+ HAPPIENESS OF LIFE PLANT TISSUE CULTURE-BT-124 Essay on Plant Tissue Culture Contents: the Definition of Plant Tissue Culture. the History of Plant Tissue Culture. the Basic Requirements of Plant Tissue Culture. the General Techniques of Plant Tissue Culture. the Basic Aspects of Plant Tissue Culture. the Cellular Totipotency, the Differentiation. the Methods in Plant Tissue Culture. the Applications of Plant Tissue Culture. the Morphogenesis. the Subculture or Secondary Cell Culture. the Soma-Clonal Variation. * the Somatic Hybrids and Cybrids. * the Micro-Propagation, * the Artificial Seed. the Cryopreservation. t+ Pt ® dee > Definition of Plant Tissue Culture: Plant tissue culture has a great significance in plant biotechnology specially in the crop improvement programmes. The term tissue culture may be defined as the process of in-vitro culture of explants (pieces of living differentiated tissues) in nutrient medium under aseptic conditions. However, in general, the tissue culture includes the term tissue culture as well as cell culture, organ culture and suspension culture also. Plant tissue cultures fundamental to most aspects of biotechnology of plants. Tt is evident now that plant biotechnology is one of the most beneficial of all the sciences. The products of plant biotechnology are being transferred rapidly from laboratories to the fields. Also, the plant tissue culture has become of great interest to the molecular biologists, plant breeders and even to the industrialists, as it helps in improving the plants of economic importance. In addition to all this, the tissueculture contributes immensely for understanding the patterns and responsible factors of growth, metabolism, morphogenesis and differentiation of plants. Related Terms: Tissue Culture: The in-vitro culture of the tissue e.g. Callus culture Cell Culture: Denotes the in-vitro culture of single or a few cells. Organ Culture: This term is used for in-vitro culturing of organs like embryo, root or shoot apices. Suspension Culture: Defined as the culture of cell and cell aggregates suspended in a liquid medium. Ex plant: The excised piece of differentiated tissue or the organ which is used for culture is called as explant. e.g., embryos, young leaf, bud, etc. Callus: The undifferentiated mass of cells is referred to as callus. The cells of callus are meristematicin nature. History of Plant Tissue Culture: G. Haberlandt, a German botanist, in 1902 cultured fully differentiated plant cells isolated from different plants. This was the very first step forthe beginning of plant cell and tissue culture. Further contributions were made by the Cell Doctrine which admitted that a cell is capable of showing totipotency. With the identification of a variety of chemicals like cytokinin, auxin, other hormones, vitamins, etc. and their role in affecting cell division and differentiation, the methods of plant tissue culture developed in a proper manner. Three other scientists Gautheret, White and Nobecourt also made valuable contributions to the development of plant tissue culture techniques. Later on, a numberof suitable culture media were developed, for culturing plant cells, tissues, protoplasts, embryos, anthers, root tips, ete. The discovery and understanding of role of plant growth hormones in the multiplication of cell also provided an extra aid for the development of in-vitro culture methods. of plants.‘Advancement First attempt of in-vitro culture of pant cet (Catare of embeyogenic tive of crises Inco coun of roms Zygotic embryo culture in Linum Caltare of roots of tomato plant Successful establishment of indefinite callus culture | Caine of Crown Gall Trssues Cusltares from stem tip. Horm Kien dacover Discovered that Auxin © Cytokinin ratio ergulatcs the ‘organ formation veep fag cag ino ge] Sacces fl ronplat (oon Successfull Anther Culture [Plants regenerated from protoplasts |Biocransformation in plant tissue culture Production od wornad | Pomato Basic Requirements and Techniques of Plant Tissue Culture: The main requirements of plant tissue culture are: (4) Laboratory Organisation (2) Culture Media (3) Aseptic Conditions 1. Laboratory Organisation: In a standard tissue culture lab, there must be a few basic facilities like: i. A Media Room for preparation, sterilization and storage of culture media. ii. Facilities for washing of lab-wares, explants, etc. iii. Space for storage of lab-wares. iv. Culture rooms or incubators where conditions of temperature, humidity and light ete. can be maintained. y. Observation and Data Collection area.2. Culture Media: The formulation or the medium on which the explant is cultured is called culture medium. It is composed of various nutrients required for proper culturing. Different types of plants and organs need different compositions of culture media. A number of media have been devised for specific tissues and organs. Some important of them are: MS (Murashige and Skoog) Medium LS (Linsmaierand Skoog) Medium Bs (Gamborg’s) Medium White's Medium, ete. Important constituents ofa culture medium are: Organic supplements: (a) Vitamins like thiamine (B.), Pyridoxin (B.), Nicotinic Acid (B;), etc. (b) Antibiotics like Streptomycin, Kanamycin; (c) Amino Acids like Arginine, Asparagine. (ii) Inorganic Nutrients: Micronutrients as Iron (Fe), Manganese (Mn), Zine (Zn), Molybdenum (Mo), Copper (Cu), Boron (B). Macronutrients include six major elements as Nitrogen (N), Sulphur (S), Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg). (iii) Carbon and Energy Source: Most preferred carbon source is Sucrose. Others include lactose, maltose, galactose, raffinose, cellobiose, ete. (iv) Growth Hormones: a, Auxins-mainly for inducing cell division. b. Cytokinins-mainly for modifying apical dominance and shoot differentiation. c. Abscisic Acid (ABA)-Used occasionally. d. Gibberellins-Used occasionally.Gelling Agents: These are added to media to make them semisolid or solid. Agar, Gelatin, Alginate ete. are common solidifying or gelling agents. Other Organic Extracts: Sometimes culture media are supplemented with some organic extracts also like coconut milk, orange juice, tomato juice, potato extract, etc. 1 ltr of MS medium = (50 ml of stock solution I) +(gml of each stock solutions IL, ILIV) 3. Aseptic Conditions: Maintenance of aseptic conditions is the most critical and difficult aspect of in- vitro culturing experiments. Aseptic condition mean the conditions free from any type of microorganisms {so as to prevent the loss of experiment by contamination). For this, sterilization (i.e., complete removal or killing of microbes) is done. The most common contaminants in culture are fungi and bacteria. Measures to be taken for maintaining asepsis during tissue culture are: i, Sterilization of the culture vessels using detergents, autoclaves, etc, ii, Sterilization of instruments like forceps, needles etc. by flame sterilization.iii. Sterilization of culture medium using filter sterilization or autoclaving methods. iv. Surface sterilization of explants using surface disinfectants like Silver Nitrate (19%), H.O: (10-12%), Bromine water (1-29), Sodium Hypochlorite solution (0.3-0.6%), ete. ‘The whole procedure of plant tissue cultureis to be carried out essentially under aseptic conditions. So, the overall design of the laboratory must focus on the maintenance of aseptic conditions. Secondly, the worker is also required to have proper knowledge of operating various equipment's like pH meter, balance, laminar air flow, microscope, ete. While performing the tissue culture experiments there must present the first aid kits and fire extinguishers in the laboratory to avoid any mishap or accident. In addition, proper attention should be given while handling the toxic chemicals and all the chemicals should be kept in correct labeled containers and bottles. 4. General Technique of Plant Tissue Culture: General technique of plant cell, tissue and organ culture is almost the same with a little variation for different plant materials. There are certain basic steps for the regeneration of a complete plant from an explant cultured on the nutrient medium (Fig, 1). These basic steps for in-vitro culturing of plants are:Pig. 1. Steps in general technique of Plant tise culture. (a) Selection and Sterilisation of Explant: Suitable explant is selected and is then excised from the donor plant. Explant is then sterilized using disinfectants. (b) Preparation and Sterilisation of Culture Medium: A suitable culture medium is prepared with special attention towards the objectives of culture and type of explant to be cultured. Prepared culture medium is transferred into sterilized vessels and then sterilized in autoclave. (c) Inoculation: Sterilized explant is inoculated (transferred) on the culture medium under aseptic conditions. (d) Incubation: Cultures are then incubated in the culture room where appropriate conditions of light, temperature and humidity are provided for successful culturing. (e) Sub culturing: Cultured cells are transferred to a fresh nutrient medium to obtain the plantlets.(@P Transfer of Plantlets: After the hardening process (i.e., acclimatization of plantlet to the environment), the plantlets are transferred to green house or in pots. Equipment in Tissue Culture Lab: + Autockave * Refrigerator + Weighing Balance + Deep freeze + Magnetic stirrer * Incubator + pH meter * Hot air oven + Laminar Air flow Hood (Fig. 2) + Microscopes + Light meter » Rotary Shaker * Glasswares: + Spirit lamps + Measuring cylinders + Micropipettes. * Forceps, meedies, spatul ‘Sterilized Chamber Fig. 2. Laminar Air Flow Hood. 5: Basic Aspects of Plant Tissue Culture: In plant tissue culture technique, an explant is taken, it is cultured on a nutrient medium under certain conditions and finally we obtain a whole new plant. How does it happen? The answer to this question lies in the inherent capacities of plant cells that are differentiation and cellular totipotency. 6. Cellular Totipotency: The potential of a plant cell to grow and develop into a whole new multicellular plant is described as cellular totipotency. In other words, the property of a single cell for differentiating into many other cell types is called as totipotency. This is the property which is found only in living plant cells and not in animalcells (exception being stem cells in animals). The term totipotency was coined in 1901 by Morgan. During culture practice, an explant is taken from a differentiated, mature tissue. It means, the cells in explants are generally non- dividing and quiescentin nature. To show totipotency, such mature, non-dividing cells undergo changes which revert them into a meristematic state (usually a callus state). This phenomenon. of reverting back of mature tells to dividing state is called dedifferentiation. Now, these dedifferentiated cells have the ability to form a whole plant or plant organ. This phenomenon is termed as re-differentiation. Epa’ Calas (Mature, Non-dividing | Dedifferentiation | (nqitverentiated and Redifferentiation | New Planiteis | land differentiated Celts) ‘Meristeratic cells} Nain tiated jetty Dedifferentiation and re-differentiation are the two inherent phenomena involved in the cellulartotipotency. Regarding this, it is clear that the cell differentiation is the basic event for development of plants and it is also referred to as cyto-differentiation. To express its totipotency, a differentiated cell first undergoes the phenomenon of dedifferentiation and then undergoes the re-differentiation phenomenon (Fig. 3). Usually the dedifferentiation of the explant leads to the formation of a callus. However, the embryonic explants, sometimes, result in the differentiation of roots or shoots without an intermediary callus state.Fig. a. Scheme depicting the Lotipetancy of plant calls, Thus, from the above account it is clear that unlike animals (in which differentiation is irreversible usually), the plants have such a quality that even highly mature and differentiated cells have an ability to revert back to meristematic state. The property of totipotency of plant cells indicate that even the undifferentiated cells of a callus carry the essential genetic information required for regeneration of a whole plant. It is also clear that all the genes responsible for dedifferentiation or re- differentiation are present within the individual cells and they become active for expression under adequate culture conditions. As totipotentcells are the basis of whole plant tissue culture techniques, so, by the exploitation of this potential of plant cells, biotechnologists are trying to improve the crop plants and other commercially important plants, Totipotency in Different Plant Parts: The somatic cells in plant body are totipotent. It is to be noted here that only the living plant cells have the ability to regenerate and the dead cells which lack cytoplasm and nucleus (tracheid’s, vessel elements, ete.) are not totipotent at all.Different plant parts have different totipotent abilities. For example, in tobacco plant, the type of bud formed by in-vitro culture of the epidermis of different regions of the plant are different in their form, Another example to add here may be given about the totipotency of crown-gall cells which have the capacity to grow as an un-organised mass of cells under normal conditions, however whole plants can be recovered from them in culture. Thus, it is clear that totipotency is not similar in all plant parts. Applications of Totipotency: Cellulartotipotency of plants cells has proved to be a boon to mankindas it is the basis of plant tissue culture. The plant tissue culture exploits this unique property of plant-cells to attain commercial benefits. Various applications of cellular totipotency are: i, It has potential applications in the crop plant improvement. ii. Micro-propagation of commercially important plants. iii. Production of artificial or synthetic seeds. iv. It helps in conservation of germplasm (genetic resources). vy. This ability is utilized for haploid productions. vi. Applied in producing somatic hybrids and cybrids. vii. Helps in cultivation of those plants whose seeds are very minuteand difficult to germinate. viii. Also helps to study the cytological and histological differentiations. ix. For high scale and efficient production of secondary metabolites. x. The genotypic modifications can also be possible. 7. Differentiation: Whilestudying totipotency, it is stated that the dedifferentiation and redifferentiation processes result in the differentiated plant organs, finally producing a whole plant. In case of plants, the differentiation is reversible but in animals, it is irreversible.The term differentiation describes the development of different cell types as well as the development of organised structures like roots, shoots, buds, etc., from cultured cells or tissue. Differentiation may also. be defined in simple words as the development change of a cell which leads to its performance of specialised function. However, normally morphological characteristics. For example, differentiation accounts for the origin of different types of cells, tissues and organs during the formation of a complete multicellular organism (or an organ) from a single- celled zygote. Actually, the development of an adult organism starting from a single cell occurs as a result of the combined functioning of cell division and cell differentiation. Various techniques of tissue culture provide not only a scope of studying the factors governing totipotency of cells but also serves for the investigation of patterns and factors controlling the differentiation. Types of Differentiation: As stated earlier also, the plant cells have a tendency to remain in a quiescent stage which may be reverted to the meristematiestage. This process is termed as dedifferentiation and as a result of this, a homogeneous undifferentiated mass of tissue i.e., callus is formed. There callus cells then differentiate into different types of cells or an organ or an embryo. On this basis, the differentiation may be of the following types: (a) Cytodifferentiation (b) Organ Differentiation (ce) Embryo Genic Differentiation a. Cytodifferentiation: The differentiation of the cells is an important event of the development of plants. The differentiation of different types of cells from the cultured cells is known as cytodifferentiation. When an undifferentiated callus re-differentiates into whole plant, it first undergoes cytodifferentiation. Amongst different cytodifferentiations, the differentiation into vascular tissues has received maximum attention. However, it is important here to mention that the cells of mature xylem elements and phloem cells cannot be re- differentiated or cannot be reverted back to the meristematic state due to lack of cytoplasm in them.Although, in initial stages of their development, they can be reverted to meristematic cells. Xylogenesis is the differentiation of parenchymatous cells (of callus) into xylem-like cells of vascular plants. Phloem differentiation is the formation of phloem-cells from parenchyma in culture. Factors affecting cytodifferentiation: (i) Physical factors like light, temperature and pH are effective at optimum levels. (ii) Chemical factors. a. Low Nitrogen content increases vascularization b. High Ca ions stimulates the formation of tracheid’s and sieve tubes. ce. Sucrose in high concentration results in pronounced xylem differentiation. (iii) Hormones: Some hormones play important role in cytodifferentiation. These are: a. Auxin plays major role in vascularization. b. Cytokinin promotes cytodifferentiation. c. Gibberellins along with auxins promote it. d. Abscisic acid inhibits it usually. b. Organ Differentiation: It is synonymous to organogenesis or organogenic differentiation. It refers to the development or regeneration of a complete organised structure (or whole plant) from the cultured cells/tissues (Fig. 4).tN Pig. 4 Gig cei Organogenesis literally means the birth of organ or the formation of organ. It may occur either by shoot bud differentiation or by the formation of root. Organogenesis commences with the stimulus produced by the components of culture medium, the substances initially present in the original explants and also by the compounds preduced during culturing. Among different organs, which can be induced in plant tissue culture are included the roots, shoots, flower buds and leaves. Regenerations into flower buds and leaves occur ina very low frequency. However, the roots and shoot bud regenerations are quite frequent. Out of all these types of organogenie differentiation, only the shoot bud differentiation can give rise to the complete plantlets therefore, it is of great importance in tissue culture practices. The initiation of roots is termed as rhizogenesis while the initiation of shoots is called as caulogenesis and these two phenomenaare affected by alterations in the auxin : cytokinin ratio in the nutrient medium. A group of meristematic cells called as meristemoids is the site of organogenesis in callus. Such meristemoids are capable of producing either a root or a shoot.Organogenesis may occur either through callus formation or through the direct formation of adventitious organs (like adventitious shoot). Latter mode of organogenesis does not involve the intervening callus phase. Shoot bud differentiation was first of all demonstrated by White (1939). Further, in 1944, Skoog indicated that organogenesis could be chemically controlled. Shoot bud differentiation refers to the formation of shoot buds from the cultured cells by providing appropriate culture conditions and nutrient medium. The chemical and physical factors required for shoot bud differentiation vary for explants from different plant species. Factors affecting organogenesis: (i) Auxin: Cytokinin ratio in medium is an important factor affecting root/shoot bud differentiation in most plants, (ii) Usually Gibberellic acid inhibits organogenesis. (ii) Physiological state and size of explant play important role in organ differentiation. (iv) Genotype of the donor plant plays a crucial role. (iv) Physical factors like light, temperature, moisture, etc., play effective role in organogenesis. ¢. Embryo Genic Differentiation: The embryos formed from the somatic cells of plant in culture under in-vitro conditions are called as somatic embryos. When the somatic cells of plant organs result into the regeneration into embryos, then the process is called as somatic embryogenesis or embryo genic differentiation or embryogenesis (Fig. 5):Fig. 5. Somatic Embryo (S.E) Differentiation Somatic embryos are also referred to as embryoids, and they can be obtained either indirectly (with formation of callus) or directly from the explant without intervening callus formation. However, direct embryogenesis is not a normal process because the medium requirement for this is complex. Somatic embryogenesis under in-vitro conditions was first of all observed by Steward et. al. (1958) in carrot (Daucus carota). Thereafter, somatic embryoids have been induced in many plants namely Citrus, Coffea, Zea mays, etc. To obtain embryoids, there is a requirement of two nutrient media, first for initiation and the other medium for proper development of the embryoid. ‘The development of somatic embryo passes through the stages like globular, heart-shaped, torpedo-shaped and finally giving rise to the cotyledonary stage of somatic embryo. A somatic embryo does not have any vascular connection with the explant or callus therefore it can be separated easily. Somatic embryogenesis is not used very frequently for propagation of plants because, the technique is usually difficult and also, there is a high risk of occurrence of mutations. Another major drawback of somaticembryogenesis isthat there are greater chances of loss of regenerative capacity on repeated sub- culturing. Factors affecting Embryogenesis: a. Physiological condition and type of explant. b, Genotype of donor plant. ¢. Growth regulators: i, Auxinis essential for embryo initiation ii, Cytokinin promotes embryogenesis iii. Gibberellins inhibit embryo genic differentiation iv. Abscisic Acid (ABA) suppresses it. d. Nitrogen and Oxygen concentration e. Physical factors like temperature and light. 8. Methods in Plant Tissue Culture: There are different methods of culturing plant material. These methods differ on the basis of explants used and their resultant products, Some of the most popular and advantageous methods in plant tissue culture are discussed below: 4. Cell Culture: Cell culture is actually, the process of producing clones of a single cell. The clones of cell are the cells which have been derived from the single cell through mitosis and are identical to each other as well as to parental cell. First attempts for cell culture were made by Haberlandt in 1902. However, he failed to culture single cell but his:attempts stimulated other workers to achieve success in this direction. The method of cell culture is meritorious over other methods of culturing because it serves as the best way to analyse and understand the cell metabolism and effects of different chemical substances on the cellular responses, Single cell culturing is of immense help in crop improvement programmes through the extension of genetic engineering techniques in higher plants.The method of cell culture is done by following three main steps: (a) Isolation of single cell from the intact plant by using some enzymatic or mechanical methods. (b) In-vitro culturing of the single cell utilizing micro chamber technique, or micro drop method or Bergmann cell plating technique (Fig. 6). Pig. 6. Steps in Bergmann cell plating technique. (c) Testing of cell viability done with the phase contrast microscopy or certain special dyes. It is important to note here that the cell cultures require a suitably enriched nutrient medium and it should be done in dark because light may deteriorate the cell culture. Large scale culturing of plant cells under in-vitro conditions provides a suitable method for production of large varieties of commercially important phytochemicals. 2. Suspension Culture: A culture which consists of cells or cell aggregates initiated by placing callus tissues in an agitated liquid medium is called as a suspension culture. The continuous agitation of the liquid medium during a suspension culture is done by using a suitable device called as shaker, most common being the platform/orbital shaker.Agitation with shakeris important because it breaks the cell aggregates into single cell or smallergroups of cells and it helps in maintaining the uniform distribution of single cell and groups of cells in the liquid medium. A good suspension is the one which has high proportion of single cells than the groups of cells, Changes in the nutritional composition of medium may also serve as a useful technique for breakage of larger cell clumps (Fig. 7). Fig. 7. Diagrammatic summary of Suspension Culture. The general technique of suspension culture involves basically two types of cultures: batch culture and continuous cultures. Abatch cultureis a suspension culture in which cells grow in a finite volume of the culture medium and as a result, medium gradually depletes. On the other hand, a continuous suspension culture is the one which is continuously supplied with nutrients by the inflow of fresh medium but the culture volume is normally constant. 3- Root Culture: Pioneering attempts for root culture were made by Robbins and Kotte during 1920s. Later on, many workers tried for achieving successful root cultures. In1934, it was White who successfully cultured the continuously growing tomato root tips. Subsequently, root culturing of a number of plant species of angiosperms as well as gymnosperms has been done successfully. Root cultures are usually not helpful for giving rise to complete plants but they have importance's of their own. They provide beneficial information regarding the nutritional needs, physiological activities, nodulations, infections by different pathogenic bacteria or other microbes, etc. 4. Shoot Culture: Shoot cultures have great applicability in the fields of horticulture, agriculture and forestry. The practical application of this method was proposed by Morel and Martin (1952) after they successfully recovered the complete Dahalia plant from shoot-tips cultures. Later on, Morel realized that the technique of shoot culturing can prove to be a potent method for rapid propagation of plants Ge. Micro propagation). In this technique, the shoot apical meristem is cultured on a suitable nutrient medium. This is also referred to as Meristem Culture (Fig. 8).‘fom mansion te Fig. 8. Regeneration of plants through Meristem Culture, The apical meristem of a shootis the portion which is lying beyond the youngest leaf primordium. Meristem tip culture is also beneficial for recovery of pathogen-free specially virus-free plants through the tissue culture techniques. Various stages in this culture process are the initiation of culture, shoot multiplication, rooting of shoots and finally the transfer of plantlets to the pots or fields. 5: Protoplast Culture: A protoplast is described as a plasma membrane bound vesicle which consists of a naked cell formed as a result of removal of cell wall. The cell wall can be removed by mechanical or enzymatic methods. In-vitro culturing of protoplasts has immense applications in the field of plant biotechnology. It not only serves for genetic manipulations in plants but also for biochemical and metabolic studies in plants. For protoplast culture, firstly the protoplasts are isolated from the plants utilizing some chemical or enzymatic procedure. At present, there are available a number of enzymes which have enabled the isolation of protoplasts from almost every plant tissue. After isolation ofprotoplasts, they are purified and then tested for their viability. Finally the purified viable protoplasts are cultured in-vitro using suitable nutrient medium which is usually eithera liquid medium or a semisolid agar medium. 6. Haploid Production: Haploid plants are those which contain half the number of chromosomes (denoted by n). Haploids can be exploited for benefits in the studies related to experimental embryogenesis, cytogenetics and plant breeding. Haploids have great significance in field of plant breeding and genetics. They are most useful as the source of homozygous lines. In addition, the in-vitro production of haploids also aids for induction of genetic variabilities, disease resistance, salt tolerance, insect resistance, etc. Presently, attention is being focused on improving the frequencies of haploid production in their advantageous utilization far economic plant improvement. There are two approaches for in-vitro haploid production. These are: (a) Androgenesis: The technique of production of haploids through anther or microspore culture is termed as androgenesis. It is a method par excellence for the large scale production of haploids through tissue culture. Androgenesis technique for haploid production is based on the in-vitro culture of male gametophyte i.e., microspore of a plant resulting into the production of complete plant from it. It is achieved either by another culture or by: microspore (pollen) culture. The technique of another culture is quicker for practical purposes and is an efficient method for haploid production. But sometimes during another culture, the plantlets may originate from different other parts of anther also (along with from the pollens).On the other hand, microspore culture is free from any uncontrolled effects of the anther wall or other tissues. Microspore culture is ideal method for studying the mutagenic and transformation patterns (Fig. 9).Fig. 9. Androgenesis for Haploid Production, (b) Gynogenesis: It is an alternative source of in-vitro haploid production, It refers to the production of haploid plant from ovary culture or ovule culture. The method of gynogenesis for haploid production has been successful, so far, ina very few plants only, hence it is not a very popular method for in-vitro production of haploids, Thus, androgenesis is preferred over gynogenesis. 7. Embryo Culture: The technique of embryo culture involves the isolation and growth of an embryo under in-vitro conditions to obtain a complete viable plant. First success for embryo culture was made by Hannig in 1904 when he isolated and cultured embryos of two crucifers namely Cochleria and Raphanus. Embryo culture is used widely in the fields of agriculture, horticulture and forestry for production of hybrid plants. This technique allows the detailed study about the nutritional requirements of embryos during different developmental stages. Also, it helps for identifying the regeneration potential of embryos, Embryo culture is advantageous for in- vitro micro propagation of plants, overcoming seed dormancy and for production of beneficial haploid plants.8. Endosperm Culture (Triploid Production): Endosperm tissue is triploid therefore the plantlets originating by the culture of endosperm are also triploid. In majority of flowering plant families (exceptions being Orchidaceae, Podostemaceae, Trapaceae which lack endosperm) the endosperm tissues are present. Endosperm is formed after the double fertilization of one male nucleus with two polar nuclei. Immature endosperm has more potential of growth in culture especially among the cereals. Endosperm culture has provided a novel strategy for plant breeding and horticulture for the production of triploid plantlets. It is an easy method for production of a large number of triploids in one step. Moreover, it is much more convenient that the conventional techniques like chromosome doubling by crossing tetraploids with diploids fortriploid induction. Full triploid plants of endosperm origin have been produced ina number of plant species like Populus, Oryza sativa, Emblica officinalis, Pyrus malus, Prunus, ete. The triploid plants are usually seedless therefore this technique is most beneficial for increasing the commercial value of fruits like apple, mango, grapes, watermelon, etc. In addition to all the above described applications, endosperm culture is helpful for studying biosynthesis and metabolism of certain natural products also. 9. Applications of Plant Tissue Culture: 1, Germplasm conservation mainly in the form of cryopreservation of somatic embryos or shoot apices, ete. 2. Large scale production of useful compounds and secondary metabolites by using genetically engineered plant tissue cultures, 3. Technique of micro propagation for enhancing the rate of multiplication of economically important plants. 4. Eradication of systemic diseases in plants and raising disease free plants. 5. Soma-clonal variations are useful sources of introduction of valuable genetic variations in plants.6. Helps plants in imparting resistance to antibiotics, drought, salinity, diseases, ete. 7. Somatic hybrids and eybrids overcome species barriers and sexual incompatibility and produce hybrid plants with desired combination of traits. 8, Embryo culture helps in overcoming seed sterility and dormancy. g. Haploid production in culture helps to solve various problems of genetic studies and thus aids the plant breeders for producing new varieties. 10. Production of synthetic seeds via somatic embryo differentiation for commercially important plants helps to achieve increased agricultural production. 11, Large scale production of biomass energy. 12. Plant tissue culture aids in producing the genetically transformed plants. 13. Early flowering can be induced by in-vitro culturing of plants so as to attain commercial benefits. 14. Triploids as well as polyploid plants can also be produced by tissue culture techniques for uses in plant breeding, horticulture and forestry. 15. Seedless fruits and vegetables. can be produced by following the endosperm culture method which add to their commercial values. 16, Increased Nitrogen fixation ability can be achieved through association of tissue culture techniques with genetic engineering, 17. Callus cultures are useful in plant pathology as they act as an effective tool in the study of mechanism of disease resistance and susceptibility. 18. Different tissue culture techniques help us to study various biosynthetic processes, physiological changes and cytogenetic changes. 10. Morphogenesis: Literal meaning of the term morphogenesisis origin of form. It includes all the activities which are involved in the formation of a complete individual starting from the cells/tissues in culture. Various internal as well as external factors affect the morphogenic potential of the cells.The study of such factors and their effecton the regeneration of cells constitutes the morphogenesis. The appearance of the plants may be altered by the changes in growth environment, The causal factors of such changes may be studied properly by morphogenesis. Morphogensis may also be termed as developmental morphology and it may be defined as “the branch of biology that deals with the causes and activities during the organization of a complete individual plant.” Another definition of morphogenesis can be given as the study of various factors which affect the organic form and describe the growth patterns of the individual. All those anatomical and physiological events which are involved in the growth and development of an organism are included in the morphogenetic studies. Differences in the motphogenetic phenomena lead to the differences in the form of characteristic organs or structures. In simple words, morphogenesis may be described as the developmental pathways in differentiation as a result of which the recognizable tissues are formed. Cell culturing has blossomed into such a technology which is capable of solving, a number of problems with an impact on morphogenesis of plants. This is so because, through the elegant system of plant cell/tissue culture, it is possible to have an effective control of exogenous factors which affect the differentiation starting with the callus or a single cell or tissue. Factors of Morphogenesis: There are many such factors which influence the morphogenic potential of cells by affecting important developmental activities. For e.g., i, Several experiments have shown that certain growth factors (like cytokinin) induce the somatic embryos in culture by changing the cell polarity. ii. Some chemical factors promote asymmetricdivisions in the cultured cells thus resulting in difference in form. iii. Differentconcentrations of 2, 4-D, auxins, etc. alter the development of organ in culture by causing the altered cell elongation or rate of cell division. iv. With the increase in culture duration, the organogenic differentiation shows decline. All such examples show that different factors have different type of effect on differentdevelopmental activities. Such dynamic and causal aspects of organic forms are studied in morphogenesis.Alist of the factors having great importance in deciding the morphogenic pattern is given below: i. Physical factors like light, temperature, pH. ii. Chemical factors like C/N ratio, oxygen content, carbohydrates (sugar) concentrations, etc. iii, Growth hormones like Auxin, Cytokinin, Gibberellins, ABA, etc. iv. Water v. Culture duration vi. Mechanical strains, pressures, bending, etc., may cause changes in plane of cell division, thus may cause difference in morphogenic potential. vii. The regenerability of explant may be affected by factors like the organ from whieh it is taken, the physiological state of the explant, its size, ete. viii. Electrical stimulation, ix, Changes in gene expression in cells in culture, x. Genotype and status of nucleicacids in different cells. xi. Repeated subculture of a totipotent callus may also result in reduction or complete loss of morphogenetic potential. Role of Morphogenesis: As described above, morphogenesis includes all those events which occur during the growth and development of an organism. Thus, it is very important for providing a satisfactory explanation of various biological growth patterns. It also aims on studying and correlating different environmental factors with changes in the morphogenetic patterns and/or potential. Major contributions for morphogenetic studies in India have been provided by Department of Botany, University of Delhi. Numerous experiments on a wide variety of subjects were performed by the department to study the morphogenesis by utilizing tissue culture techniques. De-novo organ initiation and formation under aseptic culture form a fascinating subject to study the effect of different factors on the morphogenetic patterns.Different objectives of morphogenesis are summarized below: i. To study the biological growth patterns like polarity, symmetry, differentiation, ete. ii. To provide an explanation to all such growth patterns. iii, To relate morphogenesis with other major biological sub sciences, iy. Advantageous utilization of tissue culture techniques to study morphogenetic patterns. v. Determining interacting influence of growth hormones in shoots or root regeneration. vi. To study the effects of repeated sub-culturing on the regeneration potential of a totipotent callus, vii. To analyse the growth regulation in differentiation of plant species by trial and error, viii. Study of effect of osmotic inhibition using high levels of sucrose during shoot formation. ix. Study of role of light and temperature in flowering, x. Exploring the correlation between nuclear state and potential for differentiation and regeneration. 11. Subculture or Secondary Cell Culture: The process of transferring the cultured cells in a fresh nutrient medium is called as sub-culturing, and the cell cultures which are sub-cultured (i.e., inoculated in a separate medium) are called as subculture or secondary cell cultures or secondary cell lines. Tt is important to subculture the organ and tissues to fresh medium to avoid the condition of nutrition depletion and drying of medium. It is possible to maintain the plant cell and tissue cultures for indefinitely long time durations if they are regularly subculture in a serial manner (Fig. 10).Fig. 10. Scheme of Subculturing 12. Soma Clonal Variation: Soma clonal variations may be defined as those variations which occur in the cultured cells/tissues or plants regenerated from such cells in-vitro. Soma clonal variations are usually heritable for qualitative as well as quantitative characters of plants. Soma clonal variants have proved as an alternate tool to plant breeding for production of improved varieties of plants. Gene mutations and changes in the structure, number of chromosomesare the main causes of production of soma clonal variants. A number of new varieties of cereals, oil seeds, fruits, tomatoes, etc., possessing disease resistance, better quality, better yield ete., have been generated through soma clonal variations, Some of those crop species are potato, tomato, oats, wheat, rice, maize, datura, carrot, soybean, ete. In simple words, the variability which is generated by the use of a tissue culture cycle is termed as soma clonal variation. Soma clones is a general term which is used to describe those plants which have been derived from any type of somatic cell cultures. 13. Somatic Hybrids and Cybrids: Somatic hybridization may be described as the production of hybrid cells by the fusion of protoplasts of somatic cells derived from two different plant species /varieties. It is immensely helpful for generating new and improved hybrid varieties of plant that may have characters of a completely different species. For example, ‘Pomato’ is a somatic hybrid which is produced by the fusion of protoplast of somatic cells from potato and tomato which are totally different species. A cybrid is a cytoplasmically hybrid cell which has the cytoplasm of both fusing cells but nucleus of only one fusing cell. The process of production of a cybrid is called cybridisation. Steps involved in somatic hybridization/cybridization (Fig. I) are given below:rec et a. Protoplast isolation from parent plant using any mechanical or enzymatic method. b. Fusion of isolated protoplasts derived from two different parents either by utilizing chemical fusogens (like NaNO,, Polyethylene Glycol or high pH-high Car: treatment) or by the electro-fusion method. c. Selection of the hybrid cells is done after fusion process. d. The selected somatic hybrids/cybrids are then verified for hybridity. This is done to check whether the hybrid is carrying the desired characteristics of both parents or not. e, On successful verification and characterization, the somatic hybrids or cybrids are cultured for regenerating into the plantlets with desired characters, Somatic hybridization overcomes the sexual incompatibility barriers and it enables to produce interspecific as well as inter-generic crosses in plants. It helps in imparting disease-resistances and improving the quality characters in plants. It is. an immensely beneficial tool for study of cytoplasmic genes and. their expressions.14. Miero-Propagation: Tissue culture helps in the rapid propagation of plants by the technique of micro-propagation or clonal propagation in-vitro. The asexually produced progeny ofa cell or individual is called as clone and the clones have an identical genotype. Micro propagation is the technique of in-vitro production of the clones of plants i.e., it produces the progeny plants which have an identical genotype as. their parents, by cell, tissue or organ culture (Fig, 12). It helps in the production of plants in large number starting from a single individual. It serves as an alternate method to conventional vegetative propagation methods, Pig. 12. Micropropagation of plants. Micro propagation may be achieved by shoot tips, axillary buds, adventitious buds, bulbs or somatic embryos. A number of plant species are being clonally propagated in vitro, specially the commercially valuable plants like orchids and forest trees. Some of the important plants that have been micro propagated on large scales are: Orchids like Cymbidium, Dendrobium, Aranda, Vanda, Odontoma, Vanilla, etc, Forest trees like Tectona grandis, Biota, Cedrus deodara, Eucalyptus, Picea, Pinus, etc. The technique of micro propagation generally involves fourstages. Each of these stage has its own requirements. The stages in general technique of micro-propagation are described below: Stage I. Initiation:This stage also involves the preparatory process for achieving better establishment of aseptic cultures of explant. Suitable explant is selected from the mother plant. Then, the explant is sterilized and transferred to the nutrient medium for culture. State II, Multiplication: ‘This is the most important stage of micro propagation. In this stage, there occurs the proliferation or multiplication of shoots (or embryoids) from the explant on medium. It occurs either by the formation of an intermediary callus or by induction of adventitious buds directly from the explant. Stage IL. Sub-culturing: ‘The shootsare transferred to rooting medium (sub-cultured) to form roots. As a result, complete plantlets are obtained. Stage IV. Transplantation: In this stage, the regenerated plantlets are transferred out of culture. These are grown in pots followed by field trials. Advantages of Micro Propagation: 1. Rapid multiplication of disease free plants. 2. Rapid multiplication of commercially important plants. 3. Maintenance of genetic uniformity. 4. Technique does not depend on seasons and is capable of producing plants all round the year. 5. Technique is valuable in cases where only limited explant is available. Limitations of Micro Propagation: 1. The technique is costly. 2. It requires proper skill. 3. Many tree crops, including gymnosperms, cannot be multiplied by clonal propagation. 4. Clonal propagation in some cases may lead to the formation of off-types rather than clones, after many generations.5. If culture is contaminated, then the pathogen gets multiplied to very high levels and becomes difficult to handle. 15. Artificial Seed: These are also called a: thetieseeds. These are living seed like structures which are capable of giving rise to plants when sown in the field. An artificial seed is made of a somatic embryo (S.E.) encapsulated with a protective layer of a gel which protects it from desiccation or microbial attack (Fig. 13). Fig. 13. Outline of preparation of synthetic seeds, 16. Cryopreservation: It means preservation at ultralow temperature: This technique is used mainly for long term storage of germplasm and thus helps in conservation of nature also. Plant tissues and organs are cryopreserved usually in liquid Nitrogen which has a temperature of 196°C. Cryopreservation technique has proved to be one of the most reliable methods for long term storage and preservation of plant germplasm in the form of pollens, shoot-tips, embryos, callus, protoplasts, etc. Although, this is a very advantageous technique but it suffers from a major difficulty of fermation of ice-crystals during freezing and/or thawing, These ice-crystals may cause damage to the preserved material. To prevent the formation of ice-crystals during cryopreservation, some special chemicals are used which are called as cryoprotectants. A few common cryoprotectants are glycol, sucrose, proline, Dimethyl Sulfoxide (DMSO), Polyethyleneglycol (PEG), ete... Vascular differentitation: Vascular tissues, xylem and phloem, are differentiated from meristematic cells, procambium, and vascular cambium. Auxin and cytokinin have been considered essential for vascular tissue differentiation; this is supported by recent molecular and genetic analyses. Xylogenesis has long been used as amodel for study of cell differentiation, and many genes involved in late stages of tracheary element formation have been characterized. A number of mutants affecting vascular differentiation and pattern formation have been isolated in Arabidopsis. Studies of some of these mutants have suggested that vascular tissue organization within the bundles and vascular pattern formation at the organ level are regulated by positional information. Totipotency? ‘Totipotency is the genetic potential of a’plant cell to produce the entire plant. In other words, totipotencyis the cell characteristic in which the potential for forming all the cell types in the adult organism is retained. Expression of Totipotency in Culture: The basis of tissue cultureis to grow large number of cells in a sterile controlled environment. The cells are obtained from stem, root or other plant parts and are allowed to grow in culture medium containing mineral nutrients, vitamins and hormones to encourage cell division and growth. As a result, the cells in culture will produce an unorganised proliferative mass of cells which is known as callus tissue. The cells that comprise the callus mass are totipotent, Thus a callus tissue may be in a broader sense totipotent, i.e., it may be able to regenerated back to normal plant given certain manipulations of the medium and the culturalen- vironment. Truly speaking, totipotency of the cell is manifested through the process of differentiation and the hormones in this process play the major role than any other manipulations. In the fifties, F. Skoog and C.O. Miller of U.S.A. discovered a new plant growth hormone kinetin from herring sperm DN A. With a correct concentration ratio of auxin and cytokinin in tobacco cultures, Skoog was able to demonstrate the role of kinetin in organogenesis. When the ratio of kinetin to auxin was higher, only shoot developed. This is known as caulogenesis. But when the ratio was lower, only roots were formed. This is known as rhizogenesis. Around the same period, F. C. Steward and his colleagues at Cornell University of USA, devised a method for growing carrot tissue by excising small disc, from the secondary phloem region of carrot root and placing them in a moving liquid medium under aseptic conditions. In presence of coconut milk in the medium, the phloem tissue began to the grow actively.In moving liquid medium somesingle cells and small groups of cells were loosened from the surface of growing tissue. When these isolated cells were grown separately it was found that some single cells developed somatic embryos or embryoids by a process that occurs in normal zygotic embryo Tt is also observed in some experiment that cells of some callus mass frequently differentiate into vascular elements such as xylem and phloem without forming any plant organs or embryoids. This process is known as histogenesis or Cyto- differentiation. Thus the totipotent cells may express themselves in different way on the basis of differentiation process and manipulation. Where the totipotent cells are partially expressed or not expressed, it is obvious that the limitation on its capacity for development must be imposed by the microenvironments. The totipotency of cells in the callus tissue may be re- tained fora longer period through several subcultures. Practically, it is observed that the ex- plant first forms the callus tissue in the calliis inducing medium and such callus tissue is maintained through some subcultures. After then it is generally transferred to another medium which is expected to be favourable for the expression of totipotent cells. Actually, the regeneration medium is standardized by trial and error method, In more or less suitable medium, the totipotent cells of the callus tissue give rise to meristematicnodules or meristemoids by repeated cell division. This may subsequently give rise to vascular differentiation or it may form a primordium capable of giving rise to a shoot or root. Sometimes the totipotent cell may produce embryoids through sequential stages of development such as globular stage, heart shaped stage and torpedo stage ete. After prolonged culture, it has been observed that calluses in some species (e.g. Ntcotiana tabacum, Citrus aurantifolia etc.) maybe- come habituated. This means that they are now able to grow on a standard maintenance medium. which is devoid of growth hormones. The cells of habituated callus also remain totipotent and are capable to regenerate a plant withoutany major manipulation,Totipovent cell 4 Differentiation 4 i Organogenesis Histogenesis or ‘| Cytodifferentiation eS ee 1 Caulogenesis ‘Rhizogenesis Caulorhizogenesis Xylem, Phloem, ete: plantlet suidasy ouy root Shoot ean root Atypical crown gall tumourcell has the capacity for unlimited growth independent of exogenous hormones. It shows totally lack of organ genic differentiation. So such tissue is considered to have permanently lost the totipotentiality of the parent cells. In some plant species, the crown gall bacterium (Agrebactenum tumefaciens) induces a special type of tumour, called teratomas, the cells of which possess the capacity to differentiate shoot buds and leaves when they are grown in culture for unlimited periods. Thus it is clear that the mode of expression of totipotency of plant cell in culture varies from plant to plant and also helps us to understand the process of differentiation in vitro. Importance of Totipotency in Plant Science: The ultimate objective in plant protoplast, cell and tissue culture is the reconstruction of plants from the totipotent cell. Although the process of differentiation is still mysterious in general, the expression of totipotent cell in culture has provided a lot of information's. On the ather hand, the totipotentiality of somatic cells has been exploited in vegetative propagation of many economical, medicinal as well as agriculturally important plant species. Therefore, from fundamental to applied aspect of plant biology, cellular totipotency is highly important. Recent trends of plant tissue culture include genetic modification of plants, production of homozygous diploid plants throngh haploid cell culture, samatic hybridization, mutation etc. The success of all these studies depends upon the expression of totipotency. In many cases, successful and exciting results have been obtained. Plant breeders, horticulturists and commercial plant growers are now more interested in plant tissue culture only for the exploitation of totipotent cells in culture according to their desirable requirement. Totipotent cells within a bit of callus tissue can be stored in liquid nitrogen for a long period. Therefore, forgermplasm preservation of endangered plant species, totipotency can be utilized successfully. CULTURE MEDIA: Growth and morphogenesis of plant tissues in vitro are largely governed by the composition of the culture media. Although the basic requirements for culturing plant tissue is same but in practice there are some specific nutritional requirements for promoting optimal growth from different kinds of explants in case of different plant species. Components of Medi: “Table 14: Companion imp of thre banal medinfor plan tee cultures Nurahigeand —— Gamberg ata Ingreiret Show (MS) median (8) eda it's a SLND, =— ~ NO) euNO AMO e210 The principal components of most plant tissue culture media are inorganic nutrients (macronutrients and micronutrients), carbon sources, organic supplements, growth regulators, vitamins, amino acids and gelling agent forsolid or semisolid media. The various culture media formulated for plant tissue culture are Murashige and Skoog’s medium, Gamborg’s medium, White's medium, ete. which differ (Table 16.1) mainly in quantity of the components. (@) Inorganic Nutrients: A variety of mineral elements (salts) supply the macro- and micronutrients required in the life of a plant. Elements required in concentration greater than 0.5 m mol/i referred to as macronutrients and those less than 0.05 m mol/1 as micronutrients. The macronutrients include six major elements like Nitrogen (N), Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg) and Sulphur (S) which are present as salts in various media, The micronutrients are iron (Fe), Manganese (Min), Zine (Zn), Boron (B), Copper (Cu), Molybdenum (Mo). Among these iron is used in the medium as chelated form with EDTA (ethylene-diamino- tetra-acetic acid). (ii) Carbon and Energy Source: Plant cells. and tissues in the culture medium lack autotrophic ability and therefore need external carbon for energy. The most preferred carbon source in tissue culture media is sucrose; glucose supports equally for good growth while fructose is less efficient. (iii) Vitamins: The plant cells and tissues are capable of synthesizing different vitamins but in in vitro condition they are being produced at sub-optimal level. Hence it is necessary to supplement the media with required vitamins such as Thiamine (B,), Nicotinic acid (B;), Pyridoxine (Bc), Pantothenic acid (B;) and myoinositol. Except these other vitamins like Biotin, Folic acid, Ascorbic acid are also used in some media. (iv) Amino Acids: In vitro grown plant cells or tissues are capable of synthesizing amino acids but the addition of amino acids to media is important for stimulating cell growth and protoplast culture and for establishment of cell lines. Glutamine, asparagine, glycine, arginine, cysteine are the commonly used amino acids. (v) Growth Regulators: Three broad classes of growth regulators mainly auxins, cytokinins and gibberellins are used in tissue culture. The growth, differentiation and organogenesis of tissues become feasible only on the addition of one or more of these classes of hormones to a medium,Various kinds of auxins like IAA (Indole-3-Acetic Acid), NAA (Naphthalene Acetic Acid), 2, 4-D (2, 4-dichlorophenoxy acetic acid) are used to induce cell division, elongation of stem, internodes and rooting. Cytokinins like BAP (Benzyl amino purine), Kinetin (Furfurylamino purine), Zeatin or 2iP (Isopentynyl adenine) are used which are mainly concerned with cell division, modification of apical dominance and shoot differentiation in tissue culture. ‘The ratio of auxin and cytokinin is important with respect to morphogenesis in the culture system. For embryogenesis, callus initiation and root initiation the requisite ratio of auxin to cytokinin is high, while the reverse leads to organogenesis. and shoot proliferation. Gibberellins (GA,) are occasionally used growth regulators to induce plantlet formation from adventive embryos developed in culture. (vi) Other Organic Supplements: Culture media are often supplemented with variety of organie extracts which have the constituents of an undefined nature e.g. casein hydrolysate, coconut milk, malt extracts and fruit juice. (vii) Activated Charcoal: The addition of activated charcoal to culture media is reported to stimulate growth and differentiation by reducing toxicity by removing toxic substances (e.g. Phenols). (viii) Antibiotics: For transformation experiments addition of antibiotics to culture media is required to prevent the growth of Agrobacterium which retards the cell or tissue growth. (ix) Gelling Agent: Gelling or solidifying agents are commonly used for preparing semisolid or solid tissue culture media to provide solid surface area for growth. Agar (a polysaccharide obtained from seaweeds), Gelatin (commercially avai- lable as Phytagel, Alginate or Gelrite) are commonly used solidifying agents at a concentration of 0.8-1.0% (which are not any kind of nutrient), depending on the type of tissue and the species of plant and culture methods. Media Preparation: For media preparation it is convenient to use stock solutions of major salts (20X), minor salts (200X), organic supplements (200X), growth regulator (1m.mol or 10m mol). For final media preparation the stock solutions are mixed together in appropriate quantities. All the stock solutions are stored in proper plastic or glass containers at low temperature. After preparation of media the pH is adjusted at 5.5-6.0 and agar is mixed. whenever it is required. The media is poured into the desired type of culture vessels (culture tubes, conical flasks, petriplates, etc.), properly plugged with non-absorbent cotton and autoclaved. PLANT TISSUE CULTURE MEDIA: Basically, the plant tissue culture media should contain the same nutrients as required by the whole plant. It may be noted that plants in nature can synthesize their own food material. However, plants growing in vitro are mainly heterotrophici.e. they cannot synthesize theirown food. Composition of Media: The composition of the culture media is primarily dependent on two parameters: 1. The particular species of the plant. 2. The type of material used for culture i.e. cells, tissues, organs, protoplasts. Thus, the composition of a medium is formulated considering the specific requirements of a given culture system. The media used may be solid (solid medium) or liquid (liquid medium) in nature. The selection of solid or liquid medium is dependent on the better response of a culture. Major Types of Media: The composition of the most commonly used tissue culture media is given in Table 43.1, and briefly described below.Gycine 3 Fote acs = eer lena pH 58 - White’s medium: This is one of the earliest plant tissue culture media developed for root eulture. MS medium: Murashige and Skoog (MS) originally formulated a medium to induce organogenesis, and regeneration of plants in cultured tissues. These days, MS. medium is widely used for many types of culture systems. B5 medium:Developed by Gamborg, B5 medium was originally designed for cell suspension and callus cultures. At present with certain modifications, this medium is used for protoplast culture. N6 medium: Chu formulated this medium and itis used for cereal anther culture, besides other tissue cultures. Nitsch’s medium: This medium was developed by Nitsch and Nitsch and frequently used for anther cultures. Among the media referred above, MS medium is most frequently used in plant tissue culture work due to its suecess with several plant species and culture systems. Synthetic and natural media: Whena medium is composed of chemically defined components, it is referred to as a synthetic medium. On the other hand, if a medium contains chemically undefined compounds (e.g., vegetable extract, fruit juice, plant extract), it is regarded as a natural medium. Synthetic media have almost replaced the natural media for tissue culture. Expression of concentrations in media: The concentrations of inorganic and organic constituents in culture media are ustially expressed as mass values (mg/l or ppm or mg 1+). However, as per the recommendations of the International Association of Plant Physiology, the concentrations of macronutrients should be expressed as mmol/I-and micronutrients as pmol/l-. Constituents of Media: Many elements are needed for plant nutrition and their physiological functions. Thus, these elements have to be supplied in the culture medium to support adequate growth of cultures in vitro. A selected list of the elements and their functions in plants is given in Table 43.2.The culture media usually contain the following constituents: 1, Inorganic nutrients 2. Carbon and energy sources 3. Organic supplements 4. Growth regulators 5. Solidifying agents 6. pH of mediumInorganic Nutrients: The inorganic nutrients consist of macronutrients (concentration >0.5 mmol/I-) and micronutrients (concentration <0.5 mmol/I-). A wide range of mineral salts (elements) supply the macro- and micronutrients. The inorganic salts in water undergo dissociation and ionization. Consequently, one type of ion may be contributed by more than one salt. For instance, in MS medium, K: ions are contributed by KNO, and KH:PO,while NO, ions come from KNO, and NH.NOs. Macronutrient elements: The six elements namely nitrogen, phosphorus, potassium, calcium, magnesium and sulfur are the essential macronutrients for tissue culture. The ideal concentration of nitrogen and potassium is around 25 mmol I+ while for calcium, phosphorus, sulfurand magnesium, it is in the range of 1-3 mmolL-. For the supply of nitrogen in the medium, nitrates and ammonium salts are together used, Micronutrients: Although their requirement is in minute quantities, micronutrients are essential for plant cells and tissues. These include iron, manganese, zinc, boron, copper and molybdenum. Among the microelements, iron requirement is very critical. Chelated forms of iron and copper are commonly used in culture media. Carbon and Energy Sources: Plant cells. and tissues in the culture medium are heterotrophic and therefore, are dependent on the external carbon for energy. Among the energy sources, sucrose is the most preferred. During the course of sterilization (by autoclaving) of the medium, sucrose gets hydrolysed to glucose and fructose. The plant cells in culture first utilize glucose and then fructose. In fact, glucose or fructose can be directly used in the culture media. It may be noted that for energy supply, glucose is as efficient as sucrose while fructose is less efficient. Itis a common observation that cultures grow better on a medium with autoclaved sucrose than on a medium with filter-sterilized sucrose. This clearly indicates that the hydrolysed products of sucrose (particularly glucose) are efficient sources of energy. Direct use of fructose in the medium subjected to autoclaving, is found to be detrimental to the growth of plant cells. Besides sucrose and glucose, other carbohydrates such as lactose, maltose, galactose, raffinose, trehalose and cellobiose have been used in culture media but with a very limited success.Organic Supplements: The organic supplements include vitamins, amino acids, organic acids, organic extracts, activated charcoal and antibiotics. Vitamins: Plant cells. and tissues in culture (like the natural plants) are capable of synthesizing vitamins but in suboptimal quantities, inadequate to support growth. Therefore the medium should be supplemented with vitamins to achieve good growth of cells. The vitamins added to the media include thiamine, riboflavin, niacin, pyridoxine, folic acid, pantothenic acid, biotin, ascorbic acid, myoinositol, Para amino benzoic acid and vitamin E. Amino acids: Although the cultured plant cells can synthesize amino acids to a certain extent, media supplemented with amino acids stimulate cell growth and help in establishment of cells lines. Further, organic nitrogen (in the form of amino acids such as L-glutamine, L-asparagine, L- arginine, L-cysteine) is more readily taken up than inorganic nitrogen by the plant cells. Organic acids: Addition of Krebs cycle intermediates suchas citrate, malate, succinate or fumarate allow the growth of plant cells. Pyruvate also enhances the growth of cultured cells. Organic extracts: Tt has been a practice to supplement culture media with organic extracts such as yeast, casein hydrolysate, coconut milk, orange juice, tomato juice and potato extract. It is however, preferable to avoid the use of natural extracts due to high variations in the quality and quantity of growth promoting factors in them. In recent years, natural extracts have been replaced by specific organic compounds e.g., replacement of yeast extract by L-asparagine; replacement of fruit extracts by L-glutamine. Activated charcoal: Supplementation of the medium with activated charcoal stimulates the growth and differentiation of certain plant cells (carrot, tomato, orchids). Some toxic/inhibitory compounds (e.g. phenols) produced by cultured plants are removed (by adsorption) by activated charcoal, and this facilitates efficient cell growth in cultures. Addition of activated charcoal to certain cultures (tobacco, soybean) is found to be inhibitory, probably due to adsorption of growth stimulants suchas phytohormones.Antibiotics: Tt is sometimes necessary to add antibiotics to the medium to prevent the growth of microorganisms. For this purpose, low concentrations of streptomycin or kanamycin are used. As far as possible, addition of antibiotics to the medium is avoided as they have an inhibitory influence on the cell growth. Growth Regulators: Plant hormones or phytohormones are a group of natural organic compounds that promote growth, development and differentiation of plants. Four broad classes of growth regulators or hormones are used for culture of plant cells- auxins, eytokinins, gibberellins (Fig. 43.1) and abscisic acid. They promote growth, differentiation and organogenesis of plant tissues in cultures. coon = HN-CHy Smuctures of satacted plant growth regulators. Auxins: Auxins induce cell division, cell elongation, and formation of callus in cultures. At a low concentration, auxins promote root formation while at a high concentration callus formation occurs. A selected list of auxins used in tissue cultures is given in Table 43.3.‘Toa 43.3 A selected list of plant growth ‘regulators used In culture medla Growth cegutsion (Chemical aame Among the auxins, 2, 4-dichlorophenoxy acetic acid is most effective and is widely used in culture media. Cytokinins: Chemically, cytokinins are derivatives of a purine namely adenine. These adenine derivatives are involved in cell division, shoot differentiation and. somatic embryo formation, Cytokinins promote RNA synthesis and thus stimulate protein and enzyme activities in tissues. The most commonly used cytokinins are given in Table 43.3. Among the cytokinins, kinetin and benzyl- amino purine are frequently used in culture media. Ratio of auxins and cytokinins: The relative concentrations of the growth factors namely auxins and cytokinins are crucial for the morphogenesis of culture systems. When the ratio of auxins to cytokinins is high, embryogenesis, callus initiation and root initiation oceur. On the other hand, for axillary and shoot proliferation, the ratio of auxins to cytokinins is low. For all practical purposes, it is considered that the formation and maintenance of callus cultures require both auxin and cytokinin, while auxin is needed for root culture and cytokinin for shoot culture. The actualconcentrations of the growth regulators in culture media are variable depending on the type of tissue explant and the plant species. Gibberellins: About 20 different gibberellins have been identified as growth regulators. Of these, gibberellin A, (GA,) is the most commonly used for tissue culture. GA, promotes growth of cultured cells, enhances callus growth and induces dwarf plantlets to elongate. Gibberellins are capable of promoting or inhibiting tissue cultures, depending on the plant species. They usually inhibit adventitious root and shoot formation. Abscisic acid (ABA): The callus growth of cultures may be stimulated or inhibited by ABA. This largely depends on the nature of the plant species. Abscisic acid is an important growth regulation for induction of embryogenesis. Solidifying Agents: For the preparation of semisolid or solid tissue culture media, solidifying or gelling agents are required. In fact, solidifying agents extend support to tissues growing in the static conditions. Agar: Agar, a polysaccharide obtained from seaweeds, is most commonly used as a gelling agent for the following reasons. 1. Itdoes not react with media constituents. 2. It is not digested by plant enzymes and is stable at culture temperature. Agar at a concentration of 0.5 to 1% in the medium can form a gel. Gelatin: Tt is used at a high concentration (10%) with a limited success. This is mainly because gelatin melts at low temperature (25°C), and consequently the gelling property is lost. Other gelling agents: Bio-gel (polyacrylamide pellets), phytagel, gelrite and purified agarose are other solidifying agents, although less frequently used. It is in fact advantageous to use synthetic gelling compounds, since they can form gels ata relatively low concentration (1.0 to 2.5 g |»). pH of medium: The optimal pH for most tissue cultures is in the range of 5.0-6.0. The pH generally falls by 0.3-0.5 units after autoclaving. Before sterilization, pH can beadjusted to the required optimal level while preparing the medium. Itis usually not necessary to use buffers forthe pH maintenance of culture media. Ata pH higher than 7.0 and lower than 4.5, the plant cells stop growing in cultures. If the pH falls during the plant tissue culture, then fresh medium should be prepared. In general, pH above 6.0 gives the medium hard appearance, while pH below 5.0 does not allow gelling of the medium. Preparation of Media: The general methodology for a medium preparation involves preparation of stock solutions (in the range of 10x to 100x concentrations) using high purity chemicals and demineralized water. The stock solutions can be stored (in glass or plastic containers) frozen and used as and when required. Most of the growth regulators are not soluble in water, They have to be dissolved in NaOH or alcohol. Dry powders in Media Preparation: ‘The conventional procedure for media preparation is tedious and time consuming. Now a days, plant tissue culture media are commercially prepared, and are available in the market as dry powders. The requisite medium can be prepared by dissolving the powder in a glass distilled or demineralized water. Sugar, organic supplements and agar (melted) are added, pH adjusted and the medium diluted to a final volume (usually 1 litre). Sterilization of Media: The culture medium is usually sterilized in an autoclave at 121°C and 15 psi for 20 minutes. Hormones and other heat sensitive organic compounds are filter- sterilized, and added to the autoclaved medium. Selection of a Suitable Medium: In order to select a suitable medium for a particular plant culture system, it is customary to start with a known medium (e.g. MS medium, B5 medium) and then develop a new medium with the desired characteristics. Among the constituents of a medium, growth regulators (auxins, cytokinins) are highly variable depending on the culture system. In practice, 3-5 different concentrations of growth regulators in different combinations are used and the best among them are selected. For the selection of appropriate concentrations of minerals and organic constituents in the medium, similar approach referred above, can be employed.Medium-utmost Important for Culture: For tissue culture techniques, it is absolutely essential that the medium preparation and composition are carefully followed. Any mistake in the preparation of the medium is likely to do a great harm to the culture systemas a whole. Plant hormones and growth regulators are chemicals that affect: 1 Flowering. 2 Aging. 3 Root growth. 4 Distortion and killing of organs. 5 Prevention or promotion of stem elongation. 6 Color enhancement of fruit. 7 Prevention of leafing, leaf fall or both. 8 Many other conditions, Very small concentrations of these substances produce major growth changes. Compound Effect/Use Stimulates cell division and elongation, Gibberellic acid (GA) Creaks dormancy, speeds germination Ripening agent; stimulates leaf and fruit Ethylene gas (CH2) abeeIESIOn Stimulates apical dominance, rooting, Indoleacetic acid (IAA) nd learabselesian Indolebutyric acid Stimulates root growth (IBA) Naphthalene acetic Stimulates root growth, slows acid (NAA) respiration (used as a dip on holly) Growth retardants Prevent stem elongation in selected (Alar, B-9, Cycocel, crops (€.g., chrysanthemums,Arest) poinsettias, and lilies) Distorts plant growth; selective and nonselective materials used for killing unwanted plants Hormones are produced naturally by plants, while plant growth regulators are applied to plants by humans. Plant growth regulators may be synthetic compounds, such as IBA and Cycocel, that mimic naturally occurring plant hormones, or they may be natural hormones that were extracted from plant tissue, such as IAA, Herbicides (2,4-D, etc.) Applied concentrations of these substances usually are measured in parts per million (ppm) and in some cases parts per billion (ppb). These growth-regulating substances most often are applied as a spray to foliage or as a liquid drench to the soil around a plant's base. Generally, their effects are short- lived, and they may need to be reapplied in order to achieve the desired effect. There are five groups of plant-growth-regulating compounds: auxin, gibberellin (GA), cytokinin, ethylene, and abscisic acid (ABA). For the most part, each group contains both naturally occurring hormones and synthetic substances. AUXIN: causes several responses in plants: 1. Bending toward a light source (phototropism). 2. Downward root growth in response to gravity (geotropism). 3. Promotion of apical dominance (the tendency of an apical bud to produce hormones that suppress the growth of the buds below it on the stem). 4, Flower formation. 5, Fruit set and growth. 6, Formation of adventitious roots.Auxin is the active ingredient in most rooting compounds in which cuttings are dipped during vegetative propagation. GIBBERLIN: stimulate cell division and elongation, break seed dormancy, and speed germination. The seeds of some species are difficult to germinate; you can soak them ina GA solution to get them started. CYTOKININS: Unlike other hormones, cytokinins are found in both plants and animals, They stimulate cell division and often are included in the sterile media used for growing plants from tissue culture. If a medium's mix of growth-regulating compounds is high in cytokinins and low in auxin, the tissue culture explant (small plant part) will produce numerous shoots. On the other hand, if the mix has a high ratio of auxin to cytokinin, the explant will produce more roots. Cytokinins also are used to delay aging and death (senescence). ETHYLENE: Ethylene is unique in that it is found only in the gaseous form. It induces ripening, causes leaves to droop (epinasty) and drop (abscission), and promotes senescence. Plants often increase ethylene production in response to stress, and ethylene often is found in high concentrations within cells at the end of a plant's life. The increased ethylene in leaf tissue in the fall is part of the reason leaves fall off trees. Ethylene also is used to ripen fruit (e.g., green bananas). ABSCISIC ACID: Abscisic acid (ABA) is a general plant-growth inhibitor, It induces dormancy and prevents seeds from germinating; causes abscission of leaves, fruits, and flowers; and causes stomata toclose, High concentrations of ABA in guard cells during periods of drought stress probably play a role in stomatal closure. STERILIZATION TECHNIQUES: A) Dry Heat Sterilization — Principle and Uses What is dry heat sterilization? It is the process of killing bacterial spores and microorganisms using a high temperature. This type of sterilization method is used on items that cannot get wet such as powders, oils, and the likes Commonly used instruments for dry heat sterilization are the following: 1,Hot air oven . Microwave - Radiation . Flaming Incineration/burning . Glass bead sterilizer . Bunsen burner NOOhwN Dry heat sterilization has many types such as the following: Hotalroven, Itis a common form of dry heat sterilization used in the workplace. It is basically an oven that can hold several objects. It is closed for a specific timeframe to sterilize the objects inside it. The desired temperature should be achieved to fully maximize the sterilization process. Hot air oven sterilizes the object inside it without causing harm to the objects and to the environment. (1, 2, and 3)The principle of hot air oven dry heat sterilization Sterilization is achieved by means of conduction. The heat in the oven is absorbed by the item inside it and passes towards the center of the item layer by layer. For the item to be fully sterilized, it needs to reach the required temperature. What dry heat sterilization does is it inflicts damage by oxidizing molecules leading to the organism's death. Resistant spores are killed by exposing them ata higher temperature for a long period of time. There are two types of hot air oven. These are the following: 1. Static air hot air oven — The oven is heated using the coils on the bottom. It would take some time for the oven to achieve its desired temperature. There is also a possibility that the desired heat will not be eventually distributed throughout the oven. 2. Forced air hot air oven — it has a motorized blower that evenly distributes the heat throughout the oven. Another advantage of this hot air oven is that it does not take long to heat the entire oven. What are the advantages of dry heat sterilization? itis a non-toxic way of sterilizing things Itis safe for the environment. It gently and thoroughly penetrates materials It is compatible with metal and sharp objects because it is non-corrosive. It has a low operating cost. (4, 5) What are the disadvantages of dry heat sterilization? Such method of sterilization takes time because of the slow rate of heat penetration. It requires an extremely high temperature to thoroughly kill resistant microbes, which makes it not suitable for rubber and plastic materials. Overexposure to heat may ruin the items being sterilized for. (5, 6)Things commonly sterilized using dry heat sterilization method Metal instruments Glassware Syringes Paper-wrapped items Powder Fats Anhydrous oils (7) Dry heat sterilization features 1. It does not require human intervention. What the human needs to do is to simply set the required temperature and time. As a matter of fact, some dry heat sterilization equipment only requires you to choose a particular setting and after which the machine will do all the necessary work. . .Dry heat sterilizers come with a special ventilation system. The air in the chamber is heated evenly thereby preserving sterilized objects. The temperature inside the sterilizer is supported and the possibility of overheating is eliminated. 3. It comes with a reserve system dependent on the thermostat and works independently from the primary system. 4. You can sterilize both packed and unpacked objects. (3, 5, 7, and 8) Difference between dry heat sterilization and moist heat sterilization Both dry heat and moist heat are used to sterilize objects. However, there is a huge difference between the two. Dry heat uses dry air of high temperature while moist heat sterilization uses alow temperature and a high pressure of water. Dry heat sterilization takes more time than the heat moist sterilization. The two comes with advantages and disadvantages. Moist heat sterilization requires low temperature and less time to complete. It is also low cost and non-toxic. However, it cannot sterilize heat-sensitive instruments.STERILIZATION TECHNIQUES: Since it is using water, the instruments or items being sterilized remain wet which increases the possibility of rusting. Repeated exposure to moisture may damage the items being sterilized. The advantages and disadvantages of dry heat sterilization are already mentioned above. WET HEAT (Autoctaving) The method of choice for sterilisation in most labs is autoclaving; using pressurised steam to heat the material to be sterilised. This is a very effective method that kills all microbes, spores and viruses, although for some specific bugs, especially high temperatures or incubation times are required Autaclaving kills microbes by hydrolysis and coagulation of cellular proteins, which is efficiently achieved by intense heat in the presence of water. The intense heat comes from the steam, Pressurised steam has a high latent heal; at 100degC it holds 7 times more heat than water at the same temperature. This heat is liberated upon contact with the cooler surface of the material to be sterilised, allowing rapid delivery of heat and good penetration of dense materials. At these temperatures, water does a great job of hydrolysing proteins.., so those bugs don't stand a chance. DRY HEAT (Flaming, baking) Dry heating has one crucial difference from autoclaving. You've guessed it — there's no water, so protein hydrolysis can't take place. Instead, dry heat tends to kill microbes by oxidation of cellular components. This requires more energy than protein hydrolysis so higher temperatures are required for efficient sterilization by dry heat. For example sterilisation can normally be achieved in 15 minutes by autoclaving at 121degC, whereas dry heating would generally need a temperature of 160degC to sterilize in a similar amount of time. FILTRATION Filtration is a great way of quickly sterilizing solutions without heating. Filters, of course, work by passing the solution through a filler with a pore diameter that is too small for microbes to pass through. Filters can be scintered glass funnels made from heat-fused glass particles or, more commonly these days, membrane filters made from cellulose esters. For removal of bacteria, filters with an average pore diameter of 0.2um is normally used But remember, viruses and phage can pass through these filters so filtration is not a good option if these are a concern. SOLVENTSEthanol is commonly used as a disinfectant, although since isopropanol is a better solvent for fat it is probably a better option. Both work by denaturing proteins through a process that requires water, so they must be diluted to 60-90% in water to be effective. Again, a it's important to remember that although ethanol and IPA are good at killing microbial cells, they have no effect on spores. RADIATION UV, xrays and gamma rays are all types of electromagnetic radiation that have profoundly damaging effects on DNA, so make excellent tools for sterilization. The main difference between them, in terms of their effectiveness, is their penetration. UV has limited penetration in air so sterilisation only occurs in a fairly small area around the lamp. However, it is relatively safe and is quite useful for sterilising small areas, like laminar flow hoods. X-rays and gamma rays are far more penetrating, which makes them more dangerous but very effective for large scale cold sterilization of plastic items (e.g. syringes) during manufacturing. So those are some of the main methods for sterilization | can think of. If I've missed any, please feel free to let me know in the comments section. Sterilisation Instruments: Routinely Used Instruments for Sterilisation: a. Physical Methods — Dry Heat: i, Hot air oven: The oven is heated by electricity and has a thermostat to maintain the air temperature constant (160°C for 1 hour). It is the best method to sterilize dry glass water, cotton wool plugged test tubes, Petri dishes, flasks, pipettes, swabs, powder, fat or grease). All glassware’s should be dry, and wrapped in Kraft paper to soak any water drop on the glassware. The oven should be loaded in such a way that the air can circulate through the load. The temperature should be raised slowly in the course of 1-2 hours to reach 160°C—which must be maintained for 1 hour. This is the holding period. Finally, the oven is allowed to cool gradually for 2 hours before the door is opened, otherwise the glassware’s may crack due to sudden atmospheric cooling. Infrared radiation: The infrared rays are directed on the object to be sterilised at a temperature of 180°C as a means of sterilising surgical instruments,