Selection and genetic transformation of a fast-growing cell line in cotton (Gossypium hirsutum) for transgene expression studies

A cell line, named CCL-1 has been obtained in cotton which proliferates on phytohormone free MS medium containing high KNO3. This cell line can be maintained as a friable callus on solidified medium and also readily converted to cell suspensions and maintained in liquid medium. CCL-1 cell suspensions were transformed using a disarmed Agrobacterium strain with binary vectors containing selectable marker genes- nptII, hptII, bar and ALS(dm) to check the efficacy of these marker genes for selecting transformed cell lines. Transformation was monitored by reporter genes, either GUS or GFR Transformed cell lines could be readily obtained with all the marker genes, hpt being the most efficient marker for unambiguous selection in 10 day time period. Transformed cell lines showed expression of the reporter genes. We developed a protocol for using these cell lines for insect feeding bioassays by using control CCL-1 and a transformed derivative line containing the cry1Ac gene. The most optimal feeding method is to mix lyophilized transformed cells with an artificial diet to achieve various concentrations of the toxin. The most useful selectable markers for insect feeding bioassay studies are bar and ALS(dm) as these do not interfere with insect growth. CCL-1 will be a convenient tool for studying transgene expression in cotton and will be particularly useful for testing a large number of genes for antifeedant/insecticidal activity.