I am working on invitro protocol optimization on ginger. I face many challenges on finding clean ex-plants. The major problem is the systemic bacteria that exudates out from plant tissue to that of the media and contaminate the media first and the ex-plant at the end. I am currently using 0.3% Mancozeb (fungicide), Berekina (have active chlorine concentration) and 70% ethanol. The source of the ex-plant is rhizome bud for direct organogenesis. So, please assist me how can I remove the systemic bacteria that contaminate the media and ex-plants?
Wash your ex plants i.e.rhizome buds in running tap water until it gets cleaned. Wash with tween 20 (detergent). for around 20-25 minutes.Treat your explants with Bavistin (0.1-0.5%) for 30 min - 2 hour.At times one night incubation is also required depending on the nature of your explant. Wash with double distilled water in combination with tween-20 again for 15 minutes followed by washing for around 5-6 times with double distilled water. Then in LAF treat your explant with 0.1%HgCl2 followed by 70% ethanol( 30 sec - 3 minutes) and finally with autoclaved doubled distilled water for 3-4 times. you can also use sodium hypochlorite in place of mercuric chloride.
Good luck
Shiwani Kaushal
Dear Mihiret,
I am working on tissue culture of saffron using its corms as explant and i also had the same issue as you have because when you are using an underground part as an explant there are more chances of contamination and using 70% ethanol and bleach or HgCl2 is not enough. I didnt get bacterial contamination but everytime i got fungal contamination. I started to use carbendazim in different concentrations before 70% ethanol and bleach (with a drop or 2 of tween) and finally reduced its contamination from 50-60% to 5%.
Wash the explants in running tap water for 30 mins to 1 hour followed by washing with 5% tween 20 for 10 mins. Then wash it with 0.5% carbenbazim for 5 mins followed by double distilled water and then in LHF wash it with 70% ethanol for 2-3 mins and 50% commercial bleach (with a drop or 2 of tween 20) for 2-3 mins. Rinse it with sterile water 3-4 times.
Regards
Asif Khan
Dear all,
Thank you very much for your assistance. I will try all the methodology. But, for Khan carbenbazim is an antibiotics i think, isn't it? There is no access to this chemical (not available in our laboratory). The purchasing process is also very long to get in short period of time. Could you tell me other chemical that have similar function to this e.g kanamycin, stereptomycin, gentamycin?
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