Abstract
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.
Highlights
Plant transformation systems have evolved dramatically since the first successful reports in 1980s
The aberrant segregation in this event could be due to gene silencing or insertional mutation leading to the reduced transmission of transgene through male or female gametes. This is the first demonstration of Agrobacterium mediated direct germline transformation of meristem explants through organogenesis without selection
Base on an average of 32.3% germline transmission and the range of 0.5%–2.2% T0 putative transformation frequency, the marker-free germline transformation frequency in this experiment would be in the range of 0.16%–0.71%
Summary
Plant transformation systems have evolved dramatically since the first successful reports in 1980s. Transgenic cotton plants were initially produced through somatic embryogenesis of hypocotyl or cotyledon cultures (Firoozababy et al, 1987; Umbeck et al, 1987) or suspension cell cultures (Finer and McMullen, 1990) These systems are tedious, laborious, genotype-dependent and have long. The major breakthrough of cotton transformation technology was the development of Agrobacterium-mediated transformation of cotton meristem tissues of imbibed mature seeds using spectinomycin selection. This meristem transformation system was rapid, high throughput and genotype flexible (Chen et al, 2014). It was concluded that tissue culture parameter optimization and Agrobacterium improvement contributed to the development of genotype flexible and high throughput meristem transformation systems (Chen et al, 2014; Ye et al, 2016)
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