Transgenic cotton:: factors influencing Agrobacterium-mediated transformation and regeneration

Various aspects of transformation and regeneration processes were examined in efforts to improve the efficiency of production of transgenic cotton (Gossypium hirsutum L.). Green fluorescent protein (GFP) proved to be a valuable tool in elucidating the timing and localization of transient gene expression and in visualizing conversion of transient events to stable transformation events. By day 4 after infection, there was maximal transient activity in the cells at the cut edge of Agrobacterium-infected cotyledon disks. We were able to visualize conversion of some of these events to stable transformation by day 8. The effects of Agrobacterium strains, acetosyringone, and temperature on stable transformation were also evaluated. Strain LBA4404 proved to be significantly better than EHA105. Acetosyringone increased significantly the stable transformation efficiency in cotton. Cocultivation at 21 degreesC, compared to 25 degreesC, consistently resulted in higher transformation frequencies. GFP expression in stably transformed callus was useful in studying the efficiency of selection during early stages of culture. We found that the survival of individual callus lines on selection medium was influenced by their original size and initial transgene expression status. Larger-size calluses and calluses expressing the transgene (GFP) had a higher rate of survival. Survival could be improved by an additional two-week culture on medium high in cytokinin and low in auxin before transfer to a medium to induce embryogenesis. However, this treatment delayed embryogenesis. Various other important aspects of the regeneration process are described and an overall scheme for producing transgenic cotton is presented.