Tissue Culture��Dr. M. M. V. Baig�Head, �Department of Biotechnology,�Yeshwant Mahavidyalaya, �Nanded�

UNIT –III: BIOTECHNOLOGY (11periods)

Tissue culture:

• Introduction
• Basic aspects of tissue culture, media,
• culture techniques,
• cellular totipotency.
• Applications of tissue culture:
• Micropropagation,
• Production of disease free plants,
• production of secondary metabolites,
• Anther culture and production of haploids,
• protoplast culture and somatic hybridization,
• synthetic seeds

4. Tissue culture: Basic concepts, technique of tissue culture (Steps involved) callus culture, differentiation and morphogenesis, plantlets, protoplast culture and Anther culture

5. Applications of Tissue culture.

Introduction:-

protoplast, cell, tissue, or organ are cultured in vitro on artificial media under aseptic conditions.

This method of tissue culture has been practiced mainly for

(i) preserving germplasm (genetic material) of valuable plant varieties, (ii) rapid multiplication of genetic stocks (clonal propagation),

(iii) for obtaining pathogen free plant materials and

(iv) for other application.

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Tissue Culture

Haberlandt (1902) a German botanist was the first to give an idea about the cell and tissue culture. He tried to grow the leaf cells but could not succeed.

White (1932) was successful in culturing the roots of tomato.

Gautheret Nobecourt and White (1939) were successful in raising callus culture.

Skoog and Millar (1950) explained the role of auxin and cytokinins. They demonstrated the development of callus into organ with the help of auxin and cytokinin.

Steward (1964) raised entire carrot plant from phloem cell from root.

Guha and Maheshwari (1966) developed embryos from anther cultures of Datura plant.

Carison and Co-workers (1972) and Y.P.S. Bajaj (1977) successfully demonstrated fusion of protoplasts from two cells.

Carison and co-workers produced first somatic hybrid between Nicotiana glauca and N. langsdorffii by fusion of their protoplasts. Since then several new somatic hybrids have been obtained using the technique of protoplast fusion.

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• Basic aspects of tissue culture:
• a) Media: The most important of these media are M S media (Murashige and Skoog, 1962). The pH of the medium is usually adjusted between 5.00 and 6.00.
• The constituents of the medium include

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• (i) inorganic nutrients C, H, O, N, P, S, Ca, K, Mg, Fe, Mn, Cu, Zn, B, Mo, Cl , Mn, Cu, Zn, B, Mo and Cl

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• (ii) organic nutrients vitamins, amino acids and sugars like glucose, sucrose, fructose, casein hydrolysate, coconut milk, corn milk, malt extract, tomato juice and yeast extract.

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• (iii) growth hormones auxins, cytokinins and gibberellins(Indole – 3 – butyric Acid), NAA (Naphthalene Acetic Acid) and IAA (Indole –3 – Acetic Acid,Kinetin, gibberellin is GA3

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• (iv) agar

• b) Cellular totipotency: It is a capacity of a single cell to regenerate the complete and differentiated organism from which it derived.

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• c) Culture techniques: The culture techniques involves following steps:
• i) Ex plant : The plant part used for the purpose of tissue cultures is called as ‘explant’, Nodal or internodal segments of stem, apical or axillary bud, leaf, leaf disc, petiole, anther, pollen, flower bud, petal, inflorescence, ovule, ovary, root and even isolated epidermal peel, gland and trichome

• b) Cellular totipotency: It is a capacity of a single cell to regenerate the complete and differentiated organism from which it derived.

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• c) Culture techniques: The culture techniques involves following steps:
• i) Ex plant : The plant part used for the purpose of tissue cultures is called as ‘explant’, Nodal or internodal segments of stem, apical or axillary bud, leaf, leaf disc, petiole, anther, pollen, flower bud, petal, inflorescence, ovule, ovary, root and even isolated epidermal peel, gland and trichome

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• ii) Callus culture: The sterilized explants are cultured aseptically on a nutrient medium into an undifferentiated mass of cells called callus.
• iii) Cell culture: Cell suspension cultures are initiated by transferring callus to liquid nutrient medium.

• d) Differentiation and morphogenesis:
• Since the undifferentiated callus cells are all parenchymatous in nature, the differentiation of these cells into a variety of cells is required during the formation of whole plant. Any morphological changes into callus is called morphogenesis and development of organs like root, shoot, leaves etc from callus is called as organogenesis.

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• e). Plantlets
• A plantlet is a very small plant still attached somewhere to its parent plant where these plantlets obtain food from the parent plant until they are established. In tissue culture studies plantlets are referred to very minute regenerated plants from callus.

Applications of tissue culture:

A few applications are described as below:

a) Micropropagation: Regeneration of whole plant through tissue culture is popularly called as micropropagation. By this method within a short time and space, a large number of plantlets can be produced. This micropropagation is also popularly known as clonal propagation. The major benefits of this method include -

• Rapid multiplication of superior clones and maintenance of uniformity.
• Multiplication of disease free plants
• Multiplication of sexually derived sterile hybrids.

• b) Production of disease free plants: Developing disease free or virus free plants is one of the most significant applications of tissue culture. The technique has applications in diverse areas such as rapid clonal multiplication of vegetatively propagated crop plants, virus elimination and germplasm preservation.

• c) Product of secondary metabolites: A plant cell produces two types of metabolites primary and secondary. Primary metabolites are produced a result of primary metabolism. Carbohydrates, lipids and proteins are primary metabolites and they take part in synthesis of cell components.

• Anther cultures and production of haploids: Anther is fertile part of stamen of androecium (a male reproductive organ). It is diploid tissue. Microspore mother cells or pollen mother cells present in side anther are also diploid. As a result of microsporogenesis (meiosis) tetrads of microspores or pollen tetrads are formed. Pollen grains from tetrad are haploid. During embryogenesis, embryos are formed from pollen grains and these embryos are haploid. Haploid plants can be produced from these haploid embryos. Haploid plants have single set of chromosomes that may be useful for improvements of many crops plants. Chromosome set of these haploid can be doubled by mutagenic chemicals like colchicine (Fig 10.3).

• e) Somatic embryogenesis: The process of embryo development is called as embryogenesis. Under certain experimental conditions, the cells of angiosperm sporophyte behave like a zygote and develop into embryo like structures. Since embryo like structures are derived from the sporophytic or somatic cells of the plant, a phenomenon is known as somatic embryogenesis. Somatic embryogenesis is also a non-sexual development of embryos.

• f) Protoplast culture and somatic hybridization:
• Protoplast is a cell minus cell wall or naked plant cell. Protoplast is biologically active and most significant material of cell. Plant cell wall acts as a physical barrier and protects cytoplasm from microbial invasion. Cell wall consists of complex mixture of cellulose, hemi cellulose, pectin, lignin, lipids, proteins etc (Fig 10.4). For dissolution of different components of cell wall it is essential to have the respective enzymes.
• Isolation of protoplasts: The protoplast can be isolated either by mechanical or enzymatic method.
• Mechanical method: The mechanical method involves isolation of protoplasts by cutting plasmolysed tissues with sharp razor. The protoplasts are released from the cut ends of the cells.
• Enzymatic method: By using this method protoplasts can be isolated from any part of the plant body but it is easier to isolate protoplasts from mesophyll of leaf or pollen mother cells or tetrads.

• g) Protoplast culture and regeneration:
• From the protoplast solution of known density about 1 ml protoplast suspension is poured on sterile and cooled down nutrient medium in Petridishes. The plates are incubated at 25oC in dim light.
• The protoplast regenerates a cell wall, undergo cell division and form callus. The callus can be sub cultured. The embryogenesis begins from callus when it is placed on nutrient medium. The embryos develop into seedlings and finally mature plants.

h) Protoplast fusion and somatic hybridization:

The fusion of protoplasts of genetically different lines or species has also been possible. For example plants that show physical or chemical incompatibility in normal sexual crosses, may be produced by the fusion of protoplasts obtained from two cultures of different species. This technique of hybrid production through the fusion of protoplasts from different genetic backgrounds is known as somatic hybridization or parasexual hybridization. Production of somatic hybrids involves several steps.

Interspecific hybrids:

• Datura inoxia x D. candida
• D. inoxia x D. discolour.

Intergeneric hybrids:

• Atropa belladona x Petunia hybrida
• Solanum tuberosum x Lycopersicum esculentum.