Dear José,

interesting problem. Does this low yield only occur in the clean bench? If so, vibrations from the fan may induce lysis of the fragile protoplasts. Try putting your dish on a piece of foam to reduce the vibrations.

 kind regards, Harrie Verhoeven

It is very interesting observation. What is the cell wall composition of filamentous brown algae? Why not you give a try of treating of thallus separately in cellulkase and then alginate lyase. This will help you to know which enzyme has detrimental effect on your sample.  

Nice observation Harrie! I ll try that. But i think that the vibration couldn t be the problem becuase i observe that greenish color in the shaker incubator after add the enzymes in the clean bench. But anyway, nice observation.

Dr. Reddy. Thanks for your answer. I also checked the effect of the concentration of cellulase and alginate lyase on the protoplast yield. But all the optimization process i didnt make in the clean bench. In that experiments i didn t see that problem, even when i used higher concentrations of enzymes that i m currently using. The cell wall composition of brown algae are mainly alginate (with G and M blocks varying according the type of tissue or growth phase), fucoidans and low percentage of cellulose.

PD: The only modification that i ve made when i perform protoplast isolation in the clean bench is that I clean the sample (i m not using axenic material yet) with Betadine 1% during 30 seconds and then i wash it during 6 minutes in sterile seawater. Finally, i make three more washing steps of 30 seconds each one. May the betadine be the problem?

Dear José,

Betadine is not really advised to sterilise your samples. It contains a lot of supporting chemicals: disodium phosphate, glycerol, sodium hydroxide, citric acid, modified lauryl alcohol and potassium iodine to the main agent, which is pvp-iodine. This results in a highly adhesive film, which is not readily washed away. It may explain the massive loss of viable protoplasts in your clean bench experiments. I would suggest to try sterilization with a low percentage of bleach or hydrogen peroxide in stead. Hope this helps.

I would also like to suggest you to handle your specimen so carefully that the sterilizing agents does not kill the sample. I have experienced in the past when surface injured sample treated with betadine got severely affected.

Perhaps the variability in protoplast yield is caused by variation in the starting material?  I had an analogous situation years ago when isolating protoplasts from greenhouse-grown tomato seedlings.  Some isolations went well and others did not, just like you describe for brown algae.  It turned out to be related to the number of sunny and cloudy days the seedlings were exposed to 3 or 4 days prior to protoplast isolation.  I ended up with 2 different enzyme cocktails - one for cloudy days and one for sunny days (higher cellulase and macerase levels were required). This worked okay but I eventually got access to a growth chamber where I could grow the seedlings.  From then on the yields were extremely consistent and required only a single enzyme cocktail.

Another thought is to routinely include a non-"clean bench" isolation along with the clean bench isolation.  Since it is only a 6 hour incubation contamination is not an issue.  This might help determine if the problem is something about the clean bench process.

When you figure it out be sure to let us all know.

For suppress the protease activity you can put your solution 10mn at 55°C.

Be careful with the batch of cellusase and amount of activity that could change between manufacturers.

Thank you so much to everybody for your answers. I ll follow your recommendations and I ll let you know the results :)!!!

Hi everybody!

I think the problem was the Betadine treatment. Without using it, i didn t see this greenish color from my explants and i also recovered a lot of protoplast.

Thanks everybody for your suggestions!