Transient Induction of Plant Regeneration

Recalcitrance for in vitro regeneration in some species or genotypes of common crops limits the use of biotechnology tools such as doubled haploid production and somatic embryogenesis. There is therefore an urgent need to develop a generic tool to improve plant regeneration processes in a germplasm-independent manner.

Embryo-expressed transcription factors like the AP2 domain protein BABY BOOM and the CAAT-box binding factor LEAFY COTYLEDON1 have been used to enhance plant regeneration in a range of crops when stably expressed from a constitutive promoter. Although this transgenic approach has been highly successful, it precludes the routine commercial utilization of these plants. We aim to overcome this problem by transiently activating endogenous BBM/LEC1 gene expression using CRISPR-dCas9 technology. This will be done by identifying transcriptional activator or repressor binding sites in the arabidopsis BBM/LEC1 promoters using a CRISPR/Cas9 deletion series, followed by transcriptional regulation of LEC1/BBM gene expression from these cis-regulatory elements using dCas9 fusion proteins.