Direct Germline Transformation of Cotton Meristem Explants With No Selection

Regeneration of transgenic plants without selectable markers can facilitate the development and commercialization of trait stacking products. A wide range of strategies have been developed to eliminate selectable markers to produce marker-free transgenic plants. The most widely used marker free approach is probably the Agrobacterium-based 2 T-DNA strategy where the gene-of-interest (GOI) and selectable marker gene are delivered from independent T-DNAs (Darbani et al., 2007). The selectable marker gene is segregated away from the GOI in subsequent generations. However, the efficiency of this 2 T-DNA system is much less than the traditional 1 T-DNA system due to the inefficiency of T-DNA co-transformation and high rate of con-integration between the GOI and selectable marker gene T-DNAs. In contrast, no selection transformation utilizes a single T-DNA carrying the GOI and thus eliminates the need to remove the selectable marker insert and potentially provides a viable alternative marker-free system. In this study, we reported the successful regeneration of transgenic cotton plants through Agrobacterium inoculation of seed meristem explants without the use of selective agents. Regeneration of putative transgenic plants were identified by GUS histo-chemical assay. The germline transmission of transgene to progeny was determined by segregation of pollen grains, immature embryos and T1 plants by GUS expression. The results were further confirmed by Southern analyses. The marker-free transformation frequency in this no selection system was similar to current meristem transformation system with selection (0.2%-0.7%). The strategy for further improvement of this system and its implication in improving cotton transformation pipeline and in developing transgene-free genome editing technology is discussed.