Just separate the small plantlets from the calli and transfer it to regeneration media with selective agents (antibiotics). And don't rely on this single transformation experiment, go ahead and transform more explants, so that you have more batches of the transform cells, if one get contaminated or not successful, you will have more to save your time rather than repeating the whole process again.

Hi Yumna,

Do you see just small leaves or shoot like structure in your callus?

If you see something shoot like is there then let it grow till approx 1-2 cm then only disect it from callus and transfer it into rooting media.

if you are not in hurry then I will suggest don't cut the leaves at this stage to check presence of transgene. let it grow into complete plant then only confirm transgene by PCR. 70-80% transgenic shoots only grow normally in selective media nontransgenic shows abnormal growth.

As suggested by Zahid Hussain do repeated transformation to save your time.

Hope this will help you.

Zahid Hussain Thank you for your response. I have placed more batches of explants for safe side. Though my calluses are already on regeneration media with selective agent. I have been using BAP 1mg/L and IAA 1mg/L. Would you recommend to use any other auxin or cytokinin?

Asmita Gupta Thank you for your response. I have only small leaves on most of the calluses, only 1 or 2 calluses have small shoots. I have only 1 semester left to complete the experiment, that's why I wanted to do the confirmation asap.

First confirm your calli with selection media (SM). Add different concentrations of desired antibiotic in selection media. As the high concentration of antibiotic and long time exposure is lethal for callus growth and can produces yellowish or brown symptoms. I think, first confirm your transformed calli. After selection step wash your calli with cefixime, double distilled water etc. and then transformed to regeneration media (direct or indirect).

Hi Yumna,

In that case you can just take small part of leaves from the shoot and extract its DNA (by crude method)

and check by PCR. According to my experience in transgenic plants small shoots will further grow into plantlet and if your shoots are already in selection media 80% chance is that they will be positive transformant.

Sohail Ahmad Jan I did that too. I placed them on different concentrations of selective reagent, and before that I washed them with cefotaxime. After that, I got growth from few explants and they turned into calluses and them small leaflets, still placed on selection medium. Thank you for your response.

Asmita Gupta Thank you. I'll do the confirmation procedure soon.

The attached article showing different steps of an efficient gene transformation method.