I followed "Floral dip" ( Agrobacterium in 5%sucrose and 0.05% Silwet L 77 solution.) method for Agrobacterium-mediated
transformation of Arabidopsis thaliana and in next day after floral dip shoots died. This happen for several times.
What may be the reason
Does this concern the inflorescence, leaves or both?
Possible reasons could be a too high temperature and/or too much light. Important is to use either short day or long day and temperature around 21C, do not use continuous light. Further, the silwet could be reduced to 0.03%.
In case the leaves die, try to avoid submerging the rosettes in the Agro solution. In case specifically the inflorescence dies, make sure to have enough distance between lamps and the plants, and/or reduce light intensity.
I hope the suggestions are helpful.
What protein-construct does your agrobacterium carry? Any images? Need further details to gauge as to what is going on?
Dear Dr. Eric, yes it consists both Inflorescence & leaves. After inoculation I kept them in dark condition for 16-24 hours and when I see them at that time all shoots, Inflorescence wilt, decayed :(
Dr Biswas, photos attached.
Thank you all.
Dear Akila,
I also sometimes observed this problem. Make sure you don´t dip longer than 20-30 seconds. It might also help to buffer the dipping solution with MES to pH5.7. Also make sure the plants have plenty of humidity during the dark incubation after dipping and water thoroughly in the first week.
Dear Dr. Eric,
Thank you very much for your kind support :) I'll follow the your suggestions in next round
Dear Akila,
I would reduce the surfactant to 0.025 % and I do not recommend such an extended darkness because it supposes an additional stress for the plant. If you can, you may dip just before the lights switch off (or a few hours before) in order to couple with the "natural" darkness phase of the long day photoperiod. Bests.
Dear Akila,
In our lab, we transform al afternoon and after dipping around 1', took out the big drops of culture and put the pot inside a plastic (transparent) bag all around the night. Additionally, we only transform really young flowers (absolutely green, we could not see any white of the petals), and cut out all the others (in that way we obtained no quimeras). The transformed plants should be in good health condition and between 4-6 weeks-old. DO Agrob= 0.8 (in 1/2 MS, 5% sucrose and 0.2 % silweet).
I can send you my detailed protocol but I think is really similar the protocol en Arabidopsis: A laboratory manual, from Weigel and Sambrooks
https://books.google.com.ar/books?id=IfZAMNPWVk4C&redir_esc=y
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