Rooting Conifer Genetic Research: An Accessible and Efficient Transformation System

Conifers serve as the cornerstone of global forest ecosystems, yet their genetic transformation faces notorious challenges. To overcome the intrinsic resistance of conifer adventitious roots to Agrobacterium/Rhizobium-mediated transformation, we systematically and iteratively engineered the binary vector by introducing chimeric Ri plasmid derived T-DNA borders, a phosphomimetic VirG mutant (VirGN54D) and a hyperactive replication origin mutant (pVS1 R106H). This optimised system enabled efficient, cross-species genetic transformation in diverse plants, including the recalcitrant gymnosperm Ginkgo biloba, thus establishing a broadly applicable genetic toolkit for plant research. Leveraging this system, we established a non-aseptic efficient root transformation system for Pinus tabuliformis. Given that conifers are evolutionarily ancient gymnosperms, this readily accessible system provides an unprecedented window to probe fundamental questions in functional genomics and evolutionary mechanisms, such as the dynamics of long-distance macromolecular trafficking. Transgenic deployment of Arabidopsis florigen FT in Chinese pine roots uncovered conserved protein level long-distance mobility, despite lacking functional FT orthologs in conifer genomes. Conversely, the conifer age biomarker DAL1 exhibited no detectable mobility even when engineered fusion with tRNA-like sequence (TLS) tag, which has been shown to facilitate long-distance mRNA transport in angiosperms, exposing lineage-specific constraints on mobile mRNA. Our work provides a versatile genetic tool for challenging plant species and offers new insights into long-distance signalling evolution. The streamlined protocols and universal vector system address critical bottlenecks in conifer biotechnology and open avenues for functional genomics in other non-model plants.