Abstract
Genome editing allows precise DNA manipulation, but its potential is limited in many crops by low regeneration efficiencies and few transformable genotypes. Here, we show that expression of a chimeric protein including wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) dramatically increases the efficiency and speed of regeneration in wheat, triticale and rice and expands the number of transformable wheat genotypes. Moreover, GRF4-GIF1 induces efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. By combining GRF4-GIF1 and CRISPR-Cas9 technologies, we were able to generate large numbers of edited wheat plants. The GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects, likely due to post-transcriptional regulatory mechanisms operating on GRF4 in adult tissues. Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus suggesting that this strategy can be expanded to dicot crops.
Competing Interest Statement
JFP and JMD are co-inventors in patent US2017/0362601A1 that describes the use of chimeric GRF-GIF proteins with enhanced effects on plant growth (Universidad Nacional de Rosario Consejo Nacional de Investigaciones Cientificas y Tecnicas). JFP, JD, DMT and JMD are co-inventors in UC Davis provisional patent application 62/873,123 that describes the use of GRF-GIF chimeras to enhance regeneration efficiency in plants. Vectors are freely available for research, but commercial applications may require a paid non-exclusive license. There is a patent application from KWS/BASF (WO 2019 / 134884 A1) for improved plant regeneration using Arabidopsis GRF5 and grass GRF1 homologs. None of the authors of this manuscript is part of the KWS/BASF patent or is related to these companies. The KWS/BASF patent focuses on a different cluster of GRF genes than the one described in our study and does not incorporate the GIF1 cofactor or the generation of GRF-GIF chimeras.