Review and Validation of Plant Gene Function Research Methods Bypassing Tissue Culture

The genetic transformation of plants has provided fundamental insights into plant biology. However, the genetic transformation systems for most horticultural plants remain incomplete. Genome editing has significantly contributed to the improvement of crop traits, but it heavily relies on effective genetic transformation. Currently, reducing costs and improving the efficiency of genetic transformation are crucial for promoting the widespread application of genome editing in plants. Here, we review the advances in plant genetic transformation research, performing analysis of three methods for plant gene function analysis that bypass tissue culture: Agrobacterium rhizogenes-mediated root genetic transformation, developmental regulators (DRs)-mediated genetic transformation, and virus-mediated genome editing. We analyzed transformation efficiency in strawberry and citrus using the A. rhizogenes infiltration method, employing GFP to label different subcellular locations to investigate the morphology of microfilaments, nuclei, and peroxisomes in strawberry cells. Sequence analysis revealed that a series of developmental regulators critical for enhancing genetic transformation efficiency in specific species are highly conserved across different plant species. Additionally, we successfully edited the endogenous Pds gene in Cas9-overexpressing transgenic tobacco using TRV and CLBV containing the gRNA module. These three methods offer the benefits of being cost-effective and time-efficient, providing valuable technical insights for the application of plant genome editing.