Dear Shirin,

You could try this:

 Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Zhiyang Zhai, Ha-il Jung, and Olena K. Vatamaniuk

 Enzyme Solution: 0.5 M sucrose, 10 mM MES-KOH [pH 5.7], 20 mM CaCl2, 40 mM KCl, 1% Cellulase (Onozuka R-10), 1% Macerozyme (R10). Filter-sterilize and freshly use.

You may also try reducing the incubation time and slightly increasing the osmolarity of your usual buffer by adding more mannitol, but be careful to reduce the osmolarity very slowly once the protoplasts have been released.

Good luck!

Simon .

The age of the leaves also matters and the plant should be healthy. check this out and retry. Luck

As mentioned by Dr. Vats, the age of leaves is very crucial. Go for younger leaves with bright green color. Also, regardless of protocol you use, the protoplasts are very fragile and it's really important to carry out all steps after adding the digestion solution very slowly and add/mix/transfer solutions/suspensions in small quantities.

I used this reference in the past with good success (little optimization): Abel, S. and Theologis, A. (1994) Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. Plant J. 5, 421–427. 

Getting the enzyme concentrations right is also important and in my experience  it is company/batch dependent. Best luck.

It is very common to obtain broken protoplasts using enzymatic method. So do not worry and try conducting viability test for the remaining protoplasts.

Thanks everyone for your helpful tips, I will try again considering all the good points you mentioned.

Hi Shirin,

I used the protocol from Yoo et al. successfully (http://www.ncbi.nlm.nih.gov/pubmed/17585298). I prepare the enzyme solution freshly each time and I have never experienced troubles during this step. It might also be that what you think are broken protoplasts is simply debris from the tissue. Washing the isolated protoplasts carefully (e.g. as described in the protocol, or on a sucrose cushion) can help you remove debris.

As Bulat has mentioned already, the handling of protoplasts is really crucial. Always treat your protoplasts gently.

good luck

Hi Shirin,

1. How do you know that majority of your protoplasts were broken? You were looking at 'enzyme solution after 1 hour' treatment. In the enzyme solution, you might have  observed many debris, which was from digested cell walls. Instead, you should check the yield after you done, counting their numbers with a hemocytometer.  

2. If yield is low, you should check several things (which other members have mentioned):

(1) the age of the leaves

(2) the enzymes (ex. cellulose R-10 and Macerozyme), different company/batch may produce different results

(3) protoplasts are very fragile, handle them with extra care

(4) I saw you store your enzyme solution at -20C. I always use freshly-prepared enzyme solutions (prepare at the same day). In a few occasions, I stored the leftover at 4C overnight for second day use (for another batch of protoplast isolation). They still worked well.

Thank you so much Dr. Yuan,

I just observed the enzyme solution under the microscope, and this is what i got. So i automatically guessed that more of them should look round and intact.

Thanks again, i'll try running this experiment soon again and will keep all those points in mind :)

Thanks, Shirin.

I saw your posted picture. Yes, you still have several steps to go, including washes with different buffers, centrifugations to remove debris, then concentrate them to obtain a higher density (intact) protoplasts populations.

For transformation, in protocols, it usually suggests a specific protoplast number you should use for better results. So, you need to count them with a hemocytometer (see below picture 'D', you count the protoplast number in a square for calculation). Once you have the number (per ml), you can calculate how much volume you should use for your experiment.  

In a few cases, I experienced very few yield (don't know the reasons). I had to re-isolate a new batch of protoplasts. So, constantly to maintain enough plants (in the best conditions) in the greenhouse/growth chambers is also important if you plant to isolate many batches of them.

@ Shirin,

There is also a method to isolate Arabidopsis protoplasts by using tapes. They call "Tape Arabidopsis sandwich' method. See attached paper. I have not tried that, but the authors claimed that it is a quick and efficient method.

Dr. Yuan,Thanks for all the information you shared with me, i really appreciate.

As a matter of fact we already do use the sandwich method in our lab and my lab mates say the yield is so much better than the older method indeed. And regarding to my broken cells problem, i figured, the enzyme solution is very sensitive to any external chemical, and i had treated my plants with some pesticide a week before the proto isolation which i guess caused the problem. now I have moved to the next level and i loose many cells after PEG treatment (transfection). as everyone mentioned, protos are super sensitive and i have to practice handling them more carefully and gently.

Thanks again everyone :)

Yes dear, spraying with pesticides may cause such problem. Sandwich technique is very helpful, 

@ Shirin,

I am not sure whether or not the pesticide caused broken protoplasts. My plants also grew in a greenhouse, and the manager spread pesticide periodically too. But, I don't know whether we have the same kind of pesticide.

Hi Shirin,

Have you checked and controlled temperature during the enzymatic digestion step. Temperature too high can have serious consequences on the viability of protoplasts. You should also try to reduce the concentration of cellulase and the stirring speed ?

Thanks again for your kind replies,

aw! then I don't know if the pesticide was not causing the problem, it must have been something else.

I'd like to know how high a temperature are we talking about Dr. Roger? because I do the digestion step at room temperature which in case of our lab would be around 32-37 degree centigrade.

I suggest you try 25 degrees at first without changing other parameters (if possible in a controlled culture chamber).

My protoplast isolations were also performed at room temperature. In our lab the temperature was controlled, but it fluctuated around 25-28oC.

Although I never pursue the subject that whether or not the temperature affects protoplast isolation, some other researchers seem to think it might. See this attached paper (p.93, yellow highlights).

**This paragraph is from the paper:

"The results also confirmed that increased incubation temperature from 28°‒31°C significantly reduced the time needed for rapid release of protoplasts. In this protocol, optimal incubation temperature was 28 ‒ 29°C and pH of 5.7 ‒ 5.9 for better protoplast yield."  

Hello :)

Thanks Dr. Roger and Dr. Yuan. I finally succeed and got transfected protoplasts after second try. I tried to handle the protos super carefully and used cut tips all the time. And it worked. I will share my results with you through another question to ask you about your idea of how your interpret the signals. I also attach one photo here :)

Cheers

Hello Shirin,

The picture reminds me of the microscopic observation that we made after staining with FDA. I think it is a membrane labeling. What do you think ?