I'm just starting to get into TC and I had a few questions. nepaholic answered some of them on another site, but I was wondering if you'd share some information. Here are a few questions: 1) How do you sterilize your tissue? Here's a method that I've read: a) Dip the cutting in the 90% alcohol quickly. Hold it above the alcohol to let any excess drain. b) Drop the cutting in 3% peroxide for 2 minutes c) Drop the cutting in 10% bleach mix for 10 minutes d) Drop the cutting in sterile water for 10 minutes e) Drop the cutting in 2% PPM (plant preservative mixture) solution for 30 to 60 minutes f) Transfer to media
2) What size cuttings do you put on the media? Do you cut up the tissue and then sterilize each piece or do you take a large piece, sterilize it and then cut it up into smaller pieces? 3) Do you use any hormones like BAP or IBA? If so, in what ratio (i.e. 1ml/L)?
I have sterilized leaves from alfalfa, violets cephallotus and juvenile flower stalk from VFT with success, followed the next procedure.
1. Choose a juvenile piece of plant(explant) which is growing and wash thoroughly in running water, next for flower stalk of VFT, dip it in bleach solution( bleach 30 mL + water 70 mL with + one or two drops of liquid detergent) for 15 min. Dip in sterile water for 10 min( I use a bottle with sterile water and then I will introduce the explant in the bottle). Now transfer the explant into flask with medium.
The success depend of explant and the sterilization procedure. in general is more easy sterilize flower stalk than leaves, the concentration bleach solution is critical, for that you make try with different bleach solutions and times(usually 10 o 15 min)
Bleach 10 mL + water 90 mL= 10 % Bleach solution Bleach 20 mL + water 80 mL= 20% Bleach solution Bleach 50 mL + water 50 mL= 50% Bleach solution
For example, in cephallotus leaves I used a 50% bleach solution for 15 min, the explant resisted this treatment. Before I used 20% and 30% bleach solution without success.
The method that you read should work, I suggest in point c) that you try with different bleach solutions beginning with 20% but no more that 50%.
2) What size cuttings do you put on the media? this is your second question. If you have a plant growing in vitro you do not need sterilize again, only to take the plant out the flask and cut it up into smaller pieces and each piece transfer into a flask, like this photographs.
take the plants out
Cut the plant in pieces
transfer into the flasks
Matt, finally I do not use BAP or IBA in this case, only 1/3 MS.
Matt is quite difficult to me write in english, I will hope that you understand me.
I had no problems at all understanding your english. It is quite good. Thank you for your time and your response. It has helped a lot. Tomorrow I am going to try my first attempt at tissue culture. I hope that I have moderate success.