Plant regeneration seems to be a problem associated with Agrobacterium mediated transfer because of which people are working towards viral vectors. Can someone elaborate what exatly the problems are in context with plant regeneration for agrobacterium mediated transfer?
Article Geminivirus Vectors Deliver Reagents for Plant Genome Engineering
"The transformed cells resistant to the antibiotic kanamycin remained vigorous whereas non-transformed cells were necrotised and died on selection medium"
This was one of our findings in our recent research.
The critical points could be:
1. Starin & Itslevel of response to selective marker
2. Infection time
3.Use of acetosyringone
4. Disinfectant used
5. Regenaration media used
6. Frequent changing of regenaration media during different level of growth
7. Photoperiod.
Hope this might be helpful.
Regards
NZ
Dear Subin,
There are good protocols for many crops, from annuals to perennials; cereals & legumes to fruit trees. It has been time tested and works well. One has to produce many transformants and get them to the greenhouse because depending on the point of insertion and the number of insertions some plants may not look normal. So, those need to be screened out. When there are no protocols, like in new crops, one has to check protocols for closely related species and can get clues to develop new protocol.
Good luck
RP
Use LBA 4404 strain of Agrobacterium rather using EHA 105. This will help to avoid Agrobacterium Overgrowth in the selection medium after agroinfection. Frequent subculture to new selection media will help to avoid Agrobacterium contamination. Use timentin disodium salt along with clavulanic acid instead of using carbenicillin alone. this will help to reduce vigourous growth of agrobacterium in the TC media.
All the best.
Hi. Respected , my own thinking is that, that during co-cultivation the explant is so small and have less energy to grow, even if it is grown without co-cultivation it wil not grow in a good way, the explant must be larger than normal, because explant energy is utilized by the bacterium that is why it is no more to grow.Thanks
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