Unlocking regeneration potential: harnessing morphogenic regulators and small peptides for enhanced plant engineering

Abstract

Plant genetic transformation is essential for understanding gene functions and developing improved crop varieties. Traditional methods, often genotype-dependent, are limited by plants' recalcitrance to gene delivery and low regeneration capacity. To overcome these limitations, new approaches have emerged that greatly improve efficiency and genotype flexibility. This review summarizes key strategies recently developed for plant transformation, focusing on groundbreaking technologies enhancing explant- and genotype flexibility. It covers the use of morphogenic regulators (MRs), stem cell-based methods, and in planta transformation methods. MRs, such as maize Babyboom (BBM) with Wuschel2 (WUS2), and GROWTH-REGULATING FACTORs (GRFs) with their cofactors GRF-interacting factors (GIFs), offer great potential for transforming many monocot species, including major cereal crops. Optimizing BBM/WUS2 expression cassettes has further enabled successful transformation and gene editing using seedling leaves as starting material. This technology lowers the barriers for academic laboratories to adopt monocot transformation systems. For dicot plants, tissue culture-free or in planta transformation methods, with or without the use of MRs, are emerging as more genotype-flexible alternatives to traditional tissue culture-based transformation systems. Additionally, the discovery of the local wound signal peptide Regeneration Factor 1 (REF1) has been shown to enhance transformation efficiency by activating wound-induced regeneration pathways in both monocot and dicot plants. Future research may combine these advances to develop truly genotype-independent transformation methods.

Keywords: Agrobacterium rhizogenes; Agrobacterium tumefaciens; biolistic bombardment; cut‐dip‐budding; genotype‐flexible transformation; in planta transformation; leaf transformation; somatic embryogenesis; tissue culture‐free transformation.

Figure 1

Maximum likelihood phylogenic tree of WUS and WOX orthologs from representative embryophytes. (a) Simplified WUS/WOX phylogeny (see Figure S1 for full tree). (b) WUS/WOX5/7 subclade. (c) WOX2 subclade. The full phylogeny groups WUS/WOX proteins into three superclades: The Ancient T1WOX, Intermediate T2WOX, and Modern T3WOX. WUS/WOX clades and individual WUS/WOX proteins are color‐coded. Numbers on each branch represent likelihood estimates, and support <50 are hidden for clarity. Yellow and purple stars label branches of the WUS/WOX5/7 and WOX2 subclades, respectively. WUS/WOX orthologs known to enhance transformation with and without pleiotropic effects are indicated by arrowheads, black and blue, respectively. Note the discrepancy in WUS/WOX nomenclature, that is, protein names are based on the NCBI protein repository, and names in parenthesis are names as described in the corresponding text and Table 1. Protein IDs and accession numbers are provided in Table S1. At, Arabidopsis thaliana; Md, Malus domestica; Os, Oryza sativa; Pp, Pinus pinaster; Sb, Sorghum bicolor; Ta, Triticum aestivum; Zm, Zea mays.