RESUMEN
Female inflorescence is the primary output of medical Cannabis. It contains hundreds of cannabinoids that accumulate in the glandular trichomes. However, little is known about the genetic mechanisms governing Cannabis inflorescence development. In this study, we reported the map-based cloning of a gene determining the number of inflorescences per branch. We named this gene CsMIKC1 since it encodes a transcription factor that belongs to the MIKC-type MADS subfamily. Constitutive overexpression of CsMIKC1 increases inflorescence number per branch, thereby promoting flower production as well as grain yield in transgenic Cannabis plants. We further identified a plant-specific transcription factor, CsBPC2, promoting the expression of CsMIKC1. CsBPC2 mutants and CsMIKC1 mutants were successfully created using the CRISPR-Cas9 system; they exhibited similar inflorescence degeneration and grain reduction. We also validated the interaction of CsMIKC1 with CsVIP3, which suppressed expression of four inflorescence development-related genes in Cannabis. Our findings establish important roles for CsMIKC1 in Cannabis, which could represent a previously unrecognized mechanism of inflorescence development regulated by ethylene.
RESUMEN
Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is getting more attention recently. Several candidate genes involved in cannabinoid biosynthesis have been elucidated using omics analysis. However, the gene function was not fully validated due to few reports of stable transformation for Cannabis tissues. In this study, we firstly report the successful generation of gene-edited plants using an Agrobacterium-mediated transformation method in C. sativa. DMG278 achieved the highest shoot induction rate, which was selected as the model strain for transformation. By overexpressing the cannabis developmental regulator chimera in the embryo hypocotyls of immature grains, the shoot regeneration efficiency was substantially increased. We used CRISPR/Cas9 technology to edit the phytoene desaturase gene and finally generated four edited cannabis seedlings with albino phenotype. Moreover, we propagated the transgenic plants and validated the stable integration of T-DNA in cannabis genome.