Highly efficient adventitious root regeneration and Agrobacterium rhizogenes-mediated hairy root transformation in pear

Traditional cross-breeding of pears is often time-consuming due to prolonged juvenile phases, whereas target gene manipulation is an effective approach to accelerate breeding with desired traits and is widely used in various species. However, the transformation system represents a bottleneck for genetic improvement due to reduced efficiency using the Agrobacterium tumefaciens transformation method, particularly in Asian pear species. Therefore, there is a pressing need to establish an efficient genetic transformation approach for pears. Compared to A. tumefaciens, A. rhizogenes presents quicker transformation, but it has not been widely utilized in pear until now. In this study, we examined the influence of various factors on this process using leaves of tissue cultured seeding from two varieties. Optimal circumstances for induction of adventitious roots in ‘Shanli No.1’ include 1/4MS with 1.0 mg/L IBA and 15 g/L sucrose, while for ‘Conference,’ the conditions include MS with 0.6 mg/L IBA and 7.5 g/L sucrose. A three-week period of continuous darkness is optimal for ‘Shanli No.1’ and ‘Conference.’ During hairy root transformation, ‘Shanli No.1’ leaves underwent pre-cultivation, infection with OD₆₀₀ = 0.7 bacteria for 10 min, followed five days co-cultivation, and then placed on rooting medium with 1/4MS, 30 g/L sucrose, and 500 mg/L timentin. For ‘Conference,’ the leaves were directly infected with OD₆₀₀ = 0.1 bacteria for 20 min, co-cultured for three days, and subsequently placed on rooting medium with 1/8 MS, 30 g/L sucrose, and 125 mg/L timentin. Subsequent marker gene screening pinpointed mCherry fluorescent protein as the optimal choice for hairy root transformation in pears. We successfully used the established system to confirm the gene function of PbrMYB169, a documented lignin regulator. This study aimed to establish adventitious root regeneration and an Agrobacterium rhizogenes-mediated hairy root transformation system in ‘Shanli No.1’ and ‘Conference,’ demonstrating its potential for genetic improvement and gene functional analysis in pears.