A rapid regeneration and visible-marker-assisted transformation platform supporting gene function studies in Zanthoxylum armatum

Zanthoxylum armatum , commonly known as green Sichuan pepper, is an economically important woody spice tree species with valuable medicinal and edible properties. However, the lack of an efficient regeneration and genetic transformation system has limited functional studies and genetic improvement in this species. To address this, we developed a rapid in vitro regeneration and visible-marker-assisted Agrobacterium -mediated transformation platform for Z. armatum using stem segments from one-year-old seedlings. Our optimized regeneration protocol achieved a shoot induction rate of 88.33% within two weeks, a rooting rate of 85.01% within 30 days, and approximately 93.33% survival of regenerated plantlets after acclimatization. Using the RUBY reporter, key transformation parameters were systematically optimized, including Agrobacterium concentration, infection duration, co-cultivation time, and cefotaxime concentration. The highest RUBY-positive transformation frequency reached 56.37% under optimized conditions. Leveraging this system, three DsRed2-linked target constructs were introduced into Z. armatum explants, and reporter fluorescence, PCR detection, and RT-qPCR analysis supported successful transgene delivery with 2.56- to 5.38-fold upregulation in transcript levels. Together, this study provides a practical regeneration and visible-marker-assisted transformation platform, offering a useful technical basis for uncovering the genetic basis of important traits and future genetic improvement in Z. armatum .