Establishment of in situ genetic transformation system for Jatropha curcas using the cambium as receptor

Jatropha curcas L. is a perennial woody plant. Its seeds have a high oil content, making it widely recognized as one of the most promising feedstock crops for biodiesel production. With the advance of high-quality genomic sequence data and stable genetic transformation system, J. curcas is also regarded as a model plant for studying perennials. However, its genetic transformation system relies on tissue culture techniques, resulting in a time-consuming, costly, technically challenging, and contamination-prone transgenic process. This study established a genetic transformation system for J. curcas employing the RUBY reporter gene, using the cambium as explants. We developed three methods that do not require tissue culture: cut-induced hypocotyl rooting system (CHR), girdling-induced branch rooting system (GBR) and cut-induced hypocotyl budding system (CHB). The results demonstrated that both CHR and GBR successfully induced transgenic hairy roots, with GBR significantly outperforming CHR in multiple metrics. GBR achieved a maximum positive transformation efficiency of 54.18 %, producing 9.80 roots per explant and shortening the root-initiation time to approximately 15 days than CHR. Results indicate that younger seedlings are more conducive to root transformation, and the bacterial lawn method is superior to the bacterial suspension method. Furthermore, the CHB method was applied to induce organ regeneration from the cambium via smearing a bacterial lawn onto the transverse section of the decapitated hypocotyl, resulting in RUBY -transformed buds. This method achieved a maximum positive transformation efficiency of 8.52 % and only took 45 days. In conclusion, an efficient genetic transformation system was established in this research for in planta hairy roots and regeneration buds in J. curcas using the cambium as the receptor, providing a reliable technical platform for functional gene studies in woody plants. • GBR outperformed CHR in hairy root transformation, achieving 54.18% efficiency and transgenic roots within 15 days. • CHB enabled direct in situ transgenic shoot regeneration from hypocotyl cambium in J. curcas for the first time. • A tissue culture-free platform was built using cambium enabled transgenic root and shoot regeneration without in vitro steps.