PLANT TISSUE CULTURE (PTC)

PLANT TISSUE CULTURE (PTC)

28.      PLANT TISSUE CULTURE (PTC)

PTC is a technique of maintaining and growing plant cells, tissues or organs especially on artificial medium in suitable containers under controlled environmental conditions.
The part which is cultured is called explant. The capacity to generate a whole plant from any cell/explant is called cellular totipotency. Gottlieb Haberlandt first initiated the tissue culture technique in 1902. The technique of PTC name largely helped the green revolution by improving the crop yield and quality.
28.1.    Hormones used in plant tissue culture
1.    Auxin newline (Indole-3-acetic acid, Indole-3-butyric acid, potassium salt-Naphthalene acetic acid 2, 4-Dichlorophenoxyacetic acid P-Chloro Fleroxy acetic acid).
2.    Gibberellin (Gibberellic Acid)
3.    Cytokinins (6–Benzylaminopurine, 6–Dimethylally aminopurine (Zip), Kinetin).
4.    Abscisic Acid (ABA)
5.    Polyamines (Putrescine, Spermidine)
28.2.    Environmental Conditions
There are three important aspects
1.    Nutrient medium
2.    Aseptic conditions
3.    Aeration of the tissue.
28.2.1.    Nutrient Medium
The composition of PTC medium can vary depending upon the type of plant tissue or all that is used for culture.
A typical (generalized) nutrient consists of inorganic salts (both micro and macro elements), a carbon source (usually sucrose), vitamins (eg. nicotinic acid, thiamine, pyridoxine and myoinositol), amino acids (eg. arginine) and growth regulators (eg. auxin-like 2, 4–D and cytokinin, gibberellins).
Other compounds like casein hydrolysate, coconut milk, malt extract, yeast extract, tomato juice etc. may be added for specific purposes.
An optimum pH (usually 5, 7) is also very important. The most extensively used nutrient medium is MS medium which was developed by Murashige and Skoog in 1962.
Usually, a gelling agent agar (a polysaccharide obtained from a red algae Getidium amansi) is added to the liquid medium for its solidification.
28.2.2.    Aseptic conditions (Sterilization)
Nutrient medium contains sugar which increases the growth of microorganisms such as bacteria and fungi. These microbes compete with growing tissue and finally kill it.
It is essential to maintain aseptic conditions of tissue culture. Thus sterilization means complete destruction of microorganisms so that complete aseptic conditions are created for in vitro culture.
28.2.3.    Aeration of the tissue
Proper aeration of the cultured tissue is also an important aspect of culture technique. It is achieved by occasionally stirring the medium by starting or by the automatic shaker.
Plant material the Explant
Any part of a plant taken out and grown in a test tube under sterile conditions in special nutrient media is called explant.
28.3.    Methods of plant tissue culture \rightarrow two major methods
1.    Type of in-vitro growth \rightarrow Callus and suspension cultures.
2.    Type of explant \rightarrow single cell culture, shoot and root cultures, somatic embryo culture, meristem culture, another culture and haploid production, protoplast culture and somatic hybridisation, embryo culture, ovule culture, ovary culture etc.
28.4.    Types of PTC
28.4.1.    Callus and suspension cultures.

28.5.    Subculturing \rightarrow If tissue cultures are kept in the same culture vessel, they die in due course of time. Therefore, cells/tissues are regularly transferred into new culture vessels containing fresh media. This process is called subculturing.
It is important to note that during subculture, only a part of the culture from a vessel we transferred into the new culture vessel.
28.6.    Single cell culture (Cell cloning)
Cells derived from a single cell through mitosis constitute a clone and the process of obtaining clones is called cloning.
28.7.    Shoot and Root cultures
Shoot culture is promoted by a cytokinin like BAR. However, root cultures is promoted by an auxin like NAA.  
The shoot and root cultures are generally controlled by auxin – cytokinin balance.
Usually, an excess of auxin promotes root – Culture, whereas that of cytokinin promotes shoot culture.
28.8.    Somatic embryo culture
Embryo forms from the somatic cell.
The pattern of development of a somatic embryo is comparable to that of a zygotic embryo.
The embryo has no connection with pre-existing vascular tissue within maternal callus.
Induced by the high concentration of an auxin as 2, 4-D.
28.9.    Endosperm culture
It is unique because it supplies nutrition to the developing embryo. It is also triploid in its chromosome constitutions. Triploid plants are used for the production of seedless fruits. The technique of endosperm culture involves the following.
1)    The immature seeds are dissected under aseptic condition. Endosperm along with embryos are excised.
2)    The excised endosperm are cultured on a suitable medium and embryos are removed after initial growth.
3)    The initial callus phase is developed.
4)    The shoots and roots may develop and complete triploid plants are fattened for further use.
28.10.    Anther culture and Haploid production
An individual/cell having the chromosome number found in the gametes of the species is called haploid. A haploid has only one copy of each chromosome.
When the chromosome number of a haploid plant is doubled by using colchicine, the plants of normal chromosome number for particular species are obtained.
In nature, haploid plants originate from unfertilized egg cells, but in the laboratory, they can be produced from other male and female gametes. 
This technique was developed by Guha and Maheshwari (1964) who cultured mature anthers of Datura inoxia.


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