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Introduction of plant biotechnology
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Introduction of plant biotechnology



1. What is Plant Biotechnology? ...

1. What is Plant Biotechnology?
2. Broad Categories of Biotechnology
3. Characteristics of Biotechnology
4. Relation of Biotechnology with other Branches of Sciences
5. Plant Tissue Culture

6. Organogenesis
7. Introduction Single Cell Culture
8. Introduction to Callus Culture
9. Suspension Culture and Its Principle
10. Introduction to Somatic Embryogenesis
11. Shoot-Tip and Meristem Culture
12. Micro Propagation
13. Anther, Pollen and Ovule Culture (Haploid Production)
14. Embryo Culture
15. Invitro Pollination
16. Meaning of Somaclonal Variation
17. Somatic Hybridization and Cybridization
18. Biopesticides
19. Cryopreservation
20. Secondary Metabolites
21. Types of Biofertilizers



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Introduction of plant biotechnology Introduction of plant biotechnology Document Transcript

  • Page1
  • Page2 ABOUT THE AUTHOR Dr HARI KRISHNA RAMA PRASAD. SARIPALLI M.Sc,M M M,M.A., M.Sc IT., M.Phil, DASM (IBAM), DMLT, RDBMS, Ph.D (BioTech). has been Asst. Prof. of Dept. of Biology and Biotechnology at College of Natural and Computational Sciences, Aksum University, Axum, Ethiopia, North East Africa. He has 14.7 years of experince in academics, administration and research in various institutes like K. L. University, St. Ann’s College for women, Southren Institute of Medical Sciences, Hindu College of Pharmacy, St. Joseph’s College of Nursing and St. Ann’s College of Nursing. He received his B.Sc degree in Chemistry, Botany, Zoology from Andhra Loyola College, Affiliated to ANU, India (where he learned fundamentals of microbiology with Prof. Madhavarao), M.Sc degree in Microbiology from Campus College of ANU in the year 1997. He obtained his research degree M.Phil in Botany and Microbiology from Campus College of ANU with Prof. Vijaya Lakshmi.M, and his Ph.D in Biotechnology from RPSC-MU,Patna with Prof. Madan Prasad and also guided by Prof. Vijaya Lakshmi.M. A part from life sciences, he has versatile academic degrees like Marketing Management, Medical Sociology, Information Technology, Medical Lab Technology, Alternative Medicine and Database Management System. Dr. H K R Prasad. Saripalli is recipient of the South Asian Foundation fellowship (2003), University rank in Microbiology programme(1997). While at St.Ann’s College, he has received the Best Teacher Award(selected by students and management). He is a co-author, with Prof. R.P.Singh, of the text Biological Chemistry and Microbiology; with Prof. T. Pullaiah, of the texts, Emerging trends in Biological Sciences(2009), Recent Trends in Plant Sciences (2005); and other texts, Diversity of Microbes and Cryptogams and Gymnosperms, plant anatomy, ecology and biotechnology. He was elected to the Board of studies, Industrial microbiology, JMJ College (ANU). He is a founder and a member of the Scientific Advisory Board of Association of Global Science Innovations (AGSI). He published thirty research papers in both national and international peer reviewed journals.
  • Page3 Introduction of Plant Biotechnology Contents 1. What is Plant Biotechnology? 2. Broad Categories of Biotechnology 3. Characteristics of Biotechnology 4. Relation of Biotechnology with other Branches of Sciences 5. Plant Tissue Culture a. Brief History of Plant Tissue Culture b. Culture Technique in Plant Tissue Culture c. Totipotency d. Classification of Plant Tissue Culture Technique 6. Organogenesis a. General Account of Organogenesis b. Protocol for Organogenesis in Tobacco Callus c. Role of Growth Regulators in Organogenesis d. Factor Affecting the Organogenesis 7. Introduction Single Cell Culture a. Methods of Single Cell Isolation b. Methods of Single Cell Culture c. Importance of Single Cell Culture 8. Introduction to Callus Culture a. Principles of Callus Culture b. Protocol of Callus Culture c. How the Callus Tissue is formed? d. Application of Callus Culture 9. Suspension Culture and Its Principle a. Protocol of Suspension Culture b. General Account of Suspension Cultures c. Types of Suspension Cultures d. Synchronization of Suspension Culture e. Importance of Suspension Cell Culture 10. Introduction to Somatic Embryogenesis a. Embryoid b. Principles of Somatic Embryogenesis c. Protocols for Inducing Somatic Embryogenesis in Culture d. Somatic Embryogenesis in Dicotyledonous and Monocotyledonous Culture e. Induction of Embryogenic Cell Suspension f. Factors Affecting the Embryogenesis g. Practical Application of Somatic Embryogenesis h. Importance of Artificial Seeds 11. Shoot-Tip and Meristem Culture
  • Page4 a. Application of Shoot-tip or Meristem Culture b. Method of Virus Elimination 12. Micro Propagation a. General Procedure of Microprogation b. Micropropagation Techniques c. Factors Affecting Shoot Multiplication d. Advantages and Limitations of Micropropagation 13. Anther, Pollen and Ovule Culture (Haploid Production) a. Androgenesis b. Principle of Development of Androgenic Haploids c. Pathway of Microscope Division in Androgenesis d. Protocol for Anther Culture e. Protocol for Pollen Culture f. Advantages of Pollen Culture over Anther Culture g. Production of Homozygous Diploid Plants h. Application of Haploids in Plant Breeding i. Importance and Implications of Anther and Pollen Culture j. Ovule Culture – Meaning, Principle and Protocol k. Importance of Ovule Culture 14. Embryo Culture a. Types of Embryo Culture b. Technique of Embryo Culture c. Protocol for Embryo Culture d. Application of Embryo Culture 15. Invitro Pollination 16. Meaning of Somaclonal Variation a. Mechanisms Causing Somaclonal Variation b. Molecular Basis of Somaclonal Variation c. Isolation of Somaclonal Variants d. Application of Somaclonal Variation 17. Somatic Hybridization and Cybridization a. Methods of Protoplasts Fusion b. Selection of Somatic Hybrids and Cybrids c. Practical Applications of Somatic Hybridization and Cybridization d. Limitation of Somatic Hybridization 18. Biopesticides a. Advantages and Disadvantages of Biopesticides 19. Cryopreservation 20. Secondary Metabolites a. Advantages of Plant Cell Culture for Production of Secondary Metabolites 21. Types of Biofertilizers a. Large Scale Production of Biofertilizers b. Large Scale Production of Blue Green Algae c. Biofertilizers – Introduction, Meaning and Concept
  • Page5 1. What is Plant Biotechnology? The origin of Biotechnology can be traced back to prehistoric times, when microorganisms were already used for processes like fermentation. In 1920’s Clostridium acetobutylicum was used by Chaim Weizman for converting starch into butanol and acetone, latter was an essential component of explosive during World War- II. This raised hopes for commercial production of useful chemicals through biological processes, and may be considered as the first rediscovery of biotechnology in the present century. Similarly, during World War-II (in 1940’s) , the production of penicillin (as an antibiotic discovered by Alexaner Flemming in 1929) on a large scale from cultures of Penicillium notatum marked the second rediscovery of biotechnology. The third rediscovery of biotechnology is its recent reincarnation in the form of recombinant –DNA technology, which led to the development of a variety of gene technologies and is thus considered to be greatest scientific revolution of this century. Biotechnologies, as world indicate, is the product of interaction between the science and technology. Definition of Plant Biotechnology: 1. Biotechnology is the application of biological organisms, system or processes to manufacturing and service industries. 2. Biotechnology is the integrated use of biochemistry , microbiology and engineering science in order to achieve technological application of the capabilities of micro-organism, cultured tissue cells and part thereof. 3. Biotechnology is “a technology using biological phenomenon for copying and manufacturing various kinds of useful substances.” 4. Biotechnology is “the controlled use of biological agents such as micro-organisms or cellular components for beneficial use. (U.S National Science Foundation)
  • Page6 2. Broad Categories of Biotechnology The new biotechnology may be classified into the following four broad categories: 1. Techniques for cell and tissue culture likely to produce substantial impact on agriculture. 2. Technological development associated with fermentation processes, particularly those in the chemical sector which include the enzyme immobilization technique. These techniques are already creating some impact in several industrial branches. E.g. Production of enzymes and amino acids. 3. Techniques that apply microbiology for the screening, election and cultivation of cells and micro-organisms. 4. Techniques for the manupulation and transfer of genetic material. 3. Characteristics of Biotechnology Any technological revolution usually has the following five characteristics: 1. A drastic reduction in the cost of several products and services. 2. A dramatic improvements in the technical properties of processes and products. 3. Social and political acceptability in the sense that innovation is socially accepted but it involves modification in the legislative and regulatory patterns of society and some changes in management and labour attitude. 4. Environment acceptability. 5. Pervasive effects brought the economic system. Recent advances in biotechnology have been exploited in a variety of ways both for production of industrial, important biochemical and for basic studies in molecular biology.
  • Page7 4. Relation of Biotechnology with other Branches of Sciences Following are some of the field s where biotechnology innovations are playing important roles: 1. Tissue Culture Techniques in Biotechnology: An important aspect of all biotechnology processes is the culture of either the microorganism or plant or animal cells or tissues and organs in artificial media. While members in culture are used in recombinant DNA technology and in variety of industrial processes, plant cells and tissues are used for a variety of genetic manipulation. For example, another culture is used for haploid breeding, gametic and somatic cell or tissue cultures are used for tapping gametoclonal and Somaclonal variation or for production of artificial seeds. Transformation of protoplast in culture leads to production of useful transgenic plants. 2. Gene Technology as a Tool for Biotechnology: Most biotechnology companies make use of gene technology or genetic engineering which involves recombinant DNA and gene cloning. Most recently, extensive use of newly discovered polymerase chain reaction (PCR) has also been made for gene technology. 3. Hybridization and Monoclonal Antibodies in Biotechnology: Rapid progress has been made in hybridoma technique and monoclonal antibodies which is extremely used in human health care. Enzyme conjugated antibodies are being used for detection of viruses both in plants and animals using ELISA test. Immunotixins are being produced from gene fusion so that the toxic drugs meant for killing tumour cells may be carried to the target sites with the help of specific antibodies. 4. Biotechnology in Medicine: In the field of medicine, insulin and interferon synthesized by bacteria have already been released for use. A large number of vaccines for immunization against deadly diseases, DNA probes and monoclonal antibodies for diagnosis of various diseases, and human growth hormone and other pharmaceutical drugs for treatment of disease are being released. 5. Biotechnology and Protein Engineering: Protein engineering will lead to production of superior enzymes and storage proteins. Biochemistry has also provided us with remarkable in the form of immobilized enzymes system, which allowed the production of variety of substances. E.g. High- fructose corn syrup using an immobilized enzyme, glucose isomerase. 6. Biotechnology in Agriculture: Biotechnology has also revolutionized research activities in the area of agriculture which include following:
  • Page8 i) Plant cell, tissue and organ culture. ii) Genetic engineering leading to transformation followed by regeneration of plants to give transgenic plants carrying desirable traits like disease resistance, insect resistance and herbicide resistance. iii) Somatic hybrids between sexually incompatible species permitting transfer of desirable traits from wild or unrelated species to our crop plants. iv) Transgenic animals produced in mice, pigs, goats, chicken, cows, etc. It is suggested that some of these will eventually be used as bioreactor to produce drugs through their milk, blood or urine, this area has sometimes been described as molecular farming. 7. Biotechnology and Environment: Biotechnology methods have been devised for some environmental problems like i) Pollution control ii) depletion of natural resources for non-renewable energy. iii) Restoration of degraded lands and iv) biodiversity conservation. For instance, microbes are being developed to be used as bio pesticides, bio fertilizers. Biosensors etc and for recovery of metals, cleaning of spilled oils, etc.
  • Page9 5. Plant Tissue Culture The term “Plant tissue culture” broadly refers to the in vitro cultivation of plant parts under aseptic conditions. Such parts as meristems, apices, axillary buds. Young inflorescence, leaves, stems, and roots have been cultured. A controlled aseptic environment and suitable nutrient medium are the two chief requirements for successful tissue culture. These essential nutrients include inorganic salts, a carbon and energy source, vitamins and growth regulators. The basic technology can be divided into five classes, depending on the material being used: Callus, organ, meristem, and protoplast and cell culture. The technique of embryo, ovule, ovary, anther and microspore culture are used and can yield genotypes that cannot easily be produced by conventional methodology. a. Brief History of Plant Tissue Culture It was Gottlieb Haberland (1902) who in the first decade of this century pioneered the field of plant tissue culture. His idea was to achieve continued cell division in explanted tissue grown on nutrient medium. Following the discovery and use of auxins, the work of Gautherel, Nobecourt and White ushered in the second phase of plant tissue culture over 30 years ago. These and other workers determined the nutritional and hormonal requirements of the cultured plant tissues. It was observed that the whole plant could be successfully regenerated from undifferentiated tissues or even single cells in culture. Rapid advances in diverse aspect of plant culture have been made during the last few years and plant tissue culture techniques have been extensively applied to agriculture and industry. Condensed Chronology of Important Development in the Plant Tissue Culture: Year Worker Contribution 1902 C.Haberlant First attempt to culture isolated plant cells in vitro on artificial medium 1922 WJ Robbins and W. Kotte Culture of isolated roots ( for short periods) ( organ culture) 1934 P R White Demonstration of indefinite culture of tomato roots ( long period) 1939 R J Gautheret and P Nobecourt First long term plant tissue culture of callus, involving explants of cambail tissues isolated from carrot. 1939 P R White Callus culture of tobacco tumor tissues from intersepcific hybird of Nicotina glaucum X N.longsdorffi 1941 J Van Overbeek Discovery of nutritional value of liquid endosperm of coconut for culture of isolated carrot embryo. 1942 P R White and A C Experiments on crownn-gall and tumor formation in
  • Page10 Braun plants, growth of bacteria free crown-gall tissues. 1948 A Caplan and F C Stewart Use of coconut milk plus 2, 4-D fro proliferation of cultured carrot and potato tissues 1950 G Morel Culture of monocot tissues using coconut milk. 1953 W H Muir Inoculation of callus pieces in liquid medium can give a suspension of single cells amenable tosubculture. Development of technique for culture of single isolated cells. 1953 W Tulecke Haploid culture from pollen of gymnosperm ( Ginkgo) 1955 C O Miller, F Skeog and others Discovery of cytokinins. E.g. Kinetin, or potent cell division factor. 1955 E ball Culture of gymnosperm tissues ( Sequoia) 1957 F Skoog and C O Miller Hypotheses that shoot and root initiation in cultured callus is regulated by the proportion of auxins and cytokinins in the culture medium. 1960 E C Cocking Enzymatic isolation and culture of protoplast. 1960 G Morel Development of shoot apex culture technique. 1964 G Morel Use of modified shoot apex technique for orchid proportion. 1966 S G Guha and S C Maheshwari Cultured anthers and pollen and produce haploid embryos. 1974 J P Nitsch Culture of microspores of Datura and Nicotina, to double the chromosome number and to harvest seed from homozygous diploid plants just within five months. 1978 G Melchers Production of somatic hybrids from attached to plasmid vectors into naked plant protoplast. 1983 K A Barton , W J Brill and J H Dodds Bengochea Insertion of foreign genes attached to plasmid vectors into naked plant protoplast. 1983 M D Chilton Production of transformed tobacco plants following single cell transformation or gene insertion. b. Culture Technique in Plant Tissue Culture Plant cells are cultured in a suitable nutrient medium composed of inorganic salts, carbon source, vitamins, and growth regulators and organic supplements. In general , plant parts, tissue and cells can grow on media containing only the salts, of nitrogen and other essential elements, sucrose, certain amino acids, vitamins and growth factors. In several plants, the formation of shoot in vitro is promoted by higher levels of cytokinins relative to auxins while the reverses promote the root development. Some commonly employed cytokinins are kinetin and 6- benzylamino- purine and some commonly used and effective auxins are IBA , NAA and 2,4-D. These tend to induce rapid callus proliferation. Higher levels of 2,4-D strongly suppresses the oliogenesis. The most
  • Page11 commonly used culture media are Murahige and Skoog medium, Gamborg et. al medium and White’s medium. The procedure for establishing the culture is as follows. A 2-4 mm3 sterile segment excised from stem or root of the plant is placed on 30 ml of nutrient agar or liquid medium and incubated t 20- 30 0C in light. Within few days, cell proliferates and callus culture is obtained. In this, dividing cells form a layer of meristem and build a globular mass of non-dividing parenchyma. Alternaively, they may form small meristematic zones interspersed in non-meristematic regions, yielding a sort of nodulated callus. After 2-3 subcultures, small bits from soft callus can be cut and incubated into liquid medium where they give rise to suspension culture. Cell clones can be raised in the same manner as in case of micro-organisms, by plating a suspension of cells on agar plates. Colonies are formed, each representing a clone. They can be picked up individually and inoculated in liquid medium. c. Totipotency Capacity of higher organism cell to differentiate into entire organism, totipotent cell contains all genetic information necessary for complete development. When an explant from differentiated tissue is used for culture on a nutrient medium, the non- dividing quiescent cells first undergo certain changes to achieve a meristematic state. The phenomenon of the reversion of mature cells to the meristematic state leading to the formation of callus is called “dedifferentiation”. The component cells of the callus have the ability to form a whole plant a phenomenon described as “dedifferentiation”. These two phenomenons of dedifferentiation and “redifferentaition” are inherent in the capacity described as “cellular totipotency”, a property found only in plant cell and not in animal cells. In other words, while a differentiated plant cell retain its capacity to give rise to whole plant, an animal cell loses its capacity of regeneration after differentiation. Although, generally a callus phase is involved before the cell can undergo redifferentaition leading to regeneration of whole plant, but rarely, the dedifferentiated cells give rise to whole plant directly without an intermediate callus phase. d. Classification of Plant Tissue Culture Technique I) Embryo Culture: For embryo culture, embryos are excised from immature seeds, usually under a ‘hood’, which provides a clean aseptic and sterile area. Sometimes, the immature seeds are surface sterilized and soaked in water for few hours, before the embryos are excised. The excised embryos are directly transferred to a culture dish or culture tube containing synthetic nutrient medium. Entire operation is carried out in the ‘laminar flow cabinet’ and the culture plates or culture tubes with excised embryos are transferred to a culture room maintained at a suitable temperature, photoperiod and humidity. The frequency of excised embryos that gives rise to seedlings
  • Page12 generally varies greatly and medium may even have to be modified made for making Interspecific and Intergeneric crosses within the tribe Triticeae of the grass family. The hybrids raised through culture have been utilized for i) Phylogenetic studies and genome analysis. ii) Transfer of useful agronomic traits from wild genera to the cultivated crops and iii) to raise synthetic crops like triticale by producing amphiploids from the hybrids. Embryo culture has also been used for haploid production through distant hybridization followed by elimination of chromosomes of one of the parent in the hybrid embryos cultured as above. A popular example includes hybridization of barley and wheat with Hordeum bulbosum leading to the production of haploid barley and haploid wheat respectively. Haploid wheat plants have also been successfully obtained through culture of hybrid embryos from wheat X maize crosses. Application of Embryo Culture: i) Recovery of distant hybrids. ii) Recovery of haploid plants from Interspecific crosses. iii) Propagation of orchids. iv) Shortening the breeding cycle v) Overcoming dormancy. In addition ovule and ovary can also be cultured. II) Meristem Culture: In attempts to recovery pathogen free plants through tissue culture techniques, horticulturists and pathologists have designated the explants used for initiating cultures as ‘shoot –tip’, tip, meristem and meristem tip. The portion of the shoot lying distal to the youngest leaf primerdium and measuring up about 100 µm in diameter and 250 µm length is called the apical meristem. The apical meristem together with one to three young leaf primordia measuring 100-500 µm constitute the shoot apex. In most published works explants of larger size (100-1000 µm long) have been cultured to raise virus- free –plant. The explants of such a size should be infact referred to as shoot-tips. However, for purpose of virus or disease elimination the chances are better if cultures are initiated with shoot tip of smaller size comprising mostly meristematic cells. Therefore, the term ‘meristem’ or meristem-tip’ culture is preferred for in vitro culture of small shoot tips. The in vitro techniques used for culturing meristem tips are essentially the same as those used for aseptic culture of plant tissues. Meristem tips can be isolated from apices of the stems, tuber sprouts , leaf axils , sprouted bunds o cuttings or germinated seeds. Application of Meristem Culture: i) Vegetative propagation ii) Recovery of virus free stock. iii) Germplasm exchange iv) Germplasm conservation
  • Page13 III) Anther or Pollen Culture: Angiosperms are diploid the only haploid stage in their life cycle being represented by pollen grains. From immature pollen grains we can sometimes raise cultures that are haploid. These haploid plants have single completes set of chromosomes. Their phenotype remains unmasked by gene dominance effects. In china, several improved varieties of plants have been grown from pollen cultures. When pollen grains of angiosperm are cultured, they undergo repeated divisions. In Datura innoxia the pollen grains from cultured anther can form callus when grown on a media supplemented with yeast extract or casein hydrolysate. Similarly, when isolated anthers are grown on media containing coconut milk or kinetin, they form torpedo- shaped embryoids which in due course grow into small haploid plantlets. The usual approach in anther culture is that anthers of appropriate development stage are excised and cultured so that embryogenesis occurs. Alternatively pollen grains may be removed form the anther, and the isolated pollen is then cultured in liquid medium. Cultured anthers may take upto two months to develop into plantlets. Application: Pollen culture or anther culture is useful for production of haploid plants. Similarly, haploid plants are useful in plant breeding in variety of ways as follows: i) Releasing new varieties through F1 double haploid system. ii) Selection of mutants resistant to diseases. iii) Developing asexual lines of trees or perennial species. iv) Transfer of desired alien gene. v) Establishment of haploid and diploid cell lines of pollen plant. IV) Tissue and Cell Culture: Single cells can be isolated either from cultured tissues or from intact plant organs, the former being more convenient than the latter. When isolated from culture tissues, the latter is obtained by culturing an organised tissue into callus. The callus may be separated from explant and transferred to fresh medium to get more tissue. Pieces of undifferentiated calli are transferred to liquid medium, which is continuously agitated to obtain a suspension culture. Agitation of pieces break them into smaller clumps and single cells, and also maintains uniform distribution of cells clumps in the medium. It also allows gases exchange. Suspension cultures with single cells can also be obtained from impact plant organs either mechanically or enzymatically. Suspension cultures can be maintained in either of the following two forms i) Batch culture: are initiated as single cells in 100-250 ml flasks and are propagated by transferring regularly small aliquots of suspension to a fresh medium. ii) Continuous culture: are maintained in steady state for long periods by draining out the used medium and adding fresh medium, in this process either the cells separated from the drained medium are added back to suspension culture or addition of medium is accompanied by the harvest of an equal volume of suspension culture.
  • Page14 Application of Cell Culture: i) Mutant selection ii) Production of secondary metabolites or biochemical production. iii) Biotransformation iv) Clonal propagation v) Somaclonal variations
  • Page15 6. Organogenesis The main objective in plant cultures is to regenerate a plant or plant organ from the callus culture. The regeneration of plant or plant organs only taken place by the expression of cellular totipotancy of the callus tissues. Scattered areas of actively dividing cells, known as meristematic centres, develop as a result of differentiation and their further activity results in the production of root and shoot primordia. These processes can be controlled by adjusting the cytokinins: auxin ratio in culture medium. The production of adventitious roots and shoots from cells of tissue is called organogenesis. Definition of Organogenesis: “The development of adventitious organs or primordia from undifferentiated cell mass in tissue culture by the process of differentiation is called organogenesis. “The formation of roots, shoots or flower buds from the cells in culture in manner similar to adventitious root or shoot formation in cuttings is called organogenesis. Caulogenesis: Type of organogenesis by which only adventitious shoot bud initiation take place in the callus tissue. Rhizogenesis: Type of organogenesis by which only adventitious root formation takes place in the callus tissues. Organoids: In some culture tissues, an error occurs in development programming for organogenesis and an anomalous structure is formed. Such anomalous organs like structures are known as Organoids. Although Organoids contain the dermal, vascular and ground tissues present in plant organs, they differ from true organ in that the Organoids are formed directly from the periphery of the callus tissue and not from organised mersitemoids. Meristemoids: Meristemoid is localized group of meristematic cells that arise in callus tissue and may give rise to shoots and or roots. They are also termed as nodules or growth centres. Cytodiffrentiation: In plant tissue culture, during growth and maturation of callus tissue or free cells in suspension culture, few dedifferentiated cells undergo cytoquiescece and cytosenescence and this twin
  • Page16 phenomenon are mainly associated with redifferentaition of vascular tissues, particularly tracheary elements. The whole developmental process is termed as cytodifferetiation. a. General Account of Organogenesis In vitro organogenesis in the callus tissue derived from small piece of plant tissue, isolated cells, isolated protoplasts, microspores etc can be induced by transferring them to a suitable medium or a sequences of media that proliferation of shoot or root or both. The suitable medium is standarize by trial and error method. Organ formation generally follows cessation of unlimited proliferation. Individual cells or groups of cells of smaller dimensions may from small nests of tissue scattered throught the cells of smaller dimension may from small nests of tissue scattered throught the callus tissue, so called meristemoids which become transformed into cyclic nodules from which shoot bud or root primordia may differentiate. In most calli, initiation of shoots buds may procede Rhizogenesis or vice versa or the induced shoot bud may grow as rootless shoot. Shoot bud formation may decrease with age and subculture of the callus tissue, but the capacity of the rooting may persist for longer period. In some calli, rooting occurs more often than other form of organogenesis. During organogenesis, if the roots are first formed, then it is very difficult to induce shoot bud formation from the same callus tissue. But if the shoots are first formed, it may form root later on or may remain as rootless condition unless and until the shoots are transformed to another media or hormone less medium or condition that induce root formation. In certain cases, shoot and root formation may occur simultaneously. But the organic connection between two different organ primordia may or may not be established. Therefore, organic connection between soot and root primordia is essential for the regeneration of complete plantlet from the same culture. Shoot formation followed by rooting is the general characteristics of organogenesis. The callus tissue may cases shows a high potential for organogenesis when initiated but gradually a decline sets in as subculture proceeds with eventual loss of organogenic response. The loss of potential for organogenesis may be due to either a genetic or physiological change induced by either prolonged cultural conditions or the composition of the culture media. The effects in callus tissue are reflected in changes of chromosome structures or number such as anuploidy, polyploidy, cryptic chromosomal rearrangement etc. It is generally observed that shoot bud formation take place from the diploid cells of callus tissue. At early stage of culture, the callus tissue exhibits maximum number of diploid cell. According to physiological hypothesis, subculture often leads to loss of many endogenous factors or morphogens present at the critical stages of growth. Such factors present in the callus tissue at the initial stages may not be synthesized at all or synthesized only in insufficient quantities at later stages. as a result , callus tissue fails to exhibit the potential for organogenesis at later stages. as a result , callus tissue fails to exhibit the potential for organogenesis or embryogenesis. However, if these factors are supplemented to the medium during subculture, then restoration of organogenicpotential should be regained. Generally, high concentration of cytokinin brings about shoot bud initiation, whereas high levels of auxin favour rooting. Certain phenolic compounds peroxidise, and accumulation of higher amount of starch before shoot induction, and synthesis of enzymes of EMP pathway and pentose phosphate pathway, are playing important role in organogenesis.
  • Page17 b. Protocol for Organogenesis in Tobacco Callus This is an experiment in which mature tobacco stem is initiated to give rise to callus tissue. Under appropriate hormonal condition callus is induced to form either root or shoot primordia. The protocol is given below: 1. The upper part of the stem of 3-4 ft tall tobacco plants are harvested and cut into 2 cm long internodes segment. 2. Surface sterilization of tissue is done by immersing the stem pieces in 70% v/v ethanol for 30 seconds, followed by 15 minutes incubation in sodium hypochlorite. Then tissue is washed in several changes of sterile distilled water. 3. The stem explants are taken in a sterilized petri dish and cut longitudinally in two equal pieces and inoculated onto Murashige and Skoog’s solid medium supplemented with 2 mg indole acetic acid (IAA) and 0.2 mg kinetin. The cultures are then incubated at 25 0C with an illumination of about 2000 lux. 4. Organogenesis in callus culture can be stimulated by transferring tobacco callus onto MS medium with different auxin or cytokinins rations. Shoot primordia develop within three weeks of transfer of callus to MS medium with IAA at 0.02 mg and kinetin at 1 mg/ lambada. Root formation occurs within 2-3 weeks of transfer of callus to MS medium supplemented with 0.2 mg / lambada. 5. Callus tissue which is white or yellow in colour, begins to form in two weeks and after six weeks it should be sub cultured to fresh medium. 6. After 6 weeks, rootless shoots can be excised and placed onto the root inducing medium with 0.2 mg/lambada, IAA and 0.02 mg/ lambada kinetin. 7. It is possible to transplant tobacco plantlet to soil. It should be noted that aseptic procedure is not required for the transplantation of plantlets. The plantlets are removed from the culture vessels and are care should be taken not to damage root or shoot system. The plantlets are carefully washed with tap water to remove the residual agar medium. Individual plantlets are separated out and transplanted into pots containing seedling compost. The soil is watered. The pot is covered with small inverted polythene bag. This will reduce the amount of water lost by plantlets due to transpiration. After 7 days several small holes are made in polythene bags and gradually enlarged during next 2-3 weeks. At this stage, tobacco plantlets should be sufficiently “hardened off” to allow the complete removal of plastic bag. They can be grown to maturity in green house. c. Role of Growth Regulators in Organogenesis Of the many factors that influence organogenesis in vitro, the most important single factor seems to be the phytohormones. Skoog and Miller ( 1957) found that when the concentration of cytokinins are high relative to auxin, shoots are induced, when the concentrations of cytokinins
  • Page18 are low relative to auxin, roots are induced, and at intermediate concentration the tissues grow as unorganised callus. This basic concept has been used to regenerate a wide variety of dicotyledonous plants. In general monocotyledonous plants do not show a pronounced response to cytokinins and need high concentration of auxin such as 2,4-D to obtained changes in development of cultured tissue. Other plant hormones particularly abscisic acid and gibberellins have some dramatic action on in vitro organogenesis. Endogenous ethylene retards organ initiation during early stages of culture but in later stages it helps shoot initiation. Phytohormones are regarded as primary morphogens. According to this, hypothesis responding cells or group of cells are competent to react to the hormones but are not committed to a particular development fate. When the cells are treated with hormones, the cells start to move a specific development pathway. Alternatively, hormones the cells start to move a specific developmental pathway. Alternatively, hormone responsive cells are already determined and that hormones stimulate the expression of the committed state. Increased levels of phosphate (PO4) in the medium is reported to countered the inhibitory effect of auxin and promote bud initiation in absence of cytokinins. Casein Hydrolysate or tyrosine also induce kinetin type bud formation even in presence of higher level of IAA in medium. Similarly, polyamines, BAP, 2-iP, 6-Tetrahydropyrane-adenine and zeatin are found to induce shoot bud initiation. d. Factor Affecting the Organogenesis In vitro organogenesis is controlled by a number of factors other than phytohomes such factors are discussed below: 1. Size of Explant: Organogenesis is generally dependent upon size of explant. The large explant consisting parenchyma, vascular tissues and cambium have greater regenerative abilitythan the smaller explant. Small group of homogenous tissues taken from epidermal or subepidermal layer could directly give rise to complex organs like flower or bud or roots. 2. Source of Explant: The most suitable part of the plant for starting culture will depend on species. Leaves and leaf fragment of many plant species like Begonia, Solanum, Nicotina, Crepis , etc have shown capacity to regenerate shoot buds. Bulb scale of Hillium, sps, regenerate adventitious bulbelts, flower stem explant of Tulip asps. Regenerate shoot bud , inflorescence axis of Haorthia sp. Also forms shoots and root section of Convovulus sp. Produce shoot bud in culture. 3. Age of the Explant:
  • Page19 Physiological age of explant is important for in vitro organogenesis. In Nicotiana species, regeneration of adventitious shoot is only noted if the leaf explant is collected from vegetative stage i.e. before flowering. Leaf explants of Echeveria sp. That are collected from young leaves only produce roots, whereas older leaves initiate only shoot buds and leaves of medium age produce both shoots and roots. 4. Seasonal Variation: Bulb scales of Lilium speciosum regenerate bulblets freely in vitro when explant is taken during spring and autuma period of growth but same explant collected from summer or winter season does not produce any bulblets. 5. Oxygen Gradient: In some cultures, shoot bud formation takes place when the gradient of available oxygen inside the culture vessel is reduce. But rooting requires a high oxygen gradient. 6. Quality and Intensity of Light: The blue region of spectrum promotes shoot formation and red light induce rooting. The treatment of blue light followed by treatment of red light also stimulates the organogenesis phenomenon. In some cultures artificial fluorescence light favours rooting and inhibits in others. In case of Pisum sativum shoot bud initiation takes place in dark followed by sudden treatment of lights. Normally, organogeneses in culture take place with an illumination of 2000- 3000 lux. However, the callus tissue of Nicotina tabacum also produces shoot bud or embryo when tissue is exposed to high intensity of light of 1000-15000 lux. 7. Temperature: Most tissue culture are grown successfully at twp. Around 25 0C. In number of bulbous species optimum temperature may be much lower of about 15-18 0C. Increase in temp upto upto 330C may be associated with rise in growth of tobacco callus but for shoot bud initiation a lower temp of about 18 0C may be optimum. 8. Culture Medium: Medium solidified with agar favours bud formation although there are some reports about the development of leaf shoot buds on culture grown in a liquid medium. 9. PH of the Medium: The PH of the culture medium is generally adjusted between 5.6 and 5.8 before sterilization. The pH may have a determining role in organogenesis.
  • Page20 10. Ploidy Level: Variation in chromosome number i.e anuploidy, polyploidy, etc of plant cell in culture has been well documented. With the increase in chromosome instability there is a general decline in morphogenetic potentiality of callus tissue. So the most important factor in maintaining organogenic potential of callus tissue is the maintenance of chromosome stability. Frequency of subculture can affect the chromosome stability of cell culture. So in order to maintain chromosome stability, cultures are subcultured frequently and regularly. 11. Age of Culture: A young culture frequently produces organs. But the organogenic potential may decrease and ultimately disappear in old culture. In certain cultures of some plants, the plant regeneration capacity may retain indefinitely for many years.
  • Page21 7. Introduction Single Cell Culture A flowering plant body is made up of a wide range of innumerable cell types which are successfully integrated both in terms of structure and function. The plant body aquires this cellular diversity either due to permanent alteration in the genetic composition of the cells or due to more adaptation of the cells to perform a peculiar function in the plant body without affecting their genetic make-up. It is amazing that all such cell types have been derived from a single celled zygote by equatorial division. With advancement in technology it is possible today not only to culture single cells but also to induce the cell division and to raise a whole plant from it. The advantage of single cell culture over callus or cell suspension culture or intact organ culture is that single cell culture system is an ideal system for studying cell metabolism, the effect of various substances on cellular responses and to obtain single cell clone. Free cells in cultures permit quick administration and withdrawal of diverse chemicals or substances, thereby making them easy targets for mutant selection. Cell line selection technique can be usefully applied to produce high- yielding cultures as well as plants with superior agronomic traits. Definition of Single Cell Culture: Single cell culture is method of growing isolated single cell aseptically on a nutrient medium under controlled conditions. Methods of Single Cell Isolation A) From Plant Organ: The most material for the isolation of single cell aseptically on a nutrient medium under controlled conditions. a. Method of Single Cell Isolation: A) From Plant Organ: The most suitable material for the isolation of single cells is the leaf tissue since a more or less homogeneous population of cells in the leaves offer good candidates for raising defined and controlled large scale cell culture. From such intact plant organs single cells can be isolated using mechanical or enzymatic methods. i) Mechanical Method: Mechanical isolation involves tearing or chopping surface sterilized explant to expose the cells followed by scrapping of the cells with a fine scalpel to liberate the single cells hoping that it remained undamaged. Gnanam and Kulandaivelu ( 1969) developed a procedure to isolate mesophyll cells, active in photosynthesis and respiration, from mature leaves of several species of dicot and monocot. The procedure involves mild maceration of 10g leaves in 40 ml of
  • Page22 grinding medium (20 µ mol sucrose, 10µ mol Mgcl2, 20 µ mol tris –HCL buffer, Ph7.8) with a mortar and pestle. The homogenate is passed through two layer of muslin cloth and cell thus released are washed by centrifugation at low speed using the same medium. The mechanical isolation of free parenchymatous cells can also be achieved on a large scale. ii) Enzyme Method: Takebe et.al. (1968) treated tobacco leaf tissue with enzyme pectinase and obtained a large number of metabolically active cells. The potassium dextran sulphate in the enzyme mixture improved the yield of free cells. Isolation of single cells by the enzymatic method has been found convenient, as it is possible to obtain high yields from preparation of spongy parenchyma with minimum damage or injury to the cells. This can be accomplished by providing osmotic protection to the cells while the enzyme macerozyme degrades the middle lamella and cell wall of the pranchymatous tissue. Applying enzymatic method to cereals (Hordeum vulgare, Zea mays) has proven rather difficult since the mesophyll cells of these plants are apparently elongated with number of interlocking constrictions, thereby preventing their isolation. B) From Cultured Tissue: The most widely applied approach is to obtain a single cell system from cultured tissue. Freshly cut piece from surface sterilized plant organs are simply placed on a nutrient medium consisting of a suitable proportion of auxins and cytokinins to initiate cultures. Explant on such a medium exhibit callusing at the cut ends, which gradually extends to the entire surface of the tissue. The callus is separated from an explant and transferred to a fresh medium of the same composition to enable it to built up a mass tissue. Repeated sub-culture on an agar medium improves the friability of the callus, a pre-requsite for raising a fine cell suspension in a liquid medium. The piece of undifferentiated and friable callus are transferred in a continuously agitated liquid medium consisting of flask or suitable vials. Agitation is done by placing the culture medium exerts mild pressure on small pieces of tissue, breaking them into free cells and small cell aggregates. Further, it augments the gases exchange between the culture medium and the culture air and also ensures uniform distribution of cells as wells as clumps in the medium. b. Methods of Single Cell Culture There are five important methods which are widely employed for culturing single cells. The methods are: 1) The filter – paper raft nurse technique. 2) The Petri dish planting technique. 3) The micro-chamber technique. 4) The nurse callus technique. 5) The micro droplet technique.
  • Page23 1. The Filter-paper Raft Nurse Technique: i) Single cell are isolated from suspension cultures or a friable callus with the help of a micropipette or microspatuala. ii) Few days before cell isolation, sterile 8X8 mm square of filter paper are placed aseptically on the upper surface of the actively growing callus tissue of the same or different species. iii) The filter paper will be wetted by soaking the water and nutrient from the callus tissue. iv) The isolated single cell is placed aseptically on the wet filter paper raft. v) The whole culture system is incubated under 16 hrs cool white light (3000 lux) or under continuous darkness at 25o C. vi) The single cell divides and redivides and ultimately forms a small cell colony. When a cell colony reaches a suitable size, it is transferred to fresh medium where it gives rise to the callus tissue. The callus tissue on which the single cell is growing is called the nurse tissue. Actually the callus tissue supplies the cell with not only the nutrients from the cultures medium but sometimes more that is critical for cell division. The single cell absorbs nutrient through filter paper. The nutrients actually diffuse upward from culture medium through callus tissue and filter paper to the single cell. A callus tissue originating from the single cell is known as a single cell clone. 2. Petri Dish Plating Technique: The technique developed by Bergmann (1960) is the most popular one for plating of single cells. The techniques are as follows: i) A suspension of purely single cell is prepared aseptically from the stock cell suspension culture by filtering and centrifugation. The requisite cell density in the single cell suspension is adjusted by adding or reducing the liquid medium. ii) The solid medium (1.6 % Difco agar added is melted in water both). iii) In front of laminar air flow, the tight lid of Falcon plastic petri dish is opened. With the help of sterilized pasteurpipette, 1.5 ml of single cell suspension is put and equal amount of melted agar medium when it cools down at 35 0C is added in the single cell suspension. iv) The single cell divides and redivides and ultimately forms a small cell colony. When a cell colony reaches a suitable size, it is transferred to fresh medium where it gives rise to the callus tissue. v) The medium is allowed to solidity and petri dish is kept the inverted position.
  • Page24 vi) The cultures are incubated under 16 hrs light (3000 lux cool white) or under continuous dark at 250 C. vii) The petri dishes are observed at regular interval under the inverted microscope to see whether the cells have divided or not. viii) After certain days of inoculation, when cells start to divide, a grid is drawn on the under surface of the petri dish to facilitate counting the number of dividing cells. ix) The dividing cells ultimately form pin-head to facilitate counting the number of dividing cells. x) The plating efficiency (PE) can be calculated from the counting of cell colonies by the following formula: PE= Number of colonies per plate X 100 _________________________ Number of total cells per plate xi) Pin-head shaped colonies when they reach a suitable size are transferred to fresh medium for further growth. 3. The Micro-chamber Technique: i) A drop of liquid nutrient medium containing single cell is isolated aseptically from stock suspension culture with the help of long fine Pasteur pipette. ii) The culture drop is placed on the centre of a sterile microscopic slid (25X75 mm ) and rinsed with sterile paraffin oil. iii) A drop of paraffin is placed on either side of the cultural drop and a cover glass is placed on each oil drop. iv) A third cover glass is placed on the culture drop bridging the two raiser cover glasses and forming a micro-chamber to enclose the single cell aseptically within the paraffin oil. The oil prevents the water loss from the culture drop but permit gaseous exchange. v) The whole micro-chamber slide is placed in a petri-dish and is incubated under 16 hrs white cool illumination (3000 lux) at 25 0C. vi) Cell colony derived from single cell gives rise to single cell clone. vii) When the cell colony becomes sufficiently large, the cover glass is removed and tissue is transferred to fresh solid or semisolid medium. The micro – chamber technique permits the regular observation of the growing and dividing cell.
  • Page25 4. The Micro-droplet Technique: i) In this method single cells are cultured in a special Cuprack dishes which have two chambers- a small outer chamber and a large inner Chambers. The large chamber carries numerous numbered wells each with a capacity of 0.25-25 of nutrient medium. ii) Each well of inner chamber is filled with a micro drop of liquid medium containing isolated single cell. The outer chamber is filled with sterile distilled water to maintain the humidity inside the dish. iii) After covering the dish with lid, the dish is sealed with paraffin. iv) The dish incubated under 16 hrs white cool light at 25 0C. v) The cell colony derived from the single cell is transferred on to a fresh solid or semisolid medium in a culture tube for further growth. 5. The Nurse Callus Technique: This method is actually a modification of petri-dish plating method and paper raft nurse culture method. In these methods, single cells are plated on to a agar medium in a petri-dish as described earlier. Two to three callus masses derived from the same plant tissue are also embedded directly along with the single cells in the same medium. Here the paper barrier between single cells and the nurse tissue is removed. Cells first begin to divide in the regions near the nurse callus indicating that the single cells closer to nurse callus in the solid medium gets the essential growth factors that are liberated from the callus mass. The developing colonies growing near to nurse callus also stimulates the division and colony formation of the other cells. c. Importance of Single Cell Culture 1. Single cell culture could be used successfully to obtain single cell clones. 2. Plants could be regenerated from the callus tissues derived from single cell clones. 3. The occurrences of high degree of spontaneous variability in the cultured tissue and their exploitation through single cell culture are very important in relation to crop improvement programmes. 4. Isolated single cells can be handed as microbial system for the treatment of mutagens and for mutant selection. In practice, single cells are grown on medium containing the mutagenic compounds and the proliferation cell lines are isolated. The mutant nature of the selected cell lines can be confirmed by regenerating the plants and comparing their phenotypes with normal
  • Page26 plant. Many cell lines resistant to amino acid analogues, antibiotics , herbicides , fungal toxins etc. have been selected by this simple method. 5. Several workers have reported the synthesis of several times higher amounts of alkaloids, stearoids by cell culture than the alkaloid content in the intact plant. Therefore, large scale production of such compounds from single cells is possible. 6. Biotransformation means the cellular conversion of an ecogenously supplied substrate compounds not available in the cell or the precursor of a particular cellular compound to a new compound or the known compounds in higher amount. 7. Induction of polyploidy has been found to be very useful for plant breeding to overcome the problem of sterility associated with hybrids of unrelated plants. Polyploidy can easily be achieved by single cell culture.
  • Page27 8. Introduction to Callus Culture Higher plant body is multicellular and is made up of highly organised and differentiated structures like stem, leaf, root, etc. different tissue system present in different organs function in a highly coordinated manner. Now, if the organised tissue are diverted into an unorganised proliferation mass of cell by any means, they will form the callus tissue. In nature, sometimes callus or callus –like tissue is found to form to form in various part of intact plant either due to deep wound or due to some disease. Deep large wound in branches and trunks of intact plants results in the formation of soft mass of unorganised parenchymatous tissues which are rapidly formed on or below the injured surface of the organ concerned. Such callus tissue is known as wound callus. Wound callus is formed by the division of cambium tissues. They may also be formed by the same process from the parenchymatous cells of cortex, phloem and xylem rays. Callus like growth is also stimulated due to some disease caused by Agrobacterium tumefaciens, synchytrium endobioticum and virus, insects etc. Such callus –like outgrowth is known as gall or tumour. But the callus in tissue culture is produce experimentally from the small excise portion called the explant of any living healthy plant. In culture, the excised plant tissue losses its structural integrity and changes completely to a rapidly proliferative unorganised mass of cells which is called the callus tissue. What is Callus Tissue? Callus tissue means an unorganised proliferative mass of cells produced from isolated plant cells, tissues or organs when grown aspically on artificial nutrient medium in glass vials under controlled experimental conditions. a. Principles of Callus Culture For successful initiation of callus, three important criteria should be accomplished. i) Aseptic preparation of plant material. ii) Selection of suitable nutrient medium supplemented with appropriate ration of auxin and cytokinins or only appropriate auxin iii) Incubation of culture under controlled physical condition. Different plant parts carry a number of surface borne micro-organism like bacteria, fungus, etc. The excised plant parts called explants are at first washed with liquid detergent. Then explants are surface sterilized by the most commonly used chemical such as 0.1% w/v mercuric chloride (Hgcl2) or sodium hypochloride (0.8% to 1.6% available chloride) for a limited time ( generally 10-15 minutes). After sterilization, the explants are repeatedly rinsed with autoclaved distilled water. The surface sterilized plant material is cut aseptically into small segments (a few millimetres in size) and are transferred aseptically on a suitable nutrient medium solidified with agar.
  • Page28 Agar solidified or semi-solid nutrient medium after its preparation and sterilization by autoclave at 15 lbs, pressure for 15 minutes is used for induction of callus tissue. For most successful callus culture and for healthy callus growth usually both an auxin and cytokinins are required. The suitable temperature for in vitro callus initiation and growth is usually 25+- 2 0C. In some plant materials initiation and growth of callus take place in totally dark condition. However, in some cases a particular photoperiod (16 hrs light and 8hrs dark) is necessary for initiation and growth of callus tissues. Approximately, 2000 to 3000 lux artificial light intensity is needed. Generally, 55 to 60 % relative humidity is maintained in culture room. Once the growth of the callus tissue is well established, portions of the callus tissue can be removed and transferred directly on to fresh nutrient medium to continue growth. In this manner, callus cultures can be continuously maintained by serial subcultures. b. Protocol of Callus Culture Callus tissue can be induced from different plant parts of may plant species, however, carrot is a highly standardize material. The callus culture from exercise tap root of carrot is described below: 1. A fresh tap root of carrot is taken and washed thoroughly under running tap water to remove all surface dirt’s. 2. The tap root is then dipped into 5% “Teepol” for 10 minutes and then the root is washed. The carrot root, sterilized forceps, scalpels, other instruments, autoclaved nutrient medium Petri dishes are then transferred to laminar air flow or inoculation chamber. Throughout the manipulation sequences forceps, scalpels must be kept in 95% ethanol and flamed thoroughly before use. 3. The tap root surface sterilized by immersing in 70% v/v ethanol for 60 seconds, followed by 20-25 minutes in sodium hypochlorite (0.8% available chlorine). 4. The root is washed three times with sterilized distilled water to remove completely hypochlorite. 5. The carrot is then transferred to a sterilized petri dish containing a filter paper. A series of transverse slice 1mm in thickness is cut from the tap root using a sharp scalpel. 6. Each piece is transfer to another sterile petri dish. Each piece contains a whitish circular ring of cambium around the pith. An area of 4mm2 across the cambium is cut from each piece so that each piece contains part of phloem. Cambium and xylem size and thickness of explant should be uniform. 7. Always the lid of petri dish is replaced after each manipulation.
  • Page29 8. The closure from a culture tube is removed and flamed the uppermost 20mm of the open end. While holding the tube at an angle of 45 0, an explant is transferred using forceps onto surface of the surface of the agarified nutrient medium. Nutrient medium is Gamborg’s B5 or Ms medium supplemented with 0.5 mg/, 2,4-D. 9. The closure is immediately placed on the open mouth of each tube, Date, medium and name of the plant are written on the culture tube by a glass marking pen or pencil. 10. Culture tubes after inoculation are taken to the culture room where they are placed in the racks. Cultures are incubated in dark at 25 0C. 11. Usually, after 4 weeks in culture the explants incubated on medium with 2,4-D will form a substantial callus. The whole callus mass is taken out aseptically on a sterile petri dish and should be divided into two or three pieces. 12. Each piece of callus tissue is transferred to a tube containing fresh same medium. 13. Prolonged culture of carrot tissue products large calluses. c. How the Callus Tissue is Formed? Formation of callus tissue is the outcome of cell division and cell exoansion of the cells of explant. During the formation of callus tissue, the explant loses its original characteristics. So under the influence of exogenously supplied hormone. The explant is triggered off a growth sequence in which cell enlargement and cell division predominate to form an unorganised mass of cells. As a result , the explant undergoes an irreversible changes of its shape, size , symmetry, structural organization and cellular integrity. Depending upon the types of explant viz. leaf, stem segment, root segment etc. either enlargement in size or the swelling followed by rupture of tissue within few days of inoculation take place. This change indicates the response of explant for callus formation and is followed by the appearance of little irregular cellular masses around the cut edges or from the cut edges or from the ruptured surface. It is now explained that initial formation of cellular mass particularly at the cut end may be due to injury during excision. Some endogenous growth substances oozes out through the injured tissue at cut end and stimulates the cell division which is simultaneously induced by the exogenously supplied growth hormones. It is assumed that both endogenous product and exogenous hormones make a threshold level and their interaction causes formation of unorganised cellular growth at cut end. Auxin is required for growth and cytokinin is required for cell division. The type of tissue as a explant plays important role, such as meristematic tissue containing vascular cambium. d. Application of Callus Culture 1. The whole plant can be regenerated in large number from callus tissue through manipulation of the nutrient and hormonal constituents in the culture medium which is called as organogenesis
  • Page30 or morphogenesis. Similarly, callus can be induce to form somatic embryo which can gives rise to whole plant. 2. Callus tissue is good source of genetic or karyotypic variability, so it may be possible to regenerate a plant from genetically variable cells of the callus tissue. 3. Cell suspension culture in moving liquid medium can be initiated from callus culture. 4. Callus culture is very useful to obtain commercially important secondary metabolites. If a bit tissue from a medicinally important plant is grown in vitro and produced callus culture, then secondary metabolites or drugs can be directly extracted from the callus tissues without sacrfting the whole plant. 5. Several biochemical assays can be performed from callus culture.
  • Page31 9. Suspension Culture and Its Principle Introduction: It is a type of culture in which single cell or small aggregates of cells multiply while suspended in agitated liquid medium. It is also referred to as cell cultures or cell suspension culture. Principle: Callus proliferates as an unorganised mass of cells. So it is very difficult to follow many cellular events during its growth and developmental phases. To overcome such limitation of callus culture, the cultivation of free cells as well as small cell aggregates in a chemically defined liquid medium as a suspension was initiated to study the morphological and biochemical changes during their growth and developmental phases. To achieve an ideal cell suspension, most commonly a friable callus is transferred to agitated liquid medium where it breaks up and disperses. After eliminating the large callus pieces, only single cells and small cell aggregates are again transferred to fresh medium and after two to three weeks a suspension of actively growing cells is produced. Ideally suspension culture should consist of only single cells which are physiologically and biochemically uniform. a. Protocol of Suspension Culture 1. Take 150/250 ml conical flask containing autoclaved 40/60 ml liquid medium. 2. Transfer 3-4 pieces of pre-established callus tissue from culture tube using the spoon headed spatula to conical flask. 3. Flame the neck of conical flask, close the mouth of conical flask, with piece of aluminium foil or a cotton plug. Cover the closure with piece of brown paper. 4. Place the flasks within the clamps of a rotary shaker moving at the 80-120rpm. 5. After the filtrate to settle for 10-15 minutes or centrifuge the filtrate at 500 to 1000 rpm and finally pour off the supernant. 6. Resuspend the residue cells in a requisite volume of fresh liquid medium and despise the cell suspension equally in several sterilized flasks. Place the flasks on shaker and allow the free cells and cell aggregates to grow. 7. Resuspend the residue cells in a requisite volume of fresh liquid medium and despense the cell suspension equally in several sterilized flasks ( 150/250 ml). Place the flasks on shaker and allow the free cells and cell aggregates to grow.
  • Page32 8. At the next subculture, repeat the preveious steps but take only one fifth of the residual cells as the inoculum and despense equally in flasks and again place them on shaker. 9. After 3-4 subculture, transfer 10ml of cell suspension from each flask into new flask containing 30 ml fresh liquid medium. 10. To prepare a growth curve of cells in suspension, transfers a definite number of cells measured accurately by a haemocytometer to a definite volume of liquid medium and incubate on shaker. Pipette out very little aliquot of cell suspension at short intervals of times and count the cell number. Plot the cell count data of a passage on a graph paper and the curve will indicate the growth pattern of suspension culture. b. General Account of Suspension Cultures The movement of nutrient medium in suspension culture provides vital aeration of the medium to sustain cell respiration in the liquid medium and also encourages the callus tissue to brake up. As the cell division starts in the callus tissue, they shed and dispense directly into medium. A more friable callus tissue is an ideal material for the dispersion of cells. Increasing the concentration of auxin or adding very low concentration of cellulose and pectinase enzymes in the liquid medium are also effective for dispersion of cells. The period of incubation during which the suspension culture is developed from callus tissue is usually called as initiation passage. In general, media suitable for growing callus cultures for particular species are also suitable for growing suspension cultures provided that agar is omitted. The concentration of auxin and cytokinins used for callus cultures is generally reduced for suspension cultures. The cells within the aggregates in a different microenvironment from the free floating cells. The cells in suspension may vary in shapes and sizes. They maybe oval, round elongated coiled etc. although suspension cultures consist of thin walled cells, other posses a proportion of lignified, tracheid like elements. These usually arise in the cell aggregates. c. Types of Suspension Cultures There are two types of suspension cultures, i) Bat culture ii) Continuous Culture A) Batch Culture: a. Slowly rotating culture b. Shake culture c. Spinning culture d. Stirred culture B) Continuous Culture:
  • Page33 a. Chemostats b. Turbidostats. A) Batch Culture: These cultures are maintained continuously by propagating a small aliquot of inoculum in the moving liquid medium and transferring it to fresh medium ( 5 x dilution) at regular intervals. Generally cell suspensions are grown in flasks ( 100-250 ml) containing 25-75 ml of the culture medium. Batch suspension cultures are most commonly maintained in conical flasks incubated on orbital platform shakers at the speed of 80-120 rpm. The biomass growth in batch culture follows the fixed pattern. When the cell number in suspension cultures is plotted against the time of incubation, a growth curve is obtained depecting that initially the culture passes through a lag phase, followed by a brief exponential growth phase- the most fertile period for active cell division. The growth declines after three to four cell generations, signalling that the culture has entered the stationary phase. For subculture, the flask containing suspension culture is allowed stand still for a few seconds to enable the large colonies to settle down. A pipette or syringe with orifice fine enough to hold aggregate of two to four cells or only single cells is used. The suspension is taken from the upper part of the culture and transferred to a fresh medium. i) Slowly Rotating Cultures: Single cells and cell aggregates are grown in a specially designed flask, the nipple flask. Each nipple flask possesses eight nipple-like projections. The capacity of each flask is 250 ml. Ten flasks are loaded in a circular manner on a large flat disc of a vertical shaker. When the flat disc rotates at the speed of 1-2 rp, the cell within each nipple of the flask are alternatively bathed in a culture medium and exposed to air. ii) Shake Culture: It is very simple and effective system of suspension culture. In this method, single cells and cell aggregates in fixed volume of liquid medium are placed in conical flask. Conical flasks are mounted with the help of clip on a horizontal large square plate of an orbital platform shaker. The square plate moves by a circular motion at 60-180 rpm. iii) Spinning Culture: Large volume of cell suspension may be cultured in 10L,bottles which are rotated in a culture spinner at 120 rpm at an angle of 450 . iv) Stirred Culture: This system is also used for large scale batch culture. In this method, the large culture vessel is not rotated but the cell suspension inside the vessel is kept dispersed continuously by bubbling sterile air through culture medium. The use of an internal magnetic stirrer is the most convenient
  • Page34 way to agitate the culture medium safely. Magnetic stirrer revolves at 200-600 rpm. The culture vessel is a 5-10 litres round bottom flask. B) Continuous Culture System: In this system, the old liquid medium is continuously replaced by the fresh liquid medium to stabilize the physiological stage of the growing cells. Normally, the liquid medium is not changed until the depletion of some nutrients in the medium and the cells are kept in the same medium for a certain period. As a result, the active growth phase of the cell declines the depletion of nutrient. The cells passing through out flowing medium are separated mechanically and reintroduced in the culture. i) Chemostats: In this system, cultures vessels are generally cylindrical or circular in shape and posses inlet and outlet pores for aeration and for introduction of and removal of cells and medium. The liquid medium containing the cell is stirred by a magnetic stirrer. The introduction of fresh sterile medium, which is pumped in at a constant rate into the vessel is balanced by the displacement of an equal volume of spent or old medium and cells. Such a system can be maintained in a steady state so that new cells are produced by division at a rate which compensate the number lost in outflow of spent medium. ii) Turbostats: In this system, the input of medium is intermittent as it is mainly required to control the rise in turbidity due to cell growth. The turbidity of a suspension culture medium changes rapidly when cells increase in number due to their steady state growth. The changes in turbidity of the culture medium can be measured by the changes of optical density of the medium. In Turbostats an automatic monitoring unit is connected with the culture vessel and such unit adjusts the medium flow in such a way as to maintain the optical density or PH at chosen, present level. d. Synchronization of Suspension Culture Cells in suspension cultures vary greatly in size, shape DNA and nuclear content. Moreover, the cell cycle time varies considerably within individual cells. Therefore, cell cultures are mostly asynchromous. This variation complicates studies of biochemical, genetic physiological and other aspects of cell metabolism. A synchronous culture is one in which the majority of cells proceed through each cell cycle phase (G,S ,G2 and M) simultaneously. A) Physical Methods: i) Selection by Volume: Synchronization may be achieved on the basis of selecting the size of cell aggregates present even in the finest possible suspension cultures. Cell fractionation is employed for selection.
  • Page35 ii) Temperature Shock: Low temperature shocks combined with nutrient starvation are reported to induce synchronization of suspension culture. B) Chemical Methods: i) Starvation: The principle of starvation is based on depriving suspension cultures of an essential growth compound leading to a stationary growth phase. Resupplying the missing compounds is expected to induce resumption of cell growth synchronously. Growth hormone starvation is also reported to induce synchronization of cell cultures. ii) Inhibtiion: Synchronization is achieved by temporarily blocking the progression of events in the cell cycle and accumulating cells in a specific stage using a biochemical inhibitor. On release the block cells with synchronously enter the next stage. Inhibitors of DNA synthesis ( 5-aminourail, 5- flurodexypurine, hydroxyurea or excess thymidine) in cell cultures accumulate cells at the G1/S boundary. iii) Mitotic Arrest: Colchicine has been widely used to arrest cells at metaphase. Suspension cultures in exponential growth are supplied with 0.02% (w/v) colchicine for 4-8 hr in order to inhibit spindle formation. e. Importance of Suspenion Cell Culture 1. Suspenion cultures system is capable of controlling many significant information about cell physiology, biochemistry and metabolic events at the level of individual cells and small cell aggregates. 2. By cell plating technique different cell clones can be developed. 3. Suspension cultures can be used for production of secondary metabolites. 4. Mutagenesis studies may be facilitated by the use of cell suspension culture to produce mutant cell clones from which mutant plant can be regenerated.
  • Page36 10. Introduction to Somatic Embryogenesis In angiosperms, ovules are developed within the ovary. Within the ovules, a sac like structure known as embryo sac lies embedded into nucleus. The embryo sac represents the female gametophyte of angiosperms. The ovule contains a haploid egg cell or ovule which is female reproductive cell or female gamete. During fertilization, the male gamete fuses with egg cell or female gamete. During fertilization, the male gamete fuses with egg cell or female gamete resulting in formation of an unicellular zygote or oospore. The zygote gives rise to multicultural embryo, cells of which are diploid. Embryos derived in this sexual process are known as zygotic embryo, and process of embryo development is called embryogenesis. Sometimes, embryo is formed by the unfertilized egg and such embryos are called parthenocarpic embryo. Again sometimes, any cell of the female gametophyte or Sporophytic tissue around the embryo sac may give rise to an embryo and such embryos are called non zygotic embryos. In nature there are no instances of ex-ovule embryo development. therefore, there is no evidence of embryo development in vitro from any somatic cells of the plants. That means, in vivo somatic plant cells do not express any embryogenic potential to form embryo. Somatic Embryogenesis: In plant tissue culture, the developmental pathway of numerous well organised, small embryoids resembling the zygotic embryos form the embryogenic potential somatic cell of callus tissue or cells of suspension cultures is known as somatic embryogenesis. Embryogenic Potential: The capacity of somatic cell of a culture to produce embryoids is known as embryogenic potential. a. Embryoid Embryoid is a small, well organised structure comparable to the sexual embryo, which is produced in tissue culture of dividing embryogenic potential somatic cells. Embryos formed in cultures have been variously designated as accessory embryos, adventive embryos, embryoids and super- numeracy embryos. Types of Embryos: 1. Zygotic Embryos: These formed by fertilized egg or the zygote. 2. Non-Zygotic Embryos: a) Somatic Embryos:
  • Page37 Those formed by Sporophytic cells either in vitro or in vitro. Such somatic embryos arsing directly from other embryos or organs are termed adventive embryos. b) Parthenocapic Embryos: Those formed by unfertilized egg. c) Androgenic Embryos: Those formed by the male gametophyte. b. Principles of Somatic Embryogenesis Somatic Embryogenesis may be Initiated in two Different Ways: 1. In some cultures somatic embryogenesis occurs in absence of any callus production from “pro-embryogenic determined cells” that are already programmed for embryo differentiation. For instances somatic embryos have been developed directly from leaf mesophyll cells of orchard grass without an intervening callus tissue. Explants made from the basal portions of two innermost leaves of orchard grass were cultured on Schenk and Hildebrandt medium supplemented with 30µm, 3, 6-dichloro-0-anisic acid, plant formation occurred after subculturing the embryos on the same medium without decamba. 2. The second types of somatic embryo development needs some prior callus formation and embryoids originate from “Induced embryogenic determined cells” ( IEDCs) within the callus tissue. In most cases direct embryogenesis occurs. For direct somatic embryogenesis where it, has been induced under in vitro condition, two distinctly different types of media may be required- one medium for the initiation of the embryonic cells and another for the subsequent development of these cells into embryoids. The first or the induction medium must contain auxin in case of carrot tissue and somatic embryogenesis can be initiated in the second medium by removing the hormone or lowering its concentration. With some plants, however, both embryo embryo initiation and subsequent maturation occur on the first medium and second medium is required for plantlet development. Embroids are generally initiated in callus tissue from the superficial clumps of cells associated with enlarged with enlarged vacuolated cells that do not take part in embryogenesis. The embryogenic cells are generally characterised by dense cytoplasmic contents, large starch grains, relatively large nucleus with a darkly stained nucleolus. In suspension culture, embryoids do not form from suspended single cell, but form from cells lying at or near the surface of the small cell aggregates. Each developing embryoid of carrot passes through three sequential stages of embryo formation such as globular stage heart shaped stage and torpedo- stage. The torpedo stage is a bipolar
  • Page38 structure which ultimately gives rise to complete plantlet. The culture of other plants may not follow such sequential stages of embryo development. In general, somatic embryogenesis occurs in short term culture and this ability decreases with increasing duration of culture. The loss of embryogenic potential of culture may be due to changes in Ploidy and lose of certain biochemical properties of cultured cells. In callus culture or in suspension culture, embryoids formation occurs asynchronously. A high degree of synchronization has been achieved in carrot suspension culture by sieving the initial cell population. C. Protocols for Inducing Somatic Embryogenesis in Culture The plant material Ducus carota represents the classical example of somatic embryogenesis in culture. The protocol is described below: 1. Leaf petiole or root segments from seven day old seedlings or cambium tissue from seven day old seedling or cambium tissue from storage roots can be used as explant. The leaf petiole and root segment can be obtained from aseptically growth seedlings. Cambium tissue can be obtained from surface sterilized tap root. 2. Following aseptic technique, explants are placed individually on a semi-solid MS medium containing 0.1 mg/l, 2,4-D and 2% sucrose. Cultures are incubation in dark. In this medium explant will produce sufficient callus tissue. 3. After 4 weeks of callus growth, cell suspension culture is to be initiated by transferring 0.2g of callus to a 150 ml of Erlenmeyer flask containing 20-25 ml of liquid medium of the same composition as used for callus growth. Flasks are placed on a horizontal gyratory shaker with 125-160 rpm at 25 0C. The presence or absence of light is not critical at this stage. 4. Cell suspension are subcultured every 4 weeks by transferring 5ml to 65 ml of fresh liquid medium. 5. To induce a more uniform embryo population, cell suspension is passed through a series of stainless steel mesh sieves. For carrot, the 74 µ produce a fairly, dense suspension of single cell and small multiple clumps. To induce somatic embryogenesis, portion of sieved cell suspension are transferred to 2,4-D free liquid medium or cell suspension can be plated in semi-solid MS medium devoid of 2,4-D. for normal embryo development and to inhibit precocious germination especially root elongation , 0.1 µm ABA can be added to the culture medium. Cultures are incubated in dark. 6. After 3-4 weeks, the culture would contain numerous embryos in different stage of development.
  • Page39 7. Somatic embryos can be placed on a agar medium devoid of 2, 4-D for plantlet development. 8. Plantlets are finally transferred to pots or vermiculite for subsequent development. Somatic Embryogenesis in Dicotyledonous and Monocotyledonous Culture d. Somatic Embryogenesis in Dicotyledonous Culture: Totipotent embryogenic cells can be obtained from explants of embryogenic or young seedling tissue. Excised small tissues from young inflorescence are equally effective in including somatic embryogenesis in cultures. Other explants used are scuttelum, young roots, petiole, immature leaves and immature hypocotyl. Citrus nucellar cells have natural potential for somatic embryogenesis. Somatic embryos germinate in situ or when they are excised and cultured individually on fresh semisolid medium. a special and noteworthy feature may be the development of a fresh crop of adventive embryos which originate from single epidermal cells on the stem surface of the plantlets obtained from germinating embryos. The essential requirement for induction and promotion of somatic embryo is suspension culture. Presence of auxin- in the medium is generally essential for embryo initiation. First, the callus is initiated and multiplied on a medium rich in auxin (2,4-D, 0.5mg/l ) which induce differentiation of localized groups of meristematic cells called “Embryogenic clump” ( ECs). Second, ECs develop into mature embryos when transferred to a medium with a very low level of auxin (0.01 to 0.1mg/l) or no auxin at all. Consequently the medium with auxin is called a ‘Proliferation medium (PM) and without auxin an ‘ embryo development medium’ (EDM). In some cases, NAA, IBA , BAP , ethephon also induce embryogenesis. Somatic Embryogenesis in Monocotyledonous Culture: Many monocotyledonous plants are of agricultural and medicinal importance. Unlike dicot, the vegetative parts of monocot plant donot readily proliferate in culture. Therefore, explants are best taken from embryogenic on meristematic tissues. Selection of Explant: i) Zygotic Embryo: In case of zygotic as explant, young caryopses ( 10-15 day after pollination) or seeds are surface sterilized by normal procedure and zygotic embryos are excised aseptically and transferred to a culture vials containing MS medium supplemented with 2, 4-D in case of cereals and sucrose (2- 6%). Cultures are incubated in diffuse light or complete darkness. Culture will develop within 4- 6 weeks. ii) Young Inflorescence: Premitotic inflorescence, with primordia of individual florets just beginning to protrude is best suitable material in some cases. The inflorescence of 1-2cm in length is excised. Sterilized and
  • Page40 each inflorescence is exposed by vertical incision through the surrounding leaves and then cut into 1-2 mm thick segments. Individual segments are then cultured on medium containing 2,4-D for proliferation and initiation of embryogenic callus. iii) Young Leaf: Leaves of young seedlings obtained from seeds, germinate under aseptic condition, are removed and cut into 1-2 mm thick transverse segments starting from the level of the shoot meristem upto the leaf apex. Six to eight examples are placed on nutrient medium to obtain a callus. e. Induction of Embryogenic Cell Suspension The callus obtained from cultured explants is sliced and teased apart into small pieces which are then incubated in a liquid medium. Once a good embryogenic suspension has been established, somatic embryos can be obtained either by allowing the culture to age or by incubating the embryogenic tissue in a medium without 2,4-D. Since an unspplemented basal medium often encourages root formation, the normal practise is to add 500ml glutamine with 0.1 µm ABA to the medium to facilitate the embryo development. Factors Affecting the Embryogenesis 1. Chemical Factors: i) Auxins: Somatic embryogenesis in carrot is a classical example. It is two step process. The carrot cells first develop into a callus tissue in the medium containing the auxin namely2, 4-D (0.5 to 1mg/l). When such callus tissue is transferred to the same medium with a very low level of auxin or no auxin at all, embryoids are formed. If the callus tissue is maintained continuously in the medium containing 2, 4-D, embryoids would not form. Similarly, if the carrot cells are maintained continuously from the initial step in auxin free medium, embryoids do not develop. Therefore, the presence of auxin namely 2, 4-D (0.5 to 1mg/l). When such callus tissue is transferred to the same medium with a very low level of auxin or no auxin at all, embryoids are formed. If the callus tissue is maintained continuously in the medium containing 2,4-D , embryoids would not form. Similarly, if the carrot cells are maintained containing from the initial step in auxin free medium, embryoids do not develop. Therefore, the presence of auxin in the first step is possibly essential for the proliferation of callus tissue and for the induction of embryogenic potential cells. In second step, auxin is no longer required for the embryogenic potential cells to form embryoids. Like carrot , two –step process of in vitro development of somatic embryo is also found in Coffea Arabica. Other than 2,4-D , NAA and IBA have also been used in other culture system for induction of embryogenic potential cells. In Citrus sinensis, callus tissue is imitated from the nuclear tissue in the medium containing IAA and kinetin. Such callus when transferred to auxin-free medium causes the induction of embryogenesis. Non – requirement of auxin in medium during second step may be probably due
  • Page41 to synthesis of adequate amount of both auxin and cytokinins which they required for growth and somatic embryogenesis. A minimum of cytokinins in embryogenesis is somewhat obscure because of conflicting results. In carrot suspension culture, zeatin a type of cytokinins, stimulates embryogenesis when the cells are subcultured in auxin –free –medium. But the process is inhibited by the addition of either kinetin or BAP to the medium. The inhibitory effect of cytokinins may be due to selective stimulation of cell of the culture. Stewart et.al ( 1964) also reported the importance of coconut milk for somatic embryogenesis. iii) Gibberellins: Gibberellin has no positive effect. In carrot and citrus, gibberellin inhibit somatic embryogenesis. iv) Reduced Nitrogen: Substantial amount of reduced nitrogen (NH+4) are required for embryogenesis. In carrot culture, addition of NH4 to embryogenic medium already containing KNO3 produces near- optimal numbers of embryoids. It is therefore convenient to use (NH4) in combination with NO3- . But no other forms of inorganic reduced nitrogen have been as effective as NH4 for somatic embryogenesis. Glutamine, glutamic acid, urea and alanine are found to partial replace NH4CL as supplement to KNO3. These various nitrogen sources are not specific for the induction of embryogenesis, although, at low concentration organic forms are much more effective than inorganic nitrogen compounds. 2. Other Factors: The medium supplemented with activated charcoal has facilitated embryogenesis in several cultures. The induction of embryogenesis is achieved successfully by the addition of charcoal when auxin depletion in the medium fails to produce the desired result. It has been suggested that charcoal may absorb a wide variety of inhibitory substances as well as hormone. Optimum level of dissolved oxygen and high potassium in the medium are necessary for embryogenesis. But in citrus, certain volatile and non-volatile substances inhibit embryogenesis. g. Practical Application of Somatic Embryogenesis The potential applications and importance of in vitro somatic embryogenesis and organogenesis are more or less similar. i) Clonal Propagation: Since both the growth of embryogenic cells and subsequent development of somatic embryos can be carried out in liquid medium, it is possible to combine somatic embryogenesis with
  • Page42 engineering technology to create large-scale mechanical or automated culture systems. Such systems are capable of producing propagules repetitively with labour input. In this process of repetitive somatic embryogenesis is initiated where by somatic embryos proliferate from the previously existing somatic embryo in order to produce clones. ii) Raising Somaclonal Variants in Tree Species: Embryos formed directly from pro-embryogenic determined cells ( PEDCs) appear to produce relatively uniform clonal material, whereas the indirect pathway involving induced embryogenic determined cells (IEDCs) generates a high frequency of Somaclonal variants. Mutation during adventive embryogenesis may give rise to a mutant embryo which on germination would form a new strain of plant. For clonal propagation of tree species, somatic embryogenesis from nucellar cells may offer only rapid means or obtaining juvenile plants equivalent to seedlings with parental genotype. iii) Synthesis of Artificial Seeds: Artificial seeds are the living seeds like structures which are made experientially by a technique where somatic embryoids derive d from plant tissue culture are encapsulated by a hydrogel and such encapsulated embryoids behave like a true seeds if grown in soil and can be used as a substitute for natural seeds. Several Steps are followed for making Artificial Seeds: 1. Establishment of callus culture 2. Induction of somatic embryogenesis in callus culture. 3. Maturation of somatic embryos 4. Encapsulation of somatic embryos Maturation of somatic embryos means completion of embryo development throught some stages. Initially, embryo develops as globular shaped stage, the heart-shaped stage and finally torpedo- shaped stage. In the final stage embryo attains maturity and develops the opposite poles for shoot and root development at two extremities. This embryo then starts to germinate and produces plantlets. Two types of artificial seeds have developed, namely, , hydrated and desiccated Redenbergh et.al. (1986) developed artificial seeds by mixing somatic embryos of alfalfa, celery and cauliflower with sodium alginate , followed dropping into a solution of calcium chloride to form calcium- alginate beads. About 29-55% embryos encapsulated with this hydrogel germinated and formed seedlings in vitro. Kim and Janick (1989) applied synthetic seeds coats to clumps of carrot somatic embryos to develop desiccated artificial seed. They mixed equal volumes of embryo suspenion and 5% solution of polythene oxide, a water soluble resin, which subsequently dried to further achieved by embryo a ‘ hardening ‘ treatment with 12% sucrose or 10-6 MABA, followed by chilling at inoculum density.
  • Page43 Another delivery system for somatic embryos for obtaining transgenic plant is fluid drilling. Embryos are suspended in a viscous- carrier gel which extrudes into the soil. The primary problem in fluid –drilling is that the sucrose level necessary to permit conversion also promotes rapid growth of contaminating micro-organisms in a non-aseptic system. iv) Source of Regenerable Protoplast System: Embryogenic callus, suspension cultures, and somatic embryos have been employed as source of protoplast isolation for a range of species. Cells or tissues in these system have demonstrated the potentiality to regeneration in culture and therefore, yield protoplast that are capable to forming whole plants. v) Genetic Transformation: Repetitive embryos originate from single epidermal or sub epidermal cells which can readily be exposed to Agrobacterium. Thus the transformation technique applied to primary somatic embryos. Repetitive embryogenesis is also ideally suited to particle gun-mediated genetic transformation. Instead or recycling on Agrobacterium to mediate the transfer of genes into plant cells, the particle gun literally shoots DNA that has been precipitated onto particles of a heavy metals, into the plant cells. Embryogenic suspension cultures of the cotton and soybean, initiated cell lines following each firing of the gun. The transformed cell lines can then the induced to form an unlimited number of transformed somatic embryos through repetive embryogenesis. vi) Synthetic of Metabolites: The repetitive embryogenesis system is of potential use in the synthesis of metabolites such as pharmaceuticals and oils. Borage contains high level of Y-Linoleic acid, used as precursor of post-glandins or in the treatment of atopic eczema. Somatic embryos of borage also produce this metabolite but through repetitive somatic embryogenesis a continuous supply of Y-lenolenic acid is ensured. Which otherwise would be limited to the growing season in the zygotic embryos. The same principle can be applied for production in vitro of industrial lubricant from jojoba and leo- palmitostearin from cacao. h. Importance of Artificial Seeds 1. Have to wait upto end of reproductive phase for obtaining true seeds. But artificial seeds are available within at least one month. 2. The production of true seed is season bound at particular seasons of a year. But production of artificial seed is not time or seasonal bound. 3. Life cycle of plant could be shortened in case of plant where dormancy of seed is prolonged. 4. Artificial seeds will be applicable for large scale monoculture as well as mixed genotype plantation.
  • Page44 5. It gives the protection of meiotically unstable, elite genotype. 6. Artificial seed coating also has the potential to hold and deliver beneficial adjuvant such as growth promoting rhizobacteria, plant nutrients and growth control agents, and pesticides for precase placement. 7. Artificial seeds help to study the role of endosperm and seed coat formation.
  • Page45 11. Shoot-Tip and Meristem Culture Most of the horticultural and forest crops are infected by systemic disease caused by fungi, viruses, bacteria, Mycoplasma and nematode. While plant infected with bacteria and fungi may respond to treatments with bactericidal and fungicidal compounds, there is no commercially available treatment to cure virus infected plants. It is possible to produce disease free plants through tissue culture. Apical meristems in the infected plants are generally either free or carry a very low concentration of the viruses. The various reasons attributed to the escape of the meristems by virus invasion are: a) Viruses move readily in a plant body through the vascular system which in meristems is absent, b) A high metabolite activity in the actively dividing meristematic cells does not allow virus replication and c) A high endogenous auxin level in shoot apices may inhibit virus multiplication. Meristem –tip cultures has also enabled plants to be freed from other pathogens including Viroids, mycoplasmas, bacteria and fungi. Therefore, main objective of shoot-tip and meristem –tip culture is the production of disease free plants through micro propagation. Shoot-tip Culture: It may be described as the culture of terminal (0.1-1.0mm) portion of a shoot comprising the meristem (0.05 -0.1) together with primordial and developing leaves and adjacent stem tissue. Meristem Cultures: Meristem cultures is the in vitro culture of a generally shiny special dome like structure measuring less than 0.1mm in length and only one or two pairs of youngest leaf primordia, most excised from the shoot apex. Principle: The excised shoot tip and meristem can be cultured aseptically on agar solidified simple nutrient medium or on paper bridges dipping into liquid medium and under appropriate conditions will grow out directly into a small leafy shoot or multiple shoots. Alternatively, the meristem may form a small callus at its cut base on which a large number of shoot primordia will develop. These shoot primordia grow out into multiple shoots. Once the shoot have been grown directly from the excised shoot tip or meristem, they can be propagated further by nodal cuttings. This process involves separating the shoot into small segment each containing one mode. The axillary bud on each segment will grow out in culture to form a yet another shoot. The excised stem tips of orchids in culture proliferate to form callus from which some organised juvenile structures known as protocorm develop. When the protocorm are separated and cultured on fresh medium,
  • Page46 they develop into normal plants. The stem tips of Cuscuta reflexa in culture can be induced to flower when they are maintained in the dark. Exogenously supplied cytokinins in the nutrient medium plays a major role for the development of a leaf shoot or multiple shoots from the meristem or shoot tip. Generally high cytokinins and low auxin are used in combination for the culture of shoot tip of meristem. Addition of adenine suifate in the nutrient medium also induces shoot tip multiplication in some areas. BAP is the most effective cytokinins commonly used in shoot tip or meristem culture. Similarly, NAA is most effective auxins used in shoot tip culture. Coconut milk and gibberlic acid are also equally effective for the growth of shoot apices in some cases. Protocol: 1. Remove the young twings from the healthy plant. Cut the tip portion of the twig. 2. Surface sterilize the shoot apices by incubation in a sodium hypochlorite solution ( 1% available chlorine) for 10 minutes. The explants are thoroughly rinsed 4 times in sterile distilled water. 3. Transfer each explant to a sterilize petridish. 4. Remove the outer leaves from each shoot apices with pair of jweller’s forceps. This lessens the possibility of cutting into the softer underlying tissues. 5. After the removal of all the outer leaves, the apex is exposed. Cut off the ultimate apex with the help of scalpel and transfer only those less than 1 mm in length to the surface of the agar medium or to the surface of Filter Paper Bridge. Flame the neck of culture tube before and after the transfer of excised tips. Binocular dissecting microscope can be used for cutting the true meristem or shoot tip perfectly. 6. Incubate the culture under 16 hrs light at 25 0C. 7. As soon as the growing single leafy shoot or multiple shoots obtained from single shoot tip or meristem, transfer them to hormone free medium to develop roots. 8. The plants form by this way are later transferred to pots containing compost and kept under green house condition for hardening. a. Application of Shoot-tip or Meristem Culture 1. Virus Elimination: Plants are often infected with more than one type of virus, including some even not known. A general term virus- free is used by commercial horticulturist for plants freed of any type of virus. 2. Micro Propagation:
  • Page47 A sexual or vegetative propagation of whole plants using tissue culture techniques referred to as micropropagation. Shoot tip or meristem culture of many plant species can successfully be used for micro propagation. 3. Storage of Genetic Resources: Many plants produce seeds that are highly heterozygous in nature or that is recalcitrant. Such seeds are not accepted for storing genetic resources. So , the meristem from such plants can be stored in vitro. The materials are preserved at 196 0 C for the long term preservation of germplasm. 4. Use in Plant Breeding: In many plant breeding experiments the hybrid plants produce abortive seeds or non viable seeds. As a result, it makes a barrier to crossibility in plants where non-viable seeds are unable to develop into mature plants. Shoot-tip or meristem from such hybrid plant can be cultured to speed up breeding programme. 5. Propagation of Haploid Plants: Haploid plants derived from anther or pollen culture always remain sterile unless and until they are made homozygous diploid. Meristem or shoot-tip culture of haploid plants can be used for their propagation from which detailed genetic analysis can be done on the basis of morphologically character and biochemical assay. 6. Quarantine: There are some regulations concerning the international movement of vegetative plant material. After thoroughly checking, the plant materials may be rejected by quarantine authority if the plant material carries some diseases. Plantlets derived from shoot-tip or meristem cultures are easily accepted by the quarantine authority for international exchange without any checking. Therefore, using this technique , crop plants can be easily exchanged in crop improvement programmes that are based on materials from different parts of the world. Limitation of Tissue Culture in Plant Disease Elimination: The overall operation involving the production, multiplication and maintenance of the in vitro regenerated plants required a good knowledge of pathology. Viruses can be transmitted either mechanically , through an insect vector or through other biological agents. The advantages of the tissue culture technique in raising disease free plants can be offset by increased susceptibility of these plants to attack from more severe viruses and fungi. One of the reason for this increased susceptibility maybe the altered nutritional and physiological state of the tissue culture derived plants as a result of pathogen eradication. It is also essential to have a good knowledge of greenhouse maintenance to control the reinfection of disease free plants.
  • Page48 b. Method of Virus Elimination i) Thermo-therapy and Meri-stem Culture: Conveniently viruses are eliminated by thermo-therapy of whole plants in which the plants are exposed to temperature between 35 to 40 0C for a few minutes to several weeks depending on the host-virus combination. Thermo-therapy is based on the fact that most viruses are killed at temperature much below those which kill their host plants. Thermotherapy is usually effective against iso-metric and thread –like viruses. Thermotherapy is often combined profitably with meri-stem culture to obtain virus- free plants in general; shoot-tips are excised from heat treated plants since larger ex. Plant can be safely taken from heat treated plants. 2. Cryo-therapy: Prolonged exposure of shoot tip culture to a low temperature ( 5 0C) has proved quite successful in virus elimination. This is often called Cryo-therapy. E.g. In case of chrysanthemum, shoot tip culture exposed with 5 0C four (4 ) months yieldest 67 % plant free from chrysanthemum stunt virus (CSV) and 22% plants were fre from chrysanthemum chloritic mottle virus ( CCMV). The frequency of virus free plants increased to 73 % in case of CSV and to 49 percent in CCMV after 7.5 months exposure to 5 0C. The cold treatment is preferred as the heat treatment is less injurious to the plants and often more effective in virus elimination. 3. Chemo-therapy: Some chemicals viz. Virazole, Actinomycin-D, Cyclohexamide, etc. which affect the virus multiplication may be added into the culture medium for curing the shoot tips virus. 4. Virus Indexing: Testing of plants for the presence or absence of concerned viruses is called virus indexing. Every meri-stem tips or callous derived plants must be tested before using it is a mother plant to produce virus-free stocks. This necessitates indexing of plants several times at periodical intervals and only those individuals which give consistency negative results should be labelled as virus tested for specific viruses. a) Sap Transmission Test: In which the sap from test plants may be used to inoculate highly sensitive and healthy indicator plants of a specific virus or group of viruses. An indictor plant of a specific virus is that plant species on variety which is highly susceptible to the virus and readily develop the symptoms. The inoculated indicators plants are maintained in a green house or aphid- proof cages. b) Elisa Test (Enzyme Linked Immuno Sorbant Assay) : This test is performed by adding a drop of centrifuged sap of a test plant to a drop of anti-serum taken from the blood of rabbit, if the virus is present, the precipitation will take place due to the
  • Page49 presence of specific anti-bodies in the blood. The Elisa is one of the serological methods used to identify virus based on anti-body reaction. It is most convenient, rapid and effective test system especially when a large number of samples are to be handled.
  • Page50 12. Micro Propagation Micro Propagation: In nature, the method of plant propagation may be either asexual (by multiplication of vegetation parts) or sexual (through generation of seed ). Sexually propagated plants show a high degree from inbreds lines. Asexual reproduction , on the other hand, gives rise to plants which are genetically identical to the parent plants and thus permits perpetuation of the unique characters of the cultivars. Clonal Propagation: Multiplication of genetically identical copies of a cultivar by asexual reproduction is called clonal propagation. Micro Propagation: “Clonal propagation through tissue culture technique is called micro propagation.” “Regeneration of whole plant through tissue culture is popularly called micro Propagation.” Micro Propagation can be achieved in a short time and space. Thus, it is possible to produce plants in large numbers starting from a single individual. Use of tissue culture for microprogation was initiated by G.Morel (1960) in orchid. a. General Procedure of Microprogation In vitro micro propagation is a complicated process requiring many steps or stages Murashige (1978) , proposed four distinct stages that can be adopted for overall production technology of clones commercially. Stages I-III are followed under in vitro conditions. Where as stage IV is accomplished in greenhouse condition. Debergh and Maene (1981) suggested an additional stage O for various micro propagation systems. Establishment of a reproducible system with well characterised with well characterized stages is a pre-requisite for promotion of projection targets and schedule in the commercial of plants. 1. Stage O: This is initial step of micro propagation in which stock plants used for culture initiation are grown for at least 3 months under carefully monitored conditions. Stock plants are grown at a relatively low humidity and watered either with irrigation tubes or by capillary sand beds or mats. This stock plant preconditioning stage also includes measures to be adopted for reduction of surface and systemic microbial contaminants. 2. Stage 1:
  • Page51 Murashige defined this stage as the initiation and establishment of aseptic cultures. The main steps involved are preparation of the explant followed by the establishment on a suitable culture medium. Cultures are initiated from explants several organs but shoot tips and auxillary buds are most often used for commercial micro propagation. Procedures to surface sterilise the explant and induce a healthy growth in the culture medium defined for each species may be devised. It may also be advisable to control microbial contaminantion within explant tissues in case such efforts at stage O were not successful. Stage I lasts 3 months to 2 years and requires atleast four passages of the subculture. Usually explants carrying a performed vegetative bud are suitable for enhanced auxillary branching. When objective is to produce virus free plants from an infected individual, it becomes obligatory to use cultures derived from submillimetre shoot tips. If stock plants are tested virus- free, the most suitable explants are nodal cuttings. These are some advantages in using small sized explants for micro propagation. Small shoot-tip explants have low survival rate and show slow initial growth. Meristem- tip cultures may also result in the loss of certain horticultural traits exhibited by the presence of virus. Therefore sub-terminal or slightly older segments are desirable which can withstand the toxic effects of sterilization agents much better than the terminal cuttings. For rhizomatic plants, runner tips are commonly used. 3. Step II: This stages takes up the bulk of micro propagation activity using a defined culture medium that stimulates maximum proliferation of regenerated shoots. Various approaches followed for micro propagation include: a) Multiplication through the growth and proliferations of meristems excised from apical and axillary shoots of the parent plant. b) Induction and multiplication of adventitious meristems through a process of organogenesis or somatic embryogenesis directly on explants. c) Multiplication of calli derived from organs, tissues, cell or protoplasts and their subsequent expression of either organogenesis or somatic embryogenesis in serial subculture. Shoots obtained from these calli can be further multiplied following procedures a) and b). Passage or harvest cycle generally requires 4 weeks. Shoots are harvested from the multiplying culture to either be sold as a Stage II product or carried onto Stage III. Generally stage II lasts to 10-36 months with large number of subcultures of similar age. 4. Stage III: Shoots proliferated during stage II are transferred to a rooting medium. Sometimes shoots are directly established in the soil as micro cuttings to develop roots. Since such a possibility depends on the particular species and at present, a large number of species cannot be handled in this manner. The shoots are generally rooted in vitro. When the shoots or plantlets are prepared for soil, it may be necessary to evaluate the survival factors such as i) Dividing the shoots and
  • Page52 rooting individually ii) Hardening the shoots to increase their resistances to moisture stress and diseases. iii) Rendering the plants capablr of autotrophic development in contrast to the heterotropic state induced by culture and iv) Fulfilling requirement of breaking dormancy, especially of bulb ceops. Stage III requires 1-6 weeks. 5. Stage IV: Steps taken to ensure successful transfer of the plantlets of Stage III from the aseptic environment of the laboratory to the environment of greenhouse comprise stage IV. Unrooted stage II shoots are also acclimatised in Suitable compost mixture or soil in pots under control conditions of light, temperature and humidity inside the greenhouse. In such cases stage III is skipped. Supplying bottom head-aids to pots with plantlets or cuttings and maintenance of a dense fine- particle fog system, within the greenhouse enhances the rooting process. Complete plants can also be established in the artificial growing media such as soilless mixes, rockwood plugs or even sponges. It takes 4-16 weeks for the finished product to be ready for sale or shipment. b. Micropropagation Techniques 1. Micropropagation by Axillary and Apical Buds: Axillary and apical shoots contain quiescent or active meristems depending on the physiological state of the plant. Vascular plants with an interminate mode of growth have in their leaf axils subsidiary meristem with potential for growing into a shoot. However, only limited numbers of axillary meristems have the capacity to develop in vivo if the type of branching of a particular species display apical dominance. Shoot tips cultured on basal medium containing to growth regulators typically develop into single seedling like shoot with strong apical dominance. On the contrary , when the shoots of the same explant material are grown on culture media containing cytokinins, axillary shoots of the same explant material are grown on culture media containing cytokinins , axillary shoots develop precociously which proliferate to form clusters of secondary and tertiary shoots. These cultures can be further subdivided into smaller clumps of shoots or separate shoots, which in turn , will form similar clusters when subcultured on fresh medium. about 5-10 multiplication rates can be achieved on a regular 4-8 week micro propagation cycle, which may ultimately lead to extremely impressive rapid clonal propagation level in the range of 0.1 -3.0 X 10-6 within one year. In general, the technique of proliferation by axillary shoots is applicable to any plant that produces regular axillary shoots and responds to cytokinins such as BAP, 2 ip and Zeatin many forest and orchid tree species are good candidates for in vitro clonal propagating using axillary shoots. Apical shoots (1-5mm) are normally cultured on media containing mixture of auxin ( 0.01- 0.1mg/l) and cytokinins ( 0.05-0.5 mg/l). The level of cytokinins is raised subsequent subcultures to induce an acceptable rate or proliferation without yellowing distoration of shoots. If the presence of cytokinins in the median inhibits root development culture material is often
  • Page53 transferred to a rooting medium in stage III which contains either no or reduced level of cytokinins. 2. Micro Propagation by Adventitious Shoots: Adventitious shoots are stem and leaf structures that arise naturally on plant tissues located in sites other than at normal leaf axil regions. These structures include stem, bulbs, corms, tubers and rhizomes. Almost every one of these organs can be used as cutting in conventional clonal propagation. Similar type of adventitious shoot development can be induced in cultures by using a suitable explant from preconditioned plant material and appropriate levels of growth regulators in the medium. Bulbs and corms grow from meristem at the base of leaves and scales where they join the basal plants. These meristematic regions regenerate multiple shoots on a suitable culture medium. Continuous propagation by adventitious shoot proliferation from bulb and corms can be achieved by cultivating two vertically split piece of shoot bases. Clusters of shoots develop from around the abaxial surface of developing leaves and scales. Senescence and dormancy in such cultured material can be prevented in vitro by trimming of shoots within 2-3 mm of the basal plate. This method is found useful in cultures of Iris, Lilum and Tulipa hybrids. Clonal propagation by adventive embryo formation is another useful approach following for many important plant species. Adventive embryos can directly arise from a group of cells within the explants or from primary embryoids. Orchids produce a large number of embryos at the tip of leaves in vitro, while cultivar of citrus and mangifera develop polyembryos from the nucellar tissues. Adventitious embryos obtained in vitro by inducing embryogenesis on explants are good material for clonal propagation. 3. Micropropagation: Differentiation of plants from cultured cells via shoot-root formation or somatic embryogenesis, where applicable, can be the fastest method of shoot multiplication and cloning of plant species. However, cultures in which calli are produced tend to be of low value as a means of micro propagation. The most serious drawback in the use of callus cultures for shoot multiplication is the genetic instability of their cells, due to which the initial plant regeneration capacity of the tissue may decline with the passage of time. In vitro propagation via organogenic or embryogenic or embryogenic calli is unfavourable in some economically important species of cereals, forage legumes, citrus, and coffee. Forest and tropical palm trees. Even plant regeneration from protoplasts also requires passage through at least one callus stage. Production of many thousands of plantlets from calli either derived from cell suspension or isolated protoplasts constitutes unique cases of cloning. Such as ‘calliclones’ and ‘protoclones’. Such clones commonly exhibit Somaclonal variations. c. Factors Affecting Shoot Multiplication Morphogenesis and Proliferation rate of culture depend on various factors, influencing the relative incidence of organogenesis or embryogenesis.
  • Page54 i) Physiological Status of Plant Material: Successful cultures are rarely obtained from senescing tissues. Explant isolated from more recently produced parts of the plant are more regenerative than those from older regions. The regenerative potential of tissue culture diminished with each year of maturation. Papaya tissue cultures can be established in hot summer months, whereas flower stem explants of Tulipa gives rise to shoots only when excised during the dry storage phase. ii) Culture Media: The standard tissue culture media are more suitable for achieving stage I and II of Micropropagation. Only stage III requires some modifications. As proposed by Skoog and Miller (1957), that organ differentiation in plant is regulated by an interplay of auxin and cytokinins should work as guide when developing a new medium for a new plant for micro propagation. To induce adventitious root formation, after axillary shoot propagation, cytokinins is usually omitted and auxin added. GA and ABA in a medium also reported to inhibit root formation. Activated charcoal may also induce the formation of adventitious rots in some species. Its presence in medium reduce the light supply to in vitro regenerated shoots and helps in removing inhibition by the absorption of all such compounds released in culture. iii) Culture Environment: Although in vitro regenerated shoots are heterotrophs, the light absorbed by the photosynthetic pigments in cultured tissue, plays an important role in inducing the morphogenesis of these tissues. The optimum light intensity found for shoot multiplication in most of the species is 100 lux. The quality of light also controls the organogenic differentiation and growth of shoot in cultures. For example, blue light (467 nm) induces bud formation in tobacco shoots and even doubled the number of lettuce shoots regenerated from callus cultures. Red and far-red light induce root formation. A diurnal illumination of 1 hr day and 8 hr night is generally found satisfactory for multiplication and proliferation of shoot. Most micro propagation culture are normally maintained at 25 0C. iv) Genotype: The genotype differs for regeneration capabilities in culture. The microprogation system develops for one particular cultivar will not automatically be applicable to another even within the same species. It has been found in grape that genotype with vigorous germination and branching capacity propagated rapidly. v) In Vitro Rooting: Media having low concentration of salt have been satisfactory for rooting of shoots micropropagated at stage II. Roots are mostly induced in the presence of suitable auxin in the medium although the shoots of some plants. Narcissus and strawberry may readily root on a hormone free medium.
  • Page55 vi) Acclimatization of Plants Transferred to Soil: Micropropagation on a large scale can be successful only when plants after transfer from culture to the soil show high survival rates and cost involved in the process is low. Tissue cultured plants generally show some structural and physiological abnormalities which , include, a) Abnormal leaf morphology and anatomy, (b) Poor photosynthetic efficiency, (c) marked decrease in epicuticular wax and (d) malfunctioning of stomata. These characteristics as well as heterotrophic mode of nutrition and poor mechanism- for- water loos control further render micropropagated plants vulnerable to transplantation shock. It is essential to wash thoroughly the lower part of tissue cultured plants/ shoots before their transfer to potting mix (pumice, peat, vermiculite, soil, sand or their mixtures in different proportions). Transplanted plantlets or shoots are immediately irrigated with an inorganic nutrient solution and maintained under high humidity for the initial 10-15 days. Storage organs have been induced in cultured shoots of several species. These structure donot require hardening and can be directly transplanted in soil. a well known example chloramequat. Other example is dioscorea species. In vitro cormlet formation in gladiolus requires high concentration of sucrose. d. Advantages and Limitations of Micropropagation Advantages of Micropropagation: 1. Requires relatively small growing space. 2. The technique of micropropagation is applied with the objective of enhancing the rate of multiplication. Through tissue culture over a million plants can be grown from a small, even microscopic, piece of plant tissue within 12 months. 3. Shoot multiplication usually has a short cycle (2-6 weeks) and each cycle results in logarithmic increase in number of shoots. 4. Tissue culture gives propagules such as minitubers or microcorms for plant multiplication throughout the irrespective of the season. 5. The small size of propagules and their ability to proliferate in a soil free environment facilitate their storage on a large scale ability to proliferate in a soil free environment facilitate their storage on a large scale and also allows their large scale dissemination by suitable means of transport across international boundries 6. Stocks of germplasm can be main for many years. 7. Pathogen free plants can be raised and maintained economically.
  • Page56 8. Clonal propagation in dioecious is extremely important since seed progently yields 50% females and 50% male plants of one sex are desired commercially. For example, male plants of asparagus officinalis are more valuable then female plants. Propagation by stem cutting in male asparagus is not successful but can be achieved by tissue culture. Limitations of Micropropagation: 1. Sophisticated facilities are required. 2. Demands greater skill in handling and maintenance than conventional techniques. 3. Shoot-tip derived plants may show genetic instability, E.g. 6-35% of banana clones developed through shoot tip culture show morphological variation.
  • Page57 13. Anther, Pollen and Ovule Culture (Haploid Production) An important aspect of plant breeding is the induction of maximum genetic variability of germplasm sources to secure a wider scope for selection and introduction of better trait qualities in existing crop species. Plant breeders have worked extensively to obtain haploids either in vitro or in vitro. In nature, haploids arise as a result of parthenogenesis and these plants rarely produce characters of the male parent. In angiosperms the haploid or Gametophytic phase is extremely brief and is extremely brief and is represented by pollen grains in anther and one cells in the embryo sac of the ovule. A typical anther in cross section shows two anther lobes and each lobe possesses two pollen two pollen sacs. During microsporogenesis, pollen mother cell (PMC) inside the pollen sac form pollen tetrad by meiosis. In each pollen tetrad, four pollens are held together temporarily by their callose wall. Pollens separate as descrete unit by dissolution of callose wall. Each pollen possesses an unique genome where every gene is present as a single copy. Exploitation of this unique genetic unit and the totipotency of the plant cell is the basis of anther or pollen culture for the production of haploid plants. On the other hand, egg cell produced within the ovule is very difficult to separate from complex tissue integration. In culture, the anther swells and dehisces along its upper margin, lengthwise. This phenomenon helps to expose the pollen grain. Alternatively, huge amount of pollen grains can be isolated manually and can be cultured aseptically very easily. Therefore, pollen is more suitable material than egg cell for the production of haploid. The development and production of haploid plant in vitro is very important for the study of fundamental and applied aspects of genetics in the higher plants. Production of homozygous diploid by doubling the chromosome number of haploid in vitro makes a pure line in single step and such homozygous pure line is of great importance in plant breeding. Anther Culture: Anther culture is technique by which the developing anthers at a precise and critical stage are excised aseptically from unopened flower bud and are cultured on a nutrient medium where the microspore within the cultured anther develop into callus tissue or embryoids that give rise to haploid plantlets either through organogenesis or embryogenesis. Pollen Culture: Pollen or microspore culture is an in vitro technique by which the pollen grains, preferably at the uninucleated stage, are squeezed out aseptically from the intact anther and then cultured on nutrient medium where the microscope, without producing male gametes, develop into haploid embryoids or callus tissues that give rise to haploid plantlets by embryogenesis or organogenesis. Ovule Culture:
  • Page58 Ovule culture is an elegant experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions. a. Androgenesis Androgenesis is the in vitro development of haploid plants originating from totipotent pollen grains through a series of cell division and differentiation. There are two methods of androgenesis as under: i) Direct Androgenesis: In this type, microscope behaves like a zygote and undergoes change to form embryoids which ultimately gives rise to plantlet. ii) Indirect Androgenesis: In contrast to the direct androgenesis, the microspores, instead of undergoing embryogenesis, divide repeatedly to form callus tissues which differentiate into haploid plantlets. b.Principle of Development of Androgenic Haploids The basic principle of anther or pollen culture is the production of haploid plants exploiting the totipotancy of micro spore and the occurrence of single set of chromosome (n) in the microspore. In this process, normal development and the function of pollen cell to become a male gamete is stopped and is diverted forcely to a new metabolic pathway for vegetative cell division. For this objective, microspores , either within intact anther or in isolated state , are grown aseptically on the nutrient medium where the developing pollen grain will form callus tissue or embryoids that ultimately gives rise to haploid plantlets. The principle behind the anther culture is that without disturbing the natural habitat and environment of the enclosed anther, pollen can be grown by culturing the intact anther. In culture condition, the diploid tissue of anther will remain living without proliferation at the selective medium and , at the same time it will encourage the development of pollen by nursing and providing nutrient. The haploid embryoids or the callus tissue can be seen as the anther dehisces in culture. But there is always possibility that the diploid callus or plantlets. To avoid this problem, free pollens isolated from the anther are grown in nutrient medium. Pollens at the uninucleate stage, just before the first mitosis is most suitable for induction of haploids. Induction of haploids can be enhanced by keeping the anther or flower bud at low temperature. Low temperature causes the dissolution of microtubules, alteration in the first mitosis or maintenance of higher ratio of viable pollen capable of embryogenesis. Cold treatment may also act to help the embryogenesis of the anther which is considered to be inhibitory for the production of haploids.
  • Page59 Nutritional requirement of the excised anther are much similar than those of isolated micro spores. Rich medium should be avoided for anther culture since it proliferate the diploid tissue of anther wall. Incorporation of activated charcoal into medium has stimulated the induction of androgenesis. Iron, potato extract, coconut milk and growth regulators like auxin and cytokinins are used in anther and pollen culture due to their stimulatory effect on androgenesis. c. Pathway of Microscope Division in Androgenesis For pathways based on few initial divisions in the microscope have been identified as leading to in vitro androgenesis. i) Pathway I: The microspore divide by an equal division and two identical daughter cells contribute to the sporophyte development. Vegetative and generative cells are not distinctly formed in this pathway. ii) Pathway II: The division of uninucleated microspore is unequal, resulting in the formation of a vegetative and a generative cell. The Sporophytic arises throught further divisions in the vegetative cell while the generative cell either divide or does so once or twice before degenerating. iii) Pathway III: The uninucleate microscope undergoes a normal unequal division but pollen embryos are predominantly formed from the generative cell alone. The generative cell either does not divide at all or does so only to a limited extent. iv) Pathway IV: The division of microscope is asymmetrical as in pathway II. Both vegetative and generative cells divide further and contribute to the development of sporophyte.( Example: Datura innoxia, occasionally , Datura metal, Atropa belladonna). Irrespective of the above early pattern of microspore divisions, the embryogenic pollen grains ultimately become multicellular and burst open, gradually assuming the form of a globular embryo. This is followed by the normal stages of postglobular embryogeny until the development of plant. Alternatively, the multicellular mass liberated from the bursting pollen grain proliferates to a form a callus which may later differentiate into whole plants either on same medium or on a modified medium. d. Protocol for Anther Culture Tobacco is the ideal material for anther culture. So the basic protocol described below should be applicable to anther culture in general with modifications. The immature anthers containing
  • Page60 uninucleate pollens at the time of first mitosis are the most suitable material for the induction of haploids. 1. Collect the flower buds of Nicotiana tabacum at the onset of flowering. Select the flower bud of 17-22 mm in length when the length of the sepals equals that of the petals. Reject all flower buds which are beginning to open. 2. Transfer the selected flower buds to the laminar airflow. Each flower bud contains five anther and these are normally surface sterile in closed buds. The flower buds are surface sterilized by immersion in 70% ethanol for 10 seconds followed immediately by 10 minutes in 20% sodium hypochlorite. They are washed three times with sterile distilled water. Finally transfer the buds to sterile petridish. 3. To remove the anthers, slit the side of the bud with a sharp scalpel and remove them, with a pair of forceps, place the five anthers with the filaments to another petridish. The filaments are cut gently. Damaged anthers should be discarded as they often form callus tissue the damaged parts other than the pollens. 4. Anthers are placed on agar solidified basal MS or White or Nitsch and Nitsch medium. 5. The culture is kept initially in dark. After 3-4 weeks, the anthers normally undergo pollen embryogenesis and haploid plantlets appear from the cultured anther. In some cases, anther may undergo proliferation to form callus tissue which can be induced to differentiate into haploid plants. 6. At this stage the cultures are incubated at 24-28 0C in a 14 hrs day light regime at about 2000 lux. 7. Approximately 50mm tall plantlets are freed from agar by gently washing with running tap water and then transferred to small pots containing autoclaved potting compost. Cover each plant with glass beaker to prevent desiccation and maintain in a well-lit-humid green house. After some week, remove the glass beaker and transfer the plant to larger pots when the plants will mature and finally flower. e. Protocol for Pollen Culture Isolated pollen can be cultured by two methods. Method I: This method is described here for the culture of isolated pollen of tobacco. This technique can be considered as the basic protocol for pollen culture and involves the following procedure. 1. Selection of suitable unopened flower bud, sterilization, excision of anther without filaments are the same as described previously in anther culture.
  • Page61 2. About 50 anthers are placed in small sterile beaker containing 20 ml of liquid basal medium (MS or White or Nitsch and Nitsch). 3. Anthers are then pressed against the side of beaker with the sterile glass piston of a syringes to squeeze out the pollens. 4. The homogenized anthers are then filtered through a nylon sieve to remove that the anther tissue debris. 5. The filtrate or pollen suspension is then centrifuged at low speed ( 500-800 rpm/min) for five minutes. The supernant containing fine debris is discarded and pillet of pollen is suspended in fresh liquid medium and washed twice by repeated centrifugation and resuspension in fresh liquid medium. 6. Pollens are mixed finally with measured volume of liquid basal medium so that it makes the density of 10 3-10 4 pollen/ml. 7. A 2.5 ml of pollen suspension is pipetted off and is spread in 5 cm petridish. Pollens are best grown in liquid medium but, if necessary, they can be grown by plating very soft agar added medium. Each dish is sealed with cello tape to avoid dehydration. 8. Petridishes are incubated at 27-30 0C under low intensity of white cool light (500 lux,16 hrs). 9. Young embryoids can be observed after 30 days. The embryoids ultimately give rise to haploid plantlets. 10. Haploid plantlets are then incubated at 27-30 0C in a 16 hrs day light regime at about 2000 lux. Plantlets at maturity are transferred to soil as described in anther culture. Method 2: This method is known as nurse culture technique. Sharp et.al. ( 1972) first introduced this method. The steps are as under. 1. Selection of flower bud sterilization excision of anther, isolation of suitable pollen is the same as described previously. 2. In this method, the intact anthers are placed horizontally on the top of solid or semisolid basal medium within a conical flask. 3. A small filter paper disc is placed over the intact anther and about 10 pollen grains in the suspension are then placed on the filter paper disc. Here the intact anthers are considered as the nurse tissue. A control set is also prepared in exactly the same way except that the pollen grain on the filter paper are directly kept on solid medium. Sometimes, callus tissue derived from any part of the plant is used as nurse tissue.
  • Page62 4. With this method, pollen grains the control set did not grown at all. The pollen gains kept on nurse tissue grow and form a culture of green parenchymatous tissue in two weeks, such tissue ultimately form the haploid callus tissue. f. Advantages of Pollen Culture over Anther Culture i) Overcrowding of pollen grains in anther is eliminated and isolated pollen grains are equally exposed to nutrient medium. ii) Unwanted growth of diploid cells of anther wall and other associated tissue is eliminated. iii) The stage of androgenesis can be observed starting from single cell. iv) Various factors governing androgenesis can be better regulated. v) Pollen is deal for uptake, transformation and mutagenic studies as pollen can be uniformly exposed to chemicals and physical mutagens. vi) Pollen may be directly transformed into an embryoid. So it is very suitable for understanding biochemistry and physiology of androgenesis. vii) Higher yield of haploid plants per anther could be expected in pollen culture than the anther culture. g. Production of Homozygous Diploid Plants Haploids plants derived from either anther culture or pollen culture are sterile. These plants contain only one set of chromosomes. By doubling their chromosomes number, the plants can be made fertile and resultant plants will be homozygous diploid or isogenic diploid. These homozygous diploid plants show the normal meiotic separation. The fertile homozygous diploid plants are more important than the sterile haploid plants and can be used as pure line lines in breeding programme. Haploids plants can be diplodized by following methods. i) Colchicine Treatment: Colchicine has been utilized widely as spindle inhibitor to induce chromosome duplication and to produce polyploid plants. The young plantlets while still enclosed within the anther are treated with 0.5% colchicine solution for 24-48 hrs. Treated plantlets are planted in the medium after through washing. In case of mature haploid plantlets, 4% colchicine- lanoline pasts may be applied to the axil of the leaves. ii) Endomitosis: Haploids cells are unstable in culture and have tendency to undergo Endomitosis. i.e chromosome duplication without nuclear division. This property can be used for obtaining homozygous diploid plants. In this process, a small explant of stem from a haploid plant is
  • Page63 cultured on auxin-cytokinin added medium where the segment forms the callus tissue. During callus growth, diploid homozygous cells are produced by endomitosis. Now large number of isogenic diploid plants can be obtained by organogenesis. iii) Fusion of Pollen Nuclei: Homozygous diploid callus or embryoids may form by the spontaneous fusion of two similar nuclei of the cultured pollen after first division. In Brassica, the frequency of spontaneous nuclear fusion in microspore is high in culture. h. Application of Haploids in Plant Breeding In Vitro production of haploids can solve some problems in genetic studies since gene action is readily manifested due to a single allelic gene present in chromosome of entire genome. 1. Releasing New Varieties through F1 Double –haploid System: Haploid breeding technique usually involve only one cycle of meiotic recombination. However, many agronomic traits are polygenically controlled. One cycle of recombination is usually insufficient for the improvement of such quantitative traits since linkage between Polygenes will not release all potential variations available in the cross. To overcome these disadvantages, the Chinese developed a method combining anther culture with sexual hybridization among different genotypes of anther derived plants. The anthers of the hybrid (F1) progeny are excellent breeding material for raising pollen-derived homozygous plants (Double –haploids) in which complementary parental characteristics are combined in one generation. Double –haploids are also useful in studies related to inheritance of quantitative traits. Using double –haploid technique new varieties have been developed in barley, Brassica, rice, maize , rye, potato, pepper and asparagus. 2. Selection of Mutants Resistance to Diseases: Mutants with resistance to disease is of prime importance in crop improvement. Haploids provide a relatively easier system for the induction of mutations. Some examples of using anther culture technique in mutant successfully are tobacco mutants resistant to black shank disease and wheat lines resistant to scab. (Fusarium graminearum). 3. Developing Asexual Lines of Tree Perennial Species: Chinese workers obtained pollen –derived rubber tree taller by sic meters which could then be multiplied by asexual propagation to raise several clones. Another example of pollen –haploids in plant improvement is popular. 4. Transfer of Desired Alien Gene:
  • Page64 Chromosomal instability in haploids makes them potential tools for introduction of alien chromosomes on genes during wider crossing programmes. In rice , developing a resistance to blast requires about 12 years by conventional breeding through back crossing. Through hybridization and anther culture, this can be achieved in two years (Examples: cv . Zhonghua No.8 and 9 released by the institute of crop Breeding and cultivation in china. 5. Establishment of Haploids and Diploid Cell Lines of Pollen Plant: The anther culture technique was used to establish both haploid and diploid somatic cell lines of pollen plants in wheat and maize. Similarly, a haploid tobacco line resistant to methionine sulfoxomide was selected which turned out to be identical in phenotype and effect to the toxin produced by the pathogen Pseudomonas tabaci. i. Importance and Implications of Anther and Pollen Culture 1. Haploids derived from anther and pollen culture are useful in cytogenic studies. 2. Recessive phenotypic characters can be identified easily by comparing heterozygous diploid with haploid or homozygous diploid population. 3. Double haploid that are homozygous and fertile, are readily obtained, enabling the selection of desirable gene combination. 4. Culture of isolated pollen provides a novel experimental system for the study of factor controlling pollen embryogenesis of higher plants. 5. Study of meiotic behaviour of haploids valuable cubes to measure chromosome duplication within a species for understanding of phylogentic relationship between species. It also provides information for the interpretation of chromosome homology. 6. Genetic analysis could be performed on haploid population to establish inheritance patterns. 7. Use of haploids in production of monosomics, nullisomics and other aneuploids. This approach has been used in tobacco for the isolation of nullisomics, trisomics. j. Ovule Culture – Meaning, Principle and Protocol Ovule culture is an elegant experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions. Principle:
  • Page65 An ovule is a megasporangium covered by integument. An ovule contains a megaspore or an egg cell. After fertilization a single cell zygote is formed which ultimately leads to form a mature embryo possessing shoots and root primordia. In Vitro ovule culture helps to understand the factors that regulate the development of zygote through organised stages to a mature embryo. Alternatively, it may be possible to germinate pollen in the same culture as the excised and to induce in vitro fertilization and subsequent embryo production. Protocol of Ovule Culture: 1. Collect the open flower. If fertilized ovules are desired, collect the open flowers where anthers are dehisced and pollination has taken place. To ensure the fertilization, collect the flower after 48 hrs of anther dehiscence. 2. Remove sepals, petals, androecium, etc from the overies containing either fertilized or unfertilized ovules. 3. Soak the overies in 6% NaOCL solution. 4. Rinse the overies 3-4 times with sterile distilled water. 5. Using sterile technique, ovules are gently prodded with the help of spoon shaped statula by breaking the funicules at its junction placental tissue. 6. The spatula with ovules is gently lowered into the sterile solid or liquid medium as the culture vial is slanted about 45 0 C. 7. Damaged or unorganised ovules are rejected when possible during transfer. 8. Incubate the culture in either dark or light at 25 0C. k. Importance of Ovule Culture 1. Test Tube Pollination and Fertilization: Through ovule culture, test tube pollination and fertilization can be done. By technique, it may be possible to germinate pollen in the same culture as the excised ovule and to induce a vitro fertilization leading to the formation of mature seeds containing viable embryos. 2. Application in Hybridization: Ovule culture has been successfully employed to obtain hybrid seedlings in Interspecific and Intergeneric crosses. In several interspecific crosses, the hybrid embryo of Abelmoschus fails to develop beyond the heart or torpedo-shaped embryo. By ovule cultures, viable hybrids have been obtained in three out of five crosses attempted in Abelmoschus species.
  • Page66 Although hybrid plants have not been obtained between different species of cotton through fertilized ovule culture, but seed development and the production of fibre from the cultures ovule have been demonstrated. 3. Production of Haploid Callus: It is possible to obtain haploid callus by culturing unfertilized ovules. 4. Ovule Cultures of Orchid Plants: In nature, the seeds of orchid germinate only in association with a proper fungus. As a result numerous seeds are lost due to unavailability of proper fungus. Besides this the seed capsule of many orchids takes a long time to mature. To overcome such problems, several attempts have been made to culture the fertilized ovule of orchid in vitro. 5. Induction of Polyembryos: In horticultural practises, the artificial induction of polyembryos holds a great potential. It has been observed that the nucellus of mono-embryonic ovules of citrus can be induced to form adventive embryos in culture. 6. Virus Irradication: In the varieties of citrus which are impossible to free of virus by other means, the ovule culture has proved decisively advantages to make them virus free.
  • Page67 14. Embryo Culture In angiosperms, embryo represents the beginning of sporophyte. Normally, the fertilized egg or zygote undergoes embryogenesis in the post fertilization stage within the ovule and thus embryo is formed inside the seed. The typical seed embryo is a bipolar structure consisting of contrasting meristem at each pole- the primordial shoot or the plumule and the primordial root or radicle and one or two lateral appendages, the cotyledons. The mature embryo, therefore, possesses the basic organization of the adult plant. During seed germination a plant is produced through progressive and orderly changes in embryo. Like many other plant organs, embryo can be used as explant and cultured aseptically in the test tube containing medium. Embryo Culture: The embryo of different developmental stages, formed within the female gametophyte through sexual process, can be isolated aseptically from the bulk of maternal tissues of ovule, seed or capsule and cultured in vitro under aseptic and controlled physical in the glass vials containing solid or liquid nutrient medium to grow directly into plantlet. a.Types of Embryo Culture According to Pierik (1989), there are in principle two types of embryo culture. I. Culture of Immature Embryo: This type of embryo culture is mainly used to grow immature embryos originating from unripe or hybrid seeds which fail to germinate. Excising such embryos is difficult and generally a complex nutrient medium is required to raise them to produce plants. II. Culture of Mature Embryos: Mature embryos are excised form ripe seeds and cultured mainly to avoid inhibition in the seed for germination. This type of culture is relatively easy as embryo requires simple nutrient medium containing mineral salts, mineral salts, sugar and agar for growth and development. b.Technique of Embryo Culture 1. Surface Sterilization: Embryos of seed plants normally develop inside the ovule which in turn is covered by overies. Since they already exist in a sterile environment, disinfection of the embryo surface is unnecessary unless the seed coats are injured or systemic infection is present. Instead, mature seeds, entire ovule or fruits are surface sterilized. Surface sterilization is carried out by immersing the material in hypo chorine- containing commercial bleach ( 5-10% Clorox, 0.45% Sodium or Calcium hypochlorite) for 5- 10 min or ethanol ( 70-75%) for 5 min. A small amount ( 0.01-0.1%) of a surfactant may be added to disinfection solution. In case of infected seeds, the
  • Page68 excised embryos may be immersed in 70% alcohol plus 5-10 min exposure to 2.6% sodium hypochlorite solution. 2. Excision of Embryo: Embryo excision operation is carried out aseptically in a laminar airflow hood. A steremicroscope equipped with cool-ray flurescent lamp is required for excision of small embryo. The commonly used dissecting tools are foreceps, dissecting needles, scalpels, razor blades and Pasteur pipettes. Mature embryo can be isolated with relative ease by splitting open the seeds. Soaking a hard-coat seeds for few hours to a few days before sterilization makes its dissection easier. In case of embryos embedded in liquid endosperms, the incision is made at micropolar end of young ovule and pressure applied at opposite end to force the embryo out through the incision. 3. Embryo-endosperm Transplant: It is very difficult to culture embryo in vitro abort at very early stages of development because of lack of knowledge of nutritional requirements. The chances of development of immature or abortive embryos increases if they are surrounded by endosperm tissue excised from another seed of same species. Generally, endosperm older than the embryo by 5 days was more efficient as a nurse tissue than one of the same age as the embryo. 4. Nutritional Requirement: The nutritional requirements of an embryo during its development in vitro consisting two phase: a) Heterotropic phase- an early phase wherein the embryo is dependent and draws upon the endosperms and materal tissues and b) The autotrophic phase- a later phase in which the embryo is metabolically capable of synthesizing substances required for its growth, thus becoming fairly independent for nutrition. The media constituents for in vitro growth of young or immature embryos also differ from those of mature embryos. This often necessitates the transfer of embryos from one medium to another for their orderly growth. i) Mineral Salts: Inorganic nutrients of MS, B5 and White’s media with certain degree of modification are the most widely used basal media for embryo culture. Monnier (1978) modified the MS medium for immature embryo culture of Capsella which contains higher levels of potassium and calcium and reduced levels of ammonium (NH4NO3) and FeEDTA and double concentration of MS micronutrients. ii) Carbohydrates: Sucrose is the most commonly used source of energy for embryo culture. Addition of maltose, lactose, raffinose or mannitol may be required in embryo culture of some species. In some cases
  • Page69 glucose is found to be better than sucrose. Mature embryos grow fairly well at low sucrose concentration but younger embryos require higher level of carbohydrates. iii) Nitrogen and Vitamins: Ammonium nitrate is better than KNO3, NaNO3 and (NH4)2 HPO4. especially the presence of NH4+ in the medium has been found essential for proper growth and differentiation of embryos. Various Amino acids and their amides like aspargine, glutamine, and casein hydrolysate have been widely used in embryo culture. iv) Natural Plant Extract: The coconut milk ( CM) effectively stimulates the growth of excised young embryos of sugarcane, barley, tomato, carrot, Interspecific hybrids of Vigna and fern species. Van Overbeek et.al ( 1941) suggested that the coconut milk contains some ‘ Embryofactor’ which presumably makes up for deficiencies of certain sugars, amino acids, growth hormones and other critical metabolites of the culture medium. In addition to coconut milk, water extracts from dates, bananas, hydrolysate of wheat-gluten and tomato juice were also effective. v) Growth Regulators: Auxin and cytokinins are not generally used in embryo culture since they induce callus formation. At very low concentration GA promotes embryogenesis of young barley embryos without inducing precocious germination. ABA also has a similar effect on barley and Phaseolus embryos. vi) PH of Medium: Excised embryos grows well in a medium with a PH 5 to 7.5. Generally the medium PH is adjusted 0.5 units higher than the desired PH in order to compensate for uncontrollable change in its value during the autoclaving process. vii) Incubation Conditions: The embryo cultures are incubated at 25+-2 0C. whereas in case of species to warm temperature requires 27-30 0C incubation temperature and species occurring in cold regions or seasons require incubation temperature of 17-22 0C. Generally, an initial dark incubation ( 4 days) of embryo in culture is essential , following which they can grow to a mature stage even under continuous light regime. 5. Role of Suspensor in Embryo Culture:
  • Page70 Suspensor is actively involved in embryo development. The suspensor is an ephemeral structures found at the radicular end of the proembryo and attains maximum development by the time embryo reaches globular stage. In cultures the presence of a suspensor is critical , particularly for the survival of young embryos. The requirement of the suspensor may be substituted by the addition of GA or ABA to the culture medium. c. Protocol for Embryo Culture The following protocol for embryo culture is based on the method used for Capsella bursapastoris. With modification, this basic protocol should be applicable to embryo culture in general. 1. The capsules in the desired stages of development are surface sterilized for 5-10 minutes in 0.1% HgCl2 in a laminar air flow. 2. Wash repeatedly in sterile water. 3. Further operations are carried out under a specially design dissecting microscope at a magnification of about 90 X. The capsules are kept in a depression slide containing few drops of liquid medium. 4. The outer wall of capsule is removed by a cut in the region of the placenta; the halves are push apart with forceps to expose the ovules. 5. A small incision in the ovule followed by slight pressure with a blunt needle is enough to free the embryos. 6. The excised embryos are transferred by micropipette or small spoon headed spatula to standard 10 cm petri dishes containing 25 ml of solidified standard medium. Usually 6-8 embryos are cultured in petridish. 7. The Petri dishes are sealed with cello tape to prevent desiccation of the culture. 8. The cultures are kept in culture room at 25+- 1 0C and given 16 hrs illumination by cool white fluorescent tube. 9. Subcultures into fresh medium are made at approximately four weaks interval. In case of fresh seed or dry and imbided seeds, the schedule is slightly changed. Seeds are cleaned by 5% Teepol for 10 minutes and dipped in 70% ethyl alcohol for 60 seconds. Surface sterilization in 0.1 % HgCl2 is followed by washing in sterile water, then the seeds are decotylated using a sharp scalpel and embryos are transferred to solid nutrient medium. d. Application of Embryo Culture 1. Rescuing Embryos from Incompatible Crosses:
  • Page71 In Interspecific and Intergeneric hybridization programmes, incompatibility barriers often prevent normal seed development and production of hybrids. Although there may be normal fertilization in some incompatible crosses, embryo abortion results in the formation of shrivelled seeds. Poor and abnormal development of the endosperm caused embryo starvation and eventual abortion. Isolation of hybrid embryos before abortion and their in vitro culture may prevent these strong post-zygotic barriers. The most useful and popular application of embryo cultures is to raise rare hybrids by rescuing embryos of incompatible crosses. 2. Overcoming Dormancy and Shortening Breeding Cycle: Long, periods of dormancy in seeds delay breeding works, especially in horticultural and crop plants. Using embryo cultures techniques the breeding cycle can be shortened in these plants. For example, the life cycle of Iris was reduced from 2-3 years to less one year. Similarly, it was possible to obtain two generations of flowering against one in Rosa sps. Germination of excised embryo is regarded as a more reliable test for rapid testing of viability in seeds, especially during dormancy period. 3. Overcoming Seed Sterility: In early ripening fruit cultivars, seed do not germinate because their embryos are still immature. Using the embryo culture method it is possible to raise seedling from sterile seeds of early ripening stone fruits, peach, apricot and plum. ‘Makapuno’ coconunts are very expensive and most relished for their characteristics soft fatty endosperms in place of liquid endosperm in place of liquid endosperm. Under normal conditions the coconut seeds fail to germinate. Guzman et.al (1971) obtained 85% successes in raising field- grown makapuno trees with the aid of embryo cultures. 4. Production of Monoploid: An embryo culture has been used in production of monoploids of barley. With the cross Hordeum vulgare , fertilization occurs normally but thereafter chromosomes of H.bulbosum are eliminated, resulting in formation of Monoploid H. vulgare embryo which can be rescued by embryo cultures. 5. Clonal Micropropagation: The regenerative potentials is an essential pre-requisite in non-conventional methods of plant genetic manipulations. Because of their juvenile nature, embryos have a high potential for regeneration and hence may be for in vitro clonal propagation. This is especially true of conifers and graminaceous members. Both organogenesis and somatic embryogenesis have been induced in major cereals and forage grasses form embryonic tissues. Generally, callus derived from immature embryos of cereals has the desired morphogenetic potential for regeneration and clonal propagation.
  • Page72 15. Invitro Pollination Pollination and fertilization under in vitro conditions offer an opportunity for producing hybrid embryos among plants that cannot cross by conventional methods of plant breeding. In nature, Intergeneric or Interspecific hybridization occurs less frequently. This is due to barriers hindering the growth of the pollen tube on the stigma or style. In such cases the style or part of it can be excised and pollen grains either placed on the cut surface of overy or transferred through a hole in the wall of ovary. This technique, called intraovarian pollination, has been successfully applied in such species as Papaver sommiferum , Eschscholtiza California, Argemone Mexicana and Argemone ochroleuca. Another approach to overcome the barrier to pollen tube growth is direct pollination of cultured ovules or excised ovules together with placenta. This technique was developed at university of Delhi in papaveracea and solanaceae. Various other techniques developed to overcome the prezygotic barriers to fertility include: a) Bud pollination, b) Sub pollination, c) Heat treatment of style, d) Irradication and e) Mixed pollination. The development of seed through in vitro pollination of exposed ovules has been described as ‘test-tube fertilization’ whereas the process of seed formation following stigmatic pollination of cultured whole pistils has been referred to as ‘in vitro pollination’. Considering the fact that male gametes in plants do not float freely and are delivery by pollen tube, a general terms ‘in vitro pollination has been used for ovular pollination, overian pollination, placental pollination and stigmatic pollination under in vitro conditions. The vitro pollination can be accomplished by procedure by following appropriate sterilization procedure, suitable nutrient medium and selection of suitable explant. Application of in Vitro Pollination: In plant breeding the technique of in vitro pollination has potential applications in at least three different areas, viz, a) overcoming self-incompatibility b) overcoming cross-incompatibility, c) haploid production through parthenogenesis.
  • Page73 16. Somaclonal Variation It has been recognised that all plants regenerating from the tissue culture are not exactly the replicas of a parental form. Phenotypic variation is frequently observed amongst regenerated plants. Phenotypic changes are associated with genetic changes of an organism. The frequently genetic changes could result from mutations, epigenetic changes or combination of both mechanisms. Since genetic mutations are irreversible and likely to persist in the progeny of regenerated plants whereas epigenetic changes are not transmitted by sexual reproduction. It has been found that cells or tissues in cultures undergo frequent genetic changes resulting in new phenotypes on organogenesis or embryogenesis. Plant cell and tissue culture provides increased genetic variability. Variants selected in tissue cultures have been referred to as “calliclones” or “Protoclones”. Somaclonal Variation: According to Larkin and Scowcroft (1981), “Somaclonal variation is the genetic variability which is regenerated during tissue culture” or plant variants derived from any form of cell or tissue cultures. Gametoclonal Variations: Evans et.al.prefer the term “Gametoclonal variation” for variant clones specifically raised from gametic or Gametophytic cells. a.Mechanisms Causing Somaclonal Variation The Somaclonal variation may be attributes to i) pre-existing variation in the somatic cells of the explant or ii) Variation generated during tissue culture ( epigenetic) often both factors may contribute. The original Ploidy level of the plant or plant organ from which the explant is taken may play an important role in Somaclonal variation. Meristematic explants such as apical meristem derived from either shoot apex or axillary bud, have a lesser degree of genetic variability as compared to plants regenerated from non-meristematic explants which generally produce genetic variability. Cells of meristematic explant divide by normal mitosis and cells are maintained at a uniform diploid level. However, the cells in non-meristematic explants are derivation of the meristematic part of the plant and during their subsequent differentiation, do not divide by normal mitosis, but undergo DNA duplication and end reduplication. Endo reduplication leads to the formation of chromosomes with four chromatids, chromosomes with eight chromatids and polytene condition. When the cells of various genomic constitutions of the initial explants are induced to divide in cultures, the cells may exhibit changes in chromosome number such as aneuploids and polyploids. Organogenesis and or embryogenesis occur mostly from diploid cells. Therefore, pre-existing variation in explant tissue always rule out the Somaclonal variation in the culture. The presence of several chromosomal aberrations such as reciprocal translocation, deletion,
  • Page74 inversion, chromosome, reunion, multicentric, acentric fragments, Heteromorphic pairing etc were found among the somaclones of barley, ryegrass, garlic and oat. Besides these changes, there are examples of phenotypic variation which can be observed in plants regenerated from cultured cells or protoplasts variation which can be observed in plants regenerated from cultured cells or protoplasts where no apparent chromosomal abnormalitites are seen. b.Molecular Basis of Somaclonal Variation Variants may arise as a result of more subtle changes due to single gene mutations in cultures which have cells apparently showing no karyological changes. Recessive mutations are not detected in RO plants, but express in RI progeny. The RI progeny segregates in Mendelian 3:1 ration for the trait of interest. This further confirms the mutant nature of the variant. Somaclonal variants for single recessive gene mutations have been reported in respect of maize, Nicotina sylvestris, rice and wheat. In some cases specific genetically marked strains have aided in evaluation of plants regenerated from cell cultures. Changes in cytoplasmic genome have also been observed in somaclones. Gengenbach et. al. (1977) were succeeded in selecting resistant plants in maize combining resistance to toxin produced by Drechslera maydis with the cms T- trait. Both these traits are controlled by mtDNA. Another aspect of single gene mutation responsible for Somaclonal variation relates to transposable elements. Clourey and Kemble ( 1982) detected variation as a result of insertion of plasmid- like DNA in the mitochondrial genome of cms maize cell cultures. Somaclonal variation may also be molecular changes caused by mitotic crossing over in regenerated plants. This could include both symmetric and asymmetric variation. Single gene mutations by MCO may constitute s unique mechanism of inducing new genetic variations. Small changes in the structure of chromosomes could alter expression and genetic transmission of specific genes, such as deletion or duplication of a copy of gene or gene conversion during repair processes. Revent studies have demonstrated that changes in organelle DNA, isozyme and protein prodiles correlate with the occurance of Somaclonal variation in plants (Wheat, Rice, Potato, Maize, Barley, and Flax) . somoclones of wheat show alteration in gliadin profiles, nor loci, and qualitative as well as quantitative differencesin rDNA. c.Isolation of Somaclonal Variants Isolation and selection of Somaclonal variation is an important task. Since several changes are involved in producing Somaclonal variation in different plant species, it is very difficult to sort out the Somaclonal variants using a single selection system. A number of selection systems are now being used to select the variants. A. Selection without Selection Pressure: Unorganised callus and cells, grown in cultures for various periods on a medium that contains no selective agents, are induced to differentiate whole plants. The regenerated plants are ultimately
  • Page75 transferred to the field and screened for variation. Somaclonal variants of various crops like sugarcane, potato, tomato, granium, ceneals and grasses and lucern have been isolated for various desirable traits. B. Selection with Selection Pressure: In this method variant cell lines are screened from cultures by their ability to survive in the presence of a substance in medium that may be toxic or under condition of environmental stress. E.g. Amino acid analogue and amino acid resistance, disease resistance, herbicide resistance environmental stress tolerance, auxotrophic lines, antibiotic resistance etc. Different methods of selection and screening of Somaclonal variants are described below: 1. Analysis of Phenotypic Characters: Phenotypic variations may arise among the regenerates during culture. Such variant characters are observed thoroughly. The variants are transferred from culture flask to the field. In field, such variants plants are observed during their successive growth and development. such qualitative and quantitative characters viz, plant height, maturity date, leaf size, flowering date, yield, seed fertility, Waxiness in different plant parts flower morphology etc. are used as a parameter to sort out variants. Variants are also compared thoroughly with parental plants in all possible quantitative and qualitative phenotypic characters. Several consecutive seed generations of variants are analysed to pursue whether the variants character persist or not among the progeny. 2. Cytological Study of the Variant: The traditional methods of acetocarmine and feulgen-stained squashes of meristematic tissues of the variants permit the study of the number and gross morphology of chromosomes. So any change in chromosomes number or gross structure of chromosomes can be detected by this method. To have a better assessment of minor structural changes of chromosomes, banding technique can be used. 3. DNA Content of the Variant: DNA content of the feulgen stained interphase nuclei can be measured by cytophotometer. An uniformly diploid state of cells always maintains its fixed amount of DNA. Any material changes of chromosomes will show either higher or lower vales of DNA content. So the measurement of DNA content can be used as parameter for rapid screening of variants. 4. Gel Electrophoresis of Proteins or Enzymes: A somaclone could be variant for a number of biochemical characters. Among them gel electrophoresis of the proteins or enzymes extract from the homonized plant is a reliable parameter for detecting the variants. Any alteration in electrophoretic pattern of protein or enxymes indicates that the variants have lost or gained some specific proteins or enzymes
  • Page76 fractions. Assay of other biochemical products like pigments, alkaloids, aminoacids etc, using the sophisticated instruments have also revealed the extent of variation among the regruents. 5. Selection for Disease Resistance: Sometimes, disease resistance character may appear among Somaclonal variants where the parent is highly susceptible to particular disease. The pathogen or its toxin can be used as a selection agent during culture. If the in vitro selection is not feasible on cell, tissue or protoplast culture level, screening at seedling level is frequently possible. Behnke (1979) regenerated potato plants from callus selected for resistance to the toxin filtrate of Phytopthora infestans. Field resistance of some of the sugarcane variants has also been established. 6. Selection for Herbicide Resistance: The growth of weeds in a population of agricultural important crop is generally controlled by herbicides. As a herbicides have a short residual life , they are applied repeatedly on the crops. Some crops become susceptible to a herbicide due to its repeated application on them. The herbicide is generally added to the cell culture system and the regenerated plantlets showing the tolerance to herbicide are selected. The examples of herbicide resistant plants regenerated from cell cultures include Nicotiana tabacum, bentazone, chlorosulphon, isopropyl N-carbamate, phenmedifarm, picloram and paraquat and Medicago saliva. Herbicide tolerance can also be introduced into cells by somatic hybridization or through genen transfer technology. 7. Selection for Environmental Stress Tolerance: Salt, water-logging and drought, low and high temperatures and mineral toxicity and deficiency are frequently cited as environmental stresses. Many attempts have been made to isolate stress tolerant phenotypes in tissue culture. Selection of high sodium chloride tolerant cell lines in tobacco and regeneration of plants have been reported by Nabors et.al. Regenerated plants showed salt tolerance through two successive generations. Few attempts have been made to select for water –logging and drought resistance in cell cultures. Handa et.al. ( 1983) has repoted somaclonal variation for resistance to polyethylene glycol ( PEG) in tomato cells. Similarly, attempts were made to isolate variants to chilling stress in tomato, heat , tolerance in pear, aluminium toxicity and sorghum. 8. Auxotrophic Lines: Auxotophs are continuously used for DNA transformation, somatic hybridization and study of metabolic processes. The use of haploid cell cultures and the application of effective mutagenic treatment have made feasible the recovery of large number of auxotrophs. 9. Antibiotic Resistance: Cell lines resistance to the antibiotics streptomycin, linomycin, kanamycin, chlororamphenicol and cyclohexamide have been developed from various plant species.
  • Page77 d. Application of Somaclonal Variation 1. Somaclonal variation and gametoclonal variation represent useful source of introducing genetic variations that could be of value to plant breeders. 2. Single gene mutation in nuclear or organelle genome may give the best available variety in vitro that has a specific character. 3. Gametoclonal variation, induced mostly by meiotic recombination during the sexual cycle of F1 hybrid, results in trasngressive segregation to uncover unique gene combination. 4. Various cell lines selected in vitro may prove potentially applicable to agriculture and industry like resistance to herbicide, pathotoxin, salt or aluminium. 5. Variability in cell cultures has played a useful role in synthesis of secondary metabolites on a commercial scale. 6. Technique employed for Somaclonal and gametoclonal variation are relatively easier than recombinant DNA technique. Somaclonal variants for agronomically desirable traits in several crop plants have been raised from tissue culture. Some examples of Somaclonal variation in crop plants as well as in some horticulturally important plants are given below: Rice: Significant improvements relative to parent were observed for seed weight, seed proteins percentage, tiller number, panicle length and time of flowering. At IRRI, mutants were observed for many characters such as panicle, grain, and leaf morphology and tiller arrangement. Wheat: Variations were manifested for gliadin proteins in seed, grain colour, plant height, heading date and yield. Maize: Plants regenerated from selected cell lines were resistant both to T-toxin and to infection to Drechslera maydis causing southern leaf blight. Cytoplasmic male sterile lines are very sensitive to the T-toxin produced by Drechslera maydis. Potato: Somaclonal variants were selected for resistance to Phytopthora infestans and to its multiple races and resistance to early blight.
  • Page78 Tomato: Somaclones were isolated with variant phenotypes, such as recessive mutation for male sterility, resistance to Fusarium oxysporium, jointless pedicel , tangerine virescent leaf, flower and fruit colour. Sugarcane: Somaclonal variants have been isolated by different workers for cane yield, sugar yield and resistance to smut disease caused by Ustilago scitamini, downey mildew caused by Helminthosporium sacchari. Geranium: Skirvin and Jenick ( 1976) developed an improved scented geranium called ‘Velvet rose’ from Pelargonium species by isolating Somaclonal variant. The new cultivar has symmetrical flowers with large fertile stamens, five paired stigma and sets seed. The parential cultivar, on the contrary has asymmetrical flowers with reduced- sterile anthers, a two- paired stigma and never sets seeds.
  • Page79 17. Somatic Hybridization and Cybridization Sexual hybridization in higher plant is a valuable tool for the conventional plant breeding to improve cultivated crops. However, many desirable combinations of characters can not be transmitted through conventional methods of genetic manipulation. Secondly, conventional hybridization is limited to only very closely related species and was total failure for distantly related species as well as in sexually incompatible species. However, by using a protoplast fusion technology , it possible to fuse two genotypically different by protoplast to obtain para sexual hybrid protoplast. Definition of Somatic Hybridization: It is fusion between isolated somatic protoplasts under in vitro conditions and subsequent development of their product to a hybrid plant is known as somatic hybridization. Cybrid: Plasmid and mitochondrial genomes are inherited maternally in sexual crossings. Through the fusion process the nucleus and cytoplasm of both parents are mixed in the hybrid cell. This results in various nucleo- cytoplasmic combination. Sometimes interaction in the plastome and genome contribute to the formation of cybrid. Cybrids in contrast to conventional hybrids, possesses a nucleus genome from only one parent but cytoplamsmic gene Cybridization: The process of protoplast fusion resulting in the development of cybrid is called as Cybridization. In Cybridization heterozygosity of extra-chromosomal material can be obtained, which has direct application in plant breeding. Studies during last decade have revealed that the process of protoplast fusion may be a useful too for the induction of genetic variability and combination of traits which do not exist in nature. Isolation of Protoplast: Methods of Protoplasts isolation can be classified into three main groups. a) Mechanical : Mechanical isolation is done by cutting plasmolysed tissue with a sharp edged knife and releasing the protoplasts by deplasmolysis .The protoplasts isolated are few in number. Generally, protoplasts were isolated from highly vacuolated cells of storage tissues( Onion, bulbs, scales, radish root, mesocarp of cucumber and beet root) . b) Sequential Enzymatic (two step):
  • Page80 Takebe et al. (1968) employed sequential or two step procedure for isolating mesophyll protoplasts using commercial preparation of enzymes. The sequential approach involves initial incubation of macerated plant tissues with pectinase which, in turn, are then converted into protoplasts by cellulose treatment. c) Mixed Enzymatic Procedure: Cocking (1968) mixed two enzymes together and isolated protoplasts in one –step. In this mixed enzymatic approach plant tissues are plasmolysed in the presence of a mixture of pectinases and cellulases, thus inducing concomitant separation of cells and degradation of their walls to release the protoplasts directly. Source of Protoplasts: i) Leaves: The leaf is the most convenient and popular source of plant protoplast because it allows isolation of a large number of relatively uniform cells. Protoplasts isolation from leaves involve five basic stages: a) Sterilization of leaves, b) removal of epidermal cell layer c) Pre-enzyme treatment d) incubation in enzyme and e) isolation by filtration and centrifugation. In case of monocots, leaf material is cut into small places (1mm2) and then combined with vacuum infiltration. This procedure allows adequate infiltration of enzymes into leaf cells. As soon as the vacuum is removed the leaf piece will sink and eventually release the mesophyll protoplasts. ii) Callus Culture: Young actively growing callus is subcultured and used after two weeks for protoplasts isolation. iii) Cell Suspension Culture: A high –density cell suspension is centrifuged. After removing the supernant, cell are incubated in enzyme mixture ( cellulose + pectinase) in a culture flask placed on a platform shaker for 6 hrs to overnight depending on to the concentration of enzymes. A lower concentration of enzymes helps to prevent the formation of aggregates in the cell suspenion in order to obtain better yield. iv) Preconditioned Plant Materials: Mesophyll protoplast of some crop plants have a low morphogenetic response. This is because of the fact that the physiological state of growth of a donor plant under natural condition largely affect the regeneration potential of protoplasts in this system. on the contrary, tissue cultured regenerated plants are maintained under uniform physiological conditions and therefore provides materials preconditioned for protoplasts isolaton, and regeneration. This approach is particularly essential for regeneration of potato protoplasts.
  • Page81 Test for Viability of Protoplast: Cell wall formation, cell division, callus formation, etc. depends upon the viability of protoplast. The most frequently used staining methods for assessing protoplast viability are fluorescens diactate ( FDA), phenosafranine. FDA dissolved in 5.0 mg/ml acetone is added to the protoplast culture at 0.01% final concentration. The chlorophyll from broken protoplasts fluoresces red. Therefore, the percentage of viable protoplast in a preparation can be easily calculated. Phenosafranin, also used at final concentration of 0.01% is specific for dead protoplast. As soon as the strain is mixed with protoplast preparation, the invibale protoplasts strain red and viable protopalsts remain unstained. Protoplast Regeneration: Formation of Cell Wall: The process of cell-wall formation may be completed in two to several days although protoplast in culture generally starts regenerating a cell wall within a few hours after isolation. The regeneration of cell wall can be detected by using Calcalfluor White (CFW) florescence strain. The freshly formed cell wall is composed of loosely arranged microfibrils, the process requiring an exogeneous supply of a readily metabolised carbon source in the nutrient medium. Development of Callus or Whole Plant: Soon after the formation of wall around the protoplasts, the reconstituted cells show considerable increase in size and first division generally occurs within a week. Subsequent divisions give rise to cell colonies. After 2-3 weeks macroscopic colonies are formed which can be transferred to an osmotic free medium to develop a callus. The callus may be induced to undergo organogenic differentiation or whole plant regeneration following an appropriate procedure. a. Methods of Protoplasts Fusion Protoplasts fusion is a physical phenomenon. During fusion, two or more protoplasts come in contact and adhere with one another spontaneously or in presence of fusion inducing chemicals. An important aspect has been that incompatibility barriers do not exist during the cell fusion process at Interspecific, Intergeneric or even inter kingdom level. A) Spontaneous Fusion: During the enzymatic degradation of cell walls some of the adjacent protoplasts may fuse together to form homocaryons. These plurinucleate cells sometimes contain 2-3 nuclei, a phenomenon attributed to expansion and subsequent coalescence of the plasmodesmatal connections between the cells. The protoplasts once they are freely isolated, donot fuse spontaneously with each other. Mechanical Fusion:
  • Page82 The giant protoplast of Acelabularia have been fused mechanically by pushing together two protoplast. This fusion does not upon the presence of fusion inducing agents. However, in this procedure protoplasts are likely to get injury. Protoplast released from meiocytes in enzyme solutions readily fuse by gentle tapping in depression slide. B) Induced Fusion: Fusion of freely isolated protoplasts from different sources with the help of fusion inducing chemical agents is known as induced fusion. Normally, isolated protoplasts donot fuse with each other because the surface of the isolated protoplasts carries negative charge around the outside of plasma membrane and thus is a strong tendency for protoplasts to repel one another due to their same charges. i) NaNO3 Treatment: Equal densities of protoplasts from two different sources are mixed and then centrifuged at 100g for 5 minutes to get a dense pellet. This is followed by addition of 4 ml of 5.5% sodium nitrate in 10.2% sucrose solution to Resuspend the protoplast pellet. The suspended protoplasts are kept in water – bath at 35 0C for 5 minutes and again centrifuged at 200 g for 5 minutes. The pellet is once again kept in water bath at 30 0C for 30 minutes. The fusions of protoplast take place at the time of incubation. Finally, the protoplasts are plated in semisolid culture medium. The frequency of fusion is not very high in this method. ii) High PH or Ca++ Treatment: Kelier and Melchers ( 1973) developed a method to effectively induce fusion of tobacco protoplasts at a high temperature ( 37 0C) in media containing high concentration of Ca++ ions at a highly alkaline condition ( PH 10.5). Equal densities of protoplasts are taken in centrifuge tube and protoplasts are spun at 100 g for 5 minutes. The pellet is suspended in 0.5 ml of medium. a 4 ml of 0.05 M CaCl2, 2H2O in 0.4 M mannitol at PH 10.5 is mixed to the protoplast suspension. The centrifuge tube containing protoplast at high PH or Ca++ is placed in water bath at 30 0C for 10 minutes and is spun at 50 g for 3 to 4 minutes. This followed by keeping the tubes in water bath (37 0C) for 40- 50 minutes. About 20-30% protoplast are involved in this fusion experiment. iii) PEG Treatement: PEG induces protoplast aggregation and subsequent fusion. But the concentration and molecular weight of PEG are important with respect to fusion. A solution of 37.5% w/v PEG of molecular weight 1500 to 6000 aggregates mesophyll and cultured cell protoplasts during a 45 minutes incubation at room temperature. Fusion of protoplast takes place during slow elusion of PEG with liquid culture medium. Carrot protoplast can be fused by 28% PEG 1500 and fusion can be promoted by ca++ ion at concentration of 3.5 mM. But higher concentration of Ca++ ion has been considered beneficial. In some studies, high PH/Ca++ and PEG method have been combined. The PEG method has been modified slightly to fuse higher plant protoplast as indicated below:
  • Page83 a) PEG is more effective when it is mixed with 10-15% dimethyl sulfoxide. b) Addition of concanvalin A to PEG increases protoplast fusion frequency. c) Sea water has been used alone or in combination with PEG to fuse protoplasts. iv) Electro Fusion: Recently, mild electrical stimulation is being used to fuse protoplasts. This technique is known as electrofusion of protoplasts. Two glass capillary microelectrode are placed in contact with the protoplasts. An electric field of low strength (10kv m-3) give rise to dielectrophoretic pole generation within the protoplast suspension. This lead to pearl chain arrangement of protoplasts. The number of protoplasts within a pearl chain depends upon the population density of the protoplast and the distance between the electrodes. Subsequent application of high intensity electric impulse ( 100kv m-3) for some microseconds results in the electric breakdown of membrane and subsequent fusion. b.Selection of Somatic Hybrids and Cybrids It is a very difficult to identify the hybrid cells microsopically, therefore, some type of selection technique are required at the level of culture to recover hybrid cell and its callus tissue following fusion. Some important selection procedures are discussed below: 1. Auxin: The selection of hybrids of Nicotiana glauca and N. langsdorffi is based on auxin autotrophy of the hybrid cells. The parental protoplast or cell requires an auxin compounds in order to proliferate, whereas hybrid callus tissue needs no such requirements because cells are auxin autotrophic. Therefore, somatic hybrid cells can be isolated selectively by growth on auxin free culture medium. Auxin autotrophy of the hybrid cell is expressed only as a result of the genetic combination of two parental protoplasts. 2. Use of Genetic Complementation: Melcher and Lalib (1974) first used genetic complementation to isolate green somatic hybrids following fusion of two distinct homozygous haploid recessive albino mutants of Nicotiana tabacum. A population of albino protoplasts are fused with either a population of protoplasts isolated from a second non-allelic albino mutant or with a population of normal mesophyll protoplasts. In this process, the parental protoplasts form the albino colony whereas the hybrid protoplast will produce either light green or green colony. This can be usually distinguished at the cultural level. In addition, morphological markers can also be used in combination with the genetic complementation. 3. Use of Uncommon Amino Acid: Attempts have also been made to utilise uncommon amino acid as selective agents, conavalin which is present in some legumes, inhibits division of soybean and pea cells but sweet clover and
  • Page84 alfalfa are unaffected. Heterocaryon obtained by the fusion of protoplast from soybean with those from any one of the resistant plant will divide in presence of Conavalin. 4. Use of Cells Resistant to Amino Acid Analog: A number of cell lines resistant to amino acid analogs have been isolated and are used routinely for the selection of hybrid cells following protoplast fusion. For example , using cell lines resistant to 5-methyl –tryptophan ( 5-MT) and S-2 amino ethyl-cystein (AEC) , the Interspecific hybrids of Nicotiana sylvestris are selected after protoplast fusion using medium containing both amino acid analogs. In case of Daucus, two different cell lines have been raised for the selection of hybrid cells. A non-regenerating cell lines of D. carota is resistant to 5-MT and azetidine 3- carboxylate ( AZC), whereas a totipotancy wild type of D. capillifolius is sensitive to 5-MT. Hybrid colonies are selected by growth on 5-MT added medium and their to form plant through embryogenesis. 5. Use of Phytotoxin: Some well known fungal toxin may be used in selecting the fusion product. For example, the protoplast of cultured soybean cells resistant to Hm T toxin produced by Helminthosporium maydis race T, whereas the leaf protoplasts of Zeamays are sensitive to this toxin. It has been observed that fusion product of soybean and Zeamays survive on toxin containing medium. 6. Use of Antibiotic: The drug Actinomycin D has been used in the selection of somatic hybrids of two Petunia species. The cells from fusion product of protoplasts from P.parodii and P.hybrida can give rise to the complete plant via callus formation. The cell of P. hybrida fails to grow in the presence of Actinomycin D. Similarly, a kenamycin resistant cell line of Nicotiana Sylvestris has been used as genetic marker to identify the fusion product of N.Sylvestris and N. Knightiana. Similarly, streptomycin resistant mutant of N. tabacum are also used to recover interspecific hybrids with N.sylvestris. Cyclochexamide resistant cell of Daucus Carota can be used as a marker for the fusion with albino cell line of D. Carota. 7. Use of Auxotrophic Mutant: Auxotrophic mutants have been successfully used to isolate hybrid protoplast in Spherocarpus donnelii. Hybrids obtained by fusion of protoplasts from nicotinic acid and glucose requiring mutants are selected on the minimal media. The regenerated hybrid plants are identified on the basis of morphology and karyotype. 8. Use of Metabolic Mutants:
  • Page85 A series of nitrate reductase deficient mutants have been obtained from mutegenised haploid cells of Nicotiana tabacum cultured on medium containing chlorate and with amino acids as a nitrogen source. Cells with nitrate reductase convert chlorate to chlorite which is cytotoxic. The isolated mutants are unable to grow on nitrate containing medium and lack nitrate reductase and other molybdenum-protein containing enzymes. Chlorophyll deficient mutants have also been selected from haploid cells of Datura innoxia after radiation treatment. Similarly, metabolic mutant of Arabidopsis and proline requiring mutant of corn have been reported. 9. Using Isoenzyme Analysis: Isoenzymes are multiple molecular forms of an enzyme with a similar or identical substrate specificity occurring within the same organism. Now –a – days, isoenzyme analysis has been extensively used to verify hybridity. Isoenzymes of different constitutive enzymes exhibit the unique banding pattern or zymograms in polyacrylamide gel electrophoresis. The number of band and Rr value of isoenzyme are constant and specific for each parental plant species. The summation or intermediate banding pattern of Isoenzymes may be found in the hybrid callus tissue. This analysis thus help to select hybrid cells. 10. Use of Herbicides: Plants possess differences in their capacity to metabolize herbicides. This property can be utilised effectively for selection. For example, rice plants are resistant to propanil. This resistant is based on the ability of rice cells to metabolize propanil. 11. Chromosome Analysis of Hybrid Cell: Chromosome preparation from actively growing small cell colonies derived from protoplasts and their karyotype assay clearly indicate the hybridity. c. Practical Applications of Somatic Hybridization and Cybridization 1. Means of Genetic Recombination in Asexual or Sterile Plants: Somatic cell fusion appears to be the only approach through which two different parental genomes can be recombined among plants that cannot reproduce sexually. Similarly, protoplast of sexually sterile plants can be fused to produce fertile diploids and polyploids. There are several reports describing the amphidiploid and hexamploid plants produced from fusion of haploid protoplasts of tobacco. Protoplasts isolated from dihaploid potato clones have been fused with isolated protoplasts of Solanum brevidens to produce hybrids of practical breeding value. Haploid protoplasts from an anther- derived callus of rice cultivars upon fusion also produce fertile diploid and triploid hybrids. 2. Overcoming Barriers of Sexual Incompatibility:
  • Page86 In plant breeding programmes, sexual crossing at Interspecific and Intergeneric levels often fails to produce hybrids due to incompatibility barriers, which can be overcome by somatic cell fusion. Schieder ( 1978) obtained amphidiploid Datura innoxia (+) D. discolor and D. innoxia (+) D. stramonium, by fusing their diploid mesophyll protoplast. These hybrids cannot be produced conventionally and they are industrially important because show heterosis and higher (20-25%) scopolamine content than in the parental forms. Nicotiana repanda, N.nesophila and N.stockoni are resistant to number of disease but are not sexually crossable with N. tabacum (Tobacco). However, fertile hybrids have been reported in combination N. tabacum ( +) N nesophila and N. tabacum (+) N. Stockoni by protoplast fusion. Somatic hybridisation of dihaploid and tetraploid potato protoplast with isolated protoplasts of Solanum brevidens, S. phureja and S. penelliii resulted in the synthesis of fertile, partially amphidiploid plants possessing important agricultural traits, e.g. resistance to potato leaf virus V and Erwinia soft rot. Similarly, somatic hybrids between Brassica napus and B. nigra have been produced which is resistant to Phoma Lingam. 3. Cytoplasm Transfer: Power et.al. (1975) fused mesophyll protoplasts of Petunia with cultured cell protoplast of the crown gall of Parthenocissus and selected a line which contained the chromosomes of only Parthenocissus but exhibited some of the cytoplasmic properties of Petunia for some time. This was followed by direct application of cybridisation in agricultural biotechnology by transfer of cytoplasmic male sterility from Nicotiana techne to N. tabacum, N. tabacum to N.sylvestris and Petunia hybrida to P. axillaries. Besides cytoplasmic male sterility, the genophore of the cytoplasm codes for number of practially important traits, such as the rate of photosynthesis, low or high temperature tolerance, and resistance to disease or herbicides. In genus Brassica, two desirable traits coded by cytoplasmic genes have been genetically manipulated through Interspecific cybridisation between different species of Brassica. These traits include cytoplasmic male sterility ( cms) and resistance herbicide. Similarly, cytoplasmic genes coding for Atrazine and cms have been transferred into cabbage, rice and potato. d. Limitation of Somatic Hybridization 1. Intergeneric crosses between widely related plants which are not compatible sexually are not possible. 2. In certain wide crosses, elimination of chromosomes from hybrid cell is another limitation of somatic hybridisation. 3. In protoplast fusion experiments, the percentage of fusion between two different parental protoplast is very low.
  • Page87 4. For hybrid identification, selection and isolation at the culture level, there is no standardized method which is applicable for all material.
  • Page88 18. Biopesticides Pathogenic microorganism or their products (toxin and plant used as suppress the insect population is called Biopesticides. The concept of Biopesticides is by no means new. Over a country ago, introduced in India to fight foreign cactus and by 1938 the first Bt product had appeared in the market. Research on Biopesticides involves the isolation of naturally occurring strains of organism and the determination of their target specificity. The possibilities of micro-organism used in insect pest control are shown in following table: Sr.No Microorganism Pathogen Host 1 Bacteria i) Bacillus popilliae, ii) Bacillus thuringiensis. i) Japanese beetle,ii) Mosquitoes and Lepidopterous pests. 2 Viruses Baculoviruses Lepidoptera, mustard saw fly orthoptera, mosquito, mites 3 Fungi i) Beauveria bassiana , Aspergilus flavus,Metarhizium anisopliae,ii) ferticulium i) Homoptera, Lepidoptera, Coleoptera, Hymenoptera,Diptera.ii) Aphids, whitflies. 4 Protozoa Nomasa locustae Grass hoppers Bacillus Thuringiensis: The most widely used bacterium Bacillus thuringiensis (Bt) produces a toxic crystalline protein which when fed to susceptible insects, dissolves in the guts, damaging and eventually killing the insect. However, Bt based insecticides have some draw backs. The toxin usually have short self- life and remain effective for short periods because the toxic protein is broken down by sunlight and other climatic factor. Another limitation has been the much higher cost of Bt pesticides. Selected Commercial Microbial Insecticides: Sandoz India Ltd. has started a Bacillus thuringiensis based pesticide in 1992. The product is delivered in water dissolvable microorganules form under the brand name Delfin. It is active against caterpillars which attack cabbage and cauliflower. Hindustan Liver Ltd. have also produced a bio insecticides based on Bacillus thuringiensis using molasses as culture medium. It is found useful against the insect attacking cabbage, pigeon pea, cotton, safflower and maize as well as against black flies and mosquitoes.
  • Page89 Attempts are under way to effectively manage insect resistance by placing Bt toxin genes of several stains into the plants. The increasing availability of diverse Bt toxin may conceivably allow for management of pest resistance. By using gene transfer technique transgenic plants in maize and cotton have been developed by introducing Bt gene in these crops. There are many microorganism which attack insects. These microscope are called entomopahtogens. Entomopahtogens include many viruses, bacteria, fungi, and protozoa. Many arthropod pests are susceptible to viral diseases of the various kinds of insect viruses, two, viz, nucleopolyhedrosis viruses ( NPV) and granulosis viruses ( GV) have shown considerable promise as insect control agents in view of their specificity, safety, virulence and stability. Some of the host insect that may be controlled by these viruses include almond moth, Asiatic rice borer, bolloworm, cotton, leaf-down, cabbage loper, corn earworm, gypsy moth and codling moth, potato tuberworm, spruce budworm and Asiatic rice borer ( all through GV). Germplasm Storage: Germplasm: Germplasm may be defined as the irreplaceable genetic building material from which essential plant product can be derived. Proper germplasm storage is basic to the development of plant biotechnology that has valuable applications in agriculture and industry. The range of in vitro germplasm conservation systems involved in plant biochemistry extends from the non-cellular through protoplasts, cells and tissues to highly organised cultures such as shoot tips and plantlets. Some technique involve storage in the growing stage whereas other relate to the suspension of growth. Growth limitation ( E.g. By temperature reduction, use of retardant chemicals or hormones, and reduction in oxygene concentration) typically remains effective for cultures to be stored for about a year, and such cultures require periodic renewal. The another method is cryopreservation which often can store materials for virtually indefinite period. Methods of Conservation: 1. In- situ Conservation: This method of conservation mainly aims at preservation of land races with wild relatives in which genetic diversity exists or in which the weedy or wild forms present hybridise with related cultivars. These are evolutionary systems that are difficult for plant breeders to stimulate. The in- situ conservation of habitats has received high priority in the world conservation strategy programmes launched since 1980. 2. Ex-situ Conservation: Ex-situ conservation is the chief mode of preservation of genetic resources, which may include both cultivated and wild material. Generally, seeds or in vitro maintained plant cells, tissues and organs are preserved under appropriate conditions for long term storage as gene banks.
  • Page90 a. Advantages and Disadvantages of Biopesticides Advantages of Biopesticides: 1. Host specificity. 2. Ability to multiply in the target cells. 3. No problem of toxic residue. 4. No evidence or absence of resistance. 5. No problem of cross resistance. 6. Conventional technique or methods for applications. 7. Permanent control of pest or long persisting effect. 8. Idealy suited for integration with most other plant protection measures used in IPM programme. 9. No fear of environment pollution and hence ecofriendly. Disadvantages or Limitation: 1. High selectivity or host specificity. 2. Requirement of additional control measures. 3. The correct time of application. 4. Delayed effect or mortality. 5. Storage problem. 6. Difficulty of culturing in large quantities. 7. Short residual effectiveness.
  • Page91 19. Cryopreservation The principle underlying this technique involves bringing the plant cells and tissue cultures to a non-dividing or zero metabolism sate by subjecting them to superflow temperature in the presence of cryoprotectants. In this technique the plant material is frozen and maintained at the temperature of liquid nitrogen ( LN) which is around -196 0C. It is essential that cells during freeze –preservation be protected against cryogenic injuries. Formation of ice-crystals inside the cell can cause the rupture of organelles and cell itself. Increase in concentration of intracellular soultes to toxic levels is another potential source of cell damage. The general practise is to suspend material in the culture medium and after treating with a suitable cryopreservation transfer it to sterile polypropylene ampoules with screw- caps and freeze by one of the following methods. A) Slow-freezing Method: In this method the material is frozen at slow cooling rates of 0.5-40 C per minute, starting from 0 0 C until the temperature reaches – 1000 C and finally transferred to liquid nitrogen. It is useful for suspenion culture. B) Rapid- freezing Method: This method is simple as the vials containing the plant material are directly lowered into a tank filled with liquid nitrogen. The temperature decreases rapidly at the rate of 3000 C to 110000 C per minute. Dry ice (CO2), used instead of LN, exerts a similar effect. C) Step-wise –freezing Method: The plant material is cooled step-wise to an intermediate temperature, maintained at that temperature for 30 minutes and then rapidly cooled by ploughing it into LN. This method can be used for shoot apices, buds and suspension cultures. D) Dry-freezing Methods: Materials dehydrated by drying in oven or under vacuum demonstrate remarkable resistance to cryogenic damage. Dry seeds are able to survive freezing at super low temperature in contrast to water imbibing seeds which show susceptibility to cryogenic injuries. Similarly, dehydration of cells under vacuum also leads to a better survival of plant organs after freezing at 1960 C. Various studies have shown that freshly harvested cells or tissues may not survive super cooling and required to be conditioned by their brief cultures before freezing. Prefreezing treatment of this type proved beneficial for potato shoot apices only when they were cultured for 48 hours in the presence of 5% dimethylsulphoxide( DMSO). The process of hardening is also important as a prefreezing treatment in tissues culture. Plants grown at low temperature (40 C) for 3 days to a week before taking shoot apices for freeze preservation are reported to considerably enhance the survival frequency of the excised tissues. Along with DMSO3 glycerol and proline are also used as cryoprotectants.
  • Page92 Normally a dilute solution of cryoprotectants (5-10 % DMSO) is prepared and added gradually at interval of 5 minutes to prevent plasmolysis of the cells. Use of an ice bath while adding a cryopreservation is beneficial since room temperature may affect the viability of cells and tissues. After the last addition of cryoprotectants these should be an interval of 20-30 minutes prior to freezing. Cold Storage: The storage of culture material at low and non-freezing temperature (1-90 C) has been practised for some plants. Virus free strawberry plants could be maintained for 6 years at 40 C provided a few drops of liquid medium were added to the cultures after every three months. About 800 cultivars of grape plants have been stored for 15 years at 90 C by yearly transferred to fresh medium. The use of ABA and high levels of sucrose may help to prolong the interval between two subcultures. Low Pressure and Low Oxygen Storage: Attempts have been made to develop low pressure storage (LPS) and low oxygene storage (LOS) techniques. In LPS, the atmospheric pressure surrounding the tissue culture is reduced, resulting in partial decrease of pressure created by gases in contact with the plant material. On the other hand, in LOP the atmospheric pressure (760 mm hg) is not reduced but the inert gases are combined with oxygene to create low oxygene pressure. Applications: The principle objectives of germplasm conservation using cell and tissue cultures have been: 1. Conservation of Somaclonal and gametoclonal variation in cultures. 2. Maintain of recalcitrant seeds. 3. Conservation of cell lines producing medicines. 4. Storage of pollens for enhancing longevity. 5. Conservation of rare germplasm arsing through somatic hybridization or other methods of genetic manipulations. 6. Delaying the process of ageing. 7. Storage of meristem culture for micropropagation, micro grafting and production of disease free plants. 8. Conservation of plant material from enhanced species. 9. Establishment of germplasm bank. 10. Exchange of information as well germplasm at the international level.
  • Page93 20. Secondary Metabolites Metabolites that are not required by the producing organism for its support system are called secondary metabolites. Application of plant tissue culture for production of useful secondary compounds has been recognised since the early 1950s. Plant cells generally produce only small amounts of useful compounds. Techniques are now available to induce and select stable genetic variables arising from the cultured tissue and these can be applied for production of high amounts of vitamins, pigments, alkaloids, food flavours and useful metabolites and plant species. About 25% prescription medicines and various raw materials used in industries are obtained from plants. The impact of utilizing a specific tissue culture system for producing increased levels of certain secondary metabolites over the intact plant has been observed in cell lines producing high nicotine in Nicotiana tabacum, anthocynin in Euphorbia millii, antheaquinones in Morinda. Citrifolia, Crosus sativa and in about 18 species from genera Asperula, Galium and Sherardia, Shikonin derivatives in Lithispermum erythorhizon, phenolic compounds in Acer pseudoplatanus, 1-hydroxyquiphenylalanine ( L-dopa) in Stizolobium hassjoo, ferruginol in Salvia miltorrhiza, sanguinarine in Papaver Sommiferum, ajmalcine and serpentine in Catheranthus roseus, Ros-marinic acid in Coleus blumei, diosgenin in Dioscoarea deltoids etc. Various evidence suggest that the regulation of secondary metabolism is linked with induction of morphological differentiation in plants of some species. For example, cardiac glycoside of Digitalis are principally found in leaf cells quinine and quinine in the bark of Cinchona trees and tropane alkaloids in roots of many solanaceous species. This shows that cells not only undergo a morphological specialization during plant growth and maturation, but also differentiate in their capacity to produce specific chemicals. Investigation on cell differentiation versus secondary product synthesis using celery tissue culture revealed that cultures initiated from less differentiated tissues ( globular and heart-shaped embryos demonstrated no flavour compounds, while those initiated from better differentiated tissues torpedo-shaped embryos, petiole tissues) possessed the characteristics celery phthalide flavour compounds. It was further assumed that presence of chlorophyll in differentiated tissues may be someway associated with the synthesis of flavour compounds. a. Advantages of Plant Cell Culture for Production of Secondary Metabolites i) The rate of cell growth and biosynthesis in cultures initiated from a very small amount of plant material is quite high and the final product may be produced in a considerably short period of time. This is the contrast to large amounts of mature plant tissues processed to obtain a small quantity of drug. ii) Plant cell cultures are maintained under controlled environmental and nutritional conditions which ensure the continuous yields of metabolites. On the contrary, continuous decline in the natural habitats due to ecologically disturbed condition may make the availability of source plants uncertain unless they are clonally propagated.
  • Page94 iii) Suspension culture offers a more effective mechanism if incorporating precursors into cells than is found in whole plants. iv) New routes of synthesis can be recovered from deviant and mutant cell lines which can lead to production of novel compounds not previously found in whole plants. v) Some cell cultures have the capacity for biotransformation of specific substrates to more valuable products by means of single or multiple –step enzyme activity. vi) Culture of cell may be more economical for those plants which take long periods to achieve maturity ( E.g. Papaver bracteatum, the source of thebanine , takes two to three seasons to reach maturity).
  • Page95 21. Types of Biofertilizers The common microorganisms which have shown positive increase in crop yield and can possibly be used as Biofertilizers are as below: Contributing Plant Nutrient Microorganisms Crop Benefited A) Nitrogen 1) Symbiotic Organisms a) Rhizobium (Symbiosis with Legumes) Pulse Legumes: Chickpea, Pea, Lentil, Moong, Urad Bean, Pigeon Pea Oil Legumes: Groundnut, Soyabean Fodder Legume: Berseem, Lucern b) Azolla (Water Fern) Azolla Ap. + BGA Symbiosis Rice 2) Associative Symbiotic Organisms a) Azospirillum Sorghum, Perl Millet, Rice, Wheat, Finger Millet, Maize, Kodo Kutaki, Tomato, Chilli 3) Non Symbiotic Organisms a) Azatobacter (Heterotrophs) Cereals: Wheat, Rice, Maize, Sorghum, Sugarcane Vegetables: Onion, Brinjal, Tomato, Cabage Flowers: Chrysanthemum, Marygold b) Blue Green Algai (Photoautotrophs) Rice B) Phosphorus 1. "P" solubilizers and mineralizers For All Crops a) Fungi - Aspergillus, Penicillum b) Bacteria - Bacillus Psudomonas 2) "P" Absorbers (Root
  • Page96 Fungus Symbiosis) a) Versicular Arbuscular Mycorrhiza (VAM) b) Ectomycorrhizae c) Endomycorrizae Clomus Linseed, Maize, Wheat, Soyabean a. Large Scale Production of Biofertilizers Large Population of viable cells of effective strains of specific nitrogen fixing bacteria can be supplied through carrier based powder form of biofetlizer for cultivator use. Biofertilizers production technology includes isolation of bacteria, selection of suitable effective strain, preparation of mother or seed culture, inoculants isolation of bacteria, selection of suitable effective strain, preparation of mother or seed culture, inoculant production, carrier preparation and their mixing, followed by curing, packaging, storage and despatch. The production of microbial inoculants of Rhizobium, Azotobacter and Azospirillum involves following steps except the both or liquid medium used is different for different organisms. The medium used for respective organism is as follows: i) Rhizobium :Yeast Extract Mannitol ii) Azotobacter : Ashby’s medium iii) Azospirillium: Medium formulated by Okon et al. ( 1977) iv) Phosphate solubilising bacteria: Pikiyskaya’s medium. i) Preparation of Mother or Starter Cultures: Starter cultures of selected strains are obtained after ascertaining their performance in green house and at field levels. The pure culture of efficient strain of nitrogen fixing organism is grown on respective agar medium on slant and maintained in the laboratory. A loopful of inoculum from the slant is transferred in a 250 ml capacity conical flask containing liquid medium. keep the conical flak on rotary shaker for 3-7 days depending whether they are fast growing or slow growing. The content of these flasks usually attain a load of 10 5- 10 6 cells per ml called mother culture or starter culture. This mother cultures are further multiplied in larger flasks. 2. Preparation of Broth Cultures: Prepare liquid medium for respective organisms. Distribute equal quantity in big conical flasks (1000 ml). Sterilize it in autoclave for half an hour at 15 lbs pressure. After sterilization each flask containing suitable broth is inoculated with the mother culture in 1:5 proportions aseptically. Keep the flaks on rotary shaker for 96-120 hours until the viable count per ml reaches to 109 cells. The broths become thicker in consistency. This broth culture with population of 109 cells or ml should not be stored more than 24 hours or stored at 40 C temperature.
  • Page97 3. Preparation of Carrier: The carrier should have following characters: a) It should have high organic matter above 60%. b) Low soluble salts less than 1%. c) High moisture holding capacity 150 to 200% by weight. d) Provide a nutritive medium for growth of bacteria and prolong their survival in culture as well as on inoculated seed. Lignite or peat is used as carrier in the preparation of Biofertilizers. The carriers are crushed and powdered to 200 to 300 mesh. Peat or Lignite powder is neutralized by addition of 1% calcium carbonate (CaCO3) and sterilized at 15 lbs pressure for 3-4 hours in autoclave. 4. Preparation of Inoculate i.e. mixing: The sterilized and neutralized lignite or peat is mixed with high count broth culture in galvanised trays. About 1 part by weight of broth is required to 2 part of dry carrier. Final moisture content varies from 40 to 50% depending upon quality of carriers. 5. Curing or Maturation: After mixing the broth cultures and lignite or peat powder in 1:2 proportion in the galvanised trays then it is kept for curing at room temp ( 28 0C) for 5 to 10 days. After curing it is sieved to disperse the concentrated pockets of growth and to break the lumps. 6. Filling and Packing: After curing, sieved powder is filled in polythene bagas of 0.5 mm thickness leaving 2/3 space open for aeration of the bacteria. Bag is weighted for desired quantity. Then the bag is packed by sealing. The polythene bag used for filling microbial inoculant should be printed with following information. a) Name of Inoculants b) Direction for use c) Name of crops d) Date of Manufacture. e) Date of expiry. 7. Quality Checking: Check viable count in the carrier based inoculants by dilution plate method at the time of manufactures. The viable cells count in the carrier based inoculants should be maintained as per ISI specifications. 8. Storage:
  • Page98 The inoculants shall be stored by the manufacture in a cool place away from direct heat preferably at a temp of 150 C and not exceeding 300 C +- 2 0C for six months. For long survival of microorganisms the bag are stored in cold storage at 40 C temp. b. Large Scale Production of Blue Green Algae The method recommended for large scale production of BGA are simple, rural oriented, open air method and inexpensive and can be practised on farmers field incurring negligible cost. Laboratory or sophisticated equipments are not required as in the case of other Biofertilizers. c. Biofertilizers – Introduction, Meaning and Concept Introduction: The increasing population puts considerable pressure on land and other natural resources of the country causing damage to the ecological base of agriculture and serious socio-economic problems. The increased crop production over years has accelerated the removal of plant nutrient four times during the last four decades putting four fold pressure on soil resources. The replenishment of nutrients lost in crop removal through the use of chemical inputs is not considerable advisable as their use on a long run has been found to decelerate the biological activities in the soil causing impaired soil health, consequently, increasing awareness is being created in favour of adopting biological routes of soil fertility management for preventing soil degradation and for sustaining optimum crop production. In this context, the production and use of Biofertilizers in agricultures assume considerable importance. Biofertilizers: Biofertilizers or microbial inoculants can be generally defined as preparations containing live or latent cells of efficient strains of nitrogen fixing, phosphate solubilising or cellulolytic microorganisms used for application to seed, soil or composting areas with the objective of increasing the extent of the availability of nutrient in a from which can be easily assimilated by plants. Concept: Microorganism induce many biochemical transformation in soil. These include mineralisation of organically bound forms of nutrients, exchange reactions, fixation of atmospheric nitrogen and various other changes leading to better availability of nutrients already present in the soil. The group of microorganism responsible for nitrogen fixation, phosphate solubilisation and compost decomposition are being put to beneficial use in the form of Biofertilizers. Biological nitrogen fixation contributes maximum ( 67.5%) towards enriching earth surface with nitrogen. Microorganism capable of assimilating nitrogen are marked with “ nif” genes. They synthesise nitrogenase enzyme responsible for converting otherwise inert N2 to plant usable NH3. Such common useful organism are Rhizobium, Azatobacter, Azospirillium and Blue Green Algae ( BGA). There are some microorganisms capable of solubilising insoluble soil phosphours
  • Page99 while some collect available phosphorous from remote places out of reach of plant root hairs by sending elongated filaments. E.g. Vesicular Arbuscular Mycorrhiza. Few of the heterotropic organisms decompose cellulose rapidly. Such beneficial organisms are domesticated in suitable carriers which on application to soil augment crop growth and yield. These carrier based microorganisms are called Biofertilizers or appropriately called bioinoculants or microbial inoculants or microbial fertilizers.
  • Page100 ABOUT THE AUTHOR Dr HARI KRISHNA RAMA PRASAD. SARIPALLI M.Sc, M M M, M.A., M.Sc IT., M.Phil, DASM (IBAM), DMLT, RDBMS, Ph.D (BioTech). has been Asst. Prof. of Dept. of Biology and Biotechnology at College of Natural and Computational Sciences, Aksum University, Axum, Ethiopia, North East Africa. He has 14.7 years of experince in academics, administration and research in various institutes like K. L. University, St. Ann’s College for women, Southren Institute of Medical Sciences, Hindu College of Pharmacy, St. Joseph’s College of Nursing and St. Ann’s College of Nursing. He received his B.Sc degree in Chemistry, Botany, Zoology from Andhra Loyola College, Affiliated to ANU, India (where he learned fundamentals of microbiology with Prof. Madhavarao), M.Sc degree in Microbiology from Campus College of ANU in the year 1997. He obtained his research degree M.Phil in Botany and Microbiology from Campus College of ANU with Prof. Vijaya Lakshmi.M, and his Ph.D in Biotechnology from RPSC-MU,Patna with Prof. Madan Prasad and also guided by Prof. Vijaya Lakshmi.M. A part from life sciences, he has versatile academic degrees like Marketing Management, Medical Sociology, Information Technology, Medical Lab Technology, Alternative Medicine and Database Management System. Dr. H K R Prasad. Saripalli is recipient of the South Asian Foundation fellowship (2003), University rank in Microbiology programme(1997). While at St.Ann’s College, he has received the Best Teacher Award(selected by students and management). He is a co-author, with Prof. R.P.Singh, of the text Biological Chemistry and Microbiology; with Prof. T. Pullaiah, of the texts, Emerging trends in Biological Sciences(2009), Recent Trends in Plant Sciences (2005); and other texts, Diversity of Microbes and Cryptogams and Gymnosperms, plant anatomy, ecology and biotechnology. He was elected to the Board of studies, Industrial microbiology, JMJ College (ANU). He is a founder and a member of the Scientific Advisory Board of Association of Global Science Innovations (AGSI). He published thirty research papers in both national and international peer reviewed journals.