A comparison of strategies for transformation with multiple genes via microprojectile-mediated bombardment

The stable insertion and expression of multiple transgenes in crops is highly desirable, as the manipulation of complex agronomic traits and the introduction of novel biosynthetic pathways are dependent upon it. This study was performed to explore the frequency and efficiency of introducing multiple genes in soybean by using somatic embryogenesis and microprojectile bombardment transformation. The co-transformation frequency of six selectable marker or reporter genes (GusA, bleomycin resistance, glufosinate resistance, hygromycin resistance, green fluorescent protein, and kanamycin resistance) were followed throughout the T0, T1, and T2 generations. Three bombardment strategies were compared to determine the best method to generate transgenic plants that express the introduced transgenes and have a simple insertion pattern that would facilitate any downstream breeding. The plasmid bombardment treatments were (1) a six-gene-containing plasmid, (2) an equimolar treatment of five individual plasmids that collectively contained the six transgenes of interest (genes of glufosinate and hygromycin resistance were on the same plasmid), and (3) a 1:9 ratio mixture of the five plasmids, in which the plasmid containing the selectable marker used in the regeneration process, hygromycin resistance, was used in ninefold excess to all the other plasmids. Of the six bombardments performed per plasmid treatment, the results of seven independent events for the six-gene plasmid, four events for the 1:9 treatment, and a single regenerated event for the equimolar treatment indicate that containing all the transgenes on one plasmid just had an advantage in terms of frequency of a successful transformation events. Based on Southern analysis, the only events that contained all six transgenes was the one obtained by the equimolar treatment. No event was obtained that expressed all six transgenes, and certain transgenes seem to be non-randomly lost, namely gusA, bleomycin resistance, and glufosinate resistance, regardless of treatment. The addition of elements to optimize the expression of each gene cassette when multiple genes are in close proximity needs to be further investigated.