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Methods of Cryopreservation:Germplasm Bank  

2011-05-15 08:34:36|  分类: 数量遗传学 |  标签: |举报 |字号 订阅

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(i) Selection of Materials. For selecting the plant materials a number of factors are taken into account ; the important ones are, nature and density of cells in the vials/ampules to be cryopreserved ; because the cryoability of the cell cultures depends on these. Young meristematic, highly cytoplasmic and small cells which are non-vacuolated and thin walled and in small aggregates, are good materials to be selected for this purpose. Cell density in vials or amples should be high, as it shows prolonged survival at high cell density.

(ii) Addition of Cryoprotectors. Cryoprotectors are the chemicals which decrease cryodestruction. These are sugars, glycols, sugar alcohols, alcohols, polyvinylpyrrollidone, polyethylene glycol (PEG), polyethylene oxide (PEO), dextrans, hydroxystarch, glycerine, sucrose, and some amino acids (e.g. proline). Bajaj (1987) has advised to use a mixture of two or three cryoprotectants at low concentrations rather than a single cryoprotectant at a high concentration as it could be toxic. During treatment, the cultures should be maintained in ice to avoid deleterious effects.

(iii) Freezing. Freezing should be done in such a way that it does not cause intracellular freezing and crystal formation, as it is possible in sudden freezing. To avoid this problem, regulated rate of cooling or pre-freezing is done. Moreover, freezers have also been developed which allow the uniform temperature decrease at a desired rate, commonly not less than 1°C per minute (Popov, 1985), In 1987, the Institute of Cryobiology and Cryomedicine of the Ukrainian Academy of Sciences (erstwhile U.S.S.R.) devised the programme freezer which envisaged lower rate (0.5°C per minute) of temperature decrease.

(iv) Storage in Liquid Nitrogen. If the cells are not stored at sufficiently low temperature, an additional injury to the cultures may be caused. The storage temperature should be such that it stops all metabolic activity and prevents biochemical injury (Bajaj 1987). Prolonged storage of frozen materials is possible only when the temperature is lower than -130°C. This can be simply achieved with the help of liquid nitrogen, which keeps the temperature -196°C. Popove (1988) stored the cultures of carrot cells for about 5 years by doing so.

(v) Thawing. Thawing is the process of releasing the vials containing cultures from the frozen state to elevate the temperature between 35 and 40°C. It should be done quickly but without overheating. As soon as the last ice crystals disappear, the vials are tranferred into a water bath at 0°C (Popov, 1985).

(vi) Washing and Reculturing. Washing of plant materials is done to remove the toxic cryoprotectants. When low toxic or non-toxic cryoprotectants are used, the cultures should not be washed, but simply recultured. Washing becomes necessary only when cryoprotectants have toxic effects on cells. Washing follows the following procedure : dilution, resuspension, centrifugation and removal of cells. It is, however, possible that some cells die due to storage stress and the most stable ones survive. Therefore, determination of cell viability by culturing them on growth medium is essential.

vii) Regeneration of Plantlets. The viable cells are cultured on non-specific growth media to regenerate into plantlets. Bajaj (1987) has given an extensive list of works on cryopreservation of cells, tissue, and organ culture of various plants e.g. potato, cassava, sugarcane, soybean, groundnut, carrot, cotton, citrus, coconut, etc.
Methods of Cryopreservation:Germplasm Bank - 喜欢吃桃子 - wangyufeng的博客
 Potentials and prospects of cryopreservation of plant cell, tissue and organ culture and establishment of .'Germplasm Bank' (after Bajaj, 1977a).
more information links to:http://www.eplantscience.com/botanical_biotechnology_biology_chemistry/biotech_cryobiology.php
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