Q: Can we put more than 1 antibiotic in the plate for selection pressure if the agro strain has more resistant markers for better screening of transformed plants?

A: Yes- you can for selection. Make sure it is not creating unnecessary stress for the explant. I would be careful using more than one antibiotic.

A: You would likely need to adjust either timing or concentration if doing a dual selection method. This can in some cases help reduce escape rate/chimerism. Usually it’s used of some tissue stages are more resistant to a particular selection agent than others.

A: It depends on your purpose. If one selection is sufficient, there is no need to increase the selection pressure which usually affects transformation efficiency, not to mention cost. If you are doing co-transformation to select more than one construct with different markers, then yes.

Q: Selection cassette can heavily impact the transformation efficiency and selection pressure needed for success. If possible can you share the promoter use on NeoR gene?

A: You are right. Selection cassettes are very important as it determines the amount of selection agent suitable for optimal efficiency. choice of promoter and terminator elements including monocot or dicot optimized coding sequences for GOI in context with binary vector as whole and the choice of plant species one would intend to work on is critical to the success of transformation outcome. It is better to take all this into consideration early on (before you start experiments) including controls is always better to get meaning full outcome.

Response:  We are currently running both NOS and e35S promoter on nptII in tomato and have seen drastic regeneration response differences between the two. We’ve dropped our selection pressure on NOS due to the reduced regeneration efficiency but no data yet.

Response: I’m pretty sure you will do relevant controls which will help you come up with better outcome. Basically you are optimizing he workflow that is suitable for your own lab and what you have accessible to make the most of it 👍