I have attached a couple of images (cells in a hemocytometer under a cover slip). Is the structure a protoplast ? If yes, do protoplasts have non-spherical structures ? ( excuse the poor image quality). Wondering why I see paired oblong structures.
Thanks !
It look like the cell wall components are partially digested by enzymes. Since the images lack scale bar its difficult to conclude that they are protoplasts. Anyways, a well processed leaf protoplasts must be sperical.
We isolated protoplasts from different tissues of common bean. Hope this protocol may be helpful.
Nanjareddy, K., Arthikala, M., Blanco, L. et al. Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis. BMC Biotechnol 16, 53 (2016). https://doi.org/10.1186/s12896-016-0283-8
Manoj Kumar Arthikala Thank you for your input. Interesting paper giving insights into SAAT techniques. Lots of ideas to translate from the work. Could I ask how you suspect that it is partially digested ? I don't have plant experience to make such interpretation. Also, we didn't use Cellulase and Macerozyme from Yakult ( Trichoderma sp), would that affect the digestion efficiency ?
The images have grids (hemocytometer) around 250um per side. So I guess the structures I see are roughly around 20-30um ?
Yes for plant tissues you must use Cellulase and Macerozyme. These enzymes are avilable at yakult pharmaceutical Industry co. ltd. That was the reason for partial digestion of your sample.
Manoj Kumar Arthikala We used cellulase and macerozyme from A.niger ( Sigma) and seems like Cellulase / Macerozyme from Trichoderma (Yakult) is preferred. Probable cause of partial digestion. Thanks for letting me know, I'll try it out with the new enzyme source.
Initially we also had problems related to the protoplast quality with other source of enzymes. Later we resolved with Yakult for bean tissues.
Good luck with your experiments.
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