Transgene silencing and reactivation in sorghum

Amongst the important cereals, sorghum has been the most recalcitrant to genetic transformation. There are a few reports on sorghum transformation and the majority of these have reported silencing of one of the transgenes. We regenerated plants from two independent transgenic sorghum callus lines that were cotransformed with Ubi promoter :bar and Act1-D promoter :gusA gene constructs using the particle bombardment method. Southern analyses indicated integration of multiple copies of both the transgenes. T0 plants were found to express the bar gene. The gusA gene, however, was silenced. It was possible to activate gusA gene expression in T1 seedlings and in calli derived from immature T1 and T2 embryos by 5-azacytidine (azaC) treatment. In certain cases, spontaneous expression of the gusA gene was observed in T1 and T2 immature embryo-derived calli. Expression of the bar gene, as analyzed by Basta™ tolerance and Phosphinothricin acetyltransferase (PAT) assays, was detected in T0, T1 and T2 plantlets; however, the expression was reduced in the T2 progeny obtained from a homozygous T1 parent. PAT activity was also lower in the immature embryo-derived T2 calli from the same homozygous T1 parent. Again, culture on azaC increased the level of PAT activity in these calli. Moreover, in a separate set of stable transformation experiments, it was possible to recover a much higher than usual number of gusA gene expressing transgenic calli by growing the bombarded tissues in the presence of azaC. Taken together, these results suggest that methylation-based silencing is frequent in sorghum and probably responsible for several cases of transgene inactivation reported earlier for this crop.