I want to vegetatively propagate a rare, endangered, threatened species by taking the explants from the outside of the plants. Can anyone suggest the best method to sterilize the explants for tissue culture of the explants?
The obvious answer is the procedure that brings you the best amount and quality of explants usable for your purpose... Because for each kind of explant and species response may vary, it means you will need to find your best one.
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There are many sterilisation protocols. In general, you may wash the explants with tap water and subsequently treat with different concentrations of clorox (commercial bleach with ~5.25% sodium hypochlorite) containing a few drops of Tween 20 under shaking condition. After that, the explants should rinse with sterile distilled water for a few times. If the % of contamination is quite high, perhaps you can include ethanol (< 1 min) or fungicide before disinfecting with clorox.
Clean the explant under running tap water for about 5-10 min to ensure the removal of exudates/soil etc, followed by treatment with bivistin (1%) for about 5-7min, then use sterile distilled water to wash the explant followed by treatment with 70% alcohol (40sec) and finally then with Hgcl2 (0.01%) for 4-5min inside laminar flow
Hi,
The first advice is to select the youngest part of plant that probably will be not endogenously contaminated, or part covered by removable surface (seed, bud). Cleaning the explant under running tap water is the good idea, I recommend to put prts of plant to bottle, cover by piece of gauze (tissue), fixed by scrunchy and put smal amount of detergent on top, than left 10-20 min under running water .
For the youg plant material Hgcl2 (0.1-0,2%) for 4-10min is the carefull and highly effective surface desinfectants (but toxic for people!!!). Inside laminar box than remove the tissue and rinse 5x by the sterille destilled water. Than remove cut ends of plant damaged by treatment or surface parts.
Good luck!
Explants were initially dissolved in 70% ethanol for 30 seconds and then washed three times with distilled water to make. Then a solution of 5% sodium hypochlorite with bathroom shampoo for 5 minutes, we finally rinsed again with distilled water three times and explants are now ready for callus induction.
Thorough washing of explant is very important as it reduces the contaminants. Wash the explant under running tap water for several (5-10 min) followed by a brief treatment with a detergent (1%) like Citrimide or bivisting etc. for about 5 mins and then wash thoroughly with sterilized distilled water. Conc. and duration of bleach treatment depends on the type and tenderness of the expant-so this need standardization from your side. So choose sodium hypochlorite or Hgcl2 conc. very carefully and also duration of treatment too. A final dip in 70% alcohol followed by thorough rinsing with sterilized D.D. water is effective.
Similar to others, I also wash explants in running water, though for more like 2-4 hours. Then surface sterilization with commercial bleach (20% Chlorox) with a few drops of tween for about 20 min, followed by three rinses in sterile distilled water.
In addition to the previous considerable answers I suggest also the use of some biocide product, as PPM (Plant Preservative Mixture), which prevents or reduces microbial contamination in plant tissue culture. You could add it to the substrate directly or use it as sterilization solution. Visit the website for more informations.
Hi Panda
Please give more information on the type of explant that you will be using. The sterilization procedure depends on explant type...its softness or hardeness. If one knows this it will be easy for one to propose a sterilization procedure.
Hi all,
would I be able to use isopropyl alcohol in lieu of ethanol to disinfect explants?
cheers
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