Transposon mutagenesis for cotton functional genomics

To enable better reverse genetic strategies in cotton (Gossypium spp.) the creation of artificial mutations by transposon mutagenesis was investigated. The Dslox transposable element system is an enabling technology that combines the advantages of transposon-tagging using the maize Ac/Ds system to produce insertion mutants, but it also includes a lox sequence in the Ds element and another strategically placed upstream but within the T-DNA. This creates the opportunity to induce gross chromosomal rearrangements or deletions within the region flanked by the lox sites. The Dslox mutagenesis strategy gives us an unprecedented opportunity to unveil gene functions important to agronomic traits. Altogether 131 Dslox transgenic cotton (Gossypium hirsutum L.cv. Coker 312-17) lines, two Ac lines and one Cre line have been produced. Those Dslox and Ac lines were characterized at the molecular level to permit sorting into single full insertion lines. Results showed that higher than expected numbers of partial Dslox insertion events were produced. The poor recovery of full length Dslox events suggest that some factor(s) such as instability of the inserted T-DNA (event) or exceeding the physical size limits for efficient transformation are important. The Gossypium genome contains evidence of numerous transposable elements, so an endogenous mechanism leading to the transposition of Dslox without Ac would not be unexpected. Instability of the T-DNA insertion of subsequent inheritance is possible but would have occurred at a much higher rate than in other transformation projects in cotton. The large size of the Dslox TDNA (11.9 Kb) may have exceeded the physical limits for efficient transformation in cotton.