Optimization of Agrobacterium tumefaciens-mediated transformation system for Fusarium oxysporum

Abstract:

[Background] Sea island cotton is more susceptible to fusarium wilt than upland cotton. Once it occurs, it is difficult to cure, making fusarium wilt gradually become the main disease threatening the development of sea island cotton industry in Xinjiang, but the pathogenesis is still unclear. [Objective] In order to reveal the genetic variation and pathogenic mechanism of Fusarium oxysporum, the green fluorescent protein (GFP) labeled transformants of F. oxysporum were obtained and used to observe its infection pathway to sea island cotton. [Methods] Agrobacterium tumefaciens-mediated transformation, Agrobacterium tumefaciens-mediated transformation (ATMT) mediated genetic transformation of cotton fusarium wilt race st89 is carried out and the transformation conditions were optimized. [Results] The optimum conditions for ATMT transformation of F. oxysporum:150 mg/L hygromycin could completely inhibit the growth of F. oxysporum, 200 mg/L cefotaxime sodium completely inhibited the growth of Agrobacterium LBA4404, the initial concentration of Agrobacterium is OD₆₀₀ 0.2, the pre-culture time of Agrobacterium is 8 h, The concentration of conidia is 10⁵ spores/mL, the volume ratio of spore suspension of F. oxysporum to Agrobacterium LBA4404 is 1:1. The concentration of acetosyringone in co-culture is 200 μmol/mL, co-culture time is 4 d, culture temperature after transformation 25℃. Using the optimized transformation system, GFP gene is successfully transferred into F. oxysporum. Maximum transformation efficiency can be achieved 252±7.37 transformants/10⁵ spores. PCR amplification and fluorescence observation showed that GFP gene could be expressed normally. [Conclusion] The acquisition of GFP transgenic F. oxysporum laid a foundation for further study on the invasion mechanism of cotton Fusarium wilt.

Key words:Fusarium oxysporum;green fluorescent protein;Agrobacterium tumefaciens-mediated transformation