I Don't know what concentration of enzyme you are using and for how much time exactly you have placed them in the incubator. Usually upto 1% macerozyme and 3% cellulase is enough, and incubation time of 12-14 hour is enough, more digestion time usually led to disrupted plasmids and lower yields.

Also dip the tissue in 0.6M mannitol while you finely chopping the leaf tissue. fine chopping of the tissue is also the most crucial step, as finely chopped tissue have more surface area to act for the enzymes and thus led to higher yield.

Moreover, try to take the younger leaf tissue. if you still got the lower yields use the tissue cultured potato tissue to extract protoplast, since those have low lignin content, thus releases protoplast easily and had much higher yield than field grown plants.

I have been using the enzyme at lower concentration of 1% cellulase and 0.25% macerozyme and usually kept for overnight incubation or 16-18 hours. I have followed the rest of the mentioned steps for isolation. I am getting some protoplasts but not in enough number.

For effective digestion of potato protoplasts, a combination of cellulase (1-2%), pectinase (0.5-1%), macerozyme (0.5-1%), and pectolyase (0.1-0.5%). This enzyme cocktail ensures the comprehensive breakdown of the cell wall components, facilitating the isolation of viable protoplasts. Culturing conditions and purification methods also play a crucial role in optimizing the yield and viability of the isolated protoplasts [1] [2] [3] [4].

Protocol Insights

Pre-treatment: Culturing donor shoots in darkness for 7 days can increase the yield and viability of protoplasts.

Osmoticum: Elevated osmotic conditions, typically using sugars or sugar alcohols, are necessary to maintain protoplast integrity during isolation.

Sucrose Cushion Method: Purification of protoplasts using a sucrose cushion method enhances extraction efficiency.

References

1. Protoplasts Increasingly Valuable Tool in Plant Research

Grosser, J.W., Omar, A.A.

Plant Tissue Culture, Development, and Biotechnology, 2016

https://www.scopus.com/pages/publications/85136359096

2. Isolation of starch from potato using fungal enzyme

Gayal, S.G., Hadge, G.B.

Indian Journal of Microbiology, 2003

https://www.scopus.com/pages/publications/26644474376

3. Isolation and culture of protoplasts from the plum rootstock 'Marianna 2624' (Prunus cerasifera Ehrh x P. munsoniana W. Wight & Hedrick)

Andreu, P., Arbeloa, A., Terrén, N., Marín, J.A.

ITEA Informacion Tecnica Economica Agraria, 2008

https://www.scopus.com/pages/publications/77953690388

4. A more accessible, time-saving, and efficient method for in vitro plant regeneration from potato protoplasts

Moon, K.-B., Park, J.-S., Park, S.-J., (...), Kim, H.-S.

Plants, 2021

https://www.scopus.com/pages/publications/85104416713