction is the result. Host resistance is governed by dominant allele ‗R‘. In the pathogen, virulence is conditioned (governed) by recessive allele ‗a‘. The resistance reaction occurs when complementary genes in both host and pathogen are dominant. A host genotype that carries no dominant allele carries of loci is susceptible for all races of pathogen.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. B) Methods of breeding for insect resistance: Following methods are used for breeding insect resistance; 1) Introduction: Introduction of variety resistant to concerned insect in new area is quickest and easiest method of developing an insect resistant variety. 2) Selection: Selection for insect resistance (from an existing crop varieties) is practiced to isolate an isect resistant variety; a) in self pollinated crops; mass selection or pureline selection is practiced for such purpose b) in cross pollinated crops; mass selection or recurrent selection is practiced. 3) Hybridization: Insect resistance species are crossed with an agronomically superior and adapted but insect susceptible variety of the crop to develop a superior, well adapted and insect resistant variety. If a gene for resistance may be present in a related wild species, back crossing method is practiced. Resistant var (non recurrent parent) RR Susceptible var (recurrent parent) rr X F1 X recurrent parent BC1 X recurrent parent BC5-BC6 Rr (insect resistance var) If the resistance is governed by oligogenes, selection for resistance is practiced in F2 generation but in the case of polygenic insect resistance; selection for resistance is delayed till the F3 generation a relatively large number of resistant plants is selected 4) Genetic engineering: Genes conferring insect reistacne in plants have been transferred from B. thuringiensis (cry gene) and from other plants through genetic engineering
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 7.9 BREEDING FOR QUALITY IMPROVEMENT Quality trait is some aspect of produce. The quality traits are classified as; morphological traits (grain size, color), organolaptic traits ( taste, aroma, juiceness), nutritional traits ( protein, oil vit, mineral content), biological traits ( protein efficiency ratio, biological value) and other quality traits ( cooking quality of rice, keeping quality of vegetables) Breeding approaches used for improvement of quality traits are following; 1) Screening of germplasm: This may often yield a source for a quality trait especially chemical composition .e.g. high lysine germplasm line of sorghum. Since germplasm lines are inferior in yield and agronomic characteristics, screening of them is necessary. 2) Mutagenesis: A desired quality trait may be present in spontaneous/induced mutants. The mutants line may be subjected to mutagnesis and mutants lacking the undesirable features, but having desired quality trait, may be isolated 3) Hybridization: It is used to develop high yeiding varieties with desirable quality traits. It is of follwing; a) backcrossing: it is done if inferior parent has some desirablequality b) Recurrent selection: for increasing seed protein content of rajma c) Pedigree method: used when both parents are high yielding and having good agronomic characters 4) Interspecific Hybridization: it is done when wild relatives has useful quality genes. E.g. fruit color, fruit size, vit A and Vit C content in tomatoes. 5) Somaclonal vartaion: Genetic variation present in tissue culture-raised should be exploited for crop improvement and several somaclonal variants are being used as commercial varieties. 7.10 IDEOTYPE CONCEPT IN CROP IMPROVEMENT A crop ideotype is a plant model, which is expected to yield a grater quantity or quality of grain, oil or other useful product when developed as a cultivar. It is biological model which is expected to perform or behave in a predictable manner within a defined environment. IDEOTYPE BREEDING: The selection based on traits constituting ideotype is called ideotype breeding. It generates genetic diversity.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. IDEOTYPE OF CROPS: B) Rice: 1) shorter culm length (100 cm or less) 2) greater culm diameter which increases culm strength 3) lower relative internode elongation under heavy nitrogen 4) short eect leaves o f medium width 5) high tillering capacity 6) more panicle/m2 7) high harvest index 8) V-type leaves C) 1) 2) 3) 4) 5) 6) 7) 8) 9) D) 1) 2) 3) 4) 5) 6) 7) Maize stiff vertically oriented leaves above the ear maximum phyotosynthetic efficiency efficient traslocation of photosynthate into grain shorter interval between pollen shed and silk emergence small tassel size photoperiod insensitivity cold tolerance of germinating seeds and developing seedlings grain filling period should be long ear per shoot prolificacy Wheat: short strong stem erect leaves few small leaves large ear erect ear presence of awns single culm 8. Biotechnology 8.1 Scope and importance of plant biotechnology in Nepalese context, 8.2 Plant tissue culture, 8.3 Genetic engineering, 8.4 Embryo culture, 8.5 Anther or pollen culture, 8.6 Methods of gene transfer, 8.7 Molecular markers,
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 8.8 Utilization of gene of interest and gene transfer: - Haploid breeding, - Intergeneric and interspecific crosses, - Marker assisted selection, - Overcoming conventional breeding barriers - DNA finger printing - Characterization of plant genetic resources with biochemical/molecular techniques, 8.9 Recent advances related to crop improvement: - Transgenic plants (GMO’s for crop improvements and quality), - Terminator genes, - Genomics, - Biopesticide, - Biofertilizer. Biotechnology is technology based on biology, especially when used in agriculture, food science, and medicine. The UN Convention on Biological Diversity has come up with one of many definitions of biotechnology: "Biotechnology means any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use. Traditional pharmaceutical drugs are small chemicals molecules that treat the symptoms of a disease or illness - one molecule directed at a single target. Biopharmaceuticals are large biological molecules known as proteins and these target the underlying mechanisms and pathways of a malady; it is a relatively young industry. They can deal with targets in humans that are not accessible with traditional medicines. A patient typically is dosed with a small molecule via a tablet while a large molecule is typically injected. Small molecules are manufactured by chemistry but large molecules are created by living cells: for example, - bacteria cells, yeast cell,animal cells. Modern biotechnology is often associated with the use of genetically altered microorganisms such as E. coli or yeast for the production of substances like insulin or antibiotics. It can also refer to transgenic animals or transgenic plants, such as Bt corn. Genetically altered mammalian cells, such as Chinese Hamster Ovary (CHO) cells, are also widely used to manufacture pharmaceuticals. Another promising new biotechnology application is the development of plantmade pharmaceuticals. Biotechnology is also commonly associated with landmark breakthroughs in new medical therapies to treat diabetes, Hepatitis B, Hepatitis C, Cancers, Arthritis, Haemophilia, Bone
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Fractures, Multiple Sclerosis, Cardiovascular as well as molecular diagnostic devices than can be used to define the patient population. Herceptin, is the first drug approved for use with a matching diagnostic test and is used to treat breast cancer in women whose cancer cells express the protein HER2. Biotechnology in one form or another has flourished since prehistoric times. When the first human beings realized that they could plant their own crops and breed their own animals, they learned to use biotechnology. The discovery that fruit juices fermented into wine, or that milk could be converted into cheese or yogurt, or that beer could be made by fermenting solutions of malt and hops began the study of biotechnology. When the first bakers found that they could make a soft, spongy bread rather than a firm, thin cracker, they were acting as fledgling biotechnologists. The first animal breeders, realizing that different physical traits could be either magnified or lost by mating appropriate pairs of animals, engaged in the manipulations of biotechnology. What then is biotechnology? The term brings to mind many different things. Some think of developing new types of animals. Others dream of almost unlimited sources of human therapeutic drugs. Still others envision the possibility of growing crops that are more nutritious and naturally pest-resistant to feed a rapidly growing world population. This question elicits almost as many first-thought responses as there are people to whom the question can be posed. In its purest form, the term "biotechnology" refers to the use of living organisms or their products to modify human health and the human environment. Prehistoric biotechnologists did this as they used yeast cells to raise bread dough and to ferment alcoholic beverages, and bacterial cells to make cheeses and yogurts and as they bred their strong, productive animals to make even stronger and more productive offspring. Throughout human history, we have learned a great deal about the different organisms that our ancestors used so effectively. The marked increase in our understanding of these organisms and their cell products gains us the ability to control the many functions of various cells and organisms. Using the techniques of gene splicing and recombinant DNA technology, we can now actually combine the genetic elements of two or more living cells. Functioning lengths of DNA can be taken from one organism and placed into the cells of another organism. As a result, for example, we can cause bacterial cells to produce human molecules. Cows can produce more milk for the same amount of feed. And we can synthesize therapeutic molecules that have never before existed. Scope and Importance :
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. »Biotechnology is a multidisciplinary field involving biochemistry, molecular biology, genetics, plant breeding, microbiology, immunology, pharmacology, cell biology and fermentation technology. »Biotechnology is rapidly emerged field and has kept sound impact on all domains of human welfare ranging from food processing, protecting the environment to human health virtually. »The roots of modern biotechnology lie in the fermentation of foods and drinks. Large scale fermentation by fungi and bacteria for the prod of a wide variety of products. E.g. beer. »Efficient large-scale production of antibiotics is accelerated by suitable microbial strains. » Biotechnology is becoming an explosive technology through genetic engineering, development of monoclonal antibodies and hybridomas, and somaclonal variation of plant cells. » The cluster of techniques that constitute modern biotechnology include: Genetic engineering, bioprocessing, monoclonal antibodies, protein engineering, tissue culture, protoplast fusion, biological sensors, bioinformatics, biocatalytic reactors and computer linkage of reactors and processes. »Biotechnology plays a very important role in employment, production and productivity, trade, economics and economy, human health, and the quality of human life throughout the world. »Many commentators are confident that the 21st century will be the century of biotechnology, just as the 20th century was the era of electronics. A. Medical biotechnology: Production of monoclonal antibodies used for disease diagnosis (veneral, cancer, hepatitis B and other viral diseases). DNA probes for disease diagnosis (malaria). Synthetic vaccines (hepatitis B virus, E.coli vaccines for pigs, rabies virus. Drugs: insulin, bovine growth hormone. Gene therapy-for treatment of genetic diseases. DNA fingerprinting-identification of parents/criminals. B. Industrial biotechnology: Production of useful compounds (ethanol, lactic acid, glycerin, and citric acid) Production of antibiotics (penicillin, streptomycin) Production of steroid, enzymes ( -amylase, proteases & lipases) Production of single cell proteins (human feed & animal) Fuel production (ethanol, biogas) Immunotoxins production by joining a natural toxin with a specific antibody C. Animal biotechnology: In vitro fertilisation and embryo transfer: test tube babies. Hormone-induced super ovulation: rapid multiplication of superior genotype.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Production of transgenic animals for increased milk, growth rate, resistance to diseases, production of some valuable proteins in milk, blood, urine (isolation and purification of valuable biochemical is known as molecular farming). D. Environmental Biotechnology: Efficient sewage treatment. Deodorization of human excreta. Degradation of petroleum and mgmt. of oil spills. Detoxification of wasteland industrial effluents. Biocontrol of plant diseases and insect-pests by using viruses, bacteria and amoebae fungi. E. Plant biotechnology: /Agricultural biotechnology: Embryo culture/embryo rescue. Micro propagation. Clonal multiplication: meristem culture. Production of virus and other pathogen free stocks. Cryo-preservation (germplasm conservation through storage in liquid N at -1960c. Production of homozygous lines (chromosome doubling of haploid produced through anther/pollen culture). Somaclonal variant production with improved yield traits, DR, cold R, stresses R. Production of insect R, herbicide R, virus R, crops through genetic engineering. Production of quality crops through genetic engineering (oil, protein, starch composition, fruit softening, colour and longevity of flower). Use of molecular markers (AFLP, RFLPs, and RAPDs) a powerful tool for indirect selection for quantitative traits PLANT BIOTECHNOLOGY Biotechnology may be broadly defined as the use of living organisms such as plants, animals, bacteria, yeast etc or their component parts such as organs, tissues, enzymes, genes etc in the processing of materials to produce consumable goods and services. Plant biotechnology is the study of all activities for improving genetic make up, phenotypic performance or multiplication rates of economic plants or exploit plant cells or cell constituents for generating useful products and or services. it has emerged as an exciting are a of plant sciences by creating unprecedented opportunities for the manipulation of biological systems. 8.1 SCOPE AND IMPORTANCE OF PLANT BIOTECHNOLOGY IN NEPALESE CONTEXT 1) Plant biotechnology plays an important role in identification , utilization and preservation of various plant germplasms . it helps in conserving agro biodiversity in our country. 2) Biotechnological tools such as enzyme-linked immuno-sorbent assay (ELISA), polymerase chain reaction (PCR), randomly amplified polymorphic DNA (RAPD) and DNA amplification
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. finger-printing (DAF) etc can also be used in Nepal for rapid diagnosis of viral and bacterial diseases in plants. 3) Nepal has made a significant progress in the field of agricultural biotechnology specially in plant tissue culture for the last quarter century. Virus-free potato seeds have been developed by tissue culture and sand rooting technique and distributed throughout all agro -ecological zones of the kingdom. 4) Virus and greening-free citrus saplings have been produced under screen by private laboratory and nurseries with the help of tissue culture and diagnostic techniques such as biological indexing and enzyme-linked immunosorbent assays (ELISA). Disease-free banana plants have been produced by several tissue culture factories in the private sector and made available to the farmers throughout the kingdom 5) The PCR technology has also been used for biological nitrogen fixation (BNF) research that identified Rhizobium leguminosarum biovar viciae (Rlv) strains containing nodulating X gene in the mountain soils of Nepal. Rhizobium inoculants specific to different legume species have been routinely used to promote nodulation and enhance BNF in Nepal 6) Plant biotechnology is used for wide hybridization programmes and creation of genetic variability. Plant breeders can develop a new crop varieties or new crop species for benefit of human. 7) Plant biotechnology is used for rapid clonal multiplication and fast multiplication of crops. Tissue culture techniques can shorten time and can lessen labor and space requirements needed to produce a new varieties. 8) Plant biotechnology is the courses at various levels of undergraduate and graduate studies in various departments colleges like botany, genetics, plant breeding, horticulture, entomology, biotechnology etc. PROBLEMS/ CONSTRAINTS IN BPLANT BIOTECHNOLOGICAL WORKS IN NEPAL There are wide gaps between technology generation on tissue culture and dissemination of the technology to the clients or the farmers. Several protocols have been developed for tissue cu lture propagation for more than hundred plant species in Nepal but a very few of them have been widely used by the farmers. In order to popularise these technologies among the farmers, special techniques such as sand rooting have to be taught to the prospective farmers to demonstrate the simplicity and feasibility of such technologies. Unless and until these techniques are shown to be feasible and more profitable than the traditional techniques, very few people will be willing to adopt them. There is no other technique available today to control viral diseases on plants except thermo-therapy and meristem culture. Economically important plants such as potato seeds, citrus saplings, and banana and cardamom plants should be first cleaned from the viruses and then rapidly multiplied by tissue culture and distributed for planting in safe areas. In order to run such programs successfully, the farmers should be trained in the proper use of these technologies. The extension agents from both the public and private sectors should also participate in such training programs so that these technologies are readily disseminated throughout the country.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 8.2 PLANT TISSUE CULTURE Plant tissue culture refers to the in vitro cultivation of plants, seeds, plant parts (tissues, organs, embryos, single cells, protoplasts) on the nutrient media under aseptic condition.. it can be defined as the isolation, manipulation and subsequent growth of cells and tissues for micropropagation of elite materials. It is cultivation of plant organs, tissues or cells in vessels using artificial media. The tissue culture is the production of plants under sterile laboratory conditions. A variety of tissue culture techniques are used to propagate plants. In one method, growers remove a tiny piece of leaf or stem from a plant and place it in a sterile test tube on a gellike medium enriched with hormones and nutrients. A yellow-brown mass of cells called callus develops from the piece of plant. Small chunks of the callus are separated, and each piece is placed in a petri dish with a hormone and nutrient mix that stimulates the development of the callus pieces into plants. The young plants are removed from the petri dish and placed in pots with soil, or into the ground, where they grow to maturity. Steps/Techniques of plant tissue culture: 1. Explant: appropriate explant is removed from mother plant. 2. Surface sterilization: treat the explant with 1-2% solution of sodium or calcium hypochloride or with 0.1% solution of mercuric chloride to eliminate bacteria and fungi present on surface of explant. Then the explant is rinsed several times with sterilized distilled water to remove the disinfectant 3. sterilization: sterilize the instruments or apparatus dipping them on ethanol (70%) or alcohol (95%) or autoclaving 4. Nutrient medium: Inorganic salts to provide 12 elements except C,H,O and growth regulators like auxin (NAA, IAA) Kinetin is used 5. Enviromental condition: Keep medium under controlled environment 18-25 C. 6. Subculturing: Transfer organs and tissues to fresh media at every 4-6 weeks. 7. Regeneration and transfer of plants in soil. Classification/Types of plant tissue culture 1. Embryo culture 2. Meristem culture 3. Callus culture 4. Cell culture. 5. Seed culture 6. protoplast culture 7. organ culture 8. Bud culture Advantages of plant tissue culture: 1. it allows faster and large scale cloning or multiplication of new cultivars in breeding programs.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2. 3. 4. 5. it is important propagation method where in vivo vegetative propagation is inadequate. it is space saving technique compared to green house or field propagation. it is independent of season, so can be done all year round. plants free from pathogens are produced through this method 8.3 GENETIC ENGINEERING GENETIC ENGINEERING (GENE CLONING or Recombinant DNA Technology) The term gene cloning can be defined as the isolation and amplification of an individual gene sequences by insertion of that sequence into a bacterium where it can be replicated. Cloning a fragment of DNA allows indefinite amounts to be produced from even a single original molecule. Cloning technology involves the construction of novel DNA molecules by joining DNA sequences fro different sources. The product is described as recombinant DNA. The construction of such composite or artificial DNA molecule is termed as genetic engineering or gene manipulation. Genetic engineering is isolation and introduction of foreign gene into a DNA molecule of a v ector to construct a hybrid DNA molecule which is transported into the host cells where it is multiplied, doned and expressed to produce altered product. Genetic Engineering, alteration of an organism's genetic, or hereditary, material to eliminate undesirable characteristics or to produce desirable new ones. It is technology of joining of DNA segments derived from biologically different sources. Included in genetic engineering techniques are the selective breeding of plants and animals, hybridization (reproduction between different strains or species), and recombinant deoxyribonucleic acid (DNA). Steps in Genetic engineering: 1. Isolation of gene of interest 2. Insertion of gene of interest or DNA segment in a vector. The vector with an incorporated gene is called a recombinant vector. 3. Introduction of the recombinant vector or recombinant DNA into a suitable host cell by transformation. 4. Selection of transformed host cells. 5. Multiplication of recombinant DNA molecule within host cell to produce a number of identical copies of the cloned gene. Application of genetic engineering:  Production of plants resistant to insects, viruses and herbicides.  Production of novel biochemical. RECOMBINANT DNA Technology How recombinant technology works: Recombinant technology begins with the isolation of a gene of interest. The gene is then inserted into a vector and cloned. A vector is a piece of DNA that is capable of independent growth; commonly used vectors are bacterial plasmids and viral phages. The gene of interest (foreign
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. DNA) is integrated into the plasmid or phage, and this is referred to as recombinant DNA. Before introducing the vector containing the foreign DNA into host cells to express the protein, it must be cloned. Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells. Once the vector is isolated in large quantities, it can be introduced into the desired host cells such as mammalian, yeast, or special bacterial cells. The host cells will then synthesize the foreign protein from the recombinant DNA. When the cells are grown in vast quantities, the foreign or recombinant protein can be isolated and purified in large amounts. Applications for recombinant DNA Recombinant DNA technology is not only an important tool in scientific research, but it has also impacted the diagnosis and treatment of diseases and genetic disorders in many areas of medicine. It has enabled many advances, including: 1) Isolation of large quantities of pure protein In addition to the follicle-stimulating hormone (FSH) used in Follistim® AQ Cartridge and Follistim® AQ Vial, insulin, growth hormone and other proteins are now available as recombinant products. Physicians no longer have to rely on biological products (e.g. urinederived FSH), that don't possess the same level of purity and consistency of recombinant products to treat their patients. 2) Identification of mutations People may be tested for the presence of mutated proteins that may be associated with breast cancer, retino-blastoma, and neurofibromatosis. 3) Diagnosis of affected and carrier states for hereditary diseases Tests exist to determine if people are carriers of the cystic fibrosis gene, the Huntington‘s disease gene, the Tay-Sachs disease gene, or the Duchenne muscular dystrophy gene. 4) Transferring of genes from one organism to another People suffering from cystic fibrosis, rheumatoid arthritis, vascular disease, and certain cancers may now benefit from the progress made in gene therapy. 5) Mapping of human genes on chromosomes Scientists are able to link mutations and disease states to specific sites on chromosomes. TISSUE CULTURE The techniques of growing plant cells, tissues and organs in an artificially prepared medium under aseptic conditions is called tissue culture.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Types of tissue culture: a. organ culture: i. embryo culture ii. anther culture iii. pollen culture iv. ovule culture v. meristem culture b. callus culture c. protoplast culture Basic steps of tissue culture: a. source of explant b. trimming c. surface sterilization d. several washes in sterilized distilled water e. culture establishment f. culture g. sub-culture h. plant regeneration and transfer to sterile soil i. plant regeneration and transfer to non sterile soil. 8.4 EMBRYO CULTURE ―Embryo culture is the sterile isolation and growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant”. In embryo culture, young embryos are removed from developing seeds and are placed on a suitable nutrient medium to obtain seedlings. A fairly new technique in the study of plant development and physiology is embryo culture. The essence of the procedure is to remove embryonic plants from seeds before germination and to grow them in vitro on various culture media under sterile conditions. The stage of development at which the embryos can be successfully cultured varies with different plants. In some species it has been found possible to excise embryos that are almost completely undifferentiated and grow them to maturity. There are two types of embryo culture: 1. Mature embryo culture: it is culture of mature embryos derived from ripe seeds. This is done when embryos do not survive in vivo or become dormant for long period of time or done to eliminate inhibition of seed germination 2. Immature embryo culture: it is the culture of immature embryos to rescue the embryos of wide crosses. This is used to avoid embryo abortion with the purpose of producing a viable plant. Aside from its great value as a tool in the study of the development and requirements for growth of very young plants, embryo culture has had certain practical aspects. Some seeds will not
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. germinate naturally until they have had a period of after-ripening. This after-ripening often involves exposure to low temperatures, and sometimes requires several months. In some cases it has been found that embryos from such seeds, when removed and grown in test tubes, need little or no after-ripening and will begin to grow at once. This leads to a substantial shortening of the time needed to obtain mature plants from seed. In attempting to obtain plant hybrids it is sometimes found that certain desirable parent plants when crossed will not produce viable seed. Seed formation may begin, but the seeds abort before they are fully developed. In several such cases it has been found possible to remove the embryos from the hybrid seed before they begin to abort, grow them in culture, and produce mature hybrid plants that would otherwise be unobtainable. Steps involeved in embryo culture: 1. surface sterilization or disinfection 2. excision of embryo 3. culture of embryo in vitro Application of embryo culture: Production of haploids Prevention of embryo abortion in wide crosses Propagation of orchids Shortening the breeding cycles Overcoming dormancy Overcoming seed sterility. Clonal micropropagattion in conifers and members of graminae family production of virus free plants 1. Recovery of distant hybrids T. aestivum H. vulgare Hybrid embryo Embryo culture Hybrid seedlings T. aestivum secale 4x or 6x wheat carry dominant genes Kr1 and Kr2that prevent seed development 10 – 14 day old embryo Embryo Culture Hybrid seedling (9 – 12 day old)
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Triticale. 2. Recovery of haploid plants from inter specific crosses. Eg. (I) H. vulgare or T. aestivum H. bulbosum Embryo (elimination of bulbosum genome) Embryoculture Haploid plant of vulgare or aestivum. (II) Wheat (female) Maize (male) Some seed sets but caryopsis degenerates after 10 days. Embryo ( 8 –10 day old) Haploid plant wheat. 3) Production of monoploids. H. vulgare H. bulbosum Embryo Embryo culture Haploid of vulgare. 4) Shortening the breeding cycle. 5) Overcoming dormancy. 6) Propagation : eg orchids – because :seeds lack stored food and naked embryo. 7) Embryo rescue
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. In case of endosperm abortion, plants may be raised by culturing the hybrid embryos on a suitable culture medium. The embryos, in such cases, are removed from young seeds before endosperm abortion takes place; this is termed as embryo rescue. Triticum aestivum Secale spp.(Rye) Hybrid Embryo culture Triticale. H. vulgare H. bulbosum Embryo Culture Gives a plant 8.5 ANTHER OR POLLEN CULTURE “In vitro culturing of anthers containing microspores or immature pollen grains on a nutrient medium for a purpose of generating haploid plantlets.”Anther culture is a culture of plant cells derived from pollen in a synthetic medium: the progeny generated will have a single set of chromosomes. In anther culture, haploid plants may be obtained form pollen grains by placing anthers or isolated grains on a suitable culture medium. It depends on the species of crops. Solanaceae members are more responsive to this methods. Solanaceae followed by cruciferae and graminae. The Uninucleate cells should be used for anther/pollen culture. Steps: 1. select a healthy plant under controlled condition 2. temperature treatment 3. sterilize the flower or panicle 4. test the appropriate stage of microspore 5. if anthers are in proper stage, place them horizontally into the culture medium 6. place the culture at 12-16 hr light (5000-10000 lux): in responsive species, the wall tissue turns to brown. Genrally anther tissue bursts within 3-8 week 7. transfer the callus grains (embryo) to callus production medium to develop the callus
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 8. regenerate the whole plant from callus. Factors affecting anther culture: 1.Genotype 2.anther wall factor 3.Culture medium: Low CHO requiring culture: solanaceae, liliaceae High CHO requiring culture: graminae, cruciferae 4. Stage of microspore: Microspore should be in uninucleate stage. 5. Temperature treatment and light factor: Low intensity white/red light causes fast development High intensity white/red light causes slow development 6. physiological stage of donor plant Applications of anther culture:  used in haploid production  used for chromosome reduction  used for pureline development. o Improvement of crop varieties in terms of yield and/or disease resistance, cold resistance, maturity duration etc. Applications of anther culture : 1) To obtain homozygous diploid in a short period. Anther culture Haploid Colchicine Treatment Diploid. 2) Haploid (development from callus) – genetic study: gametoclonal variation 3) Selection is efficient (hybrid sorting variation) Steps: Culture F1 plants anther Haploids
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Chromosome doubling Homozygous doubled haploids (disomic plants) Grow and selection Fig. Scheme of hybrid sorting. 4) Used for the improvement of crops. eg. Disease resistant 5) Double haploid strain may be released directly as a variety or used as a parent in hybridisation program. 8.6 METHODS OF GENE TRANSFER Gene transfer is to transfer a gene from one DNA molecule to another DNA molecule. Gene insertion can be achieved using the following methods; A) Vector-mediated or indirect gene transfer 1) Ti plasmid of agrobacterium species as vector: Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used. This bacteria is known as ―natural genetic engineer‖ of plants because these bacteria have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The foreign gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted sequences. To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected and infected with Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The infected tissue is then cultured (co-cultivation) on shoot regeneration medium for 2-3 days during which time the transfer of T-DNA along with foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are transferred onto selection cum plant regeneration medium supplemented with usually lethal concentration of an antibiotic to selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred to root-inducing medium, and after another 3-4 weeks, complete plants are transferred to soil following the hardening (acclimatization) of regenerated plants. The molecular techniques like PCR and southern hybridization are used to detect the presence of foreign genes in the transgenic plants. 2) Plant viruses as vectors: Techniques are being developed to use certain DNA and RNA plant viruses eg. caulimo virus, Gemini virus etc as vectors. Cauliflower mosaic virus (CaMV; a caulimo virus) is a DNA virus. A DNA segment may be integrated into the viral DNA, which is suitably modified to serve as a vector, and the host plants may then be infected with this specially constructed virus. Following infection, the virus spreads systematically into the host plant; plant
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. cells usually have a high copy number of virus. Therefore each cell will also have a very high copy number of transferred genes. B) Vectorless or direct gene transfer In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector. The methods used for direct gene transfer in plants are: 1) Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also used to transfer DNA into cultured cells. 2) Microinjection where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometerdiameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo. 3) Electroporation: it involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient (temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA. The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus. 4) Particle gun/Particle bombardment - In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).The microprojectile bombardment method was initially named as biolistics by its inventor Sanford (1988). Two types of plant tissue are commonly used for particle bombardment- Primary explants and the proliferating embryonic tissues. 8.7 MOLECULAR MARKERS Molecular markers consists of specific molecules, which show easily detectable differences among different strains of a species or among different species. These markers are based on protein e.g. isozymes or DNA .A molecular marker is a DNA sequence that is readily detected and whose inheritance can easily be monitored.. The use of molecular markers is based on naturally occurring DNA polymorphism, which forms the basis for designing strategies to exploit for applied purpose. A marker must be polymorphic. Desirable properties of molecular marker 1. should be reproducible 2. should be easy, fast and cheap to detect 3. should evenly and frequently distributed throughout the genome 4. must be polymorphic 5. different forms of a marker should be detectable in diploid organisms.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Categories of molecular markers: A. Non-PCR-based approaches: 1. Restriction fragment length polymorphism (RFLP) B. PCR-based techniques: 1. Random amplified length polumorphic DNA ( RAPD) 2. Amplified fragment length polymorphism (AFLP) 3. Microsatellite or simple sequence repeat polymorphism (SSRP) C. Targeted PCR and sequencing: 1. Sequence tagged sites (STS) 2. Sequence characterized amplified region ( SCARs) 3. Sequence tagged microsatellites (STMs) 4. Cleaved amplified polymorphic sequences (CAPs) Applications of molecular markers: 1.used for negative selection i.e. elimination of undesirable genes 2.used for unequivocal identification of plant varieties 3.used for identifying heterotic combinations for use in production of hybrids 4.enables an effective selection for horizontal resistance 5.used for mapping of quantitative trait loci (QTL) Marker or reporter gene: Basically any DNA sequence used to distinguish between individuals, lines or varieties can be referred to as DNA marker.Marker gene permit an easy selection or identification of transformed host cells. Marker genes are divided as; selectable marker, reporter gene 8.8 UTILIZATION OF GENE OF INTEREST AND GENE TRANSFER HAPLOID BREEDING An organism having single set of chromosome is called haploid. Haploid plants naturally double their haploid chromosome set or this can be induced producing diploid plants homozygous for all genes. Crop plant breeders strive to produce homozygous or true-breeding lines. The desired trait will be carried through unchanged to subsequent generations only if the trait is present in homozygous form (i.e. having two identical alleles for a given trait). To obtain a homozygous breeding line, the breeding line would have originally been developed by self-pollination over six to eight generations, which is a very time -consuming and costly process. Nowadays homozygous lines of some plant species (e.g. tobacco, barley, potatoes, rape and wheat) can be produced from gametes, which contain only one set of chromosomes (haploid). In most cases unripe pollen is placed on a suitable culture medium, where it develops into plants with a single set of chromosomes (haploid androgenesis). Ova may also be used as the source
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. material, although this is less common (haploid parthenogenesis). Following a cultivation period of three to four weeks, the haploid plantlets are treated with colchicine, a toxin found in meadow saffron. Colchicine inhibits cell division: duplication of the chromosomes occurs, but the subsequent division into two daughter cells is suppressed. The resulting cells produce "double haploid", fully homozygous plants which produce identical offspring. Significance and uses of haploids: 1) haploids are useful source for production of homozygous lines 2) induction of genetic variability 3) induction of muatations 4) generation of exclusively male plants 5) haploid production is used for introduction of disease and insect resistance genes into cultivars. 6) Doubled haploids are used in genome mapping. 7) Haploids are used for production of aneuploids. 8) Significance in early release of varieties. Methods of haploid production: 1. Anther culture: anthers are collected during a certain stage of flowering. Sometimes they are cold treated and sterilized before culture on specific nutrient medium. After sometime the development of callus or embryoids can be obtained. 2. Culture of isolated microspores: It involves the isolation of microspores from the anthers at a particular stage of development. The delicate microspores are isolated through various purification steps, then cultured in liquid medium. In some plants small embryos may develop directly from microspores and grow into haploid plants (Microspore embryogenesis) Diploidization Haploids can be diploidized to produce homozygous plants by following methods; 1) Colchicine treatment: Colchicine is used as spindle inhibitor to induce chromosome duplication. 2) Endomitosis: haploid cells are unstable in culture and have tendency to undergo endomitosis (chromosome duplication without nuclear division) to form diploid cells INTERGENIC CROSSES AND INTERSPECIFIC CROSSES INTERGENIC CROSSES: It is cross between individuals belonging to species from two different genera. For e.g. Wheat (Triticum sp) X Rye (Secale cereale) = Triticale Radish (Raphanus sativus) X Cabbage (Brassica oleracea) =Raphanobrassica Importance: Intergenic cross is used to develop new crop species.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. The wheat genotypes produced embryos when crossed with maize, only if followed by the application of 2,4-dichlorophenoxyacetic acid (2,4-D). All plants regenerated from embryos on the B5 medium were euhaploids having a complement of 21 chromosomes. Overall frequencies of wheat haploid production were 0.2 % from the H. bulbosum cross, and 9.5 % from the maize cross. Use of the doubled haploid lines produced from F 1 hybrid plants for genetic analyses allowed an assessment of the effects of the semi-dwarfing genes Rht 1 and Rht 2 on yield performance. INTERSPECIFIC CROSSES: It is cross between individuals derived from two distict species of the same genus. e.g. Cultivated rice (Oryza sativa) X wild rice (oryza perennis) Many interspecific crosses were successful due to use of growth regulators e.g. IAA, 2,4-D etc. The of seed set in interspecific crosses is due to endosperm abortion. This may be overcome by using embryo culture. Importance: 1. it is used for the transfer of specific characters e.g. disease resistance to cultivated species. Many of the genes for rust resistance in wheat have been transferred from related from wild species. 2. It is employed for development of new crop varieties. MARKER-ASSISTED SELECTION Marker assisted selection or marker aided selection (MAS) is a process whereby a marker (morphological, biochemical or one based on DNA/RNA variation) is used for indirect selection of a genetic determinant or determinants of a trait of interest (i.e. productivity, disease resistance, abiotic stress tolerance, and/or quality). This process is used in plant and animal breeding. Marker assisted selection (MAS) is indirect selection process where a trait of interest is selected not based on the trait itself but on a marker linked to it. For example if MAS is being used to select individuals with a disease, the level of disease is not quantified but rather a marker allele which is linked with disease is used to determine disease presence. The assumption is that linked allele associates with the gene and/or quantitative trait locus (QTL) of interest. MAS can be useful for traits that are difficult to measure, exhibit low heritability, and/or are expressed late in development. Marker types 1. Morphological - First markers loci available that have obvious impact on morphology of plant. Genes that affect form, coloration, male sterility or resistance among others have been analyzed in many plant species. Examples of this type of marker may include the presence or absence of awn, leaf sheath coloration, height, grain color, aroma of rice etc. In well-characterized crops like maize, tomato, pea, barley or wheat, tens or even hundreds of such genes have been assigned to different chromosomes. 2. Biochemical- A gene that encodes a protein that can be extracted and observed; for
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. example, isozymes and storage proteins. 3. Cytological - The chromosomal banding produced by different stains; for example, G banding. 4. Biological- Different pathogen races or insect biotypes based on host pathogen or host parasite interaction can be used as a marker since the genetic constitution of an organism can affect its susceptibility to pathogens or parasites. 5. DNA-based and/or molecular- A unique (DNA sequence), occurring in proximity to the gene or locus of interest, can be identified by a range of molecular techniques such as RFLPs, RAPDs, AFLP, DAF, SCARs, microsatellites etc. Requirements of marker-assisted selection (MAS): 1) 2) 3) 4) markers should be closely linked with desired traits An efficient means of screening large populations for molecular markers should be available. The screening technique should have high reproducibility across laboratories It should be economical to use and be user friendly. Application of MAS 1. Used to enhance the expression of heterosis 2. Used to select for such traits whose phenotypic evaluation is either problematic or costly 3. used to develop disease and pest resistant plants. 4. useful to identify specific traits beneficial in improving drought resistance 5. used in improvement in qualitative characters. OVERCOMING CONVENTIONAL BREEDING BARRIERS One of the problems in the conventional plant breeding is that the range of organisms among which genes can be transferred is severely limited by species barriers. The gene transfer technologies provide a better approach for defining and manipulation of targets and speciesspecific barriers are broken. These technologies do not replace plant breeding but provide methods capable of achieving objectives not possible by other means. DNA FINGERPRINTING DNA fingerprinting refers to a conclusive test to uniquely identify the genetic materials of an organism. It helps in determining the parentage of child. It helps to determine if a suspect is really the criminal. The technique of DNA fingerprinting makes use of recombinant DNA technology and allows an examination of each individual‘s unique genetic blue print. It is developed by Alec Jeffery‘s in England in 1985. DNA Fingerprinting, method of identification that compares fragments of deoxyribonucleic acid (DNA) It is sometimes called DNA typing. DNA is the genetic material found within the cell nuclei of all living things. In mammals the strands of DNA are grouped into structures called chromosomes. With the exception of identical twins, the complete DNA of each individual is unique.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Steps: 1. Isolation of genomic DNA suitable for RFLP analysis. 2. Complete digestion of genomic DNA with an appropriate restriction enzyme. 3. Electrophoretic separation of restriction fragments in agarose gel 4. Denaturation and immobilization of separated DNA fragments within the gel or blotting the DNA fragments on a membrane. 5. Hybridization of the dried gel or membrane to radioactive or non-radioactive labeled micrsatellite complementary oligonucleotide probes. 6. Detection of hybridizing fragments by autoradiography or by chemiluminescence 7. Evaluation of banding patterns and documentation by photography.. DNA fingerprinting was first developed as an identification technique in 1985. Originally used to detect the presence of genetic diseases, DNA fingerprinting soon came to be used in criminal investigations and forensic science. The first criminal conviction based on DNA evidence in the United States occurred in 1988. In criminal investigations, DNA fingerprints derived from evidence collected at the crime scene are compared to the DNA fingerprints of suspects. The DNA evidence can implicate or exonerate a suspect. Generally, courts have accepted the reliability of DNA testing and admitted DNA test results into evidence. However, DNA fingerprinting is controversial in a number of areas: the accuracy of the results, the cost of testing, and the possible misuse of the technique. The accuracy of DNA fingerprinting has been challenged for several reasons. First, because DNA segments rather than complete DNA strands are ―fingerprinted,‖ a DNA fingerprint may not be unique; large-scale research to confirm the uniqueness of DNA fingerprinting test results has not been conducted. In addition, DNA fingerprinting is often performed in private laboratories that may not follow uniform testing standards and quality controls. Also, since human beings must interpret the test, human error could lead to false results. DNA fingerprinting is expensive. Suspects who are unable to provide their own DNA experts may not be able to adequate ly defend themselves against charges based on DNA evidence. CHARACTERIZATION OF PLANT GENETIC RESOURCES WITH BIOCHEMICAL/MOLECULAR TECHNIQUES 1. ELISA (ENZYME LINKED IMMUNOSORBENT ASSAY): ELISA is the abbreviation of enzyme-linked immunosorbent assay. It is a widely used technique for determining the presence or amount of protein in a biological sample, using an enzyme that bonds to an antibody or antigen and causes a color change. ELISA is a useful and powerful method in estimating ng/ml to pg/ml ordered materials in the solution, such as serum, urine, sperm and culture supernatant. ELISA has been widely used in the life science researches . It is highly sensitive process for the detection of specific proteins such as viral structural protein.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. The basic principle of an ELISA is to use an enzyme to detect the binding of antigen (Ag) antibody (Ab). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding. An ELISA can be used to detect either the presence of Ags or Abs in a sample, depending on how the test is designed. Steps: 1. Using special plates (ELISA plates), specific antibodies are bound through adsorption onto the base of individual wells of ELISA plate 2. Viral particles or virus fragments (antigen) bind to antibodies forming a complex 3. Enzyme coupled antibodies (conjugate) are added and bind to the already bound antigen 4. Finally enzyme activities are measured by adding enzyme substrate, the intensity of resulting color reaction is quantitatively measured using a spectrophotometer 5. The intensity of color is proportional to enzyme activity, which is proportional to the amount of conjugate and therefore to concentration of antigen. Requirements for its application are a pure preparation of virus and an antibody production system base on pure viral protein. 2. GEL ELECTROPHORESIS: Electroporation is a technique in which molecules are separated by differences in their net charge in the presence of externally applied electrical field. Agarose gel electrophoresis: since DNA has a large number of phosphate groups which are negatively charged at neutral PH. It will migrate towards the anode in an electrical field. The basic phosphodiester back bone of all DNAs is the same so they have uniform charge density. Therefore, if the DNA is placed in a porous medium such as agarose or polyacrylamide, it will migrates towards the anode at a rate which is proportional to its molecular weight. This technique which is used to separate DNA fragments of different sizes produced by digestion of DNA with restriction enzymes. 3. ANALYSIS METHODS BASED ON DNA AND RNA a) RFLP (Restriction Length polymoryphism) techniques: This technique denotes that a single restriction enzyme produces fragments of different lengths from the same stretch of genomic DNAs of different strains or related species. A restriction fragment length polymorphism, or RFLP is a variation in the DNA sequence of a genome that can be detected by breaking the DNA into pieces with restriction enzymes and analyzing the size of the resulting fragments by gel electrophoresis. It is the sequence that makes DNA from different sources different, and RFLP analysis is a technique that can identify some differences in sequence (when they occur in a restriction site). Though DNA sequencing techniques can characterize DNA
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. very thoroughly, RFLP analysis was developed first and was cheap enough to see wide application. Analysis of RFLP variation was an important tool in genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting, and paternity testing. The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis, and transferred to a membrane via the Southern blot procedure. Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe. A RFLP occurs when the length of a detected fragment varies between individual s. Each fragment length is considered an allele, and can be used in genetic analysis. Steps: 1. DNA isolation: large molecular weight genomic DNAs are isolated from several strains or related species 2. Cutting DNA into smaller fragments using restriction enzymes: these DNAs are then digested with the same selected restriction enzyme 3. Separaion of DNA fragments by gel electrophoresis: the fragments present in these digests are separated through electrophoresis 4. Transfering DNA fragments to a nylon or nitrocellulose membrane filter: the resulting gel lanes are transferred onto a suitable solid support and exposed to a suitably labeled appropriate DNA probe under conditions favoring DNA: DNA hybridization and 5. Visualization of specific DNA fragments using labeled probes : the bands to which the probe has hybridized are detected by filming them on a suitable photofil m through autoradiography. A restriction fragment length polymorphism (RFLP) is detected when the same autoradiographed band migrates to two or more different positions in the different lanes of gel. 6. Anlysis of results Uses of RFLP: 1.it permits direct identification of a genotype or cultivar in any tissue at any developmental stage. 2.indirect selection using qualitative trait 3.Tagging of monogenic traits with RFLP marker 4.RFLPs are codominat markers, enabling heterozygotes to be distinguished from homozygotes. Analysis Technique: The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest. The resulting DNA fragments are then separated by length through a process known as agarose gel
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. electrophoresis, and transferred to a membrane via the Southern procedure. Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe. An RFLP occurs when the length of a detected fragment varies between individuals. Each fragment length is considered an allele, and can be used in genetic analysis. RFLP analysis may be subdivided into single- (SLP) and multi-locus probe (MLP) paradigms. Usually, the SLP method is preferred over MLP because it is more sensitive, easier to interpret and capable of analyzing mixed-DNA samples.Moreover, data can be generated even when the DNA is degraded (e.g. when it is found in bone remains.) b) PCR (Polymerase chain reaction) technique: The polymerase chain reaction (PCR) is a technique which deals with an automated process, whereby a DNA fragment can be multiplied (or amplified) to a theoretically unlimited extent. It i s widely used in molecular biology. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. Polymerase Chain Reaction (PCR), technique in molecular biology by which a small fragment of deoxyribonucleic acid (DNA) can be rapidly cloned, or duplicated, to produce multiple DNA copies. PCR can be used to identify individuals from minute amounts of tissue or blood , to diagnose genetic diseases, and to research evolution. PCR was conceived by American biochemist Kary B. Mullis in 1983 and was later developed by Mullis and his associate Fred A. Faloona at the Cetus Corporation in Emeryville, California. There are three phases in a polymerase chain reaction. In the first phase, called denaturation, the template, or piece of original DNA, is heated to a temperature of from 90º to 95º C (194º to 203º F) for 30 seconds, which causes the individual strands to separate. In the second phase, called annealing, the temperature of the mixture is lowered to 55º C (131º F) over a 20-second period, allowing the oligonucleotide primers to bind to the separated DNA. In the third phase, called polymerization, the temperature of the mixture is raised to 75º C (167º F), a temperature at which the polymerase can copy the DNA molecule rapidly. Steps: 1.separation of DNA to be amplified (at 94-96 C) 2.the primer binds to the complimentary bases (at 50-65 C)
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 3.through the activity of Taq polymerase, assembles (gather) the new complementary nucleic acids thereby doubling the original amount of DNA (at 72 C) 4.A subsequent cycle doubles the amount of original DNA again so that the number of DNA molecule increases. Normally this cycle is repeated at 25-50 times. In this way nearly every region of the DNA can be multiplied. Application of PCR: 1. Isolation of genomic DNA 2. Amplification and quantization of DNA 3. Diagnosis of genetic disorders. In medicine, PCR is particularly useful in prenatal testing for genetic diseases 3. Analysis of homologous genes 4. Identification of forensic samples. In forensic science, PCR has revolutionized the process of criminal identification 5. PCR has been used to trace industrial waste and other products. PCR ASSOCIATED TECHNIQUE: a. Amplified fragment length polymorphism (AFLP): It combines RFLP and PCR techniques. In the first step, the genomic DNA is digested with rare and frequent restriction endonucleases. In the second step, adaptors are ligated to ends of fragments. In third steps, PCR is carried out with specially produced primers. b. c. Cleaved Amplified polymorphic sequences (CAPS) Multiple Arbitary Primed PCR (MAPP) techniques 8.9 RECENT ADVANCES RELATED TO CROP IMPROVEMENT: TRANSGENIC PLANTS (GMO’s for crop improvements and quality) Plants that carry additional, stably integrated and expressed foreign genes transferred from other genetic sources are referred to as transgenic plants. A transgenic crop plant contains a gene or genes which have been artificially inserted instead of the plant acquiring them through pollination. The development of transgenic plants is the result of integrated application of recombinant DNA technology, gene transfer methods and tissue culture techniques. The inserted gene sequence (known as the transgene) may come from another unrelated plant, or from a completely different species: transgenic Bt corn, for example, which produces its own insecticide, contains a gene from a bacterium. Plants containing transgenes are often called genetically modified or GM crops, although in reality all crops have been genetically modified from their original wild state by domestication, selection and controlled breeding over long periods of time. On this web site we will use the term transgenic to describe a crop plant which has transgenes inserted.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Transgenic plants possess a gene or genes that have been transferred from a different species. Although DNA of another species can be integrated in a plant genome by natural processes, the term "transgenic plants" refers to plants created in a laboratory using recombinant DNA technology. The aim is to design plants with specific characteristics by artificial insertion of genes from other species or sometimes entirely different kingdoms. . Varieties containing genes of two distinct plant species are frequently created by classical breeders who deliberately force hybridization between distinct plant species when carrying out interspecific or intergeneric wide crosses with the intention of developing disease resistant crop varieties. Classical plant breeders use a number of in vitro techniques such as protoplast fusion, embryo rescue or mutagenesis to generate diversity and produce plants that would not exist in nature. Why Make Transgenic Crop Plants? A plant breeder tries to assemble a combination of genes in a crop plant which will make it as useful and productive as possible. Depending on where and for what purpose the plant is grown, desirable genes may provide features such as higher yield or improved quality, pest or disease resistance, or tolerance to heat, cold and drought. Combining the best genes in one plant is a long and difficult process, especially as traditional plant breeding has been limited to artificially crossing plants within the same species or with closely related species to bring different genes together. For example, a gene for protein in soybean could not be transferred to a completely different crop such as corn using traditional techniques. Transgenic technology enables plant breeders to bring together in one plant useful genes from a wide range of living sources, not just from within the crop species or from closely related plants. This technology provides the means for identifying and isolating genes controlling specific characteristics in one kind of organism, and for moving copies of those genes into another quite different organism, which will then also have those characteristics. This powerful tool enables plant breeders to do what they have always done - generate more useful and productive crop varieties containing new combinations of genes - but it expands the possibilities beyond the limitations imposed by traditional cross -pollination and selection techniques. Transforming Plants Transformation is the heritable change in a cell or organism brought about by the uptake and establishment of introduced DNA. There are two main methods of transforming plant cells and tissues: The "Gene Gun" method (also known as microprojectile bombardment or biolistics). This technique, which is shown and explained in the animated demo section of this web site, has been especially useful in transforming monocot species like corn and rice. The Agrobacterium method, which is described below. Transformation via Agrobacterium has been successfully practiced in dicots (broadleaf plants like soybeans and tomatoes) for many years, but only recently has it been effective in monocots (grasses and their relatives). In general, the Agrobacterium method is considered preferable to the gene gun, because of the greater
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. frequency of single-site insertions of the foreign DNA, making it easier to monitor. Agrobacterium Method of Plant Transformation Agrobacterium tumefaciens is a remarkable species of soil-dwelling bacteria that has the ability to infect plant cells with a piece of its DNA. When the bacterial DNA is integrated into a plant chromosome, it effectively hijacks the plant's cellular machinery and uses it to ensure the proliferation of the bacterial population. Many gardeners and orchard owners are unfortunately familiar with A. tumefaciens, because it causes crown gall diseases in many ornamental and fruit plants. Agricultural impact of transgenic plants Outcrossing of transgenic plants not only poses potential environmental risks, it may also trouble farmers and food producers. Many countries have different legislations for transgenic and conventional plants as well as the derived food and feed, and consumers demand the freedom of choice to buy GM-derived or conventional products. Therefore, farmers and producers must separate both production chains. This requires coexistence measures on the field level as well as traceability measures throughout the whole food and feed processing chain. Genetically Modified Organism (GMO) A genetically modified organism (GMO) or genetically engineered organism (GEO) is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one molecule to create a new set of genes. This DNA is then transferred into an organism, giving it modified or novel genes. TERMINATOR GENE (gene preventing seed production): ― Any gene that produces a protein which is toxic to plants and does not allow the seeds to germinate is called terminator gene. It codes for ribosome inhibiting protein (RIP). The terminator gene encoding RIP interferes in the synthesis of all proteins in the plant cells, without being toxic to other organisms. Thus expression of RIP gene in the cells of embryo would prevent germination of seeds. It is attached with a particular type of promoter one that is active only in the late stages of seed development.‖ A gene inserted into genetically modified plants that makes them unable to produce seed after one season is called terminator gene. The terminator gene is made up of three different genes that can be spliced into genetically engineered seeds. Plants grown from these seeds do not produce fertile seeds themselves. The system was developed by researchers at the USDA and the Delta and Pine Land Company, a Mississippi-based cottonseed concern. A "terminator" gene that halts seed development, programmed to switch on specifically in the seed's embryo when the growing seed nears maturity (in genetics lingo, an "inducible, seed-specific promoter"). One example might be a non-toxic protein that blocks protein synthesis in the embryo. The plant and i ts seeds (soybeans,
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. corn kernels, etc) would look entirely normal through harvest time, but the seeds would not grow when planted The terminator gene is a specific genetic sequence inserted into a seed's DNA. Once activated by a synthetic chemical catalyst of the manufacturer's choosing, the sequence renders the seed and crop it produces sterile. Patented by the USDA and Delta and Pine Land Co., now owned by Monsanto, this terminator technology has no agricultural or economic benefits for farmers or consumers. The only motivation is to protect intellectual property rights, according to owners of the technology. They claim that it allows them to be able to recover investments on research, and produce profits from their technology, as planters must re-purchase seeds every year. Opponents claim that corporations will only use this to squeeze more money out of dependent farmers, and begin a monopoly of chemically saturated suicide seeds. Possibility of Transfer. Transgenic plants have already been shown to transfer certain genes to wild relatives or bacteria. The possibility that the terminator gene could be transferred is not denied by anyone. In fact, the tendency of genetically manipulated plants to "leak" traits is greater than others. "They learned that the transgenic plants were 20 times more likely to outcross than the mutants -they were "promiscuous," as a headline in the journal Nature put it. "Nobody knows why," Bergelson says. "We're still trying to find the mechanism that drives the pattern we saw. T here's a lot we don't understand, including how common it is." "It's inevitable that they will get out," says ecologist Joy Bergelson of the University of Chicago. "That doesn't necessarily mean that there will be negative repercussions. But there could be some. And right now we don't know enough about what they could be and when they could occur."' There is some speculation on the subject, however, despite the limited empirical evidence. Even if the terminator gene were to spread to wild weedy relatives, then it could help control the spread of genetic hybrids and accompanying artificial traits. "Moreover, if Terminator genes were packaged with other transgenic traits, they could help ensure that crop-weed hybrids would be sterile-potentially eliminating a difficult problem." In fact, some believe that an added attraction to use of the terminator gene is the possibility that it will prevent more genetic transfer from occurring. In fact, common sense recommends that the terminator gene would not spread far, because gene transfer through hybridization relies on fertile gametes of each species, the production of which is suppressed by this gene. GENOMICS Genomics is the study of how genes and genetic information are organized within genome and how this organization determines their function. Genomics is the study of the collective genetic material in an organism. It includes an examination of the following topics:  The number of genes in an organism  The function of specific genes  The influence of one gene on another  The activation and suppression of genes a
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Genomics also includes the search for genes that cause disease. Genomics are two types: 1. Structural genomics: it involves sequencing and mapping of genomes and a study of structure of all gene sequences encoded in a fully sequenced genome 2. Functional genomics: it involves a study of the functions of all specific gene sequences and their expression in time and space in organism. This scientific discipline is focused on sequencing the DNA in an organism to form a complete picture, and then identifying specific genes in that sequence which could be of interest. Genomics got its start in the 1970s, when scientists first began genetic sequencing of simple organisms, and it really took off as a field in the 1980s and 1990s, with the advent of scientific equipment to assist researchers. By sequencing the entire DNA pattern of an organism, scientists can glean a great deal of information. Complete sequences can be compared, for example, to get more information about how creatures survive in different environments. A genetic sequence can also be used as a reference base for studying other members of the same species, and for identifying genetic defects, inherited conditions, and other matters of interest, such as the e xpression of proteins and the role of ―junk‖ DNA in the body. In genomics, scientists analyze the DNA in every chromosome of the organism of interest. When a completely sequenced set of DNA has been created, this set is collectively known as a ―genome.‖ The genomes of numerous species have been sequenced, from bacteria to humans. The genome of each species is distinctly different, with varying numbers of nucelotides which can translate into huge amounts of information. Within a species, genetic variation may be minimal, but still interesting, because it can explain certain traits or tendencies. This scientific discipline is different than the study of genetics, which focuses on specific genes and what they do. Some genetics is certainly involved in genomics; for example, a scientist might want to know more about the specific location of a gene within an organism's genome, in which case he or she would use genomics techniques. Genomics looks at the collective role and function of an organism's genome, not necessarily the behaviors of individual sections. Plant genomics is study of whole genome of plants, their physical and molecular organization, evolution and functions of the myriad of constituent genes. BIOPESTICIDES Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria and certain minerals. e.g. canola oil. They are suitable preparation of virus, bacteria, fungi etc. to kill insect pests. A biopesticide is a compound that kills organisms by virtue of specific biological effects rather than as a broader chemical poison. Differ from biocontrol agents in being passive agents, whereas biocontrol agents actively seek the pest. The term biopesticide is used for microbial biological pest control agents that are applied in a similar
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. manner to chemical pesticides. Commonly these are bacterial, but there are also examples of fungal control agents, including Trichoderma spp. and Ampelomyces quisqualis (a control agent for grape powdery mildew). Bacillus subtilis are used to control plant pathogens. Weeds and rodents have also been controlled with microbial agents. Major classes of biopesticides 1.Microbial pesticides: consists of microorganism like bacteria, fungus, virus or protozoa as active ingredient. Eg.Bt (Bacillus thuringinesis) 2.Plant incorporated protectants: they are pesticidal substances that the plant produce from genetic materials that has been added to plants. Eg. Bt introduced into plant genetic materials, then plant produces substances that kill insect pests. 3.Biochemical pesticides: they are naturally occurring substances that control pests by non-toxic mechanism e.g. insect sex pheromone trap Advantages of biopesticides: 1.they are usually less toxic than conventional pesticides 2.They generally affect only the target pest and closely related organism 3.they are effective in very small quantities and often decompose quickly 4.when used as a component of IPM program, biopesticides decrease use of conventional pesticides. BIOFERILIZER The term biofertilizers may be defined as the latent cells capable of fixing nitrogen and is used with the object to increase the number of such microorganisms to supply plant nutrients. It is simply a preparation containing live and latent cells (inactive cells) of usefu l microorganisms used for application to seed, soil or composting areas. Biofertilizer is a 100% natural and organic fertilizer that helps to provide and keep in the soil all the nutrients and microorganisms required for the benefits of the plants. Classification of biofertilizers: 1.Nitrogen fixing biofertilizers: leguminous biofertilizers (Rhizobium ) and other crops biofertilizers (Azotobacter, Azosporillium, Azolla and BGA) 2.Phosphate solubilizing biofertilizers: Phosphate soluble biofertilizers (bacteria, fungi, Actinomycetes) and phosphate fixation biofertilizers (Mycorrhiza) 3.Carbonic acid dissolving biofertilizers. Advantages of biofertilizers: 1.enhances biomass production and grain yield 10-20% 2.cheap and can help to reduce consumption of chemical fertilizers 3.make available N directly to plant 4.solubilize phosphorous and increses phosphorus uptake to the plants 5.improve the soil properties and sustain the soil fertility
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 6.control and suppress soil borne disease 7.suitable for organic farming. 9. Variety Release and Seed Technology 9.1 Evaluation, 9.2 Variety release process in Nepal, 9.3 National Seed policy 1999 (2056 B.S.), 9.4 Classes of quality seed, 9.5 Seed and field standard for quality seed, 9.6 Seed priming 9.7 Seed marketing. 9.8 Seed System in Nepal 9.9. Terminator seed 9.1. EVALUATION Evaluation of a strain for release as a variety consists of various trials and tests to determine its superiority over the best existing variety in terms of yield and other agronomic traits, and its suitability for consumption. In general, there are following types of trials/tests are conducted during evaluation: 1) Station Trial (Preliminary yield trial): They are conducted for one or more years by breeders who developed new strains. Its objective is to make sure that the new strains developed by a breeder are superior in performance to the best available variety for the region 2) Multilocation Trials: They are conducted at several locations to evaluate the performance of newly developed strains at several locations distributed over a region 3) National trials: They are conducted through out the country to evaluate outstanding entries of one zone in the other agroclimatic zones to see if they perform will in other zones as well. 4) Adoptive Research Trials: They are conducted on research stations or farms of state governments. 5) Minikit Trials: They are conducted in farmer‘s fields to popularize the new variety among farmers of zones. 6) Disease and Insect Tests: Entries are evaluated for disease and insect resistance both under natural as well as artificial epidemic conditions
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 7) Quality Tests: They are conducted to determine the suitability of an entry for the various uses of its produce . 9.2 VARIETY RELEASE PROCESS IN NEPAL 1) Develop better variety: The plant breeders develop crop varieties superior to the existing ones in yielding ability, disease and insect resistance and other characteristics. The various breeding methods are used to develop such superior strains. 2) Evaluation of strains: Evaluation of a strain for release as a variety consists of various trials and tests to determine its superiority over the best existing variety in terms of yield and other agronomic traits, and its suitability for consumption 3) Identification of outstanding strains 4) Naming and Release of a variety: after identification, a variety is tested for at least one year in adoptive research trials. During this period, disease tests and quality tests are also conducted. A review board and plant breeder or agency take decision to release variety. Usually breeder suggests the name variety. 5) Production or multiplication of breeder seeds: The plant breeder make a limited seed increase of new variety; give to foundation seed production 6) Distribution of foundation seed and seed increase for commercial cultivation: Seed growers maintain genetic purity and produce quality seed. 9.3 SEED POLICY in Nepal: Objectives Bio diversity conservation and promotion of local varieties, land races and their utilization for varietal development, research. Ensure production, processing, availability and supply of quality seeds. Quality control, regulation and monitoring in seed business. Promotion of seed business with collaboration and active participation of concerned stakeholders. Self sufficiency, import substitution and export promotion. Promotion of national seed industry for competitiveness with international seed business. NATIONAL SEED POLICY 1999 (2056 B.S.) National Seed Policy 2056 (NSP) The National Seed Policy (NSP) aims at (i) making available quality seeds of various crops in required quantity, (ii) promoting export by producing quality seed, (iii) making seed business effective in terms of WTO, (iv) conserving indigenous genetic resources (land races) and coordinating with concerned organization to protect national right on them. The NSP underlines varietal development and conservation, seed multiplication, quality control, promotion of the involvement of the private sector in the seed sector, supply management, strengthening of the
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. organizations involved in the seed sector, biotechnology and other modern technology for the sustainable development of seed sector in the country. While many provisions in the National Seed Policy are positive and reformative, some require amendments in the changed contexts to enable concerned agencies and seeds suppliers to facilitate farmers to increase SRR. But National Seeds Board, central agency responsible for the formulation and implementation of this policy has not taken initiatives to amend the NSP. NAP has clearly identified the need for amending sub-sectoral policies like NSP. Main features: 1) Variety development and maintenance: Private sector will also have opportunities for the development of a variety; in this situation the monopoly of the public sector will end. 2) Seed multiplication: NSB will make a programme and plan the supply of NS, BS, FS and CS in cooperation with NARC, NSC and the private sector. 3) Quality control: Quality control will be carried out through certification and truthful labeling (quality declared seed also is now to be added). 4) Increased involvement of private sector: The developing of appropriate policies and regulations to facilitate private sector involvement in foundation certified and improved seed production and in the seed trade generally. 5) Seed supply: There will be a buffer stock of seed at the national level, which may be utilized under conditions of natural calamity. 6) Continuity of seed supply: In remote areas, seed supply will be expanded in coordination with private producers and traders including by giving some support. 7) Institutional strengthening: This will be fulfilled by the strengthening of the NSB Secretariat, and establishment of non-government laboratories, besides including institutional management in the contracting system whilst the semi-government agency involved in seed will be commercialized. 8) Bio-Technology: Advances in seed related technology have been made and are being utilized around the world. Therefore research and studies on biotechnology, genetic engineering, GMO, transgenic plant and tissues culture will proceed in Nepal also but bio -technology regulations will be prepared and implemented for the sake of the general public. SEED LAW AND REGULATIONS Seed Act 2045 and Seed Regulation 2054 Enacted in 1988, the objective of Seed Act, 2045 (1988) is to maintain the convenience and economic interest of the general public by providing seed of high quality in a well planned manner upon producing, testing and processing to improve crop production. This Act allows to produce and distribute two kinds of seed namely certified seed and truthfully labelled seed, having minimum seed standards. It also establishes the "National Seed Board" to formulate and
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. implement policies relating to seed and to give the necessary advice to the Government of Nepal on the matters pertaining to the seed sector. In order to enforce the Seed Act, 2045 the Government of Nepal formulated the "Seed Regulation, 2054‖ (1997) with the following rules: � constitution of the Variety Approval, Release and Registration Sub-Committee: the function of this sub-committee is to prepare necessary prerequisites and to make recommendations to the Board for the release and registration of new, developed varieties � constitution of the Planning, Formulation and Monitoring Sub-Committee: the function of this sub-committee is to formulate programmes for production planning, monitoring and supplying as per the national requirement � constitution of the Quality-Standards Determination and Management Sub- Committee: the function of this sub-committee is to determine minimum criteria of quality-standards for seeds prior to distribution � provisions relating to approval, release, registration and ownership of seeds: these provisions are made to process issues relating to approval, release and registration of seeds; to record of seeds; to determine right of ownership � provisions relating to seeds authentication, export and import: These provisions are made to define functions, duties and power of seed authentication body; procedures relating to authentication of seeds; function, duties and power of the central seed testing laboratory; restriction on sale and distribution of notified seeds; and export or import of seed of the notified crops � miscellaneous provisions: these provisions are made to define functions, duties and power of seed inspectors and seed analysts; authority to hear cases; powers to make manuals; and change and alternation in schedules. Enactment of Seed Regulation after a decade of Seed Act issuance shows the extent to which the Government of Nepal, particularly MOAC, has attached importance to the seed sector. When Seed Act was passed by the Parliament in 1988, there were no private traders involved in the cereal crops seed production. However, this Act has already been amended by the Parliament on 2008/01/24. Interestingly, this Act does not cover the whole country. According to Clause 2 of the Section 1, this act will come into enforcement only in those districts which are notified by the Government in the Gazette. As of now, it has come into force in 33 districts (44 percent). The amended act is merely focused on strengthening of the quality assurance system of the seeds.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. The other amendment of the act includes the inclusion of the following three categories of persons in the National Seed Board: (a) Three scientists including one woman working in the vegetable, cereals and forage seeds sub-sector in Nepal nominated by the Government of Nepal (b) Three seed entrepreneurs including one woman nominated by the Government of Nepal (c) Two seed producers and farmers one woman farmer nominated by the Government of Nepal While aforementioned provisions show government‘s positive intention to include scientists, private entrepreneurs and seed producers including women in the decision making body related to the formulation of policies on the seed sector and to give necessary advice to the government affecting the seed sector. However, provisions thereafter in the amended Act reveal that neither government intended to develop professional and independent NSB as envisaged by the Act nor to give continuities in the NSB‘s activities. According to the Act, the term of these nominated members will be of two years, but government can remove them at any time without completing two years. Seed Regulation which was approved by the Government on 1998/o1/08 is yet to be revised to reflect 2064 amendment. Quality declared seed system has begun to show results. NSB has adopted a Quality Declared Seed System, a concept developed by the Food and Agriculture Organization of the United Nations (FAO). This system places a lot of the responsibility for seed quality control with the seed producers. For example, while the seed quality control agency (SQCA) inspects about 10 per cent of all seeds, the producer is responsible for maintaining the quality of the remaining 90 per cent. One of the key reasons for the emergence of the private sector in the seed production system is the enforcement of this system. This has become a driving element behind in availing a quality-driven seed business. However, this has been a factor to contribute to the misuse of seeds. Government lacks capacity to monitor if the seed companies have appropriately used truthful labelling. Local Self Governance Act 2055 Promulgated in 1999, the Local Self-Governance Act 2055 (1999) (LSGA) authorises the local bodies such as Village Development Committees (VDC), District Development Committees (DDC) and municipalities to formulate and implement policies, programs and activities related, among others, to agriculture and rural development and to raise specified resources. Meanwhile, as per the decentralisation policy, Government has already devolved District Agriculture Development Offices to the respective DDCs. If this implies DDCs‘ accountability for the performance of the DADOs, field observations reveal that this has not happened.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. National Seed Vision 2013-2025 in Nepal: Seed Vision Purpose: To increase crop productivity, raise income and generate employment opportunities through self sufficiency, import substitution and export promotion of good quality seeds. Major Activities: Capital investment, human resources development, institutional arrangement, infrastructure development, international linkages and collaboration for; variety development and maintenance, seed multiplication, seed processing and conditioning, seed marketing, and seed use The total proposed investment is NPR 827 million per annum.  Harmonization of seed policies, rules and regulations.  Adoption of public private partnership model in the seed  sector development.  Conservation and sustainable use of indigenous genetic  resources.  Protection of farmers‘ rights and rewarding breeders.  Ensure the supply of good quality seeds in the market. Major Output:  Easy access to good quality seeds for one million farm families.  750 t of high quality seeds will have access to export market.  SRR will reach above 25% for cereals and over 90% for vegetables.  423 open pollinated and 60 hybrid varieties will be released.  Yield of rice and vegetable crops will be above 3.8 t/ha and 19 t/ha respectively.  Private sector will establish or strengthen four big seed companies.  293 highly skilled seed specialists will be developed.  255 thousand people will get additional full time employment.  Edible food availability will reach 8 million t, worth around NPR 200 billion at current price.  Nepal‘s seed sector will be able to share its experiences and knowledge to other countries. Impact:  Food security and poverty reduction  Employment generation  Contribution in biodiversity conservation and  adaptation to adverse impact of climate change  Contribution in gender equity and social inclusion 9.4 CLASSES OF QUALITY SEED
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Seed is said to be quality if it is scientifically produced under the supervision of seed certifying agency and is distinctly superior in terms of genetic purity or varietal purity, freedom from mixture of weeds and other crop seeds, seed health, high germination and vigour, seed treatment and safe moisture content etc. which are parameters to determine the seed quality. The quality seed are classified as following; a. Breeder seed: Breeder seed provides the sources for the initial and recurring production of foundation seed and is directly controlled by the originating or sponsoring plant breeder or the research institute/organization. Its production is personally supervised by a qualified plant breeder to maintain desired genetic purity. Breeder seeds are genetically pure. b. Foundation seed: Foundation seed are produced from breeder seed and it should conform to the prescribed seed certification standards. In species, where seed rates are high and seed multiplication ratio is low, usually two stages of foundation seed are recognized, namely, Fooundation stage I and foundation stage II. Foundation II is the progeny of foundation stage I and should conform to the standards prescribed for foundation stage I seed. Under a seed certification system, foundation seed also needs certification and is known as certified foundation seed. c. Certified seed: Certified seed is produced form foundation seed or in some cases from certified seed, provided this production does not exceed three generations beyond foundation sage I. It should conform to the genetic and physical purity standards prescribed for crop being certified. 9.5 SEED AND FIELD STANDARD FOR QUALITY SEED Field and seed standards are established to monitor genetic purity and physical quality of seeds of different crops. The field standards are applicable to standing seed crop and are normally grouped into following categories. 1. Land requirements: The land requirements for seed production of different crops are prescribed in relation to the previous crop to avoid contamination due to volunteer plants 2. Isolation requirements: Minimum isolations are prescribed for different species to prevent contamination of the seed crops due to mechanical admixture in self-pollinated corps and outcrossing in case of cross pollinated crops. The isolation requirements vary according to the nature of pollination of the seed crop and contaminants. 3. Minimum number of fields inspection: The varietal and analytical purity of the seed crop is affected by various contaminants at different stages of seed crop growth. Therefore number of field inspections and the stages at which the inspections should be conducted are prescribed for each crop to effectively monitor seed quality 4. Specific crop standards:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. a) Off-types: The plants which do not conform to one or more morphological characteristics of the variety under seed production. The maximum permissible limit of ff-types in seed fields of different corps is prescribed keeping in view the pollination behavior of the seed crop and the contaminants to maintain genetic purity of the variety b) Plants affected by seed borne diseases: Maximum permissible limit is prescribed of designated diseased plants for different crops to avoid contamination of seed by transmission o f seed borne disease. c) Objectionable weeds: weed species, whose seed size and shape are similar to that of seed crop and which are difficult to be separated economically by mechanical means, once mixed with seed crop. Also, such weed species which are injurious to seed crop and are difficult to eradicate once established or which may be alternate host for diseases and pest. Maximum permissible limit is prescribed for such weed species for various crops to avoid contamination d) Inseparable other crops plants: To avoid contamination of the seed crop, tolerance limit is prescribed for plants other than seed crops, whose seed size and shape are similar to that of seed crop and whose seeds are difficult to be separated economically by mechanical means once mixed with main crop seed Minimum field standard S. N. Crops Minimum isolation distance (m) Maximum Maximum Designated off-type diseased disease plants (%) plants (%) a. Cereals 1 2 3 b Maize Rice Wheat Grain Legumes FS 300 3 3 CS 200 3 3 FS 1 0.05 0.05 CS 2 0.20 0.30 FS 0.20 0.10 CS 0.50 0.50 Neck blast Loose smut 1 bean/ 10 5 0.10 0.20 - - - 2 Lentil/Mung Blackgram Chickpea 10 5 0.10 0.20 0.10 0.50 3 4 c 1. 2 3 Cowpea Pigeon Pea .Oil Seed Crops Groundnut Soyabean Rape 10 200 5 100 0.10 0.10 0.20 0.20 0.10 0.50 Fusarium wilt Anthracnose 3 3 300 3 3 200 0.10 0.10 0.10 0.20 0.50 0.50 0.10 0.20 0.50 0.50 4 d Mustard Fibre Crops 50 25 - - 0.20 0.50 Anthracnose Alternaria spot in pods sclerotinia
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1 2 Cotton Jute 50 50 25 25 0.10 0.5 0.20 1 9.6 SEED PRIMING Soaking of crop seeds in water or chemicals for some hours to enhance germination is called seed priming. Seed priming is advantageous under three specific conditions: • When seed must be quickly established before a major deadline or event. • When seed is being planted under cold soil conditions. • When a slow germinating species is being mixed with a faster germinating one. The purpose of seed priming is to reduce the germination time, make germination occur over a short period and improve stand and percentage germination. Importance of priming: Poor crop establishment is a major problem in many areas of the world, particularly for subsistence farmers in rainfed and poorly irrigated enviroments. On-fam seed priming leads to earlier germination and establishment and increased yields in a wide range of crops in many tropical and subtropical enviroments. Steps in seed priming: 1. Soak the seeds when you are ready to sow 2. Soak maize for 12-18 hrs. Sorghum for 10 hours. Cowpea for 8 hours. 3. Make sure they are not soaked longer than the given hours. If they continue to take up water they will start to germinate and you might loose the seeds! 4. Surface dry them next day by either drying them with cloth or placing in the sun. 5. Sow them the same day. 6. If you cannot sow because of bad weather, the seeds can be stored in a dry place for several days. The priming process simply involves soaking the seeds overnight or for a certain number of hours, then surface drying them and then sowing within the following day. No change to traditional sowing practices is required. If a farmer is not able to sow the following day, the primed seed can be stored in a dry condition and sown later, still performing better than nonprimed seed. The priming has proven effective for wheat, barley, upland rice, maize, sorghum, pearl millet, chickpea and mungbean. 9.7 SEED MARKETING Marketing is comprises of series of services involved in moving a product from the point of production to the point of consumption. Series of activities such as grading, cleaning, packaging and storage comes prior to the final marketing of the products. Marketing of seed is an important concern for farmers. The public sector can act as a mediator in bringing together the farmers‘ seed
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. producer groups and the seed distributors, such as private companies, merchants, local vendors and NGOs. Marketing issues can be better dealt with by establishing demand forecasting systems, whereby demand for any crop variety is known and is passed on to the producers, and the clients are aware of the sources of different seed. Making use of information networks is also important. In most communities there are informal networks or relations for the flow o f information and technologies. Problems of marketing system of seed production: 1 Lack of proper marketing system 2 Transportation problem for seed 3 Lack of seed traders 4 Lack of support price for seed production 5 Seed storage problem 6 Lack of information on marketing system 7 Availability of poor quality seed 8 Lack of awareness in selecting the quality seed Considerations to facilitate marketing of the produce: 1. Development of road and transport facilities 2. provision of crop insurance 3. provision of loans and necessary inputs 9.8. SEED SYSTEM IN NEPAL: Broadly, two types of seed system are recognized in Nepal 1. informal Seed System 2. formal seed system Informal Seed System Characterized by farmers producing and preserving their own seeds for subsequent planting. Farmers exchange small amount of seeds with other farmers as gift, and for both monetary and non monetary value. Most traditional and local land races are product of such selection and maintenance process, these land races are important genetic resources for modern plant breeding. Informal system is the system where seeds are spread from farmers to farmers. This system has an indirect linkage with the national seed system for production and distribution but significant contribution in spreading of the varieties and its effect reflected in the production and productivity at national level. In this system, the seed production process is usually managed as a part of crop production and seed distribution as per farmers‘ choice and demand among neighbors. Use of retained seeds from their own production, farmers to farmers‘ seed exchange, informal purchase, gifts etc. is the examples of informal seed distribution syste m.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Generally, farmers save seed from their previous crop but certainly the source of new variety is another farmer. Farmers may exchange seed of a new variety in a mutually advantageous interchange and in some instances, exchange seeds of same variety, often in the belief that seed performs better if it is moved from one field to another field. However, the quality of seed supplied through informal channel is always questionable. Formal Seed System Characterized by a vertically organized production and distribution of tested and released/registered varieties by public and private organizations using agreed quality control mechanism. Comprises different phases of seed cycle: Breeder, Foundation, Certified and Improved seeds. Formal system: It comprises public or private sector organizations that are linked to the national seed system (Fig. 5). NARC, DOA, NSC, Seed companies, NGOs, DISSPRO and CBSP groups etc are under this category. a) Public sector: The public sector played and has been playing an important role in seed system development. The system‘s existence was originally established as being a reliable and logical means for the delivery of public sector developed varieties. NARC and NSC under ministry of agriculture and cooperatives are the major public sector institutions working in the field of source seed production and distribution. b) Private sector: The efforts of public sector alone could not meet the overwhelming demand of improved seeds of cereal crops due to limited resources (land, budget, human etc). In the changed context, government has given more focus on the commercialization of agriculture through the partnership with private sector on seed enterprises. As a result, a program like DISSPRO under DADO initiated and covers now 63 districts under this program. This initiatives aims to make each district as self-sufficiency in cereal seeds by producing enough quantities of seeds required at local level. Sixteen DISSPRO districts have implemented an intensive commercial seed production programs located at different eco-zones. Similarly, CBSP was initiated by NARC under the funding of Hill Maize Research Program (HMRP).There are more than 170 CBSP groups producing mainly improved and truthful labeling seeds under the HMRP. There are many more CBSP groups initiated by NGOs producing rice, wheat, maize and legumes. These initiatives have demonstrated a significant contribution on seed production and delivery at the community level and its impact is reflected in crop production and productivity. Due to the foundation laid out by the CBSPs, DISSPRO and seed growers in different aspects of seed business, a number of private seed companies are emerging in the recent year. 9.9 TERMINATOR SEED Terminator technology refers to plants that have been genetically modified to render sterile seeds at harvest – it is also called Genetic Use Restriction Technology or GURT. Terminator technology was developed by the multinational seed/agrochemical industry and the United States government to prevent farmers from saving and re-planting harvested seed. Terminator has not
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. yet been commercialized or field-tested but tests are currently being conducted in greenhouses in the United States. ―Termination technology‖ acts as a physical barrier to the replanting of seeds. It enables companies to protect their large investment into the new and improved varieties of crops. Terminator seed is a sterile seed produced by a genetically modified plant to prevent farmers from reusing the seed for future crops. Impacts of terminator seeds: Terminator is a major violation of the rights of farmers to save and reuse their own seeds. Through pollen movement in the first generation, Terminator genes could contaminate farmers‘ crops - farmers might then unknowingly save and reuse seeds that are contaminated and will not germinate. This could also happen if imported grain contains Terminator genes. Farmers who depend on humanitarian food aid risk devastating crop loss i f they unknowingly use food aid grain containing Terminator genes as seed. Terminator technology helps to protect the environment by preventing the transgenic material from being transferred from generation to generation. Terminator would ensure a corporate stranglehold on seeds and result in higher seed prices at a time when farmers are experiencing the worst income crisis in the history of modern agriculture. If Canadian farmers were forced to buy Terminator seeds every year, the cost would be crippling. Disadvantages of terminator seeds: 1) ―termination technology‖ will decrease biodiversity in two ways‖. First, the transfer of genes from genetically modified plants to traditional breeds will result in the non-modified plants producing sterile seeds. Secondly, the widespread use of termination seeds will reduce interbreeding and lead to less biodiversity. 2) Many poor farmers currently utilize the traditional method of farming consisting of saving seeds and exchanging them with neighboring farms. If neighboring farms begin to buy the termination seeds, poor farmers who exchange seeds with these farms will obtain sterile seeds from their crops. In effect, they will be forced to buy termination seeds. This results in marginalization of poor. 3) Terminator is a major violation of the rights of farmers to save and reuse their own seeds. ―Termination technology‖ acts as a physical barrier to the replanting of seeds. COMMUNITY BASED SEED PRODUCTION Community based seed production is an approach of producing and distribu ting seeds with the participatory involvement of farmers‘ groups. In this approach, seed producer farmer associations are formed to multiply the seed of farmer-preferred varieties using a cost-effective approach. It is market-oriented with an unusual characteristic of placing greater emphasis on developing skills in marketing than in production. It takes account of the entire seed innovation system from initial identification of new varieties through participatory varietal selection (PVS) through to commercial seed production. It involves all stakeholders, and develops strong linkages between
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. the private sector and the community-based groups. This builds sustainable partnerships for the CBSP groups and governmental and non-governmental research and development organisations, seed traders and entrepreneurs. This has: Facilitated the development of a local seed market Provided an opportunity for local income generation through the business of selling seed Increased productivity and hence food security through the timely supply of large quantities In community based seed production approach, farmers can get seed of the varieties they prefer, community networks find suitable new varieties, involve farmers in selection, and produce seed commercially. They are market-oriented, and cost-effective because they involve all stakeholders' farmer groups, government and non-government research and development organisations, seed traders and entrepreneurs. Community-based seed organisations dealing with rice, wheat, maize, kidney bean, chickpea, mungbean, lentil, field pea and oilseed rape already operate in Nepal, and are spreading to India and Bangladesh. They boost local seed markets, open possibilities for people to start seed-trading businesses, and offer farmers a 'basket' of their favourite crop varieties from which to choose. The CBSP approach of seed supply began with the successful outcome of participatory crop improvement (PCI) projects that began in 1997 in Nepal. These projects quickly identified new, farmer-preferred varieties and an immediate challenge was to provide seeds of those varieties on a wider scale. Since 2000, the rapid formation and institutional strengthening of farmers‘ groups to produce and market seed has been widely tested in important crops such as rice, wheat, maize, and kidney bean. Dry season crops e.g. chickpea, mungbean, lentil, field pea, oilseed rape etc were included in the CBSP programme after the initiation of the rice-fallow rabi cropping (RRC) project in selected districts of Nepal terai. New community-based systems fill a big need for seed. Now, farmers can get seed of the varieties they prefer. Community networks find suitable new varieties, involve farmers in selection, and produce seed commercially. They are market-oriented, and cost-effective because they involve all stakeholders' farmer groups, government and non-government research and development organisations, seed traders and entrepreneurs. Community-based seed organisations dealing with rice, wheat, maize, kidney bean, chickpea, mungbean, lentil, field pea and oilseed rape already operate in Nepal, and are spreading to India and Bangladesh. They boost local seed markets, open possibilities for people to start seed-trading businesses, and offer farmers a 'basket' of their favourite crop varieties from which to choose. A Community-based Seed System Model Community-based seed systems (CBSS), commonly known as community seed banks (CSB), process seeds from a range of individuals or groups who share seeds among themselves. Concepts of CBSS have evolved to comprise basic de facto types of individual storage and seed exchange to the more formal and elaborate seed exchanges and seed networks that have greater geographical reach. This informal system gives farmers an active role and communities wider access to seed sources, while preserving traditional varieties that often have important social and economic characteristics. Seeds stored on-farm are a primary form of in situ preservation of genetic resources.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. The CBSS model can evolve as a means for demand-driven delivery of varieties (traditional, improved, or well-adapted varieties) and management options to increase productivity. Community seed banks Community seed banks have been widely recognised as being crucial for maintaining genetic diversity, access to seed and related traditional knowledge. Community Seed Banks (CSBs) are places of storage where indigenous seed varieties are conserved and managed by community members. These ex-situ conservation sites provide farmers with free and easy access to traditional seeds under the condition that a farmer returns twice the amount of seeds he or she borrowed. They not only reduce farmers‘ dependence on seed companies but also help conserve the agrobiodiversity of their villages. community seed bank as a community driven and community-owned effort to conserve and use both local and improved varieties for food security and to improve the livelihoods of farmers. Three types of community seed banks can be identified: i) community gene bank (solely conservation of local varieties as PGR in small quantities), ii) community seed bank (solely concerned with access and availability of cultivars) and iii) community gene cum seed bank (carries out functions of both (i) and (ii)). The term ‗community seed bank‘ should not be used if conservation and sustainable use of plant genetic resources for food and agriculture are not the major objectives. Purposes of such community seed banks are not only saving and exchanging local seeds and keeping them under the control of the farming community for easy access and use for seed security at the community level but also consolidating community roles in promoting conservation, sustainable use and improvement of important local genetic resources / traditional knowledge. Functions of CSB are wide ranging and they include: conservation of plant genetic resources; improving ease of access to local germplasm by the farming community, production distribution of quality seed, maintaining community ownership/control on plant genetic resources, etc. Community seed banks aim to promote the management and sustainable use of both local and farmer-preferred modern varieties for food security and to improve the livelihoods of farmers. IMPORTANCE AND ADVANTAGES OF A CSB It is well understood that CSB is a social system of conservation and utilization of local genetic resources, operated at local levels and run by the community. CSB ultimately helps to conserve genetic resources and associated traditional knowledge in an e volutionary way. The options of planting materials provided by CSBs to the farmers are considered an important approach to increase the production of crops at the household level. CSB plays an important role in sustainable agriculture development. The following are the major advantages and roles of CSB:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 1) CSB helps to conserve local landraces as well as improved varieties through continued utilization. 2) Samples stored in the CSB are considered safety duplicates because many of these accessions are generally stored in the National Genebank. 3) CSB supports the preservation of rare and endangered landraces. 4) All farmers have easy access to planting materials when they are needed. 5) Poor farmers do not need to store seeds for planting. 6) CSB helps to continuously increase the adaptability of local landraces because of the dynamic nature of conservation. 7) Farmers have selection options: seed bank as well as diversity blocks. 8) Farmers have access to information regarding landraces and improved varieties. 9) All local farmers have access to information on what planting materials are available at the local level. 10) The use of a CSB for technology transfer and genetic resource characterization can be effective. 10. Achievements of Plant Breeding in Nepal 10.1 Findings from Plant Breeding, 10.2 Future vision in plant breeding and genetic resources 10.1 FINDINGS FROM PLANT BREEDING 1. Development of insect and disease resistant crop variety. 2. Development of high yielding crop varieties. 3. Development of crop varieties having improved nutritional qualities to supply more balanced nutrition. 4. Development of hybrid crop varieties. 10.2.FUTURE VISION IN PLANT BREEDING AND GENETIC RESOURCES. Genetic resources: VISIONS 8) Establishment of national PGR facility to conserve genetic resources. The gene banks will be established in Tarahara, parwanipur, Lumle and Khajura in near future. 2) Promote and encourage exchange of PGR, technical information and data bas on P GR among national and international scientists. 3) Development of human resources and national capabilities to undertake national PGR activities. The additional positions for plant breeders in crop research programmes will be created and opportunities for training and post graduate studies in order to strengthen the utilization and management of PGR will be created. 4) Inventorizing and cataloguing of genetic diversity 5) Development of in-country capabilities in biochemical and molecular characterization of PGR. 6) Strengthening varietal development programmes to enhance utilization of genetic resources.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 7) Development of a National PGR system and a high level nodal Committee and functional committee with powers to secure funding and human resources development to implement national policy on PGR. 8) Development of infrastructure for ex situ/ in situ/ in vitro conservation, regeneration, characterization, evaluation and documentation. 9) Formulation of national legislation for access to PGR and their exchange in relation to convention on biological diversity. Plant breeding: VISIONS 1) Development of commercial crop varieties for different agro ecological zones and planting time. 2) Development of high yielding and insect and disease resistant crop varieties.. 3) Emphasis on participatory plant breeding programme. 4) Emphasis will be given on genetic engineering. The genes from related species will be transferred to crop 5) collaboration with foreign countries for exchange of technical information on plant breeding activites. TERMINOLOGY OF PLANT BREEDING AND GENETICS: 1) Near isogenic line: They are those lines that are identical in their genotype, except for one gene for disease resistance. The genome of any near isogenic line will most likely contain donar parent deri ved alleles at several of its loci. 2) Somaclonal variation: It refers to all types of variations which occur in plants regenerated from cultured cells or tissues. Variations are observed in morphological, cytogenetic and isozyme traits. It can be utilized for improvement of specific traits, particularly where they are lacking in available germplasm; for example, disease resistance and improvement of quality and yield in cereals, legumes, oilseeds and tuber crops and higher solid contents in fruit crops such as tomato, grapes and watermelon. 3) Cybrids: Cybrids or cytoplasmic hybrids are cells containing nucleus of one species but cytoplasm from both the parental species. They are produced in relatively high frequency by; a) irradiating ( with X-rays or gamma-rays) the protoplasts of one species prior to fusion in order to inactivate their nuclei or b) by preparing enucleate protoplasts (cytoplasts) of one species and fusing them with normal protoplasts of the other species. 4) Vybrids: A vybrid is the progeny obtained form a cross between two facultative apomictics. Vybrids show consistent and superior yields over generations.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 5) Sporogenesis: The process of formation of four haploid cells or microspores form pollen mother cells through meiosis is called microsporegenesis. The process of formation of four haploid cells or megaspores form megaspore mother cells through meiosis is called megasporegenesis. 6) Gametogenesis: The production of male gametes or sperms from generative nucleus through mitotic cell division is called microgametogenesis megaspore is known as megagametogenesis. The development of embryo sac from a 7) Isozymes: Isozymes are variant forms of an enzyme usually detectable through electrophoresis due to differences in their net electrical charges. It is one of the molecular markers. 8) Somatic embryogenesis: The development of somatic bipolar adventitive embryoids form callus cultures under certain nutritional and hormonal conditions is called somatic embryogenesis. This development follows a sequence through pro-embryoid, globular and torpedo stages. Somatic embryogenesis has four phases; a) induction b) development c) maturation d) germination. 9) Centres of diversity: Areas where cultivated plant species and/ or their wild relatives show much greater variation than anywhere in the rest of the world is called centres of diversity. Zhukovsky, in 1965, recognized 12 mega-gene centres of crop plant diversity. This serves useful guide to plant explorers to as to where to search for variation in a given species. W ithin the large centres of diversity, small areas may exhibit a much greater diversity than the centre as a whole; these areas are known as microcentres. 10) Genetic erosion: The gradual loss of variability from cultivated species, and their wild forms and wild relatives is called genetic erosion. The plant breeding activities are chief cause of genetic erosion. 11) Alien-addition lines and Alien- substitution lines: The line which has one pair of chromosomes from a related wild species in addition to the normal somatic chromosome complement (2n) of the species is called Alien- addition line. The line which has one chromosome pair from a different species in place of the chromosome pair of the recipient species is called Alien-substitution lines. 12) Variation: Enviromental variation refers to variation in characters among plants resulting from environmental influences and can be observed in genetically uniform population. e. g. difference in plant .ht. of maize. Genetic/hereditary variation refers to variation in plant characters resulting from genetic differences among individuals within a population and can be observed in genetically mixed population. Genetic variation are transmitted to the progeny. e.g. variation in seed color, awnness, disease resistance etc.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 13) Pure line: A pure line is the progeny of a single, homozygous, self pollinated plant. All the individuals within a pureline have identical genotype, and any variation present within a pureline is solely due to environment. 14) Apospory and Diplospory: Apospory is a form of apomixes in which the embryo sac develops from a vegetative cell of ovule. Diplospory is a form of apomixes in which the embryo sac develops from a megaspore. 15) Protandry and protogyny: Protandry is a form of dichogamy in which stamens mature before pistils. It occurs in crops like maize, sugarbeet. Protogyny is a form of dichogamy in which pistils mature before stamens. It occurs in crops like bajra. 16) Cleistogamy and Chasmogamy: The flowers do not open at all in case of cleistogamy, and there is anthesis. In case of chasmogamy, flowers open but only after pollination has taken place, and there is anthesis. 17) Androgenesis: It is development of plants from male gametophytes. Haploid plantlets are formed in two ways; direct androgenesis: embryo originating directly from the microspores of anthers without callusing indirect androgenesis: microspore undergoes proliferation to form callus which can be induced to differentiate into plants. 18) Inbred: Any inbred is a nearly homozygous line obtained through continuous inbreeding of a cross pollinated species with selection accompanying inbreeding. 19) Transgressive breeding: it aims at improving yield or its contributing characters through transgressive segregatiton. Trasgressive segregation refers to the appearance of such plants in an F2 generation that are superior to both the parent for one or more characters. 20) Emasculation: the removal of stamens or anthers or the killing of pollen grains of a flower without affecting in any way the female reproductive organs is called as emasculation. The purpose of emasculation is to prevent self fertilization in the flowers of line/variety to be used as the female parent. Emasculation is done using techniques such as; a) hand emasculation, b) suction method, c) hot water emasculation, d) alcohol treatment, e) cold treatment and f) genetic emasculation. 21) Southern blotting and western blotting: the process of transferring DNA fragment from electrophoretic gel to nitrocellular filter paper is known as southern blotting. When southern blotting is extended to analysis of RNA is called Northern blotting and that of protein is called Western blotting 22) GENETIC ADVANCE UNDER SELECTION:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Improvement in the mean genotypic value of the selected families over the base population is known as genetic advance (gain) under selection. Genetic advance under selection depends on: i. the genetic variability among different plants or families in the base population ii. the heritability of the character under selection iii. the intensity of selection, i.e. the proportion of plants or families selected. Genetic advance under selection may be calculated as Gs = (k) (σp) (H) K = selection differential, σp = phenotypic standard deviation of the base population H = Heritability of the character under selection. PARTICIPATORY VARIETY SELECTION What is Participatory variety selection? It is an approach to provide choices of varieties to the farmers for increasing production in their diversity of socioeconomic and agro-ecological condition. It is also a selection process of testing released or promising genotypes in farmer‘s field. PVS includes research and extension methods to deploy genetic materials at on farm experiment. Therefore, the variety has developed through PVS that can meet demand of different stakeholders. Why Participatory Variety selection? 1. Provide an opportunity to the farmers a large number of varietal choices on their own resources. 2. Enhance farmer‘s access to crop varieties and increase in diversity. 3. Increase production and ensure food security. 4. Help to disseminate the adoption of pre and released varieties in larger areas. 5. Allow to varietal selection in targeted areas at cost-effectiveness and also in less time. 6. Help seed production at community based seed. How to use participatory variety selection? PVS comprises three steps to identify preferred variety; situation analysis and identify farmers‘ needs; search for genetic materials to test in farmer‘s condition; Experimentation of on-farm research and dissemination of preferred varieties. 1. Situation analysis identifying farmer’s needs: It requires community meetings to identify, prioritize and document specific varietal traits preferred by farmers.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 2. Search for suitable varieties: Once situation analysis and identification of farmers‘ needs have been completed, a search for suitable materials will be tested to find out farmers preferred traits for inclusion in the selection. The varieties may be newly released or promising. 3. Experimentation of on farm research: Choosing the suitable pre/release varieties are essential to test in diversity of socioeconomic and agro ecological conditions. Pre/released variety along with popular variety as a check will be grown. If plot size is too large that may have risk to the resource poor farmers. Therefore, One Katha is suitable. The experimented plots are more suitable if it is along side of the road because majority farmers can visit regularly. 4.Farm walk: It needs to evaluate varietal performance across all the farmers' field. Therefrore, field visits should be organized twice with the active participation of group of farmers, multidisciplinary scientists and extension personnel during crop season. PARTICIPATORY VARIETY SELECTION  Now, agricultural land is saturated. Almost all increases in crop production will have to come from higher output per unit area  Agriculture should have a shift from a land expansion based system to a technology-based system  Seed is the first link in the food chain and seed-based technologies offer the easiest and cheapest options for increasing productivity  There is still lack of varietal selection option of farmers' preferred varieties  For the widespread of modern varieties; it needs to promote a range of crop varieties to suit specific crop production niches and socio-cultural preferences and to strengthen the farmers' seed production and supply system as there is diversity in agro-ecological and socio economic conditions  Improved seeds are primary inputs for high productivity  But, unavailability of quality seed of farmers' preferred varieties at right time with desired quantities and reasonable price of maize is one of the major constraints resulting low adoption of improved varieties and low productivity  The gap between the attainable yield (5 Mt/ha) and the farmers‘ harvest (2.2 Mt/ha) is over 3 Mt/ha in developing countries  Lack of technical know how, lack of choices of variety, improved quality seed as well as very poor formal seed supply system, lack of participatory community based seed production and marketing are factors leading stagnant yield of maize  Results indicated that PVS has contributed to adoption of improved practices, increase in yield, availability of seed locally, improving food security and income. It has also provided great opportunity to integrate research, extension, I/NGOs, CBOs and farmers together in sharing ideas and achieving a common goal  Participatory variety selection (PVS) is the selection of farmers' preferred varieties of different crops by themselves in their own fields under their own local management and practices
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal.  The main objective of PVS is to overcome the bottleneck of the diffusion of farmers' preferred varieties and to provide varietal selection options to fit in the niche environment  Once the variety is identified, farmer-to-farmer dissemination of PVS variety is the most common process of varietal diffusion  Adoption of new crop varieties in rural areas of developing countries is poor  It might be because, many varieties do not meet farmers' consumption and management preferences as most of them are tested under optimum environmental conditions of research stations with a close monitoring by researchers  A crop variety performing excellently at optimum condition is recommended to a certain larger geographical region. These tested varieties upon exposure to the real farmers' field situation not always perform well and farmers have been reluctant to adopt them  There is no guarantee that the released variety will meet farmers' requirements  Till date, farmers have to depend on other organizations for choosing the new variety seeds for their use  To overcome these problems, PVS has evolved as one of the innovative approaches to complement the conventional variety development process  Participatory research can be used to empower farmers and promote development in farmers' communities. It can also be used to increase the efficiency of formal breeding programs in producing and popularizing varieties appropriate for resource-poor farmers  The rejection of many released varieties by farmers, who do not adopt them, and the rapid and high adoption by farmers of non-released varieties are the results of PVS  Collaborative participation allows farmers to decide overall which variety or varieties they prefer as a simpler and more effective solution. PVS also increases varietal biodiversity as more varieties are adopted by farmers, when given choices, can identify varieties for specific niches.  The participatory variety selection (PVS) approach helps to identify farmers' preferred varieties in their own field under their own management within a possible shortest period of time and provides varietal selection options There are the following steps to launch PVS programme at a particular location Site selection  To identify area where farmers need improved high yielding varieties of their own choice  It can be done by participatory approaches that are in close collabo ration with extension personals, farmers, CBOs and NGOs  Farmers of the selected areas should be cooperative and having willingness towards improved varieties Need assessment for new crop varieties  Understanding of the farmers' local production condition and environments, and identification of farmers' varietal needs (what type of variety/ies do they need?)  It is carried out by different participatory methods and tools Identify crop varieties matching farmers' needs
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal.  One should search the suitable varieties from new and old releases, local landraces and from pre-released advanced materials  After identifying varieties, there should be seed increase of that variety in advance if necessary On-farm experimentation by farmers  This includes conducting PVS trials on farmers' fields to access the overall performance of the tested varieties in their own field under their own management  The innovative farmers should be selected to evaluate varieties  Generally, mother-baby (MBs) trials and farmers' fields' trials (FFTs) are used for this purpose  Five to ten varieties (based on suitability to fulfil farmers' need and seed availability) are generally used in MB trials  When a single variety (1/2-1 kg) is given to the farmer to compare it with his/her local variety using his/her own management is called Baby Trial (BT)  A baby of a single variety could be provided to 5-10 farmers  The improved variety and their local should be planted in the same field in adjacent plots so that they can compare the performance of each improved variety with their local from planting to post-harvest management  Individual farmer reports his/her own perceptions through a household level questionnaire (HLQ) through matrix ranking  Data in HLQ can be recorded as scores where the new variety is compared to their local counterpart as better, same or worse  The following points could be considered in HLQ: Germination, plant height, lodging problem, disease and insect pests problem in the field, drought resistant/tolerant, shade tolerant, prolificacy, stay green after physiological maturity, thickness of maize stover, stover liked by animals, days to maturity, ear size, cob size, shelling (%), grain colour, grain size, grain yield, stover yield, taste of different items after cooking), disease problem (rot) in storage, insect pests problem in storage and overall ranking (only for FGD) In addition to these above mentioned points, the following information can also be collected from each household  Do they plan to plant this variety next year? Why?  Have they saved the seed of this variety?  How much amount of seed of this variety do they want to plant next year?  Is there any seed demand of this variety by anyone? If yes, how much?  Which variety do they prefer, local or improved, that has been provided to them? Give overall reasons  There are two options in conducting baby trials. A baby trial is a one -on-one (Design 1) or one-on-two (Design 2) comparison under farmers' management. In this trial, farmer estimates grain yield. Mother Trials
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal.  All the varieties tested in the BTs including local check are grown together in a centrally located area of that site are referred to Mother Trial (MT)  Two sets of MTs (single farmer as a replicate) at a site managed by researchers using improved package of practices are planted in the centre of the site  The two sets can also be planted one using improved practices and second one using farmers' local practices  These MTs give opportunity to farmers, researchers and other stakeholders to see the comparative performance of tested varieties under recommended and local management practices in the same spot during field visits  In the MTs observations are taken from central rows  Researchers/recorders take observations on days to flowering, plant and ear height, insect and disease problem; grain yield and plant stand at harvest from MTs  Information collected through HLQ and FGD is tabulated, summarized, compared and conclusion is drawn  Evaluation of MTs is generally done by matrix ranking by farmers during focus group discussion (FGD) first at pre-harvest after farm walks when MBs are observed by stakeholders and second at about 2-3 months after harvest  This provides opportunity to participating farmers and other stakeholders to evaluate and identify overall performance of the tested genotypes including taste, culinary characters and insect pest problems during storage  There should be 6-8 farmers in a group. Male and female groups should be formed separately to avoid dominance of male farmers  The points included in HLQ are the same for FGD. Each group ranks the variety in matrix system where 1 is the best and 6 (last number) is the worst Wider promotion and dissemination of farmers' preferred varieties  Once the varieties are identified group should go for community based maize seed production. This will help to promote and wider disseminate farmers' preferred varieties  Small-scale farmers who can not afford improve seed each year should be trained about seed selection criteria from baby trials for next year planting V1 Farm 1 Farm 6 L Farm 2 V2 L V3 BABY TRIALS (BTs) L V4 L V2 L Farm 3 V5 L Farm 7 Farm 8 Farm 4 V3 L Farm 9 V1 L Farm 5 V5 L Farm 10 V3 L V5 L V4 L
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Farm 11 Farm 12 MOTHER TRIALS (MTs) Var. 1 Var. 2 Var. 3 Var. 4 Var. 5 Local Farm 26 Var. 1 V1 Farm 13 L Farm 19 Var. 3 Var. 4 Farm 27 Var. 5 BABY TRAILS (BTs) L V2 L V5 V3 Farm 14 V4 L Farm 18 Var. 2 Farm 15 V4 L Farm 20 V1 L Local Farm 21 V3 L V4 L Farm 17 V5 L Farm 16 V2 L L V1 L Farm 22 V2 L Farm 23 Farm 24 Farm 25 Design 1. Layout plan of a mother-baby trail of five new varieties in five farms each. V1-V5 = New varieties, L = Local variety compared with V1-V5. V1 Farm 1 V2 SC Farm 5 SC L V4 Farm 2 L Farm 3 V4 SC Farm 6 V3 BABY TRAILS SC L V3 L V4 L V2 SC L Farm 4 V2 SC Farm 7 SC L Farm 10 SC L V6 SC L Farm 8 Farm 9 SC L Farm 11 V6 SC Farm 12 V1 L MOTHER TRIAL V1 V2 V3 V4 SC(V5) V6 Farms 26 and 27 are for 2 sets of MT L BABY TRIALS V2 SC L Farm 13 V3 SC L V3 Farm 14 SC L Farm 15 V1 SC L Farm 16 SC L
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. V6 SC Farm 17 L V1 V3 SC L Farm 21 SC L Farm 18 V4 V4 SC L V1 SC L Farm 22 SC L Farm 19 Farm 23 V6 SC V6 SC L Farm 20 V2 SC L Farm 24 L Farm 25 Design 2. Layout plan of a mother-baby trail with one new variety compared with two checks (standard and local) PARTICIPATORY ON FARM RESEARCH Farmer participatory research (FPR) is an approach, which involves encouraging farmers to engage in experiments in their own fields so that they can learn, adopt new technologies and spread them to other farmers. With the scientist acting as facilitator, farmers and scientists closely work together from initial design of the research project to data gathering, analysis, final conclusions, and follow-up actions. This step, sometimes known as ―innovation evaluation‖ is essential for communication as well as for initiating diffusion. The main advantage of this approach is that farmers ―learn by doing‖ and decision rules are modified on the basis of direct experience. To shape learning, interpretations of experience must provide information about what happened, why it happened and whether what happened was satisfactory or unsatisfactory. New information, technologies and concepts may be better communicated to farmers through the FPR approach. Ways to conduct FPR ? 1. Planning meeting Initiate participatory experiments in collaboration with the local agricultural extension technician and the village head. In each village or district, invite 10 to 25 farmers. With the researcher acting as the facilitator, conduct group meetings with farmers. These half-day meetings can begin with general discussions about rice growing and related problems. Later, discussions should focus on the topic of relevance to both farmers and researchers. For instance, in pest management, the discussions may focus on the rice leaffolder, concerns about their damages, losses they could cause and methods of control. Encourage farmers to discuss whether the leaffolder needed control and whether anyone would volunteer to participate in evaluating a simple hypothesis. 2. Laying out the experiment
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Each participating farmer would mark out an area of about 100 m2 in his or her field that would that would not receive any insecticide treatment in the first 30 days after transplanting. The rest of his or her field would receive normal treatments. All other agronomic practices in both the experiment and main plots would be according to each farmer's normal practice. During the cropping season, the extension technician and researcher will make at least one follow-up visit to each participant. Researchers, extension and farmers review a farmer experiment plot. 3. Support materials Provide participating farmers with some support materials, as follows: A signboard (30 cm x 60 cm) with the farmer's name, collaborating agencies and the experimental title for display in each participant's field. Two comics strips depicting a discussion of a farmer who had done the experiment in local language A cardboard file with weekly activity sheets and a ball-point pen for recording farming activities and input costs. A set of instructions to conduct farmer participatory research. 4. Monitoring Before the experiments begin, conduct a short pre-FPR or baseline survey primarily to allow subsequent comparison of yields and costs of the practice of interest (e.g., insecticide applications in the case of leaffolder) from the experimental and main plots. Determine their practices (e.g., insecticide use patterns), and knowledge and attitude towards the problem (e.g., leaf feeding insects). The second survey could focus on farmers‘ practices, costs, perception of yield differences and benefits derived from applying the ―new‖ treatment (e.g., not using early season insecticide sprays for leaffolder control). 5. Farmer experience sharing workshop At the end of the season, organize a workshop where farmers will report their experimental results. Invite neighboring farmers and local extension technicians. Preferably a week before the workshop, help each participating farmer to prepare his report on the results. Have a one -page cost and yield comparison between treated and untreated plots. During the workshop encourage farmers to discuss possible reasons for yield differences and their plans for the next season. As a token of appreciation, give each participant a t-shirt or a cap and a certificate of participation. 6. Farmer-to-farmer spread To monitor diffusion, ask farmers in a monitoring survey for names and addresses of other farmers with whom they had directly shared the results of their experiments. Track down the farmers who heard about the experiment from the first group and had followed the simple rule
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. that was tried out in FPR. If most of them are neighbors and relative, it could suggest that the spread may be through kinship and close proximity of their neighbors. 7. Upscaling With the help of media, such as newspapers, radio and television, spread may be further enhanced. The use of these approaches may be explored in the future. Since encouraging farmers to experiment with a simple rule-of-thumb can be easily communicated in informal talks, farmerto-farmer spread of experiences from the experimentation may be further enhanced. Poster can be an effective way of disseminating improved practices PARTICIPATORY PLANT BREEDING (PPB) What is Participatory Plant Breeding (PPB)? Participatory plant breeding (PPB) is a process in which farmers and plant breeders jointly select cultivars by segregating materials in a target environment. PPB may also include activities such as germ plasm enhancement through pure line or mass selection. PPB approaches thus draw on the comparative advantages of both formal and informal systems. In recent years, has PPB also been considered as a potential strategy for enhancing biodiversity and production. The approaches that involve close farmer-researcher collaboration to bring about plant genetic improvement within a species are considered to be participatory plant breeding. Broadly, PPB is the development of a plant breeding program in collaboration be tween breeders and farmers, marketers, processors, consumers, and policy makers (food security, health and nutrition, employment). In the context of plant breeding in the developing world, PPB is breeding that involves close farmer-researcher collaboration to bring about plant genetic improvement within a species. Developing a clear vision together with the stakeholders in the breeding process is important. What are the goals do PPB? 1) Increase production and profitability of crop production through the development and enhanced adoption of suitable, usually improved, varieties. 2) Provide benefits to a specific type of user, or to deliberately address the needs of a broader range of users 3) Build farmer skills to enhance farmer selection and seed production efforts Why focus on PPB? Traditional breeder-directed breeding programs are very effective at developing varieties that can be used in farming systems that are fairly homogeneous, but less effective when the reality of the
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. farmer is more complex and risk-prone. PPB encourages two kinds of participation: Functional Participation Plant breeders can direct their research according to the needs of the specific groups of farmers (women, men, rich, poor). The physical and economic resource bases of different pe ople necessitate tailored research approaches. Farmers can assure plant breeders that they are assessing tradeoffs among traits correctly. On-farm research assures that varieties will produce well under ―real life‖ conditions. On-farm research can be managed by the researcher, by the farmer, or by both. PPB ensures greater success of adoption of innovation by the farmers. Empowering Participation Increasing farmer knowledge and skills so that farmers can participate more fully in the collaborative breeding efforts and be better at their own, personal efforts. What activities can PPB include? Identifying breeding objectives Generating genetic variability (including the provision of plants to be included in breeding program) Selecting within variable populations to develop experimental varieties Evaluating experimental varieties (PVS - participatory variety selection) Variety release Popularization (diffusion of information about new variety and and how it is managed) Seed production How do you decide what kind of breeding program is most useful? Traditional, experiment station-based breeding efforts: Areas where crop is produced over a broad, relatively uniform agro-ecological area (can be simplified and standardized) Places where farmers have access to agricultural inputs (fertilizers, chemicals, irrigation) Places where crop end-uses are broadly similar Examples: Green Revolution wheat and rice varieties, maize hybrids for temperate and mid altitude tropical zones, varieties for irrigated, commercial vegetable production. Participatory plant breeding: Areas that are not dedicated to large-scale crop production Marginal crop production areas, where environments are highly variable so that selection based on GxE interaction is not the best selection strategy Areas where agriculture is risk prone, complex (intercropping) and low input; may include the
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. development of populations or heterogeneous materials Where crop end-uses are diverse and/or locally unique Where important minor crops exist but are not the focus of formal plant breeding efforts (may include desire to conserve local biodiversity) Examples: Pigeon Pea breeding in ICRISAT video, Dr. Rosas in Honduras SOME IMPORTANT FACTS ABOUT GENETICS AND PLANT BREEDING:  Gene-for-gene relationship was given by Flor in 1951.  The term Heterosis was given by Shull in 1914.  Magnitude of heterosis is smaller in self pollinated crops than cross pollinated crops  Clean and dirty crop approach was given by Marshall in 1977.  Stratified mass selection is also known as grid method of mass selection.  Mass selection is the oldest breeding scheme available for cross pollinated crops.  Mass method of breeding was proposed by Harrington.  Pollination taking place between flowers borne by same parent is called geitonogamy.  Nepal is birth place of rice.  Inbred is nearly homozygous line.  When an inbred is crossed with an open pollinated variety, is called top cross.  Grid method of breeding was given by Gardner  Bulk method of breeding was first used by Nilsson-Ehle in 1908.  Overdominance hypothesis was given by East and Shull in 1908  Progeny test was developed by Louis de Vilmorin.  Reciprocal recurrent selection was proposed by Comstock, Robinson and Harvey in 1949.  Concept of path coefficient analysis was given by Wright in 1921.  Metroglyph analysis was given by Anderson in 1957  Partial diallele analysis was given by Kempthorne in 1957.  Self incompatibility (SI) was first reported by Koelreuter  Zhukovsky in 1965 proposed 12 mega-gene centres of crop plant diversity.  The concept of centres of origin and law of homologous series was given by N.I.Vavilov.  Term ideotype was given by Donald in 1968.  The term genetics was given by Betson.  Father of genetics is G.J. Mendel  Terms genotype and phenotype was given by Johnson in 1911.  Germplasm theory was given by Vizeman  Term chromosome was given by Waldiper  DNA is a genetic material was given by Graphiya  Term mutation was given by Hugo de Vries.  First hybrid was given by Fairchild in 1717.  Crossing over takes place in pachytene.  Formation of chiasmata and synematal complex formaton takes place in Zygotene.  Cytoplasmic genetic male sterility is used in hybrid seed production.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal.                                       Structure of chromosome can best observed at metaphase. Genes are made up of DNA only. When embryo develops from egg cell- parthenogenesis When embryo develops from antipodal cells-apogamy Clone, pureline and hybrids are heterozygous. Term biotechnology was coined by Karl Ereky, a Hungerian Engineer in 1919. The first plant breeding company was established in France in 1727. Self pollinated crops: rice, wheat, barley, oat, cow pea, pea, mung, soyabean, lentil, rajma, groundnut, gram (chickpean), linseed Cross pollinated crops: maize, bajra, sunflower, castor, niger Often cross pollinated crops: cotton, jute, tobacco, pigeon pea. Totipotency is the ability of cells to develop into complete plant. Self pollinated crops do not show inbreeding depression. Purines is made up of 2-carbon nitrogen ring structure, Adenine and Guanine Pyrimidines is made up of 1-carbon nitrogen ring structure, Thymine and Cytosine. Replacement of purines by pyrimidines and vice-versa is called transversion. Transcription is process of synthesis of RNA from DNA. Transition is replacement of purine by purine and pyrimidine by pyrimidine. Number of chromosome in endosperm is 3n Male gametes carry n chromosome number. Pureline selection is also known as single plant selection. All plants within a pureline have same genotype. Mitosis is called homotypic division and meiosis is called reductional division. A unit of mutation in a gene is called muton. NARC was established in 1991. Single seed descent selection is modification of bulk method. Law of parallel variation was given by Vavilov. Synthetic variety requires reconstitution after 5 years. Term recurrent selection was coined by Hull. Botanical Enterprises Pvt, Ltd, Godawari is one of the oldest tissue culture facilities established in the private sector in Nepal. The concept of path coefficient analysis was developed by Wright in 1921. First attempt of plant tissue culture was done by Haber landt. Evolution of any history of a species is called phylogamy. D2 statistic was proposed by Mahalanobis in 1936. Metroglyph analysis was developed by Anderson in 1957. Stability analysis was first done by Finley and Wilkinson in 1963. Bulk method of breeding was first used by Nilson-Ehle in 1908. Nepalese farmers practised the mass selection method of breeding. Mass selection is being practised by farmers in Nepal. The most popular breeding method for maize is progeny test.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal.  Origin of rice is Hindustan centre, Maize- central America or south America, Wheat- central Asia centre, potato- south America, buckwheat- China centre and gram (chickpea)Mediterranean centre of origin. Multiple Choice Identify the choice that best completes the statement or answers the question. ___B_ 1. A characteristic that an organism can pass on to its offspring is called a: a. phenotype b. trait c. genotype d. gene __C__ 2. A(n) ____ is the form of a gene that is hidden when the dominant allele is present. a. allele b. dominant allele c. recessive allele d. genotype ___A_ 3. If a recessive trait is expressed in an organism‘s phenotype, what can you determine about its genotype? a. Both alleles in the genotype are recessive. b. At least one allele in the genotype is recessive. c. You cannot determine anything about the organism‘s genotype. d. Both alleles in the genotype are dominant. ___B_ 4. Genetics is the: a. study of pea plants. b. study of heredity. c. phenotype of an organism. d. a characteristic that can be passed on. ___B_ 5. How do flowering plants reproduce? a. Ovulation b. Pollination c. Cross-breeding d. Breeding __D__ 6. An organisms genotype describes: a. the form of a trait it displays. b. its physical characteristics. c. the way it looks. d. the alleles of the gene it contains. __B__ 7. A ____ is a unit that determines traits. a. chromosome b. gene c. allele
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. d. DNA _A___ 8. For each trait, pea plants contain two forms of the same gene. These different forms of the same gene are called: a. alleles b. genotypes c. chromosomes d. All of the above D_ 9. During his experiments, Mendel found that: a. all traits do not blend b. in the first generation all of the offspring showed the dominant trait. c. in the second generation the ratio of purple flowers to white flowers was 3:1. d. All of the above ___D_ 10. Mendel studied ____ traits in pea plants. a. flower color b. pod color c. seed shape and color d. All of the above __D__ 11. If a green seed (yy) is crossed with another green seed (yy) what can you predict about the offspring? a. the phenotype will be green b. the genotype will be yy c. the recessive trait will show d. All of the above __C__ 12. If a true-breeding purple plant is crossed with a true-breeding white flowered plant, the possible phenotypes of the first generation of peas are: a. all white. b. all purple. c. 50% purple and 50% white. d. 75% purple and 25% white. ___C_ 13. When Mendel‘s first generation pea plants were allowed to self-pollinate, the ratio of purple to white flowers in the second generation was: a. 1:3 b. 1:1 c. 3:1 d. 4:1 _C___ 14. What is a true-breeding plant? a. A plant that never produces offspring with the same form of a trait when it self-pollinates. b. A plant that has been pollinated by human influence.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. c. A plant that will always produce offspring with the same form of a trait when it self-pollinates. d. A plant that will always produce offspring with a different form of a trait when it self-pollinates. ___C_ 15. The ratio of 25:5 is the same as the ratio: a. 1:5 b. 3:1 c. 5:1 d. 4:1 _C_ 16. A ____ is the form of a gene that, when present, covers up the appearance of the ____. a. recessive allele, dominant allele b. dominant gene, recessive gene c. dominant allele, recessive allele d. recessive gene, dominant gene __A__ 17. If a pea plant contains a recessive allele for wrinkled seeds and a dominant allele for smooth seeds, the organism‘s phenotype must be: a. smooth seeds. b. wrinkled seeds. c. half of the seeds are smooth and half are wrinkled. d. not really smooth or wrinkled seeds, a mixture of both traits. ___D_ 18. You find a pea plant with purple flowers. From this you know that its: a. genotype must be PP. b. phenotype can either be Pp or PP. c. genotype must be Pp. d. phenotype is purple. ___A_ 19. In order to show all of the possible combinations of alleles from parents, we use: a. punnett squares. b. phenotypes. c. genotypes. d. None of the above __C__ 20. Walter Sutton: a. is a famous scientist who strongly disagreed with Mendel‘s findings. b. came up with ideas about genetics before Mendel, but never spoke of his findings. c. discovered chromosomes, which contain genes, in grasshoppers. d. disproved Mendel‘s theories about heredity, by showing there is no way to predict traits in offspring. ___A_ 21. The works of Gregor Mendel and Walter Sutton: a. are combined in the laws of heredity. b. contradict each other. c. occurred at the same time.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. d. dealt with the same organisms. ___A_ 22. When fertilization occurs, offspring inherit: a. one homologous chromosome in a pair from each parent. b. a complete set of chromosomes from one of the parents. c. two homologous chromosomes in pairs from each parent. d. a random amount of chromosomes from each parent. ___B_ 23. ____ is the mathematical chance that an event will occur. a. Punnett square b. Probability c. Prediction d. Crossing over ___C_ 24. A punnett square is helpful in predicting: a. only genotypes of offspring. b. only phenotypes of offspring. c. both the genotypes and the phenotypes of the offspring. d. None of the above __D__ 25. A gene: a. is a segment of DNA located on chromosomes. b. determines an organism‘s traits. c. typically contains one allele from each parent. d. All of the above ___D_ 26. In the above cross, a round seed pea plant (RR) is crossed with a round seed pea plant (Rr). What are the possible genotypes for seed shape in the cross? a. RR, RR, RR, RR b. Rr, Rr, Rr, Rr c. RR, RR, RR, Rr d. RR, RR, Rr, Rr __A__ 27. What happens to alleles during meiosis? a. Alleles separate b. Alleles combine c. Alleles are created d. Nothing happens ____C 28. If a human with free earlobes (FF), and a human with attached earlobes (ff) mate. What are the possible phenotypes of the offspring? a. The offspring will either have attached earlobes or free earlobe. b. The offspring will have attached earlobes. c. The offspring will have free earlobes. d. None of the above
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. ___B_ 29. A purple flower with genotype PP is crossed with another purple flower with genotype Pp. What is the offspring‘s phenotype? a. white or purple b. purple c. white d. Pp, PP, PP, PP ___B_ 30. Yellow seeds are determined by the dominant allele (Y), while green seeds are determined by the recessive allele (y). A cross-pollination occurs between a yellow seeded plant and a green seeded plant. The offspring had yellow seeds. What is the possible genotype of the offspring: a. YY b. Yy c. yy d. Need more information to determine the genotype ___A_ 31. Sex cells carry ____ alleles for a given gene. a. 1 b. 2 c. 4 d. 8 __B__ 32. Which parent‘s genes determines the sex of a baby human? a. Mother b. Father c. Both mother and father d. Neither mother or father __C__ 33. An example of incomplete dominance is: a. tabby cats. b. ABO blood types in humans. c. pink flowers from white snapdragons crossed with red snapdragons. d. parakeet feather colors. ___A_ 34. Skin color in humans is determined by several different genes. This is an example of what pattern of inheritance? a. Polygenic inheritance b. Codominance c. Incomplete dominance d. Multiple alleles __C__ 35. The X and Y chromosomes in humans are called: a. multiple alleles. b. polygenic traits.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. c. sex chromosomes. d. codominance. __B__ 36. Which pair of chromosomes would produce a male offspring? a. XX b. XY c. XO d. AB __D__ 37.Plant fully grown height is determined by: a.its health. b. its parents‘ height. c.its nutrition. d. All of the above MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. 1) Which of these crosses will only produce heterozygous offspring? A) AA X Aa B) Aa X Aa C) aa X aa D) AA X aa E) Aa X aa 2) Translation directly converts the information stored in ______ into ______. A) RNA . . . a polypeptide B) protein . . . DNA C) DNA . . . a polypeptide D) RNA . . . DNA E) DNA . . . RNA 3) The expressed regions of eukaryotic genes are called ______. A) promoters B) exons C) caps D) introns E) tails 4) Attached earlobes are recessive to free earlobes. What genotypic ratio is expected when an individual with attached earlobes mates with an individual heterozygous for free earlobes? (Draw a Punnett square if necessary). A) 9:3:3:1 B) 2:1 C) 1:1 D) 3:1 E) 1:2:1 5) What is recombinant DNA? A) DNA that results from bacterial conjugation B) DNA that carries genes from different organisms C) DNA that is produced as a result of crossing over D) an alternate form of DNA that is the product of a mutation. E) DNA that carries a translocation
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 6) If one strand of a DNA double helix has the sequence GTCCAT, what is the sequence of the other strand? A) CUGGTU B) ACTTGC C) CAGGTA D) CAGGUA E) TGAACG 7) What is transcription? A) The manufacture of a strand of RNA complementary to a strand of DNA. B) The manufacture of two new DNA double helices that are identical to an old DNA double helix. C) The modification of a strand of RNA prior to the manufacture of a protein. D) The manufacture of a new strand of DNA complementary to an old strand of DNA. E) The manufacture of a protein based on information carried by RNA. 8) True-breeding plants ______. A) produce sterile offspring when cross-fertilized B) self-fertilize to produce hybrid offspring C) self-fertilize to produce offspring identical to the parent D) become sterile after three generations E) cannot be cross-fertilized 9) Upon completion of telophase I and cytokinesis there is(are) ______ cell(s). A) two diploid B) two haploid C) four haploid D) four diploid E) one diploid 10) What is the ultimate source of all diversity? A) meiosis B) natural selection C) mutation D) crossing over E) sexual recombination 11) What name is given to the specific location of a gene on a chromosome? A) chromaddress B) phenotype C) allele D) locus E) genotype 12) The ______ is the trait most commonly found in nature. A) dominant B) budgie C) wild type D) F1 E) recessive 14) What is the key to the recognition of a trait whose expression is determined by the effects of two or more genes (polygenic inheritance)? A) A mating between a homozygous recessive and a heterozygous individual produces offspring who all have the same phenotype. B) Pleiotropy occurs. C) A mating between a homozygous and a heterozygous individual produces more than the expected number of offspring expressing the dominant. D) The trait exhibits a continuous distribution. E) All of the alleles of the gene for that trait are equally expressed.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 15) The correct sequence of the stages of the cell cycle is ______. A) interphase, prophase, metaphase, anaphase, telophase B) telophase, prophase, interphase, anaphase, metaphase C) metaphase, interphase, prophase, anaphase, telophase D) anaphase, interphase, prophase, metaphase, telophase E) prophase, metaphase, anaphase, telophase 16) An individual who is homozygous ______. A) carries two different alleles for a gene B) is a carrier of a genetic disorder C) expresses the recessive D) expresses the dominant E) carries two copies of the same allele for a gene 17) Evidence for the spiral nature of DNA came from ______. A) X-ray crystallography studies B) base rule studies C) studies of pathogenic bacteria D) bacteriophage studies E) transforming studies 18) How many nucleotides make up a codon? A) two B) one C) three D) five E) four 19) How does prophase I differ from prophase II? A) During prophase I there is one diploid cell; during prophase II there are two haploid cells. B) Tetrads do not form during prophase I; tetrads form during prophase II. C) During prophase I chromosomes line up single file on the midline of the cell; during prophase II the chromosomes line up in double file on the midline of the cell. D) During prophase I chromatin condenses; chromatin does not condense during prophase II. E) During prophase I the nuclear envelope breaks up; during prophase II the nuclear envelope remains intact. 20) A true-breeding plant that produces yellow seeds is crossed with a true-breeding plant that produces green seeds. All of the seeds of all of the offspring are yellow. Why? A) Yellow is an easier color to produce. B) All of the offspring are homozygous yellow. C) The yellow allele is recessive to the green allele. D) The yellow allele is dominant to the green allele. E) The alleles are codominant.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 21) ______ represent sites of crossing over. A) Centrosomes B) Centromeres C) Tetrads D) Chiasma E) Synapses 22) Which of these events occurs during anaphase? A) chromosomes align on the midline of the cell B) nucleoli reappear C) cytokinesis D) centromeres divide E) tetrads form 23) In order to determine the phenotype of an individual who expresses a dominant trait, you would cross that individual with an individual who ______. A) has the genotype Aa B) is heterozygous for that character C) is homozygous dominant for that character D) is homozygous recessive for that character E) expresses the dominant character 24) The correct sequence of the events of transcription is ______. A) elongation, initiation, termination B) splicing, capping, tailing C) initiation, elongation, termination D) tailing, capping, splicing E) capping, tailing, splicing 25) RNA contains ______, whereas DNA contains ______. A) nucleotides . . . nucleic acids B) uracil . . . thymine C) cytosine . . . guanine D) a deoxyribose sugar . . . a ribose sugar E) adenine . . . guanine 26) According to Mendel's principle of segregation, ______. A) homologous chromosomes move to the same gamete B) each pair of alleles segregates into separate gametes C) more gametes carrying the dominant allele are produced than gametes carrying the recessive allele D) gametes are diploid E) gametes have one copy of each allele
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. 27) A couple has two female children. What is the probability that their next child will be male? A) 50% B) 75% C) 67% D) 33% E) 25% 28) The region of DNA where RNA synthesis begins is the ______. A) promoter B) processor C) start codon D) initiator E) terminator 29) How many autosomes do humans have? A) 44 B) 22 C) 46 D) 2 E) 23 30) What is the genetic complement of an individual with Turner syndrome? A) 2n + 1 B) n + 1 C) n - 1 D) 2n - 1 E) 4n 31) Attached earlobes are recessive to free earlobes. What is the probability of having a child with attached earlobes when an individual with attached earlobes mates with an individual heterozygous for free earlobes? A) 50% B) 25% C) 100% D) 75% E) 0% 32) A cell that completed the cell cycle WITHOUT undergoing cytokinesis would ______. A) not have completed anaphase B) have two nuclei C) be a prokaryotic cell D) be diploid E) be haploid 34) What is the key to the recognition of codominance? A) The dominant allele is not always expressed. B) The phenotype of the heterozygote falls between the phenotypes of the homozygotes. C) The alleles affect more than one trait. D) The heterozygote expresses the phenotype of both homozygotes. E) The trait exhibits a continuous distribution. 35) You are attempting to determine the identity of a criminal. The only evidence is a tiny drop of blood. How can you use this drop of blood to identify the specific individual? A) You can use gel electrophoresis to determine the length of the DNA found in the sample. B) You can use the sample to determine the individual's ABO blood group. C) You can use the sample to check for the presence of the Rhesus factor. D) You can use PCR to increase the amount of DNA available for restriction fragment analysis. E) There is insufficient material to identify the criminal. DEOXYRIBONUCLEIC ACID (DNA):
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Deoxyribonucleic Acid (DNA), genetic material of all cellular organisms and most viruses. DNA carries the information needed to direct protein synthesis and replication. Protein synthesis is the production of the proteins needed by the cell or virus for its activities and development. Replication is the process by which DNA copies itself for each descendant cell or virus, passing on the information needed for protein synthesis. In most cellular organisms, DNA is organized on chromosomes located in the nucleus of the cell. Structure of DNA: A molecule of DNA consists of two chains, strands composed of a large number of chemical compounds, called nucleotides, linked together to form a chain. These chains are arranged like a ladder that has been twisted into the shape of a winding staircase, called a double helix. Each nucleotide consists of three units: a sugar molecule called deoxyribose, a phosphate group, and one of four different nitrogen-containing compounds called bases. The four bases are adenine (A), guanine (G), thymine (T), and cytosine (C). The deoxyribose molecule occupies the center position in the nucleotide, flanked by a phosphate group on one side and a base on the other. The phosphate group of each nucleotide is also linked to the deoxyribose of the adjacent nucleotide in the chain. These linked deoxyribose-phosphate subunits form the parallel side rails of the ladder. The bases face inward toward each other, forming the rungs of the ladder. Linkage: Tendency of two or more genes to stay together during inheritance is called linkage. Complete linkage: No recombination takes place Incomplete linkage: recombination takes place Coupling phase: Dominant alleles of linked genes linked together e.g. AB Repulsion phase: Dominant alleles of one genes linked with recessive alleles of the other .e.g. Ab of aB Hardy-Weinberg Law: ―Gene and genotype frequencies in a Mendalian population remain constant generation after generation if there is no selection, mutation, migration or random drift‖. This law was developed independently by Hardy (1980) in England and Weinberg (1990) in Germany. Embryo rescue: In case of endosperm abortion, plants may be raised by culturing the hybrid embryos on a suitable culture medium. The embryos in such cases are removed from young seeds before endosperm abortion takes place; this is termed as embryo rescue Micro propagation: Production of a large number of vegetative progeny (initially very small size) through tissue culture is called micro propagation
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Objectives of micropropagation: rapid multiplication virus elimination Difference between DNA and RNA RNA DNA Generally doubled-stranded Generally thymine present and uracil absent Generally, single stranded Generally uracil present and thymine absent Pentose sugar is deoxyribose Pentose sugar is ribose MENDEL’S LAWS Mendel‘s Laws, principles of hereditary transmission of physical characteristics. They were formulated in 1865 by the Augustinian monk Gregor Johann Mendel. Experimenting with seven contrasting characteristics of pure-breeding garden peas, Mendel discovered that by crossing tall and dwarf parents, for example, he got hybrid offspring that resembled the tall parent rather than being a medium-height blend. To explain this he conceived of hereditary units, now called genes, which often expressed dominant or recessive characteristics. Formulating his first principle (the law of segregation), Mendel stated that genes normally occur in pairs in the ordinary body cells, but segregate in the formation of sex cells (eggs or sperm), each member of the pair becoming part of the separate sex cell. When egg and sperm unite, forming a gene pair, the dominant gene (tallness) masks the recessive gene (shortness). Continuing the breeding experiments, he found that the self-pollinated AA bred true to produce pure tall plants, that the aa plant produced pure dwarf plants, and that the Aa, or hybrid, tall plants produced the same three -to-one ratio of offspring. From this Mendel could see that hereditary units did not blend, as his predecessors believed, but remained unchanged from one generation to another. He thus formulated his second principle (the law of independent assortment), in which the expression of a gene for any single characteristic is usually not influenced by the expression of another characteristic. Mendel law can be summarized as following; 1) Law of segregation ( Mendel‘s first law): Two alleles of a gene remain separate and do not contaminate each other in the f1 or the hybrid. At the same time of gamete formation in the f1 two allele separate and pass in to the different gametes. 2) Law of independent assortment (Mendel‘s second law): when two genes segregate at the same time segregation is independent of each other. Alpha/Beta/Gamma Diversity At any level of organisation in the biological hierarchy, biodiversity expresses itself as the sum of within and between component diversity. For instance, if we consider a landscape with two different ecosystems, the diversity of the component species within each ecosystem is referred to
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. as alpha diversity. As we move from one ecosystem to the next within the landscape, there will be a change in the component species. This change or turnover of species is called beta diversity. The sum of alpha and beta diversities of the ecosystems is an expression of the biodiversity in the landscape, which is often considered as gamma diversity. Genetics Genetics, study of the function and behavior of genes. It is branch of biology which deals with heredity and genetic variation. In agriculture, genetic advances enable scientists to alter a plant l to make it more useful. For instance, some food crops, such as oranges, potatoes, wheat, and rice, have been genetically altered to withstand insect pests, resulting in a higher crop yield. Tomatoes and apples have been modified so that they resist discoloration or bruising on their way to market, enhancing their appeal on supermarket shelves. Gene Splicing In gene splicing, DNA cannot be transferred directly from its original organism, known as the donor, to the recipient organism, known as the host. Instead, the donor DNA must be cut and pasted, or recombined, into a compatible fragment of DNA from a vector—an organism that can carry the donor DNA into the host. The host organism is often a rapidly multiplying microorganism such as a harmless bacterium, which serves as a factory where the recombined DNA can be duplicated in large quantities. The subsequently produced protein can then be removed from the host and used as a genetically engineered product in humans, other animals, plants, bacteria, or viruses. The donor DNA can be introduced directly into an organism by techniques such as injection through the cell walls of plants or into the fertilized egg of an animal. Plants and animals that develop from a cell into which new DNA has been introduced are called transgenic organisms. Transgenic Organism Transgenic Organism, plant, animal, or bacteria that have been genetically modified to contain a gene from a different species. The new gene is inherited by offspring in the same way as the organism's own genes. The transgenic condition is achieved by injecting the foreign gene into the fertilized egg or into embryonic cells. The injected gene becomes part o f the host cell's deoxyribonucleic acid (DNA) within the chromosome and is then inherited by all the cells produced during embryonic development. It is thus present in all the cells of the resulting adult organism and is inherited by all its descendants. GENETICALLY MODIFIED (GM) CROPS: A CONTROVERSIAL ISSUES The blueprints of heredity reside in the thread like chromosomes. Each chromosome contains several hundreds genes, strung together in a row. The gene, segment of DNA molecules, has a specific position or "locus", in the string. Genes decide all the properties and capabilities of an organism. In different parts of the plants different parts of the genes are active, giving rise to different structures like the leaves, the seeds and the root.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Genetic engineering (GE) is the process of artificially modifying these blueprints and producing new forms of life by splicing, joining and manipulating the DNA. This is the process toward creating alternative life forms, that use a genetic code different from the one used by all other creatures on earth. GM crops are the creation of this miraculous technology and contain alien genes, also called transgenes, from widely different kinds of plants or animals, from bacteria or viruses, or that were created synthetically in the laboratory. Potential benefits of GM crops GM technology has several benefits such as insect resistance, pathogen resistance, herbicide tolerance, delayed ripening, longer shelf life in the super market, drought resistance, ability to fix nitrogen, ability to grow faster and bigger, higher oil content and ability to manufacture pharmaceuticals, etc. Beneifits of this technology can be broadly classified as belows. 1. Contribute in poverty reduction Every body knows that production should be increased directly or indirectly to get out of poverty and production needs to be in the hands of the poor for the benefits of the poor. GM technology is the only technology that can provide miracle genotype in the hands of poor and helps to maintain food production that will continue to match population growth.This technology can feeds more people with better food and bridge in poverty reduction by boosting production per unit area per unit time. 2. Reduce production cost Advocates of GE claims that it will lower pesticide usage and can bring higher yields and profitability to many farmers. Example, Use of herbicide and broad- spectrum herbicides resistance transgenic plants reduces the production cost for weed management under specified management regimes so that one can get higher yields with fewer inputs. 3. Reduce pesticide use Green revolution increases the markets of pesticides and fertilizers that polluted the environment and degrade cultivated land. Manipulation of foreign resistant gene against the particular pest reduces the chance of pesticide use that lower the production cost and helps to maintain pollution free environment. 4. Deliver the quality product Introduction of novel gene into the new host would maximize the production efficiency and increase the quality of end products. Example, ability to fix nitrogen in non leguminous crop, ability to grow faster and bigger in any crop, higher oil content in oil seed crop and longer shelf life in many vegetable crop would deliver the higher quality of products. Potential risk of GM crops Living organisms are highly complex; genetic engineers cannot possibly predict all of the effects of introducing new genes into them. This is because the introduced gene may act differently when working within its new host, the original genetic intelligence of the host will be disrupted; the new combination of the host genes and the introduced gene will be established that may have unpredictable effects on health, environment and socio-political circumstances. 1. Allergic reaction
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Insertion of a new gene into any organism, there would be "position effects" which can lead to unpredictable changes in the pattern of gene expression and genetic function. The protein product of the inserted gene may carry out unexpected reactions and produce potentially toxic products. The process of genetic engineering can thus introduce dangerous new allergens and toxins into foods that were previously naturally safe. Health-damaging effects caused by genetic engineering will continue forever. Unlike chemical or nuclear contamination, genetic pollution is self-perpetuating. It can never be reversed or cleaned up; genetic mistakes will be passed on to all future generations of a species. 2. Genetic contamination Crop diversity is essential to the future of our agricultural system. Conservation of crop diversity is also a means of conserving elements of cultural diversity. There is potential for GM crops to hybridize with wild relatives and forms viable offspring. Crop to wild gene flow lead to extinction of rare species through the process of swamping and outbreeding depression and this genetic contamination will exerts negative long term consequences for diversity conservation. 3. Ecological imbalance Crops are now being engineered to produce their own pesticides. Contamination of plant gene through Bacillus thurengensis (Bt) gene produces a toxic pesticide that will harm to non -target organisms also. This will promote rapid appearance of resistant insects and lead to excessive destruction of useful organisms, thus creates serious ecological imbalance. 4. Horizontal gene transfer GM crop that are likely to survive in the wild, spread their genes through gene flow and finally contaminate the natural gene pool. This movement of genes between different species across natural boundaries facilitates unnatural gene transfers between unrelated species resulting into development of "super weeds" and displaced existing species from the ecosystem with disastrous effects. In addition to movement within genomes, transgenes may move from the host organism into the DNA of other organisms resulting into horizontal gene transfer. It will connect with the emergence of more virulent or new pathogens. 5. Power shift in agriculture GE is unique in the history of agriculture and is fully controlled by private companies. Transnational corporations (TNCs) produce agrochemicals, carryout the laboratory research, field trials, produce and sales of GM crops. They spent enormous amount of money on develo ping herbicide resistant crops that are being sold to farmers as a package inclusive of both the herbicide and the seeds. It appears that GE technologies are not being developed because of their problem solving capacity, but because of the patent and thus profit it can bring to the companies. Increasing use of GM in major crops has caused a power shift in agriculture towards biotechnology companies gaining excessive control over the production chain of crops and food, and over the farmers that use their products, as well. Gene: Functionally, gene is the unit of inheritance. Structurally, gene is a segment of DNA which codes for one polypeptide, ribosomal or transfer RNA. Gene, basic unit of heredity found in the cells of all living organisms. Genes determine the physical characteristics that an organism inherits, such as the shape of a tree‘s leaf, and the color of a flower. Genes are composed of
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. segments of deoxyribonucleic acid (DNA), a molecule that forms the long, threadlike structures called chromosomes. The information encoded within the DNA structure of a gene directs the manufacture of proteins, molecular workhorses that carry out all life-supporting activities within a cell. Genome: The entire complement of genetic material (genes + non coding sequences) present in each cell of an organism or in a virus or organelle. Important date in Nepal: 15th Asar:- National Rice Day 8th May:- NARC Day 23rd December: Kisan Day 5th June: World Enviroment Day 16th October:- World Food Day Chromosome number of crops: (2n) Rice:- 24 Bread wheat: 42 Maize:-20 Tobacco / Potato:-48 Pea:-14 Pigeon Pea / Rajma: 22 Chick pea: 16 HYBRID MAIZE TECHNOLOGY What is hybrid ? Hybrid is first generation cross between two or more inbred lines. Hybrids are not qualified as varieties since they are not genetically stable i.e., they lose hybrid vigor if they are replanted, and because of segregation and uncontrolled crossing, establish new genetic combinations. The term hybrid is used in a broader sense to mean F1 resulting from the cross of at least two progenitors. The progenitors could be inbreds, partial inbreds, non-inbreds or combination of these. Hybrid varieties are first-generation offspring of a cross between parents with contrasting characters. Gene recombination occurs only as a result of sexual reproduction. In cross-pollinated crops selfpollination leads to reduction in vigor and fertility. Hybrid maize technology has evolved and passed through different stages over the past 100 years or so. A lot has been done i n theoretical ideas and concepts related to various aspects of hybrid development. This technology has made the significant impact in bringing about hybrid revolution and dramatically changing maize productivity in many countries of the world. Generally, potentially important heterotic maize populations have a better chance of forming high yielding hybrids. Source germplasm should be improved for hybrid-oriented features so that
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. probability for extracting superior lines from such populations can be increased. There should be complete records on combing ability and heterotic patterns as well as information on their strength and weakness. The parental lines should be improved for seed yield, seed quality, less prone to environmental stresses, pollen abundance and longer pollen spread in male parents. Emphasis on stresses particularly high density and abiotic stresses also facilitate improving poor inbred hybrid correlations. There should be better integration between population and hybrid research activities based on the size and diversity of activities. The performance of inbreds is important not only to superior hybrid performance but also to economics of hybrid seed production which is extremely important as one switches to two -parent hybrid maize technology where seed yields of the female inbred plays an important part. In general, hybrid-oriented pedigree maize populations are used for the extraction of maize inbred lines. Efforts in inbred line development around the world have been numerous and there is a general consensus that the frequency of obtaining usable lines is low. It is estimated that one in 10,000 inbreds show commercial usage in hybrid seed production. The problems commonly faced by breeders are weak inbreds, low productivity levels, reproductive abnormalities, a wide gap in anthesis-silking interval (ASI), poor seed set, difficulties in early generation lines and sometimes poor pollen shed. Hybrid formation among such inbreds exhibit a different set of problems such as nicking, need for staggered plantings, detasseling problems, environmental susceptibility to heat and high temperature, and sometimes to specific environmental constraints and thus affecting seed set adversely. Generally, unimproved types of germplasm deteriorate in performance rather easily as compared to the improved germplasm. The germplasm improved for breeding behavior through recurrent selection programs will perform better for inbreeding stresses and will result in producing a higher frequency of surviving lines. The net result of inbreeding stress is reduced performance in general, lowers productivity, reduced vigor, delayed silking, exposure recessive and deleterious alleles, increased ASI, and reproductive abnormalities in both male and female parents. The drastic fo rm of inbreeding is achieved by selfing, moderate by sibbing plant to plant, and mid by bulk sibbing. Normally most breeders use selfing but because of severe effect of inbreeding depression some breeders interrupt selfing by sibbing to prevent rapid fixation of genes and to maintain vigor. Inbreeding minimum refers to the average performance of random homozygous lines from the population. In most outbreeding species, inbreeding minimum is below 50% of the population mean. Inbreeding minimum is arrived when on the average 50% of both favorable and unfavorable genes have become homozygous. Some practices may be unhealthy or undesirable in inbred line development as is the case use in cutting the silks a day before pollination. In doing so one is forcing the silks to be pollinated. What is hybrid vigor or heterosis ? The term heterosis, also known as hybrid vigor or outbreeding enhancement, describes the increased strength of different characteristics in hybrids; the possibility to obtain a genetically superior individual by combining the virtues of its parents.. Heterosis is superiority of an F1 hybrid over both its parents in terms of yield or some other character. Heterosis can be classified as:
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. a. b. Mutational heterosis: it results from dominance gene action Balanced heterosis: it results from over dominance gene action. Manifestation of heterosis: a) increased yield b) increased reproductive ability c) increase in size and general vigour d) better quality e) earlier flowering and maturity f) greater resistance to disease and pests g) greater adaptability h) faster growth rate i) increase in number of a plant part. Hybrid vigor is the increase in size or vigor of a hybrid over the average or mean of its parents, the latter being referred to as the midparent value (mid-parent heterosis). Heterosis was proposed by Shull to denote the stimulation in size and vigor in a hybrid as an expression of heterozy gosis. The two terms hybrid vigor and heterosis, are synonymous and are used interchangeably. In practical usage, hybrid vigor is the increase in size and vigor of a hybrid over the best parent (high-parent heterosis). Most often we look for increased vegetative growth or grain yield; but hybrid vigor may be reflected in cell size, plant height, leaf size, root development, ear size, prolific character, grain number, seed size and others. The hybrid plant is heterozygous at many loci, but uniformity is attained, as in an inbred line. The first corn inbreds were weak and unproductive, and the hybrid seeds produced in inbred plants were small, often irregular in shape, and difficult to plant uniformity with farm equipments. There is a general tendency to use potentially important maize populations that are heterotic to each other that have a greater chance of forming high yielding superior hybrids. Source germplasm should be improved for hybrid-oriented features so that probability of extracting superior lines from such populations can be increased. It may be pointed out that diversity is important in the breeding program, it is equally important in the farmers fields. In recent years, yield increases have occurred because of the use of hybrids, increased us e of fertilizers, better weed control, higher plant densities and improved management (improved cultivars and better field husbandry). Genetic basis of heterosis is still not fully understood. Shull (1909) proposed the first theory designated as physiological stimulates or heterozygosity hypothesis. Bruce (1910) attributed heterosis to dominant favorable growth factors (dominance or the dominance of linked gene hypothesis) .Two explanations are generally offered to explain the phenomenon of hybrid vigor, but neither appears to be adequate to cover all cases. The most widely accepted explanation is based on assumption that the hybrid vigor results from bringing together favorable dominant genes. According to this theory, genes that are for vigor and growth are dominant, and genes that are harmful to individual are recessive. The dominant genes contributed by one parent may complement the dominant genes contributed by the other parent, so that the F 1 will have a more favorable combination of dominant genes than either parent. For example, let us assume that the dominant genes ABCDE are favorable for good yields, that inbred A has the genotype
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. AAbbCCddEE (ACE dominant), and inbred B has the genotype aaBBccDDee (BD dominant). Genotypes of the inbreds A and B has the genotype of the F1 hybrid, are as follows: Inbred A X Inbred B AAbbCCddEE aaBBccDDee F1 hybrid AaBbCcDdEe In this example, The F1 hybrid contains dominant favorable genes at all five loci. Hull (1945) proposed overdominance to be case of heterosis suggesting that Aa is better than AA or aa. This hypothesis was later supported by crow (1948). He also termed it as superdominance. Thus genetic mechanism (s) causing heterosis is still far from completely understood. What is inbreeding ? The result of inbreeding in a heterozygous population is to increase homozygosity. In fact, inbreeding is often defined as any system of mating that leads to an increase in homozygosity. Inbreeding occurs by mating individuals that are related by ancestry. The most rapid approach to homozygosity is through self-pollination. Heterozygosity in a population is reduced by one-half with each successive self-fertilization cycle. With self-fertilization, heterozygous alleles (Aa) will segregate into the genotypes 1AA:2Aa:1aa; homozygous alleles (AA and aa) will remain homozygous. With an increase in homozygosity there is a change in genotype frequency although gene frequency remains unchanged. Sib matings are another approach to homozygosity, but homozygosity will be reached more slowly than with self-fertilization. Ten generations of full-sib matings are normally required to reach the same level of homozygosity as three generations of self-fertilization. The maize breeder practices several generations of self-fertilization and pedigree selection to obtain inbred lines that are uniform in plant and seed characteristics. The consequence of inbreeding is the loss in size and vigor that is manifested as heterozygosity decreases. The largest decrease in vigor occurs following the first generation of inbreeding and levels off as homozygosity is approached. One factor that contributes to the loss in vigor is an increase in the frequency and homozygosity of recessive genes. Many of the recessive genes uncovered have alleles with deleterious effects. The decline in vigor with inbreeding is known as inbreeding depression. Inbreeding depression commonly occurs following increases in homozygosity in cross pollinated species. Inbred Lines In developing inbred lines, the breeder starts by selfing heterozygous plants. The original selfed plant is referred to as the S0 or I0 plant and the selfed progeny from this plant as the S1 or I1 (firstgeneration selfed) progeny. The second-generation selfed progeny is called the S2 or I2 , and so on. The source of the inbreds may be an open-pollinated variety, single, three-way, double-cross hybrids, a composite, a synthetic variety or a population improved through recurrent selection or populations developed by other procedures. Segregation for plant and ear characters occurs in the progeny of selfed plants. Undesirable plants are discarded. Plants possessing desirable characters are used for further self-pollination. Self pollination and selection continue until homozygosity is reached and the inbreds are stable for morphological and physiological characteristics. Although vigor is lost during the early generations of inbreeding, the inbred lines approach homozygosity and become stabilized about the F7 or F8 generation; no further loss of vigor is experienced. Through the inbreeding process, many undesirable recessive genes are eliminated from the progenies. To self-pollinate the plant, silks of the original S0 or I0 plants are protected from foreign pollen by covering them with specially made shoot bags, and pollen is collected from the tassel
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. and distributed over the silks of the same plant. In the S 1 generation, superior plants are again chosen for self-pollination. In succeeding generations plants are chosen between and within lines. The purpose of inbreeding is to fix genes in a homozygous condition so that the genotype of a parent inbred line may be maintained without genetic change. With the inbreeding there is a loss of vigor and individual plants in the selfed progenies exhibit many faults such as reduction in plant height, tendency to tiller, lodging, increased susceptibility, and a wide assortment of undesirable traits. The undesirable plants are discarded and the most vigorous plants are retained and selfed in the succeeding generations. During inbreeding, undesirable recessive genes that had been masked by a dominant allele in a heterozygous population are eliminated as the weak and undesirable plants are discarded. Seed stocks of inbred lines of corn are maintained by controlled self-pollination.  The seed parent inbred must be vigorous and productive so that it will produce satisfactorily seed yields when used as a female parent in the production of a single -cross hybrid.  The pollen parent must produce abundant viable pollen and shed over a long period.  Both inbreds must contribute productivity to the hybrid combination in which they are used. How are inbred lines developed? 1. Standard ear- row/ Pedigree method: It is most commonly used method. Plants are selfed in a population and the selected ears are shelled separately and planted next season ear to row. Between and within row selection is practiced during all inbreeding generations. Pedigree records are kept during all inbreeding generations. Usually the number of progenies is reduced one-half during each inbreeding generation (500 S1 , 250 S2 , 125 S3 , 63 S4 , 32 S5 , and so on). The procedure permits between and within family selection at different stages of plant growth. Resampling of superior progenies is possible, if necessary. This procedure allows several manipulations to be practiced in the field such as row length, plant density, and other stresses. It is the best method but in terms of resources use it is perhaps a costly method. This procedure has two important problems: vigor of the lines is decreased with inbreeding because of loss of favorable dominant alleles and any heterozygous loci that have over-dominant effects. Many lines are so poor in seed yield, pollen production, or some other desired agronomic attribute that they cannot be used in a program to produce single-cross hybrid seed; effective selection within the row for plants that have desired agronomic traits becomes minimal in generations beyond S3 , frequently, phenotypic uniformity is evident by the S 2 generation. Once the locus becomes homozygous in a line no further selection for segregating types is possible. Another disadvantage is when inbreeding tolerance level of the germplasm is low. In the absence of early inbred evaluation, many sister lines may be evaluated for combining ability, thus wasting resources unnecessarily. 2. Bulk method: The method consists in planting source population as a bulk. The selected plants are selfed and at harvest approximately 500 ears are selected. A balanced bulk is made of all selected ears and planted in 100 or more rows consisting of 2500 or more plants depending on the density. The selected ears are selfed the selected ears used to form the bulk. One can continue inbreeding generations as selfed bulk as desired after which each fa mily is shelled separately to develop a family structure. It is simple, requires no record keeping, easy field execution, and permits handling more germplasm volume and permits sampling more
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. plants initially. This method is particularly suitable for germpl asm with drastic inbreeding effects having low probability of extracting superior inbred lines. High density and other manipulations are possible during the inbreeding process. Another merit of this method is that switch-over to pedigree method is possible at any breeding generation. Selfed progeny evaluation for each progeny is not possible as well as selection between rows. Resampling of lines is not possible and that selection for complex low heritability traits is not effective. Also because of bulk handling, recycling among early generation lines is not possible. 3. Single hill method: The method consists in shelling S1 ears separately. A maximum of 3-5 seeds per hill are planted to represent an ear. From here one can choose one of the two options. Either identify better hills and self pollinate or identify better hills and plant long rows next season using remnant seeds. If the first option is chosen, the procedure permits very little selection between plants within the hill. The essential features of the procedure permit sampling a very large number of plants. It is resource efficient, and stress manipulations can be exercised. There is only a little opportunity for selecting sister or sub lines. The method makes very little use within family variation until and unless remnant seed is used to plant long rows. A limited number of plants can be planted in each hill. Variation in height among hills may effect expression of some hills. The method may have some use but is not practiced in general. 4.Combination pedigree/bulk: This method is a compromise between two methods pedigree and bulk. It thus harnesses benefits of both the procedures. One can follow pedigree method in the early generations followed by bulk in the selfed progenies in the subsequent generations. Alternatively one can handle by bulk method in the initial generations and then later on switching-over to the pedigree method. A combination of the two methods has several benefits including reduction in volume of work; minimal sister lines, reduce d combining ability evaluation work, and reduced efforts in keeping pedigree records and less space needed in cold storage. This method has all the disadvantages of the bulk method by not permitting between row selection and restricted resampling of good lines. 5. Selfed progeny bulk method: Selfed progeny method introduces and combines good features of both the pedigree and the bulk method. An attempt is made to bring the source population in selfed progeny structure as early as possible. Source population i s planted as a bulk making 1000 or more selfs. Select 500, shell individually, and then plant ear to row. Here, within row or within family selected ears are shelled as a bulk and not as ear to row. The remaining steps can be continued as outlined in the pedigree method. The procedure has many merits including reduced work load in the field, selection sister lines not possible, and most benefits of pedigree method. 6. Single seed descent: Each ear is shelled individually. In the following steps only one seed from each ear is advanced in each inbreeding generation. Also selection/rejection in each inbreeding generation is based on single plant performance. The method has a merit in that it permits sampling large number of plants. The selection of sister line is not possible. In terms of resource use it is highly efficient and that resampling is possible. The greatest drawback is that within the line genetic variation cannot be exploited and also not suitable when inbreeding depression is moderate to severe.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Inbred recycling: Inbred recycling involves inbred development by selfing in pedigree populations. The populations could be F2 or backcross and may involve two or more progenitors. The method has been used extensively in the developed world but in developing world the systematic efforts are lacking and it is only in recent years that breeders in the developing world have become conscious and understand merits of this procedure. Through systematic selection of progenitors and formation of populations one can greatly increase the frequency of extracting superior lines from such populations. There is also a system of inbred line development which is based on an evaluation for hybrid performance of the S0 plants or S1 lines. Genotyped that are identified for above-average hybrid performance in these tests are continued in the selfing and selection nursery. The procedure has been called "early testing," and the assumption is that the combining ability of a line is determined early in its development and will change relatively little in subsequent generations of inbreeding and selection. As part of population improvement efforts (Inbred line development from on-going population improvement): Population improvement and hybrid research are so important that each one can benefit from the other. The integration may involve several aspects which facilitate hybrid development and permits improvements of populations using inbred hybrid concepts. The integration may involve such areas as inbreeding, crossbred performance and agronomic traits that are considered important. If at least inbreeding and combining ability concepts are used in recurrent selection programs, one can select early generations lines for further inbreeding that are good combiners. Also a few top performing early generation lines could be used to develop pedigree populations for further inbreeding and for the extraction of lines. CIMMYT initiated its hybrid maize research program in 1985. A number of initiatives were started on modest scale to include such activities as interpopulation improvement, emphasis on hybrid-oriented source germplasm, and research on non-conventional maize hybrids. Mild inbreeding involving one or two generations of selfing was introduced in population improvement as a part of intra-family improvement. Based on heterotic pattern information, new heterotic groups were formed. The evolutionary process in hybrid development requires shift from non-conventional to conventional hybrids of double, three-way and single cross types. Theoretically one would expect high level of performance of single cross hybrids but this would require the development of productive and vigorous inbred lines that would make commercial seed production economically viable. Testing of inbreds  The general combining ability (GCA) of the inbreds is tested by making all possible crosses [n (n - 1)/2] in a diallel fashion. Types of testers  Open-pollinated varieties  Inbred lines (homozygous recessive at all loci)  Established inbred testers Time of testing 1.Late testing: In about fifth generation of selfing (S5 lines). 2.Early testing (S1 lines): Early testing was first proposed by Jenkins in 1935. Now a day it is more common.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. Inbred - hybrid performance and correlation Several studies have shown that correlation of an inbred trait with the same trait in hybrid is relatively high, except for grain yield. Greater relationship may occur when materials are grown in stress environments, such as high plant densities and low nitrogen level. Therefore high density and stress during inbreeding and evaluation can aid to improve the correlation for a complex trait - as yield (Russell, 1992). A need for good per se performing lines is highly essential for producing F1 seed economically. Recycling among lines with good per se and high hybrid performance should help to improve somewhat the correlation between inbred and hybrid for yield. Inbreeding generation and combining ability The debate on early versus late testing for combining ability has been going on all the time. Proponents of early testing (Jenkins, Lonnquist, Cowan, Green and Hays) support the idea that yield potential of inbred lines can be determined in early generations and that inbreeding can be carried out in superior families/lines. The proponents of late testing (Richey, 1945) on the other hand argued that combining ability of inbred lines actually changed markedly in relative yielding ability as indicated by topcrosses from one generation to the next. There is, however, consensus on early testing and the view is widely supported and accepted by most breeders. Improvement in inbreeding behaviors The frequency of good lines from any population is very low. Bank accessions and landraces which have not been subjected to inbreeding process suffer badly from inbree ding depression. Therefore, in recent years most breeders are switching over to selfed progeny selection method. This should aid in improving tolerance to inbreeding and increase the frequency of extracting superior lines from such populations. Integrating population improvement and hybrid research Two maize breeding approaches of population improvement and hybrid research cannot be isolated from each other. Each approach can help the other in more than one way. More importantly, the integration of two approaches is needed to efficient use of resources. As hybrid efforts are strengthened there is a tendency to switch over to schemes that will directly contribute to hybrid development efforts. Several national programs are also switching from intrapopulation to inter-population improvement scheme. In recent years, CIMMYT has increased its emphasis on inter-population schemes. Aspects of general importance in hybrid development Choice of germplasm - Good genetic base, high yield potential, desirable plant characteristics, resistance to the major pests and diseases in the prevalent area, good plant type and superior stalk quality Combining ability information - Source material should exhibit good general combining ability (GCA) with higher chances of extracting superior general combining lines Tolerance to inbreeding Heterotic response - To increase efficiency and to facilitate hybrid development source populations should be heterotic to each other exhibiting high level of cross -performance. Ability to produce high frequency of linesAvailability of tester parents - The tester could play several important roles including evaluating combining ability of germplasm, sorting out heterotic patterns, use in inter -population improvement programs, formation and improvement of new heterotic groups, and also could be used as fixed parent(s) to identify rapidly conventional and non-conventional hybrid
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. combinations. As a general rule, inbred testers would be used to identify single crosses, single crosses to identify three-way crosses and non-inbred progenitors to identify non-conventional hybrids. Types of hybrids to be produced Intra versus Inter population hybrids - Generally hybrids are developed using heterotic populations. Possibilities of developing intra-population inter-line hybrids should not be ignored if the source germplasm is extremely heterogeneous and is genetically broad based. Such a situation could exist in gene pools, germplasm complexes and populations which have been developed using several genetically different components. Population improvement vs. hybrid development - Both population improvement and hybrid development are complementary in many respects. One can save valuable time and program resources if breeding strategies are carefully chosen that will permit simultaneous development of hybrids and open-pollinated products. Types of hybrid 1. Non-conventional: Non-conventional maize hybrids involve the use of at least one or more parents which are non-inbred progenitors. a. Inter-varietal hybrids: Varietal hybrids produced better than the higher-yielding parent but the procedure was not accepted widely for commercial use. b. Topcross hybrids c. Inbred line X Variety d. Inbred line X experimental variety e. Inbred line X synthetic variety f. Inbred line X family g. family hybrids: From same or different populations h. Double top cross hybrid: It is the progeny of a single cross and a variety (non-inbred progenitor) i. inter-population hybrids Merits:  Identification of hybrid combinations in the shortest possible time  reduced seed cost and thereby affordable to the farmers  Less vulnerability against epidemics due to the in-built genetic variability in the material and the increased yield compared to open-pollinated varieties. Demerits:  Difficult in maintaining parents that are somewhat variable  Less uniform  Increased roguing during seed increase  Less yield disadvantage in comparison to conventional hybrids. Conventional: The conventional hybrid maize types involve only the use of inbred parents.  Single-cross: It is the product of a cross between two unrelated inbred lines. The inbred lines are chosen based on the basis of combining ability tests to produce a vigorous and productive hybrid progeny. Since the parent inbred lines are homozygous at all loci, being comparable to a pure line in this respect, single-cross plants with common parentage will be identical in genotype and uniform in appearance, although each plant will be highly heterozygous. The single -cross made from inbreds A and B is written A X B, where A is the seed parent and B the pollen parent.  Modified single-cross: A modified single cross is a hybrid progeny from a three-way cross, which utilizes the progeny from two related inbreds as the seed parent and the unrelated inbred as the pollen parent e.g. (A X A') X B.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal.  Double modified single-cross: It is the hybrid progeny from two single crosses, each developed by crossing two related inbreds e.g. (A X A') X (B X B').  Three-way cross: A three-way cross is the hybrid progeny from a cross between a single cross and an inbred line. The three-way cross differs from the modified single cross in that all three inbreds are unrelated, and it will be more diverse genetically and less uniform in appearance e.g. (A x B) X C.  Modified three-way cross: It is the progeny of a single cross as female parent and other single cross between two related inbreds e.g. (A X B) X (C X C').  Double-cross: The double-cross hybrid is the product of a cross between two single-cross hybrids. The double cross involves four unrelated inbreds. The double -cross model for hybrid corn was used from its inception by Jones in 1918 until the late 1960s. Since then it has been replaced by single and three-way crosses. For making double-cross hybrids, the single-cross parents are chosen on the basis of combining ability tests. The seeds from double-cross hybrid are uniform in size and reduce the cost of hybrid seed production, as the seed is from single -cross hybrid e.g. (A X B) X (C X D). It may be pointed out that multi-parent maize technology is quite complex requiring at least 3 or more progenitors, more steps in hybrid seed production. Quite problematic even if one of the parents is poor, more difficult to meet targets, and moreover from breeding stand point it is resource ineffective and inefficient. Theoreti cally, double crosses are expected to be more stable than single crosses over a series of environments because they are genetically heterogeneous.  Modified double cross: Parents of double crosses can also be modified as in single and three-way crosses. Number of different crosses from n number of inbreds The number of single, three-way, and double-cross combinations that can be made from n number of inbreds may be calculated using the following formulae: 1. Single crosses: n(n - 1)/2 2. Three-way crosses: n(n - 1) (n - 2)/2 3. Double crosses: n(n - 1) (n - 2) (n - 3)/8 Where n = Number of inbreds Role of plant breeder in context of global climate change? Climate change is now unequivocal. Rising temperatures, changes in rainfall, erratic weather patterns and the prevalence of pests and diseases resulting from climate change have an adverse effect on food production and food quality with the poorest farmers and the poorest countries most at risk. In addition, climate change is also expected to cause losses of biodive rsity, mainly in more marginal environments. Plant breeding is the art and science of genetically improving plants for the benefit of humankind. Plant breeding plays a particularly important role in developing varieties that are ideally adapted to changing environmental conditions.plant breeding has been developing varieties for heat, drought and flood stresses. Plant breeders have focused on identifying crops which will ensure crops perform under these conditions; a way to achieve this is finding strains of the crop that is resistance to drought conditions with low nitrogen. Plant breeder are trying to address the climate change in two ways;
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. a) environmentally friendly varieties: Improved varieties resistant to pests require fewer pesticides. b) High-yielding varieties: Increase food production per unit area and alleviate pressure to add more arable land to production systems Strategies of adaptation to climate changes that the plant breer should address may include a more accurate matching of phenology to moisture availability using photoperiod-temperature response, increased access to a suite of varieties with different duration to escape or avoid predictable occurrences of stress at critical periods in crop life cycles, improved water use efficiency and a re-emphasis on population breeding in the form of evolutionary participatory plant breeding to provide a buffer against increasing unpredictability. It is evident from this that plant breeding is vital for future agriculture to survive as it enables farmers to produce stress resistant crops hence improving food security.
    • Shrestha, J. 2014. Plant Breeding and Genetics. Nepal Agricultural research Council, National Maize Research Program, Rampur, Chitwan, Nepal. PLANT BREEDING AND GENETICS Compiled and edited by: Jiban Shrestha Nepal Agricultural Research Council National Maize Research Program, Rampur, Chitwan, Nepal January, 2014