The cell culture step Palbociclib FDA of the plant transformation proced Inhibitors,Modulators,Libraries ure itself has been shown to be a significant source of methylation changes and we evaluated whether the addition of a secondary cell culture step could inter fere with the somatic acquirement of de novo methyla tion for the recovery of phenotypes in transformed but epigenetically silenced N. attenuata lines. Explant cultures were created from hypocotyls of transgenic homozygous T2 seedlings of lines PNA 1. 2, ICE 4. 4 and ir Inhibitors,Modulators,Libraries ACX1 and were called secondary regenerants. The offspring of the secondary regenerants showed a large variability in hygromycin resistance. Most strikingly 41% of the regenerated plants produced offspring with full resist ance to hygromycin and only 24% showed a resistance loss as seen after conventional propagation of these lines.

To test if these phenotypically recovered plants also were restored in the expression Inhibitors,Modulators,Libraries of the transgene, we iso lated RNA from rosette stage plants. The gene expres sion analysis indicated much higher gene expression levels after the secondary regeneration, compared to conventionally propagated plants of the same line. Most of the regenerated lines now showed gene Inhibitors,Modulators,Libraries expression levels very similar to those of the stable expressing lines. The transgene activity of the ir ACX1 line was not tested by gene expression analysis, but in stead we determined the ability to suppress JA accumula tion after simulated herbivory, as it would be performed during an experiment. All offspring from the tested ir ACX1 regenerants showed suppressed JA accumulation, compared to wild type plants.

This indicated an actively expressed IR construct and a recov ered in trans silencing ability of the endogenous acx1 gene after secondary regeneration. To explore the durability Inhibitors,Modulators,Libraries of this recovery, we germi check details nated the subsequent generation on hygromycin containing media. Here the progression of marker gene silencing returned with the characteristic highly vari able plant to plant pattern of hygromycin sensitivity. Lines with low or variable gene expression levels had the highest probability of losing the resistance in the subsequent gen eration indicating a negative correlation between strength of transgene expression and the subsequent loss of the re sistance marker. Finally, at least one line from each of the PNA and ICE regenerants, but seven of the ir ACX1 regenerants showed enduring resistance up to the T4 generation. Discussion Erratic occurrence of unwanted transgene silencing and large differences in gene expression among iso genic plants. We suggest from our experience that test ing the subsequent generations for resistance would be the easiest way to ensure stable transgene expression in N. attenuata.