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Tissue Culture

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Definition:-

1. Developing a new seedling in a laboratory under controlled condition from a cell, tissue or piece of any plant part is called tissue culture. 2. The technique of separation of cells or tissues or organs of a plant and growing them aseptically in suitable glass container and in sterile nutrient medium under controlled conditions of temperature and light is called tissue culture.

The tissue culture technique is used for multiplying certain plants in a great number. This technique particularly used after identification of growth hormones responsible for root and shoot growth. Dr. Morvel (1960) used this technique for the first time and formulates complete procedure of tissue culture. Dr. Murashidge and Skoog in 1975 developed standard medium for tissue culture and used that technique successfully for fruit crops. Requirements of plant tissue culture Well equipped laboratory is an essential requirement of tissue culture. The laboratory should have the following facilities:

  1. Preparation of media
  2. Storage and cleaning facilities
  3. Aseptic condition of working
  4. Controlled environmental conditions for growth and development of cultures.
  5. Examination of the culture and keeping records.


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The important aspects of tissue culture are:

1. Aseptic condition:-

The complete aseptic condition is essential for tissue culture, sterilization of glass ware, working tables, medium and plant material is essential. Chemicals like mercuric acid, sodium hypochloride are used. Use dry heat (1800C – 3000 C), use of steam heat (1200 C) 15 lb pressure for 20 minutes, ultra filtration, ultra violet light, use of antibiotics are the methods of sterilization.

2. Equipments

(glassware) cylinders, beaker, funnels, Petri dishes, pipettes, oven, autoclave, filter, inoculation hood, laminar flow chamber, spirit lamp, acclimatizers, forceps, gloves, apron, refrigerator, balance, pH meter, cartridge, microscopes, automatic shaker, etc. are the equipments required for tissue culture.

3. Nutrient Medium: -

the medium should contain inorganic salts, vitamins, sucrose, growth hormones (auxins, cytokinines). The medium is solidified with 0.5-1.0% agar. Murashige and Skoog standard medium is used for tissue culture. Methodology of tissue culture The methodology of tissue culture has following steps: a) Selection of plant material:- Cultivar is selected and explant is separated. A tip of shoot meristem is normally taken as explant. Explant is cleaned and sterilized with chlorinated water. b) Preparation of synthetic nutrient medium:- A nutrient medium contains carbohydrates, amino acids, salts, vitamins, growth hormones, agar etc. c) Sterilization of culture medium:- The medium is sterilized to prevent infection and to make the medium of the aseptic. It is sterilized in autoclave at 1200 C for 20 minutes. d) Inoculation:- The sterilized explants carefully transferred to the test tube or suitable container and put on the culture medium with the help of sterilized forceps. This is known as inoculation of explant. E) Incubation:- The inoculated cultures are transferred to another room where light and temperature are strictly controlled. It is called culture room. f) Subculture:- The callus can be cut into a number of segments and sub cultured by putting them in different test tubes containing culture media. g) Organogenesis:- By adding appropriate concentration of auxins and cytokinines to the basal medium, the callus can be induced to differentiate into root and shoot and then to develop in to a new plantlet. h) Transplanting:- When the plantlets are old enough, they are transferred to soil in the pots and kept covered to maintain the humidity. This is called transplanting. i) Field cultivation:- After hardening and when the plantlets are established in the pot they are transplanted in field for cultivation.

Important applications of tissue culture:

a. Mass multiplication of commercially important plants by micro propagation. b. Production of disease free plants. c. Production of haploid and double haploid plants through anther culture. d. Industrial production of secondary metabolites. e. Induction and isolation of mutants. f. Protoplast culture used for production of clones, Somatic hybrids and direct DNA uptake. g. Production of soma clonal variations. h. Transfer of germplasm through synthetic seeds. i. Production of difficult to propagate plants. j. Overcoming dormancy of seeds. k. Obtaining the whole plant from isolated cells. l. Induction of polyembyony. m. Preservation of germplasm (Gene bank). n. Culture of hybrid embryos.

Terminology:-

1. Totipotency: - the property of cell to regenerate and to develop it into a new plant under ideal condition is know as totipotency. 2. Clone: - A group of plants multiplied vegetatively from a single plant. 3. Callus: - It is an unorganized mass of thin walled cell or a tissue in which the cells are loosely arranged and undifferentiated. 4. Culture: - It is the method of growing of cells tissues organs on nutrient medium under aseptic conditions. E.g. cell culture, embryo culture, protoplast culture, anther culture. 5. Organogenesis: - Formation of an organ such as root, shoot from the callus is called organogenesis. 6. Cultivar: A plant selected for tissue culture is called cultivar. 7. Explants: - The protoplast, cell, tissue or organ of the plant use for tissue culture is called explants. 8. Plantlets: - The plant obtained through the tissue culture technique is called as plantlet. Plant Growth Regulator:- A organic substance which when applied in very small quantity to plant’s part either accelerate, retard or modify the physiological process of plant.

Intext Questions

A) Fill in the blanks: 1. The tissue culture technique is used for certain plants in a great number. 2. The medium is solidified with agar. 3. A tip of shoot is normally taken as explant. 4. The sterilized explants carefully transferred to the test tube is known as of explant. 5. When the plantlets are established in the pot they are in field for cultivation.


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