Dear all,
I've been trying to isolate protoplasts from rice for quite a while now, but until now it has been unsuccessful. For a start my yield is quite low (10-fold lower than what is published in several articles) and in addition a large amount of debris is present (mainly broken protoplasts, chloroplasts) that I can't seem to get rid of.
I have used several protocols to try to achieve this isolation (please see below for the one I used the most), both in light-grown (12/12) and dark-grown rice plants. In most protocols I have read they use an enzyme mix with 0.6M mannitol, but I also used 0.4M mannitol as described in some protocols but to no avail.
In most of these papers however, no method of purification of the protoplasts is mentioned, but I have tried 2 different ones (one using a 20% sucrose gradient and another one with a 3 step gradient using sorbitol and sucrose). But with neither method I had a layer of protoplast which I could isolate. Out of cost considerations I have not used more expensive reagents such as Ficoll to try to achieve a sufficient separation, but I guess this wouldn’t have solved my issue anyway.
I also took a look at the resources of the Sheen labs (http://molbio.mgh.harvard.edu/sheenweb/main_page.html), in one the videos it was mentioned that the protoplast mass that collects at the bottom of the tubes after centrifugation should be easily resuspended by gentle flicking the tube, but in my case this is not the case (this video was about Arabidopsis protoplast though).
At the moment I'm getting out of ideas and frankly a little bit frustrated. Does anyone have any tips and tricks to make it work?
Thank you for your time and consideration.
Kind regards, Jonas De Saeger
Lowder, L. G. et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. pp.00636.2015 (2015). doi:10.1104/pp.15.00636
Dear Ambastha,
Thank you for your answer! This is actually one of the protocols I also used, but I also wasn't able to get a get good yield. I will check again, but I will take into account the light intensities in the growth chamber this time and I will try to order enzymes from Yakult and not from Sigma as I read that people sometimes have difficulties with their enzymes.
Apart from that, do you have other tips I should pay special attention to? Also did you use your protoplasts for the transfection of DNA and was a purification step necessary (because I sometimes have quite some broken cells I want to remove before going to the next step)?
In any case, thanks for your message!
Kind regards, Jonas
Dear Jonas,
I will recommend using enzymes from Yakult. Apart from this, the pH of the digestion buffer is a very important factor deciding the protoplast yield. Use the high-quality water (HPLC grade) for preparing protoplast digestion buffer.
In some experiment, I added Pectolyase Y-23 ,0.05% w/V, (from Yakult) which enhance the digestion process and increase the yield to double.
These are most important part in rice protoplast isolation is the condition of plants.
The plants must be between 5-7 days old ( post-germination). Grown under proper photoperiod condition at a light intensity of 80-100 photons m-2 S-1 with high humidity.
I hope it help .
Regards
Vivek
Dear Vivek,
Thank you very much for you answer, I'll try to do the protoplasting method again taking your tips and suggestions into account.
Kind regards, Jonas
Hi Jonas,
I agree with all the recommandations of Vivek.
Have you checked and controlled temperature during the enzymatic digestion step ? Temperature too high (sometimes few degrees !) can have serious consequences on the viability of protoplasts. You should also try to reduce the concentration of enzymes and the stirring speed.
Many parameters have considerable importance on the quality and yield of protoplasts.
I hope these comments will help you,
Regards, David
Dear David,
Thank you for your input, as a matter of fact I did not control the temperature during the digestion step. In most protocols I've encountered they also do not control for this and just state "incubation at room temperature". But it is indeed worth checking out, our lab here in South Korea can get quite hot, so I will definitely do the digestion step under controlled conditions next time.
Thank you for the help!
Kind regards, Jonas
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