When you filter out or centrifuge down the cells during cell suspension culture protocol, is there any method to reassociate them back into one callus? What would happen if I just plated them all together in a clump (just like how it was if they had remained a callus)
Another question (which is unrelated to the first one), after the suspensed and separated cell are planted on the plate in the last step, what will happen to the growth of the cells if they happened to be touching each other (unconnected by plasmodesmata)?
Settanan Plangsiri
For your first question, yes, you can put them together as a mass. But, what is your purpose to do that? A spread out cells can take up nutrient from a plate of medium more efficiently I guess. And more efficient to select transformed calli from a 'selection medium' (ex. contain antibiotics), if they are evenly spread out...
Yuan-Yeu Yau Thanks for the answer!!
One question... in your last sentence, you metioned using antibiotic to select the transformed plant. Does antibiotic kill non-transformed plants?
Settanan Plangsiri
We used to try carrot suspension cells for genetic transformation (a small side project). We spread the cells on a autoclaved 3M membrane (which was placed on the surface of selection medium), so we could easily move the calli when subculturing. The non-transformed callus became darken and ceased to grow, the transformed calli would continue to grow. Then, the grown callus clumps (to a size) were moved to freshly prepared selection medium for continue growing or to regeneration medium for regenerating into plantlets.
Settanan Plangsiri
This is my carrot suspension cell transformation slide. The background darkened calli were non-transgenic. The growing calli were putative transgenic.
Settanan Plangsiri
This is my cotton transgenic slide. But, these are not cotton cells from suspension culture. Those were callus cells. We directly spread the the callus mass on a membrane on a selection medium. The putatively transformed callus clumps were formed nicely. We also got transformed plantlets from those re-grown calli (and confirmed it is transgenic through GFP glowing). But, we observed quite a few adult plants derived from this method were not normal (ex. dwarf). Could be chromosome abnormality after long-term tissue-culturing (not sure, we did not follow up). We changed to standard hypocotyl transformation method.
Yuan-Yeu Yau Thanks for another answer! I really appreciate it (especially the example!)
I have another question. When you do Cell suspension culture transformation... it's not a 100% yeild right? There will always be
1. non-transformed cell aggregates
2. hybrid (both transformed and non-transformed) cell aggregates
3. transformed cell aggregates
and by putting in the antibiotic, you were able to select for only transformed cells. (For the hybrid aggregates, the non-transformed part died off leaving only transformed parts)
Is my understanding correct or is something in my explanation wrong? Thank you again!!
Settanan Plangsiri
1. I am not sure I completely understood what you meant by "it's not a 100% yield right?"
2. The 3 statements you listed are all possible
3.
That is a textbook standard theory (the textbook would tell you so). But, In reality, you might get *chimeric* cell clumps, meaning a clump of cells constitute both transgenic and non-transgenic cells. This is might due to 'protection effect'. For some reasons, the transgenic cells can protect the neighboring non-transgenic cells from dying due to antibiotic selection.
4. Several things you don't want from your generated transgenic plant lines: (1) chimeric (not a homogeneous transgenic plant), (2) multiple copies of transgene (the plant line might become gene silencing), and (3) sterile, no seed setting (no progeny), (4) transgene can not transmit into next generation