Agrobacterium rhizogenes-Mediated Hairy Root Genetic Transformation Using Agrobacterium Gel Inoculation and RUBY Reporter Enables Efficient Gene Function Analysis in Sacha Inchi (Plukenetia volubilis).

Plukenetia volubilis L., a woody oilseed plant rich in α-linolenic acid, represents a promising source of polyunsaturated fatty acids. However, the lack of an efficient genetic transformation system has significantly hindered gene function research and molecular breeding in P. volubilis. In this study, we developed a highly efficient Agrobacterium rhizogenes-mediated hairy root transformation system for P. volubilis via the use of Agrobacterium gel in combination with the visually detectable RUBY reporter for gene function analysis in roots. The results indicate that the optimal transformation method involves infecting P. volubilis seedlings with Agrobacterium gel containing acetosyringone and inducing hairy root formation in perlite. This approach resulted in more than 18.97% of the seedlings producing positive hairy roots overexpressing the RUBY gene. Using this genetic transformation system, we successfully overexpressed the antimicrobial peptide-encoding gene CEMA in hairy roots, which enhanced the resistance of P. volubilis to Fusarium oxysporum. Furthermore, by combining this transformation system with the CRISPR-Cas9 tool, we validated the regulatory role of PvoSHR in the development of root epidermal cells in P. volubilis. Unexpectedly, a 123-bp DNA fragment from the T-DNA region of the A. rhizogenes Ri plasmid was found to be knocked in to the P. volubilis genome, replacing a 110-bp fragment of PvoSHR at CRISPR-Cas9 induced double-strand DNA breaks. Conclusively, this system provides a powerful tool for gene function research in P. volubilis and provides novel insights into the development of transformation and gene editing systems for other woody plants.