Hi Hilde,

How old your protonema is when you start the preparation? Do you grow the protonema at constant light? In general, the protonema should be young (4-6 days old max). Growth conditions are very important to garantee fast growth. 

On the other hand, you will always have aggregation of protoplat after centrifugation, but then the advice is to treat them very carefully, you must add very slowly any solution (manitol 8%, for example). 

You can also reduce the spin of the centrifuge, try 180-200 g 4 min, and remove break option and choose the slowest speed to reach the max at centrifuge.

I hope it helps you! my email address is: [email protected]

regards,

Nelson

Hi Nelson,

Thanks a lot! Yes it was very helpfull!

I can't change the growth conditions from 16-8 LD to constant light because other plants are also growing in there... But I can indeed try all the other options!

Greetings,

Hilde

You might try adding some high MW dextran to your isolation medium.  We use 150,000 MW dextran when isolating BY-2 protoplasts.

Hi Hilde,

there are several reasons why protoplasts start to aggregate. One of the most annoying is using wrong tubes (some kinds of plastic are a problem) or not thoroughly cleaned glass tubes. I would suspect that your problem is not due to culture conditions in respect of day-night cycle or constant light but either due to the above mentioned issue with tubes or maybe some problems with the media and buffers you use. If your protoplasts aggregate due to the centrifugation steps, you can try to solve them very carefully by slowly rolling and inverting the tube. Sometimes this is enough to solve the clumps at least a bit.

Hope this helps! Good luck with your experiment!

Gertrud

Thank you Gertrud!

@ Hilde,

Have you found out the reasons, and know how to prevent it? If so, can you share with us? Thanks.

So far, what worked the best was centrifugation at a lower speed. I did not try other tubes yet!