PROTOPLAST CULTURE & MICROPROPAGATION :
Protoplasts are the smallest unit able to regenerate a whole plant. Therefore protoplats cultures can serve to enlarge genetic variability by introducing somaclonal variations. However, the main interest of protoplasts, is their capacity to fuse and to produce hybrids or cybrids. Naked protoplasts can accept without rejection external elements: nuclei, cytoplasmic organelles, liposomes, containing genetic information.
The hybridization programme is hampered in some cases because of sexual incompatibility, Synthetic production of hybrids has, however becomes possible through the novel technique of protoplast culture and their fusion. The isolation culture, and fusion of protoplast are one of the most fascinating fields of research, through still in developing stage. The protoplast culture technique can be suitably used for microinjection and other genetic engineering experiments. The technique are important, specially because of their far-reaching effects on crop improvement by somatic hybridization and cell modification. The protoplast culture can be regenerated into an entire plant. The discovery of enzymes which could separate the cells for isolating protoplasts and exploring the possibilities of genetic engineering.
SOMATIC HYBRIDS & CYBRIDS :
The first protoplast fusion application was a cytoplasmic transfer from one genotype to another to induce male sterility (CMS) from mitochondrial origin. These male sterile hybrids are interesting to produce F1 hybrids (Brassica, Cichorium).
Since long protoplast fusion was proposed as a novel and important method for producing hybrid plants that can be obtained by sexual means. Mechanical or enzymatic removal of cell wall from a plant cell yields, a protoplast, which is bound by plasma membrane. An isolated protoplast, under suitable culture condition can regenerate entire plant. Plasmolyzing cell prior to enzymatic treatment helps to facilitate protoplast isolation. The isolated protoplast, having lost the protective cell wall, has to be protected against the osmotic shock by keeping them in the isotonic state. 13% mannitol is a good agent in this context. A variety of enzymes are available for protoplast isolation. Most common are cellulose, pectolyase and macerozyme. The viable protoplast tend to synthesize new cell wall within few hours to few days of culture divide and re-divide, produce clumps of cells which finally produce plantlets.
PRODUCTION OF VIRUS FREE PLANTS :
In 1952, Morel and Martin were successful in regenerating a virus-free dahlia plant by the excision of some meristematic domes from virus infected shoots. Semal and Lepoivre (1992) reported that a virus-free sweet potato was producing 40T/ha in china by comparison of the 20 T/ha produced before meristem culture.
Virus eradiction is dependent on several parameters. But to take advantage of the non uniform and imperfect virus distribution in the host plant body, the size of the excised meristem should be as small as possible. For Stone (1963), only tips between 0.2 and 0.5 mm most frequently produce virus free carnation plants. The explants smaller than 0.2 mm can't survive and those larger than 0.7 produce plants that still contain mottle virus.
There are various explanations have been given: absence of plasmodesm in the meristematic domes, competition between synthesis of nucleoproteins for cellular division and viral replication, inhibitor substances, absence of enzymes present only in the cells of the meristematic zones and suppression by excision of small meristematic domes. This last proposal could explain why some potato plant showing virus particles in the meristematic domes, could regenerate a virus free plant.
SHOOT TIP MICROGRAFTING :
When meristematic tip culture fails, it is possible to graft small meristematic domes on young seedlings growing in vitro. In this way, Navarro et al. (1975) eradicted all the virus diseases from spanish Citrus orchards. This technique was also very successful in eliminating virus diseases from peach tress (Mosella et al 1980).
MICROPROPAGATION TECHNIQUES:
During the micropropagation process, the genetic stability of new shoots dependent upon their origin. Axillary shoots issue from pre-existing buds and are normally true to type, indeed the meristematic cells are genetically very stable. Adventitious shoots, such as somatic embryos, are neoformed buds developed directly on some organs, or indirectly through a callus phase formed on this organ. So, if the mother plant presents a cell mosaic or chimaeric tissues, risks of genetic variation exists. It is similar in the case of an indirect regeneration, when the callus phase is too long.
PROPAGATION BY AXILLARY SHOOTING :
This technique has proved to be the most applicable and reliable method of in vitro propagation. Axillary shoot growth is stimulated by overcoming apical meristem dominance. Commercial tissue culture laboratories are now able to propagate a large number of herbaceous ornamental species and several woody plants in this way. However, the propagation of Pelargonium, Howea and a few other horicultural plants are always difficult to propogate by axillary branching.
PROPAGATION BY DIRECT OR INDIRECT ORGANOGENESIS :
Adventitious shoots could arise directly from the tissue of explants without callus formation. Several plants of tha family gesneriaceae (Saintpaulia, Streptocarpus) regenerate directly buds on leaf explants, likewise Lilium regenerate on scales. However, more often, like for Ficus lyrata, adventitious buds appear on callus. While coffee, cocoa trees and many conifers are produced by somatic embryogenesis developed on callus or cell suspensions.
IMPROVEMENT OF AXILLARY BRANCHING :
The cost of micropropagated plantlets is also an important limitation of the techniques. In New zealend, where they produce 2-3 million micropropagated radiata pine per annum, the relative cost of micropropagated planting stock had dropped from 13,8 times the cost of seedlings in 1988 to 6,9 by 1993 (Smith, 1997). To reduce manpower costs, several improvements have been proposed.. The more simple method was in vitro layering developed by Wang(1977) to clone PVX-free potato plants.The first plantlets placed on the medium in a horizontal position developed axillary shoots. They are harvested by cutting one centimeter above the medium surface, at 3 weeks intervals. A similar technique called "hedging system" by Aitken christie and Jones (1987) was later used to produce Pinus radiata. Since 1988, Duhem was producing vary large quantities of Eucalyptus plantlets in petri dishes without anti-gibberellin but in complete darkness. Transfers from one petri to another is made by a simple squashing.
SOMATIC EMBRYOGENESIS PROPAGATION :
For genetically stable species, somatic embryogenesis offers a very fast scaling-up system, especially when it's possible to produce embryos in bioreactors. Only a few model plants are successfully produce by such technology: carrot, celery. Other applications remian at the experimental stage: coffee, oil, palms, conifers, Euphorbia pulcherrima and several other horticultural species.
Several bottlenecks limit the use of this interesting technology. one of main problems is genetic stability. Therefore, despite the clonal nature of nucellar embryos, different morphological anomalies can occur among mango somatic embryos, as it was also observed in Citrus plants derived from nucellar cultures (Litz et al 1993). Another difficulty is the loss of embryogenic capacity overtime, a phenomenon observed with different species. It is also important that somatic embryogenic lines of conifers are always originated from immature embryos.
SYNSEEDS :
Another very interesting possibility of the somatic embryogenesis technology has been developed during the past 15 years by Redenbaugh and his team (1991, 1993). They were able to encapsulate somatic embryos by hydrogel coatings (sodium alginate), producing single embryo artificial seeds. To date, some improvements offer the possibility to directly plant the artificial seeds in the greenhouse on special substrates (vermiculite, sans). This methodology will provide in future a good technique to reduce the cost of transplants.
SOMACLONAL VARIATIONS :
The production of plantlets by callus regeneration, cell suspensions, protoplast cultures could present some deviations with regard to the mother plant. This is the way to increase the genetic variability. Associated with a selective pressure (Stress to toxins, pH, salinity, cold). it's used to obtain resistant lines.Indeed after regeneration, plants can express new potentialities rarely obtained another way. Stable and profitable variants are selected and introduced in breeding programmes. In 1976, a pelargonium cv Velvet Rose was created by this technique (Reisch, 1983).
No comments:
Post a Comment