G. Shankar Since you have removed the gus gene. There is no screen marker for you to screen transformants any more. Now, you still have the kanamycin-resistant gene in the T-DNA. So, you can select the putative transformed calli on kan-containing medium. I don't recommend using PCR for screening, since the calli still have some Agrobactierium (didn't eradicate by antibiotics), and PCR can give false positive.

Dear G. Shankar

I think this article can help you

Article The maize Knotted1 gene is an effective positive selectable ...

The optimum concentration of the antibiotic in the medium can allow only the transformed cells to grow. After 2 months you can run PCR using positive and negative control.

Thanks Yuan-Yeu Yau, Mehdi Babaei and Rahman Sher Khan.

G. Shankar I don't know what kind of plant species callus you have. We used to select putative transformed carrot callus clumps using kanamycin selection. As shown in the attached picture, fresh and light colored carrot callus clumps (as opposed to the dead dark-colored calli) were observed. We just moved the new callus clumps to regeneration plates for regeneration. But, unavoidable, chimeric (transformed and non-transformed) callus clumps can be expected. This can be due to nptII enzyme, which provides cross-protection for non-transformed callus cells. There is also a concern of residual Agrobacerium presence. There is no really perfect way to pre-screen bona fide transformants in your case. You can remove those putative callus clumps to plates and do PCR. PCR on calli would most likely give you positive band. Or, after regenerating them into small plantlets and screen them with PCR (I prefer the later), then final confirm with Southern analysis.

G. Shankar Here is cotton callus selection on kanamycin. Agrobacteria was applied directly to cotton callus and then selected on kan-containing medium.