Highly efficient Agrobacterium rhizogenes -mediated hairy root transformation for gene function analysis in cotton without tissue culture

• This study proposes a novel, rapid, universal, efficient and nonsterile Agrobacterium rhizogenes -mediated cotton hairy root transformation system (ARM). • The obtained transgenic hairy roots are suitable for subcellular localization observation. • Hairy roots can be used for in-depth exploration of gene functions under abiotic stress conditions. Efficient genetic transformation technologies are crucial for exploring plant gene functions and promoting molecular breeding. However, the widely used genetic transformation technology mediated by Agrobacterium tumefaciens is time-consuming and genotype-dependent, which limits the high-throughput functional characterization of cotton genes. The transformation system mediated by Agrobacterium rhizogenes (ARM) presents a rapid and effective alternative, but previous ARM techniques in cotton suffered from low efficiency and complicated operation. Here, we optimized the traditional ARM method suitable for nonsterile environments. The two-step ARM technology we used effectively enhanced the transformation efficiency within the upland cotton. The entire process only takes one month, and this system is applicable to various upland cotton varieties, with a maximum transformation efficiency of up to 100%. Results have shown that the ARM method only produces transgenic roots rather than whole transgenic plants. The obtained transgenic hairy roots can be employed to endogenous gene silencing and gene overexpression, enabling subcellular localization analysis and in-depth exploration of gene functions. In summary, we have first described a rapid, universal, efficient, and nonsterile ARM system in cotton, offering a reliable foundation for the cotton gene functional study and the advancement of genetic improvement breeding.