This study represents the first investigation of the effect of multiple transgenes on factors related to persistence in alfalfa. The research was part of an on-going objective to improve field persistence and winter hardiness in alfalfa through genetic engineering. Previous research demonstrated that the overexpression of genes known to be related to abiotic stress tolerance, such as superoxide dismutase (Sod) and alcohol dehydrogenase (Adh), had a positive effect on persistence in alfalfa.

A series of F1 families were generated through paired crosses between two transgenic BC1 genotypes and five primary transformants. Parent genotypes contained either a TR’1-TR’2-regulated Adh gene or one of three 35S-regulated Sod cDNAs: mitochondria-MnSOD (mitMnSOD), chloroplast-MnSOD (chlMnSOD) and chloroplast-FeSOD (FeSOD). Eleven F1 families were characterized using Southern hybridization, PCR and SOD isozyme assays.

Transgenes followed the expected Mendelian segregation patterns, without reciprocal differences, in all F1 families except for one, which showed less Adh transgene inheritance than expected. Comparison of Adh transgene inheritance from other genotypes suggested that this phenomenon was specific to one Adh-transgenic parent genotype.

Novel SOD isozymes and increased SOD activity were detected in most genotypes with Sod transgenes. Comparison of the levels of SOD activity between segregation classes within families indicated that the presence of the Adh transgene did not influence the level of SOD activity. However, various forms of isozyme inactivity or transgene silencing were detected. Transgene silencing observed for MnSOD isozymes in mitMnSOD-chlMnSOD genotypes was likely due to the high level of sequence similarity between the mitMnSOD and chlMnSOD transgenes, which differed only by the transit peptide sequences. One FeSOD-transgenic parent had a non-detectable level of transgenic FeSOD activity that was inherited by all of its F1 progeny. De novo transgene silencing was detected in one FeSOD segregant of a cross between a FeSOD-transgenic parent with detectable levels of transgenic FeSOD activity and an Adh-transgenic parent.

While the occurrence of transgene silencing may be an important factor limiting the potential benefits of pyramiding similar transgenes in alfalfa, the observed maintenance of transgenic SOD isozyme activity in the presence of the Adh transgene provides a positive step forward in the attempt to simultaneously overexpress transgenes in alfalfa. This research has provided the preliminary framework necessary for the genetic improvement of persistence in alfalfa.