Introduction Tissue culture consists of growing plants cells as relatively on organized masses of cells on an agar medium (callus culture) or as a suspension of free cells and small cell masses in a liquid medium (suspension culture). Tissue culture is used for vegetative multiplication of many species and in some cases for recovery of virus-free plants. It has potential application in production of somatic hybrids, organelle and cytoplasm transfer, genetic transformation and germplasm storage through freeze-preservation. The various applications of plant tissue and cells cultural are as below:
Tissue culture is well suited for quick vegetative propagation of plant species. It is used for asexual propagation in many species of fruit and timber trees and also used for obtaining disease free and virus-free plants. The major difficulty in the use of this technique in clonal multiplication is the occurrence of genetic variation among the regenerated plants. This problem can be reduced to a large extent by using young tissue cultures, preferably during the first few subcultures.
Biochemical mutants are far more easily isolated from cell cultures than from whole plant populations. This is because a large number of cells, 106-109, can be easily and effectively screened for biochemical mutant cells. Biochemical mutants could be selected for disease resistance, improvement of nutritional quality, adaptation of plants to stress conditions, e.g. saline soils, and to increase the biosynthesis of plant products used for medicinal or industrial purposes.
Plants regenerated from tissue and cell cultures show heritable variation for both qualitative and quantitative traits; such a variation is known as somaclonal variation. Somalconal variation has been described in sugarcane, potato, tomato etc. Some variants are obtained in homozygous condition in the plants regenerated from the cells in vitro (R0 generation), but most variants are recovered in the selfed progeny of the tissue culture-regenerated plants (R1 generation). Somaclonal variation most likely arises as a result of chromosome structural changes, e.g., small deletions and duplications, gene mutations, plasma gene mutations, mitotic crossing over and possibly, transposons. Somaclonal variation may be profitably utilized in crop improvement since it reduces the time required for releasing the new variety by at least two years as compared to mutation breeding and by three years in comparison to back cross method of gene transfer. Cereal grains are deficient in lysine; maize (Zea maize) is also deficient in trytophan, while wheat (T.aestivum) and rice (O.sativa) are deficient in threonine. Pulses are deficient in methionine and trytophan. Amino acid analogue-resistant cells may be expected to show a relatively higher concentration of that particular amino acid. For e.g., carrot (D.carota) and tobacco (N.tabacum) cell lines resistant to trytophan analogue 5-methyl trytophan show a 10-27-fold increase in the level of trytophan. Similarly, rice cells resistant to lysine analogue 5-(B-aminoethyl)-cysteine, show much higher levels of lysine. This technique may prove useful in the development of crop varieties with a better-balanced amino acid content. Many pathogenic bacteria produce toxins that ae toxic to plant cells. Plant cell cultures may be exposed to lethal concentrations of these toxins and resistant clones isolated. Plants regenerated from these resistant clones would be resistant to the disease producing pathogen. This technique should be applicable to all the pathogens, which produce the disease through the action of toxin. An e.g., an application is in the case of wildfire disease of tobacco (N.tabacum) produced by Pseudomonos tabaci. Tobacco cells resistant to methionine sulfoximine, which is similar to the toxin produced by the pathogen, were isolated. Plants regenerated from these clones were resistant to wildfire disease, although to a somewhat lesser degree. The technique can be applied to those cases only where the disease is the result of a toxin produced by the pathogen. But many of the pathogens do not seem to produce a toxin, or the toxin does not appear to be the primary cause of the disease. Plant cells resistant to 4-5 times the normally toxic salt (NaCl) concentration have been isolated. Attempts to insolate such cells are being made. Similarly, attempts are being made to isolate clones that would produce more substances of medicinal or industrial value. Protoplasts can be isolated from almost every plant species and cultured to produce callus. Protoplasts of two different species may be fused with the help of polyethylene glycol. There is some evidence that gene transfer may be achieved by feeding cells with DNA in case of eukaryotes, such as, Drosophila, Neurospora, cultured mammalian cells and in some plants. Genetic changes may be brought about by DNA or by radiation-killed pollen grains. This raises the possibility of genetic modification of plant cells with the help of both homologous (from the same species) and heterologous (from a different species) DNA. It is also proposed that DNA plant viruses, such as cauliflower (B.oleracea) mosic virus and potato leaf roll virus, plasmids (e.g., Ti plasmid of Agrobacterium) and transposons, may be used as the carriers of genes for genetic modification of plant cells. In some cases, it may be desirable to transfer only organelles or the cytoplasm into a new genetic background. This may be achieved through the use of plant protoplasts. Chloroplasts have been transferred, and other organelles including nucleus may be transferred. Tissue cultures may be frozen and stored in liquid nitrogen at –1960C for long-term storage of germplasm. This would be of great value in the conservation of germplasm of those crops which normally do not produce seeds, e.g. root and tuber crops, or where it may not be desirable to store seeds. For freeze-preservation, the cells are cooled at a slow rate and are then transferred to liquid nitrogen for storage. Thawing of the cells must be very rapid for increased survival. A cryoprotectant, such as dimethylsufoxide (DMSO), is used to protect the cells from injury due to freezing and thawing. The technique of freeze-preservation i.e., crybiology, of plant cells is still in the developing stages.
Tissue culture techniques are being exploited to enhance crop production and to aid crop improvement efforts. Faster clonal multiplication is being exploited on commercial scale for many horticultural species e.g. oil palm, mentha, roses, carnation etc. Tissue cultured somatic tissues are now routinely being used for conservation of those species whose seeds are recalcitrant or ones which do not produce seed at all. Embryo culture has helped in rescuing hybrid embryos enabling the recovery of many interspecific hybrids and haploid plants. Shoot tip (meristem) culture plays a vital which is of great importance in germplasm exchange, and the development of serological techniques for the detection of viruses in plant materials is a great help to the efforts in this direction. |
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