Hypocotyl acupuncture: an efficient and practical protocol for transformation and genome editing in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge is a valuable medicinal plant, yet its genetic manipulation has been limited by the lack of efficient and practical transformation methods. Here, we report a simplified, tissue culture-independent strategy enabling Agrobacterium-mediated gene delivery under non-sterile conditions, termed hypocotyl acupuncture. The method employs needle-assisted inoculation of intact seedlings at the hypocotyl, completely bypassing callus induction, regeneration, and repeated subculture. Following inoculation, EGFP-positive hairy roots are selectively cultivated for approximately 4 months and then induced to regenerate shoots within one additional month, yielding transgenic plantlets in approximately 5 months. Systematic optimization achieved a transformation efficiency of approximately 55% (defined as the percentage of explants producing EGFP-positive regenerants). ACCC10060 was selected as the optimal strain based on efficiency and root morphology. CRISPR/Cas9-mediated genome editing generated mutation frequencies of 53.8%-88.9% with no off-target effects. Targeted mutagenesis of SmCPS1, a gene encoding a key rate-limiting enzyme in tanshinone biosynthesis, resulted in altered root pigmentation. The method was successfully extended to Astragalus membranaceus, Platycodon grandiflorus, and Isatis indigotica, achieving transformation efficiencies of 25%-55%. This study provides an efficient, practical, and broadly applicable tool for genome editing and functional genomics in medicinal plants.