New Flowering and Architecture Traits Mediated by Multiplex CRISPR-Cas9 Gene Editing in Hexaploid Camelina sativa

Adapting plants to sustainable cropping systems is a major challenge for facing climate change and promoting agroecological transition. Camelina sativa is an emerging oilseed crop species with climate-resilient properties that could be used in double-cropping systems, in particular as a summer catch crop. The availability of early-flowering camelina is essential in such cropping systems to allow full completion of the growth cycle during summer. Targeted induced gene variation (TIGV) was used in camelina on several flowering repressor genes identified in Arabidopsis to obtain early-flowering lines. Multiplex editing of 15 target genes representing FLOWERING LOCUS C, SHORT VEGETATIVE PHASE, LIKE HETEROCHROMATIN PROTEIN 1, TERMINAL FLOWER 1 and EARLY FLOWERING LOCUS 3 induced combinatorial mutations that were screened for early-flowering phenotypes. Certain mutants showing a stable early-flowering trait after five generations also presented additional phenotypes: determinate flowering, shorter stature and/or basal branching. Different combinations of mutations had a positive or negative impact on yield. This work demonstrates that efficient multiplex CRISPR is achievable in hexaploid plants like camelina, providing valuable genetic diversity for better selecting lines adapted to new cropping systems.