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水稻转化要素之三:种子消毒

已有 3206 次阅读 2008-6-26 19:35 |个人分类:生活点滴

Surface Sterilization of Plant Explants and Orchid Seed
To avoid bacterial and fungal growth, which is detrimental to culture growth, explants are surface-sterilized before they are used to establish in vitro axenic cultures. The most common disinfectants are listed below with the concentration and exposure times that preserve the explant, but at the same time destroy any microbial contamination.
Explant Sterilization
 
  1. Wash explants in a mild detergent before treatment with the disinfecting solution. (Herbaceous material may not require this step).
  2. Rinse explants thoroughly under running tap water for 10-30 minutes.
  3. Submerge explants into the disinfectant solution. Seal bottle and gently agitate.
  4. Under sterile conditions, decant the solution and rinse explants several times with sterile distilled water.
 
COMMONLY USED DISINFECTANTS
FOR PLANT TISSUE CULTURE
Disinfectant
Product No.
Concentration (%)
Exposure (min)
Calcium hypochlorite
9-10
5-30
Sodium hypochlorite*
0.5-5
5-30
Hydrogen peroxide
3-12
5-15
Ethyl alcohol
70-95
0.1-5.0
Silver nitrate
1
5-30
Mercuric chloride
0.1-1.0
2-10
Benzalkonium chloride
0.01-0.1
5-20
*Commercial bleach contains about 5% sodium hypochlorite, and thus may be used at a concentration of 10-20%, which is equivalent to 0.5-1.0% sodium hypochlorite.
 
 
Sterilization procedures may be enhanced by:
  1. Placing the material in a 70% ethyl alcohol solution prior to treatment with another disinfectant solution. The use of a two-step (two-source) sterilization procedure has proven beneficial with certain species.
  2. Using a wetting agent, such as Tween 20 or 80 (Product Nos. P 1379 or P 1754, respectively), can be added to the disinfectants to reduce surface tension and allow better surface contact.
  3. Conducting the sterilization process under vacuum. This results in the removal of air bubbles and provides a more efficient sterilization process.

Orchid Seed Sterilization

    Orchid seeds are very small and contain little to no food reserves. A single seed capsule may contain 1,500 to 3,000,000 seeds. Sowing the seed in vitro makes it possible to germinate immature seed (green pods). It is much easier to sterilize green capsules than individual seeds after the capsule has split open. Lucke (1971) indicated that orchid seed can be sterilized when the capsule is about two-thirds ripe. Listed below are the estimated normal ripening times of capsules for various orchid species (Lucke, 1971).
 
Green capsule sterilization:
  1. Soak the capsule in a 100% bleach solution for 30 minutes.
  2. Dip the capsule into 95% alcohol, and flame.
  3. Under aseptic conditions, open the capsule and scrape out the seed.
  4. Carefully layer the seed over the surface of the culture medium.
Dry seed sterilization:
  1. Collect seed and place in either a small flask or bottle, or place in a shortened pipet which has one end sealed with cotton. Seal the other end of the pipet with cotton, once the seed has been placed in the pipet.
  2. Prepare a solution containing 5-10% commercial bleach containing a few drops of Tween 20.
  3. Add the bleach solution to the flask, or draw up the solution into the pipet. Swirl the flask containing the seed and bleach or repeatedly draw and aspirate the bleach solution in and out of the pipet.
  4. Sterilize the seed for 5-10 minutes.
  5. Remove the bleach solution and rinse the seed with sterile tissue culture grade water.
Transfer the seed to sterile culture medium.


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