Introduction of plant biotechnology

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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

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  2. 2. 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.
  3. 3. 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
  4. 4. 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
  5. 5. 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)
  6. 6. 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.
  7. 7. 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:
  8. 8. 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.
  9. 9. 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
  10. 10. 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
  11. 11. 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
  12. 12. 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
  13. 13. 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.
  14. 14. Page14 Application of Cell Culture: i) Mutant selection ii) Production of secondary metabolites or biochemical production. iii) Biotransformation iv) Clonal propagation v) Somaclonal variations
  15. 15. 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
  16. 16. 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.
  17. 17. 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
  18. 18. 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:
  19. 19. 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.
  20. 20. 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.
  21. 21. 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
  22. 22. 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.
  23. 23. 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.
  24. 24. 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.
  25. 25. 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
  26. 26. 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.
  27. 27. 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.
  28. 28. 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.
  29. 29. 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
  30. 30. 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.
  31. 31. 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.
  32. 32. 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:
  33. 33. 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
  34. 34. 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.
  35. 35. 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.
  36. 36. 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:
  37. 37. 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
  38. 38. 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.
  39. 39. 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
  40. 40. 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
  41. 41. 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
  42. 42. 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.
  43. 43. 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.
  44. 44. 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.
  45. 45. 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,
  46. 46. 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:

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