i am working with gene editing in bell pepper. i have done agro transformation by floral dip method. (LBA4404 carrying plasmid, at final pellet dissolved in 5% sucrose containing 0.02% silwet ). i used different time for dipping like 5sec, 10sec, 30sec, 1min. but 3-4 days after the transformation, flowers flown down by truing the flower stem yellow color. kindly suggest, what can be the reason of the cause or how can resolve the problem.
It could be the dipping solution itself. Are all the flowers falling off post-dip or just some of them?
If it's most or all of the flowers then you'll need to make some changes. I would suggest decreasing the silwet, it can be a bit damaging. You can test this out without the Agro just to simplify things a bit. Also, after the dip, keep the plants overnight in high humidity and away from direct light. Once you find conditions that lead to most buds staying on, then re-try with the Agro added.
Good luck!
Amy Klocko
Madam, Thank you for the suggestion,
As i tried initial flower of the plants (5 plants=10 flowers), all the flowers flown down and falling started after 3days of agro.
Which stage of the flower is best for floral dip.
To me, these flowers all look too mature for a floral dip. In Arabidopsis, the thought is Agro gains access to the very young ovule (before it has fully developed and closed) and transforms the egg cell. Older flower buds will have more layers of cells/tissues between the floral exterior and the egg.
I'm not familiar with the floral progression of pepper plant but it seems if you can visually identify petals etc. the bud is fairly developed. Stage 1 looks about as mature as I'd try.
My best suggestion would be to inoculate the plant (avoiding leaves as much as practical) while the buds are exceedingly tiny. You can directly pipet your bacterial solution onto the buds and thus avoid dipping the entire shoot.
Dheeravathu Venkateshwarlu As Respected prof Amy Klocko mentioned all the possible reasons and provide very useful suggestion. I am adding a little bit, reduce the dipping time and only dip the floral parts avoiding leaves or stem. secondly you can also cut already mature (opened) flowers and dip the rest which are not yet fully opened... best of luck
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