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Plant tissue culture has a great significance in plant biotechnology especially 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 culture is fundamental to most aspects of biotechnology of plants. It 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 meristematic in 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 for the 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 number of suitable culture media were developed, for culturing plant cells, tissues, protoplasts, embryos, anthers, root tips, etc. 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. The first plant from a mature plant cell was regenerated by Braun in 1959. Foundation of commercial plant tissue culture was laid in 1960 with the discovery for a million fold increase in the multiplication of Cymbidium (an orchid) which was accomplished by G.M.Morel. In India, the work on tissue culture was initiated during 1950s at University of Delhi. This initiation is credited to Shri Panchanan Maheshwari who was working there in the Department of Botany. Discovery of haploid production was a land-mark in the development of in-vitro culturing of plants. Shri S.C. Maheshwari and Sipra Guha made a remarkable contribution in the development of plant tissue culture in India. Later on the development in the composition of nutrient media and genetic engineering served as a basis for further success in the plant tissue culture techniques. Gottleib Haberlandt was the first person to make attempts for plant tissue culture, i.e., he developed the concept of in-vitro culture of plant cells and is aptly regarded as the father of tissue culture. Thereafter, there happened some dramatic advances in tissue culture techniques. Some of the early classical contributions in the field of plant tissue culture are tabulated below:
- MS (Murashige and Skoog) Medium
- LS (Linsmaier and Skoog) Medium
- B5 (Gamborg’s) Medium
- White’s Medium, etc. Important constituents of a culture medium are: Organic supplements: (a) Vitamins like thiamine (B 1 ), Pyridoxin (B 6 ), Nicotinic Acid (B 3 ), etc. (b) Antibiotics like Streptomycin, Kanamycin; (c) Amino Acids like Arginine, Asparagine. Inorganic Nutrients: Micronutrients as Iron (Fe), Manganese (Mn), Zinc (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). Carbon and Energy Source: Most preferred carbon source is Sucrose. Others include lactose, maltose, galactose, raffinose, cellobiose, etc. 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 etc. 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)+ (5ml of each stock solutions II, III.IV) 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:
- Sterilization of the culture vessels using detergents, autoclaves, etc.
- Sterilization of instruments like forceps, needles etc. by flame sterilization.
- Sterilization of culture medium using filter sterilization or autoclaving methods.
- Surface sterilization of explants using surface disinfectants like Silver Nitrate (1%), H 2 O 2 (10-12%), Bromine water (1-2%), Sodium Hypochlorite solution (0.3-0.6%), etc.
(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. (f) 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:
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. 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 quiescent in 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. Dedifferentiation and re-differentiation are the two inherent phenomena involved in the cellular totipotency. 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.
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: Cellular totipotency of plants cells has proved to be a boon to mankind as 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: It has potential applications in the crop plant improvement.
- Micro-propagation of commercially important plants.
- Production of artificial or synthetic seeds.
- It helps in conservation of germplasm (genetic resources).
- This ability is utilized for haploid productions.
- Applied in producing somatic hybrids and cybrids.
- Helps in cultivation of those plants whose seeds are very minute and difficult to germinate.
- Also helps to study the cytological and histological differentiations.
- For high scale and efficient production of secondary metabolites.
- The genotypic modifications can also be possible. Differentiation: While studying 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 meristematic stage. 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:
- Cytodifferentiation
- Organ Differentiation
- Embryo Genic Differentiation 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
with the stimulus produced by the components of culture medium, the substances initially present in the original explants and also by the compounds produced 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 in a very low frequency. However, the roots and shoot bud regenerations are quite frequent. Out of all these types of organogenic 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 phenomena are 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:
- Auxin: Cytokinin ratio in medium is an important factor affecting root/shoot bud differentiation in most plants.
- Usually Gibberellic acid inhibits organogenesis.
- Physiological state and size of explant play important role in organ differentiation.
- Genotype of the donor plant plays a crucial role.
- Physical factors like light, temperature, moisture, etc., play effective role in organogenesis.
c. 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. 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.
(b) In-vitro culturing of the single cell utilizing micro chamber technique, or micro drop method or Bergmann cell plating technique (Fig. 6). (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. Meristem Culture: The apical meristem of shoots of angiosperms and gymnosperms can be cultured to get the disease free plants. Meristem tips, between 0.2-0.5 mm, most frequently produce virus-free plants and this method is referred to as meristem-tip culture. This method is more successful in case of herbaceous plants than woody plants. In case of woody plants, the success is obtained when the explant is taken after the dormancy period is over. After the shoot tip proliferation, the rooting is done and then the rooted plantlet is potted. Bud Culture: Buds contain quiescent or active meristems in the leaf axils, which are capable of growing into a shoot. Single node culture, where each node of the stem is cut and allowed to grow on a nutrient media to develop the shoot tip from the axil which ultimately develops into new plantlet. In axillary bud method, where the axillary buds are isolated from the leaf axils and develop into shoot tip under little high cytokinin concentration.
Callus Culture: Callus is basically more or less un-organised dedifferentiated mass of cells arising from any kind of explant under in vitro cultural conditions. The cells in callus are parenchymatous in nature, but may or may not be homogenous mass of cells. They are meristematic tissue, under special circumstances they may be again organised into shoot primordia or may develop into somatic embryos. The callus tissue from different plant species may be different in structure and growth habit. The callus growth is also dependent on factors like the type of explant and the growth conditions. After callus induction it can be sub-cultured regularly with appropriate new medium for growth and maintenance. 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. 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 (i.e. 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).
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 for 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). (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, in a very few plants only, hence it is not a very popular method for in-vitro production of haploids. Thus, androgenesis is preferred over gynogenesis. 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
