Additional virulence genes influence transgene expression: transgene copy number, integration pattern and expression

Additional copies of virulence (vir) genes do enhance transformation frequencies in multiple species; the mechanisms that play a role in this enhancement are not understood. In this report, transformants produced using additional copies of helper plasmids harboring pCH30 (virG), pCH32 (virG and virE), and pCH42 (virE) were evaluated for gene expression, copy number, and complexity of integration patterns. The vir plasmids increased transformation efficiency 2-fold with pCH42, 3-fold with pCH30, and 4-fold with pCH32. One to six transgene copy numbers were detected, and irregular transgene integration patterns were observed in all plasmid combinations (pAGM280 only, pAGM280 + pCH30, pAGM280 + pCH32 and pAGM280 + pCH42). There were no differences in transgene copy number and/or integration patterns between transformants produced with or without additional vir genes. Additionally, no relationship between transgene copy number and expression or between transgene integration patterns and expression was observed. All transgenic plants resulting from the additional vir plasmids were able to express the foreign genes. In contrast, forty percent of transgenic plants regenerated without additional vir plasmids did not express the foreign gene even though the foreign genes were present. These results demonstrate a link between an increase in copy number of the vir genes and stable transgene expression and increased plant transformation efficiency.