Hi all, I have been trying to isolate protoplast from in-vitro grown young leaves (by shredding into small pieces)of large cardamom (A. subulatum) belonging to Zingiberacea family in enzyme solution containing 2% cellulase RS and 0.5% Macerozyme R-10 (both Yakult pharmaceutical, japan) and other different enzyme concentration along with 0.5M mannitol, 5mM cacl2.2h2o, pH: 5.8 at different temperature range from 25-37oC(water bath/incubator), both shaking and static but the isolated protoplasts are very few along with the few undigested cells. I have tested enzyme in spinach, carrot at 37oC, it worked well and released protoplast within 1hr. But same condition is also not working in large cardamom. The enzymes hardly affect on maceration (as it remain intact in tissues when observed in microscope) and digestion of cell wall hardly takes place. Kindly help.
Hi
Several factors are involved in release of protoplast, it is not necessary for protoplast to be released within 1 hour only it may take long time. There are some reports on cardamom where the protoplast were released after 18-20 h (refer this: https://updatepublishing.com/journal/index.php/josac/article/view/4546)
Further you need to optimize the procedure:
1. Try to take as young leaves as possible so that the wall do not thickens and become easy to break and take less starting material.
2. use some additives that mediate the release of protoplast such as DTT
3. increase the enzyme dosage or try use other enzymes also check the optimum temperature at which the enzyme is most active and the ph of the solution.
All the best!!
Hope the information might help you!!
Thank you Yashika Rajeja for the reply. Some of your suggestion, like, to take the leaves as young as possible may help me in isolating the protoplast since I have been using in-vitro plants leaves which may be mature enough to release protoplast. Suggestion to add DTT in enzyme solution is also a good one. I have also tried many other protocol including Geetha et al which you have mentioned above and incubated for 18-20 hrs at different temperatures and pH but could get only few protoplast. One again, thanks.
Maybe the cell wall composition of your species a bit different from the ones that worked well?
Perhaps roots or stems would work as an alternate starting material. Are the leaves waxy at all?
What do you need the protoplasts for? Perhaps a different approach could work for your experimental outcome.
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