I’m working on somatic embryogenesis in the liquid medium. Now, I want to transfer the proliferated calluses to a new medium. Before subculturing, I want to count the number of cells per ml of liquid medium. Therefore, I need a protocol of subculturing and cell counting of plant cells in tissue culture conditions. I would be grateful if you could kindly help me with a suitable protocol.
Regards
Here is one way:
Plant Cell Rep. 2005 Mar;23(10-11):665-72. Epub 2004 Dec 10.
Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader.
Lamboursain L, Jolicoeur M.
https://www.ncbi.nlm.nih.gov/pubmed/15747158
@ Rohollah,
We counted plant protoplasts (cells without cell wall) with a haemocytometer (see attachment #1). According to this link below, I think that you can also count plant suspension cells with a haemocytometer (see attachment #2**). In this case, you need to buy a haemocytometer (see attachment 3). Then, count your cells under a microscope (attachment 4).
**Attachment #2 is from one of these slides display at: https://www.slideshare.net/ThanujaInturi/plant-tissue-culture-by-thanuja
Thanks to everyone who participated in answering my question. Really very useful to all.
With best regards
Phillip Morris is right, counting cells in suspension culture is a real pain. If you really need cell counts, avoid chromic acid as it is a carcinogen... If enzymes are unaffordable, you could try EDTA at pH 9, and 45 ºC during 45 minutes (in a 1.5 mL tube) and later vortex during 2 or 3 min... This procedure helped me in 2015 to get small clumps (5-10 cells) relatively easy to count moving the macro and micro in light microscope, the initial aggregates were huge with more than 50 cells, practically callus clusters swimming in liquid medium even if they were "suspension cultures", this data using Taxus globosa S. cells.
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