A highly efficient soybean transformation system using GRF3-GIF1 chimeric protein

Abstract

Expression of GRF3-GIF1 chimera significantly enhanced regeneration and transformation efficiency in soybean, increasing the number of transformable cultivars. Moreover, GmGRF3-GIF1 can be combined with CRISPR/Cas9 for highly effective gene editing.

Figure 1

GROWTH‐REGULATING FACTOR (GRF)3 – GRF‐INTERACTING FACTOR 1 (GRF3–GIF1) chimera boosts soybean regeneration and transformation (A) Subcellular localization of Glycine max (Gm)GRF3 and GmGIF1. Scale bar: 10 μm. (B) Schematic overview of the vectors utilized in this study. The target sites of GmPDS11 and GmPDS18 are additionally displayed at the bottom. (C) Explants of “DN254” transformed with pBWA(V)‐EV, pBWA(V)‐GRF3‐GIF1, pBWA(V)‐rGRF3‐GIF1, pBK2‐Cas9‐EV, pBK2‐Cas9‐GRF3‐GIF1, and pBK2‐Cas9‐rGRF3‐GIF1 after shoot induction for 1 week (first line) and 4 weeks (second line). Herbicide‐resistant multiple shoot buds in explants transformed with GRF3‐GIF1 or rGRF3‐GIF1 chimera, and little shoots buds in explants transformed without chimera (third line). Representative shoot buds induced from explants transformed with the above vectors (fourth line). (D) Transformation efficiencies achieved using different constructs when transforming six soybean genotypes. (E) Genomic polymerase chain reaction analysis of the wild type (WT) and mutants. (F) Gene editing efficiencies achieved by combining GRF3‐GIF1 or rGRF3‐GIF1 with the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system. (G) Albino phenotypes of gmpds knockout mutants (Scale bar: 1 cm). (H) The general shape and (I) size of the SAM in the regenerated shoots (genotype “DN254”) transformed with the vectors shown above. (J) The transcripts of marker genes in the regenerated shoot (genotype “DN254”) transformed with pBWA(V)‐EV, pBWA(V)‐GRF3‐GIF1, and pBWA(V)‐rGRF3‐GIF1. (K) A proposed model for the regulation of soybean regeneration through the miR396‐GRF3‐GRF1 regulatory network. LP, leaf primordium; SAM, shoot apical meristem. Values are given as mean ± SD (n ≥ 3). Asterisks denote Student′s t‐test significance (*P < 0.05, **P < 0.01) from the control vector.