A Cotyledon-Derived Agrobacterium rhizogenes-Mediated Transformation System for Regenerating Transgenic ‘Carrizo’ Citrange Plants

The citrus genetic transformation system is essential for genetic improvement and functional studies of the crop. Agrobacterium rhizogenes-mediated transformation in citrus has enabled efficient and rapid generation of transformed root tissues compared to conventional A. tumefaciens-mediated transformation. However, suitable explants and transformation conditions in this system still require further optimization to obtain ample clean transformed roots, which is a prerequisite for subsequent shoot regeneration when stable transgenic plants are desired. In this study, we established an integrated cotyledon-derived, A. rhizogenes-mediated hairy root transformation and plant regeneration system for ‘Carrizo’ citrange. Both cotyledon and epicotyl explants yielded transformed hairy roots; however, cotyledon explants exhibited higher hairy root induction and hairy root transformation efficiency under their respective optimized conditions. A 3 h AS pre-induction time, a 30 min infection time, and a 2,4-D concentration of 0.1 mg/L were identified as the optimal conditions for cotyledon transformation, and the hairy root induction rate, GFP-positive hairy root frequency, and hairy root transformation efficiency reached an average value of 69.22 ± 3.13%, 74.21 ± 1.08%, and 51.36 ± 1.99%, respectively. Transformed hairy roots were further regenerated into shoots via a callus-mediated regeneration pathway, followed by micrografting and subsequent grafting to recover intact plants. Finally, regenerated plants were successfully obtained and molecularly validated. This study reports a highly efficient A. rhizogenes-mediated transformation and regeneration system for citrus.