Hello All,
I really need some help with my protoplast media. I am trying to repeat this cycloheximide chase done in Kang 2003. They reference Tao 2005, where "transfected protoplasts were cultured overnight (14 h) in auxin-free K3 medium".
Based on my backtracking, I believe that the K3 medium is KAO'S medium No.3. from KAO et al., 1974? Does anyone know if this is correct?
What media have others tried to resuspend there protoplast in and then add drugs/reagents?
I would really really appreciate any help or advice!
Thank you!
I bought this media: https://www.rpicorp.com/products/biochemicals/plant-growth-regulators/kao-michayluk-1-l.html?ga_list_name=Category%20%28Grid%20View%29
Yes, you are correct, K3 is Medium 3 in Table 1 of -
Kao, K.N., Constabel, F., Michayluk, M.R., Gamborg, O.L. Plant protoplast fusion and growth of intergeneric hybrid cells (1974) Planta, 120 (3), pp. 215-227. (file attached).
That said, tracking down an actual formulation can get tricky for a number of reasons - often not referenced (like the Kang paper), changes made that are not reported, typos, and other errors.
The most important factor before starting the experiment is that your protocol results in the isolation of high quality protoplasts. The quality of the source tissue has a huge effect on protoplast quality. With a protocol to produce high quality protoplasts the experiment should proceed smoothly.
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