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

<i>Plukenetia volubilis</i> 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 <i>P. volubilis</i>. In this study, we developed a highly efficient <i>Agrobacterium rhizogenes</i>-mediated hairy root transformation system for <i>P. volubilis</i> via the use of <i>Agrobacterium</i> gel in combination with the visually detectable <i>RUBY</i> reporter for gene function analysis in roots. The results indicate that the optimal transformation method involves infecting <i>P. volubilis</i> seedlings with <i>Agrobacterium</i> 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 <i>RUBY</i> gene. Using this genetic transformation system, we successfully overexpressed the antimicrobial peptide-encoding gene <i>CEMA</i> in hairy roots, which enhanced the resistance of <i>P. volubilis</i> to <i>Fusarium oxysporum</i>. Furthermore, by combining this transformation system with the CRISPR-Cas9 tool, we validated the regulatory role of <i>PvoSHR</i> in the development of root epidermal cells in <i>P. volubilis</i>. Unexpectedly, a 123-bp DNA fragment from the T-DNA region of the <i>A. rhizogenes</i> Ri plasmid was found to be knocked in to the <i>P. volubilis</i> genome, replacing a 110-bp fragment of <i>PvoSHR</i> at CRISPR-Cas9 induced double-strand DNA breaks. Conclusively, this system provides a powerful tool for gene function research in <i>P. volubilis</i> and provides novel insights into the development of transformation and gene editing systems for other woody plants.