Agrobacterium-mediated root organogenesis and open treatment system (AgroROOTs) for root functional genomics studies in cotton

The plant root system plays crucial roles in nutrient uptake, stress tolerance, and overall plant health. However, research on root functions has significantly lagged behind that of aboveground organs due to the lack of efficient research methods. To address this limitation, we developed AgroROOTs, a tissue culture-independent, Agrobacterium -mediated Root Organogenesis and Open Treatment system for cotton. Initially, Agrobacterium rhizogenes K599 induced 26.6 % of transgenic hairy roots in soil-grown cotton seedling hypocotyls within one month. Optimization of transformation parameters and vacuum treatment of hypocotyl batches further enhanced the transformation efficiency to 33.3 %, enabling the production of large-scale transgenic roots. This approach successfully generated transgenic roots in multiple cotton cultivars, demonstrating its genotype independence. Additionally, we established a Rhizobium -independent root induction system using A. tumefaciens to deliver genes for root organogenesis, thereby simplifying the transformation process. Furthermore, composite plants generated through AgroROOTs were easily cultured in hydroponic systems, facilitating the enrichment, treatment, and observation of transgenic roots for diverse applications over an extended period. Using AgroROOTs, CRISPR/Cas9-mediated genome editing generated predictable mutant results for heritable target modifications. Incorporating a site-specific recombinase system, we successfully tested a virus-mediated mobile genetic switch from shoots to roots, enabling precise gene expression control and mobile signal studies within this system. Additionally, AgroROOTs provides a feasible platform for studying root exudates. Overall, AgroROOTs offers a straight forward and practical approach for characterizing root function in cotton, thereby expanding the toolkit for root biology research and genetic engineering. • A fast, simple and tissue culture-independent Agrobacterium -mediated root transformation system was established for cotton. • Optimization and vacuum treatment enhanced transformation efficiency, enabling high-throughput root production. • Developed a Rhizobium -free root induction system using A. tumefaciens , reducing Ri syndrome and streamlining transformation. • Established hydroponic cultivation of composite plants for long-term monitoring and stress studies on transgenic roots. • CRISPR/Cas9 editing, recombinase-mediated gene switches, and root exudate studies were successfully demonstrated.