Dear kumar, you are exactly right. It is possible to regenerate Chimeric transgenic/wild-type shoots from callus since the shoot are usually multicellular origin.

If you regenerate transgenic shoots through adventive shoot regeneration and you suspect you get chimera, you can do the following:

(1) You may want to grow your suspect chimeric T0 shoots (containing transgenic and wild type tissues) on a selective medium to retard (arrest) the growth of wild-type tissues and promote the transgenic ones. Through a series of subcultures in selective media, you may end up with majority transgenic tissues in the selected T0 shoots, or

(2) You may want to use part of the chimeric T0 shoots as explant and induce second round of regeneration from the T0 explant on selective medium. The fast growing, regenerated shoots on selwcted medium from the explant isolated from T0 chimeric shoots may be mostly consisted of transgenic tissues, or

(3) You may want to grow chimeric shoots into plantlets, transfer the plantlet to soils and grow the plant to maturity. Harvest the seeds from the evaluated trees (T0:1 seeds). Grow the seeds on selective medium and select seedlings that are resistance to selective agent. These seedlings are probably the transgenic T1 generation.

Good luck in your research.....

Dear Sudarsono,

Thank you very much for your suggestion.

Hi, Krishna,

During selection of the resistance calluses, split the callus into smaller pieces each subculture and use higher content of selection reagent.

best!

In my opinion, there are two ways that you can apply for getting homozygous plants in T0 or T1.

(1) self-pollination and,

(2) increasing the concentration of selection reagents.

I think the self-pollination is the safer strategy to obtain homozygous plant.

Dear Kumar,

I agree with the answer of S. Sudarsono, Just making stringent selection you will increase the efficiency of your transformation system when you are starting from multicellular origin tissue.

good luck,

Raúl