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TISSUE CULTURE TECHNOLOGY

This technique employs the isolation of a few cells from the concerned plant that are grown and maintained in artificial conditions using nutrient media containing certain plant hormones, The cells either grow in the suspension form (where shaking facilitates aeration, mixing of medium and breakage of cell aggregates) or undifferentiated mass of cells called callus, in flasks or tubes. The cultured cells can be used in various ways in the field of agriculture and forestry:

(i)  The valuable metabolites (e.g., quinine used for the treatment of malaria, jasmine for fragrance) are obtained commercially by growing the cells in large bioreactors.

(ii)  Alternatively, these cultured plant cells can be induced to develop shoots and then roots to produce complete plantlets by selecting correct hormone (auxins and cytokinins) concentrations (ability of a plant cell to regenerate into whole plant is known as totipotency). These plantlets after removal from the culture vessels, finally, can be grown in the fields. Thus, a large number of clones of slow-growing rare plants can be obtained rapidly either by avoiding the formation of embryo (e.g., ornamental plants, fruit trees like banana). (This technique is known as micropropagation) (Fig. 19.1)

Fig. 19.1 Micropopagation of plants.

(iii) The cultured cells after the formation of somatic embryos can germinate to produce whole plant, e.g., oil palm, date palm etc. These somatic embryos can be encapsulated within gel like substances (calcium alginate) containing nutrients, fungicides and bactericides to check the microbial contamination and covered with a waxy coating to prevent the drying of these artificial seeds. Such seeds are known as artificial seeds.

(iv)  This technique is also used for the germplasm conservation in the form of cultured cells or tissues (instead of seeds) and production of transgenic plants (genetically modified) after genetic manipulation of the cultured cells.

(v) Sometimes, in interspecific crosses the hybrid embryos in the seeds are unable to grow completely because of degeneration of endosperm (part of the seed containing stored food) or some seeds lack stored food (e.g., orchids) or seeds are not able to germinate due to the presence of some inhibitors in the seeds. But, by embryo culture technique the excised embryo can be in vitro allowed to complete the development and finally grown into plantlets.

(vi Using anther (contains male gametes) culture technique, that was first used in India in producing haploid Datura plants, large number of haploid plants can be obtained from the pollen grains. These haploids (having one set of chromosomes) are sterile and are used to produce superior quality homozygous (having two copies of the same set of chromosomes) plants by treatment with colchicines (inhibits the spindle formation during cell division so that all the chromosomes remain in the same cell and not distributed into the two daughter cells) that are used in the plant breeding programme, e.g., in cereals. Haploids can also be used in the isolation of mutants (recessive mutants would be expressed easily as they have just one set of genes) and chromosome number in these haploid mutants can be doubled by treatment with colchicine. Haploids can also be produced by culturing the unfertilized ovaries or ovules.

(vii) Many types of plants can also be regenerated from protoplasts that are produced by digesting away the cell wall by the enzymes pectinase and cellulase and then maintaining them in a suitable osmotic medium, e.g., potato, tobacco, tomato etc.

(viii) Somatic hybrids can be produced by fusion of protoplasts of two species in the presence of polyethylene glycol (PEG) or by exposure to a very brief high voltage electric current that cannot be produced normally by sexual hybridization process. After cell wall formation, the hybrid cells divide to produce plantlets, e.g., somatic hybrid (that produced fertile flowers) between a non-flowering variety and a flowering variety of potato, somatic hybrid produced between rice and carrot etc. These somatic hybrids may be used for gene transfer, production of useful allopolyploids (e.g., the allopolyploid AABB produced from the diploid hybrid AB), etc.

(ix) Somaclonal Variation: In in vitro culturing of cells, the genetic variability increases with duration of culture to give rise to different kinds of cells. This property is known as somaclonal variation and may arise due to changes in chromosome number or structure, spontaneous mutation, alteration of gene expression, gene amplification, etc. From these different kinds of cells, cells with desirable properties can be screened out, e.g., disease resistance (potato plants resistant to Early Blight disease caused by the fungus Alternaria solani and Late blight disease caused by the fungus Phytophthora infestans), herbicide resistance, improved nutritional quality, adaptation to stress (saline soil tolerance in Citrus, low temperature tolerance in tobacco, toxic metals), increased biosynthesis of plant products of medicinal or industrial importance etc.