A Simple and Efficient Agrobacterium rhizogenes-Mediated Root Transformation System for Highbush Blueberry

Highbush blueberry (Vaccinium corymbosum) is an important horticultural crop of significant nutritional, therapeutic, and economic value. However, the development of elite cultivars via genetic transformation has been severely restricted by labor-intensive tissue culture requirements and low transformation efficiency. Here, a simple, efficient, and tissue culture-independent Agrobacterium rhizogenes-mediated hairy root transformation system was established for the highbush blueberry cultivar "Bluerain." By eliminating the need for aseptic manipulation, new shoots generated from semilignified stem segments were subjected to vacuum-assisted A. rhizogenes strain K599 infiltration. Approximately 3 months post-infiltration, high biomass hairy roots was successfully induced, and the transformation efficiency reached 74.4% following stepwise optimization. This in planta protocol was further successfully applied to the cultivars "O'Neal," "Legacy," and "Emerald," yielding transformation efficiencies range from 26.7% to 40.0%. This optimized genetic transformation approach provides a valuable tool for the functional characterization of root-specific gene and for accelerating clonal propagation and genetic improvement in highbush blueberry.