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 (Go ssypium hirsutum L.). Green fluorescent protein (GFP) proved to be a valuab le tool in elucidating the timing and localization of transient gene expres sion and in visualizing conversion of transient events to stable transforma tion 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 transf ormation by day 8. The effects of Agrobacterium strains, acetosyringone, an d temperature on stable transformation were also evaluated. Strain LBA4404 proved to be significantly better than EHA105. Acetosyringone increased sig nificantly the stable transformation efficiency in cotton. Cocultivation at 21 degreesC, compared to 25 degreesC, consistently resulted in higher tran sformation frequencies. GFP expression in stably transformed callus was use ful 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 stat us. Larger-size calluses and calluses expressing the transgene (GFP) had a higher rate of survival. Survival could be improved by an additional two-we ek culture on medium high in cytokinin and low in auxin before transfer to a medium to induce embryogenesis. However, this treatment delayed embryogen esis. Various other important aspects of the regeneration process are descr ibed and an overall scheme for producing transgenic cotton is presented.