Plant tissue culture, that is, plant sterile culture technology, is based on the theory that plant cells are totipotent, using isolated organs such as roots, stems, leaves, shoot tips, flowers and fruits, and tissues such as cambium, epidermis, cortex, marrow cells, endosperm, etc. ) or cells (such as megaspores, microspores, somatic cells, etc. ) and protoplasts were cultured in sterile and suitable artificial medium, light, temperature, etc.
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Plant tissue culture:
In a broad sense, it is also called in vitro culture, which refers to the technology of separating tissues, organs or cells, protoplasts and so on. They are cultured under artificial control through aseptic operation to obtain regenerated complete plants or produce other products with economic value.
Tissue culture in a narrow sense refers to the regeneration of plants from different parts of plants, such as cambium, parenchyma, mesophyll and endosperm. It also refers to the callus of various organs cultivated in the process of culture, and the callus differentiates again to form regenerated plants.
Plant tissue culture step
The first step is to remove the unused parts of the collected plant materials and wash the needed parts carefully, such as with a suitable brush. Cut the material into appropriate sizes, that is, it is appropriate to put people in a sterile container. Put a faucet and let the water flow for several minutes to several hours. The washing time depends on the cleanliness of the material. Float-prone or fine materials can be washed in gauze bags. When the pollution is serious, running water washing is particularly useful. When washing, you can add washing powder to clean it, and then rinse the washing powder with tap water. Washing powder can remove dirt slightly attached to the surface of plants, remove lipid substances and facilitate direct contact with disinfectant. Of course, the most ideal cleaning substance is the surface active substance-Tween.
The second step is to soak and disinfect the surface of the material. To finish it in a clean workbench or inoculation box, prepare a sterilized beaker, glass rod, 70% alcohol, disinfectant, sterile water, watches, etc. Soak in 70% alcohol 10 ~ 30 seconds. Because alcohol can make the surface of plant materials wet, 70% alcohol has strong penetration and is easy to kill plant cells, so the infiltration time should not be too long. There are also some special materials, such as if, bud, booting stage with bud and bud, dormant bud with multi-layer scales and so on. And it is mainly made of internal materials, which can be treated with 70% alcohol for a little longer. Under aseptic conditions, the treated materials will be peeled off after the alcohol volatilizes, and the internal materials will be used.
The third step is to treat with disinfectant. There are many kinds of surface disinfectants, and 1-2 can be selected according to the situation. See table. Step 4, rinse with sterile water for about 3 minutes each time, and rinse for about 3- 10 times according to the type of disinfectant used. The washing function of sterile water is to avoid the side effects of disinfectant killing plant cells. Attention: ① Alcohol has strong permeability, and young wood is easy to lose green in alcohol, so the soaking time should be short to prevent alcohol from killing plant cells. 2 Mature materials, especially seeds, can be soaked in alcohol for a long time, for example, seeds can be soaked for 5 minutes. (3) The penetration of mercuric chloride is weak, and it is generally soaked for about 10 minute, which does little harm to plant materials. (4) Bleaching powder is easy to cause plant materials to lose green, so young materials should be used with caution. ⑤ Adding Tween 20 or 80 (a wetting agent) with the concentration of 0.08-0. 12% to the disinfectant can reduce the surface tension of plant materials and achieve better disinfection effect.