Efficient In Planta Induction of Transgenic Hairy Roots in Macadamia Seedlings and Mature Trees Using Visual Reporters

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.