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.

Severe physiological fruit abscission significantly limits yield potential in macadamia. Polygalacturonase (PG), a key hydrolytic enzyme in pectin degradation, plays a critical role in fruit abscission. However, in the macadamia genome, the PG gene family and the members involved in fruit abscission remain poorly understood. In this study, 56 PG gene family members, which were unevenly distributed across 13 of the 14 chromosomes, were identified in the macadamia genome. Phylogenetic analysis clustered these genes into seven clades, with most members found in clades D and E. The MiPGs contained 3–11 exons and 2–10 introns, and except for those in clades E and G, most contained conserved domains I–IV and were predicted to be localized exclusively to the cell membrane. MiPG promoter analysis revealed numerous light-, phytohormone-, and stress-responsive cis-elements. Expression profiling during fruit development showed that twelve MiPGs were either undetectable or expressed at low levels in the fruit abscission zone, whereas eight were highly expressed. MiPG9, MiPG37, and MiPG53 were significantly upregulated during abscission induced by a combination of girdling with defoliation and ethephon treatments. Moreover, transient MiPG37 overexpression in lily petals promoted premature abscission, suggesting that this gene plays a pivotal role in macadamia fruit abscission. These findings advance the functional characterization of macadamia PG genes and highlight a subset of candidate genes for further genetic manipulation to improve fruit retention.

Macadamia (Macadamia spp.) is an economically important nut crop whose severe recalcitrance to genetic transformation substantially hinders progress in functional genomics and molecular breeding. To overcome this critical technical bottleneck, this study established a highly efficient and broadly applicable in planta hairy root genetic transformation system with integrated visual screening. This system utilizes an Agrobacterium rhizogenes-mediated transformation method, employing multiple visual reporter gene systems (DsRed2, eGFP, RUBY, and AtPAP2) to achieve antibiotic-independent and non-destructive screening of transgenic roots. Notably, the system innovatively incorporates the air layering (marcotting) technique to extend in planta genetic transformation to branches of mature trees in the field. By circumventing the stringent sterile conditions required for conventional in vitro tissue culture, this approach achieves a largely genotype-independent transformation across open-pollinated seedlings with diverse genetic backgrounds (A4, GR1, HAES900, and O.C.). The transgenic hairy root induction frequencies ranged from 39.25% to 47.38%, although the GR1 genotype exhibited a notable developmental stage-dependent decline in transformation efficiency. Furthermore, transgenic hairy roots were successfully induced on mature tree branches, with a maximum induction rate of 28.2%. Gene expression analyses confirmed the stable, high-level expression of the target transgenes in all the transgenic hairy root lines. This in planta transformation system provides a reliable in vivo experimental platform for the rapid functional validation of candidate genes and the investigation of root biology in Macadamia. Moreover, it establishes a novel strategy for plant regeneration via root-to-shoot organogenesis, offering a promising avenue for the genetic improvement of recalcitrant woody plants.

Macadamia (Macadamia spp.) is an economically important nut crop whose severe recalcitrance to genetic transformation substantially hinders progress in functional genomics and molecular breeding. To overcome this critical technical bottleneck, this study established a highly efficient and broadly applicable in planta hairy root genetic transformation system with integrated visual screening. This system utilizes an Agrobacterium rhizogenes-mediated transformation method, employing multiple visual reporter gene systems (DsRed2, eGFP, RUBY, and AtPAP2) to achieve antibiotic-independent and non-destructive screening of transgenic roots. Notably, the system innovatively incorporates air layering (marcotting) technique to extend in planta genetic transformation to branches of mature trees in the field. By circumventing the stringent sterile conditions required for conventional in vitro tissue culture, this approach achieves genotype-independent transformation across open-pollinated seedlings with diverse genetic backgrounds (A4, GR1, HAES900, and O.C.), yielding transgenic hairy root induction frequencies of 39.25% to 47.38%. Furthermore, transgenic hairy roots were successfully induced on mature tree branches, with a maximum induction rate of 28.2%. Gene expression analyses confirmed stable, high-level expression of target transgenes in all transgenic hairy root lines. This in planta transformation system provides a reliable in vivo experimental platform for the rapid functional validation of candidate genes and the investigation of root biology in Macadamia. Moreover, it establishes a novel strategy for plant regeneration via root-to-shoot organogenesis, offering a promising avenue for the genetic improvement of recalcitrant woody plants.