I have three T0 plants (sblings) which were regenerated from same calli and expressing transgene simillar to actin level however when I analyze over 15 T1 plants I was not able to get any expression at mRNA level. I confirmed the presence of this gene at DNA level in all T1 plants. (This is abit weird but possible, in normal case it has to be mendelian segregation and I have to get 1:2:1 in genotypic level). My idea is to transfer these T1 plants to hydroponic media supplemented with 5-Azacytidine. Another way I can collect T2 seeds and germinated them MS media+ 5-Azacytidine as well. Is anyone one have any idea if it can work or any other suggestions ?
(The gene trasformed by biolistic transformation and driven by constitutive Ubi1+1i promoter )
If the all theT1 plants (grown without the selection agent) has the transgene, that means the T0 plants are homozygous for the transgene. (If they are derived from the same callus, they are clones.) So the half of T2 plants will contain the transgene. If You don't have transcription at T1, You won't have in T2 either. Somehow your plants deactivated the transgene.
Thanks for your reply. Recently, I reactivated the gene expression by germinating T2 seeds in Azacytidine media. The siliencing was highly probable because of the copy number of transgene or the gene caused methylation somehow.
Can Baysal
How many copies in your R0 plants? Usually, people collect 1 putative transgenic line from each callus. Because the lines are the same clones when they derived from a single callus as Ákos Mendel mentioned. How many *independent* lines you have (from different transgenic calli)? You need to have a few transgenic lines with different transgenic events, because some of these lines might become sterile.
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