I used leaves as explants and their direct shoot regeneration is good but the height of the shootlets is quit small and didnt grow more without died. so, please suggest, how to get good quality / vigor of the plant . I used 3% sugar, MS medium, .8% agar and TDZ+IBA+PVP combinations were used
Picture is so bad, plase have a nice camera having zero macro. The volume of the media seem very low. You added 5 plantlets into one jar. this is also too much. Transfer these shoots removing agar residue into another jar. Add 30-40 mL MS or LS medium per jar including lower TDZ than you used in combination with 0.5 or 1.0 mg/L GA3. Are these bienial or perenial plants?. If they are biennial, appearence is ok. Good luck
Hi Prakash,
I am sure that the amount of media is more than enough. One may just make rough calculations. 20 ml of medium contains 0.6 g of sucrose which would allow a DW increase of 0.4g (most DW are carbohydrates; an estimated 1/3 of sugar taken up is catabolized to CO2; this in turn may be used for photosynthesis), so 4 g of FW. Inorganics in MS are adapted to the amount of sucrose. There may of course be exhaustion of specific minerals but I expect another problem (see opinion papers by me in the trade journal Prophyta). To make better shoots, the only solution in my opinion is liquid medium , TIB or double layer. Dahlia grows like your plantlets and we found severalfold higher growth when using a shallow layer of liquid medium.
Kind regards, Geert-Jan
Hi Prakash. I think the amount of medium and too many plantlets in a single jar is not the real problem.I see the solution of your problem in liquid medium. Probably subculturing the plantlet in liquid medium will solve this issue. Even in normal circumstances also we generally recommend subculturing after a certain period of culture.
Procedure of suculturing in liquid medium: prepare the medium without agar agar and pour the sterilized medium in test tube. Prepare 'M' shaped structure out of 'Whatman' or a good quality filter paper from a 2 inch long and 1/2 inch wide strip, immerse the 'M' in test tube in such a way that both the legs of 'M' are in liquid medium, so that entire 'M' is wet with medium. Now pull out the individual plant from jar very carefully so that roots are not broken and place it in the central part of 'M' (the centre of 'V' portion) and close the tube. Of course every activity is to be done in LAF.
Good luck with your experiment!
Do plantlets use up in-jar CO2 during extended tissue culture? Is this why subculturing proves worthwhile? I've seen Mason jar lids of tyvek or similar material that allowed gas trasmission while maintaining some sterility.
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