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Tissue Culture
Meristems and root apices of plants can be axenically cultured on special tissue culture media to generate a mass of undifferentiated cells known as 'callus' and from tiny bits of this callus material, numerous calluses could be generated.
Individual cells from a macerated callus can often be regenerated into new calluses by growing them further on special media. From these callus cultures, new plants can be raised initially by transferring plantlets into small pots and then into natural soil once they have adjusted to the surroundings.
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This technique, known as early as 1930, has now reached a stage of commercial application by generating clones of plants which are uniform in certain traits such as freedom from seed-borne diseases, viruses, frost damage, salt tolerance and many other attributes which cannot be achieved by plant breeding methods. There are various types of tissue cultures which are frequently used-callus cultures, cell suspension culture, organ culture, meristem tip culture and protoplast cultures. In the case of protoplasts, the cell walls are removed by lysozyme or suitable cell-wall dissolving enzymes and they are cultured in a suitable medium, a technique which facilitates manipulation of cell units without the interference of cell walls.
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Some examples of tissue culture application in agriculture are as follows: Cassava (Manihot utilissima) is normally propagated by pushing a large section of a mature plant's stem (stake) into soil. These stakes are bundled and moved from place to place or country to country which present quarantine problems because disease causing germs may move with cassava stakes.
The Centro International de Agricultural Tropical (CIAT) and the International Institute of Tropical Agriculture (UTA) bred new varieties of cassava which possess resistance to diseases and insects and developed disease-free lines through, meristem cultures for shipment under aseptic conditions to other African countries.
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CIAT has now an in vitro cassava germplasm with 700 meristem-cultured accessions in the bank. Similarly, haploid plants were raised from anthers (anther culturing) and homozygous plants, produced in one generation, a process that requires five or six generations by conventional plant breeding methods.
The International Rice Research Institute (IRRl) obtained salt tolerant variants of Taichung 65 variety by tissue culture techniques with 20% higher yield than the parent which was most suitable to salt stressed conditions. IRRI also developed strains of Taichung 65 variety which overcame aluminium toxicity. In Asia due to low temperatures at high altitudes where rice is cultivated, yields are invariably low. By anther culture technique, IRRl has attempted to develop cold tolerant strains of rice.
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Disease-free potato plantlets and tubers can be developed by tissue culture technology. The International Crop Research Institute for the Semi-Arid Tropics (ICRISAT) is using meristem cultures to produce disease-free groundnut germplasm. The Indian Agriculutral Research Institute (IARI) has been able to overcome the vexing problem of male sterility in papaya (Carica papaya)by tissue culture techniques.
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Azolla is a water fern which can be successfully harnessed as a nitrogen input in rice cultivation because the system fixes nitrogen through an alga Anabaena azollae inhabiting its fronds. Protoplast fusion and hybrid cell generation techniques could be used to cross an Azolla that has low yields but tolerates high temperature with an Azolla that has high yields but likes cold weather.
If this can be achieved, Azolla strains could be used which can generate 400 kg N /ha in high temperature tropical fields. In the area of biological nitrogen-fixation, the possibilities of exploiting tissue and cell culture techniques are still open. Nitrogen-fixing bacteria and blue-green algae can be forced into isolated protoplasts or calluses and plantlets regenerated. The plants developed from such callus cultures with nitrogen-fixing bacteria could possibly develop into nitrogen fixing plants. Chloroplasts can be made to take in nitrogen-fixing blue-green algae.
One of the primary bottlenecks in these attempts is the physiological barriers between the protoplasts of an eukaryote and a prokaryote. Experiments have been done to transfer nif genes from a simple prokaryote (Klebsiella pneumoniae) to a simple eukaryote (Saccharomyces cerevisiae). The results so far obtained indicate that while the nif operon has been moved from the bacterium into the yeast cell, the expression of the desired trait, namely, nitrogen fixation or nitrogenase activity has not been achieved, thereby suggesting that a better understanding of the physiological steps needed for expression of nitrogenase activity by the recombinant yeast cells is a prerequisite to achieve the desired goal to transfer nif to higher plant species.
In India, tissue culture techniques have been satisfactorily used to rapidly multiply elite cultivars of sugarcane, turmeric, ginger, rubber, mustard, cardamom, citrus, pineapple, pomegranate, almond, banana, apple, Disoscorea, Bougainvillea, teak, bamboo, sandal, eucalyptus, rosewood and pine. Tissue culture propagation ensures preservation of extinct species and freedom from diseases.
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