Embriogênese somática e validação do sistema CRISPR/CAS9 em Coffea canephora

Abstract

Coffea canephora is the second most cultivated species of the genus Coffea, representing about 25% of the Brazilian production. It is used especially in the soluble coffee industry and in blends with C. arabica. Due to its high genetic variability, breeding programs have sought to identify, select, and propagate superior genotypes. However, conventional breeding takes a lot of time and the use of biotechnological tools can help to speed up this process. Thus, this study aimed to test different protocols for direct somatic embryogenesis induction of C. canephora aiming its application in future genetic transformation studies, the validation of the application of a multiplex strategy of the CRISPR-Cas9 system by silencing the CcPDS gene and obtaining plants with reduced caffeine content through silencing via CRISPR-Cas9 of the genes XMT, MXMT, and DXMT of the caffeine biosynthesis enzymes. To test the induction of direct embryogenesis, explants from in vitro leaves and leaves from the greenhouse of C. canephora clone 22 were used, submitted to three different induction treatments. Medium 3 was the most efficient, with 96.25% induction efficiency and an average of 33.99 embryos per greenhouse explant and 67% induction efficiency and an average of 66.93 embryos per in vitro leaf explant. For genome editing experiments, embryogenic calluses from C. canephora clone 14 were used and different multiplex gene cassettes, in which sgRNAs were under the regulation of U6 promoters from C. canephora and Arabidopsis thaliana. Plants and embryos showing the albino, variegated and green phenotypes were obtained by the transformation with the cassettes containing sgRNAs for the CcPDS gene, mutations were observed in 71.4% of the analyzed plants, most of them were homozygous. Six plants possibly mutated for caffeine synthesis were analyzed and five were effectively transformed. Of these, only one showed a mutation in the regions recognized by the sgRNAs, heterozygous mutations in the second target of the XMT gene, and a mutation also is heterozygous in the two targets of the DXMT gene. No mutations were observed in the MXMT gene. With these results, it is possible to conclude that direct embryogenesis is a much faster strategy for regenerating coffee embryos and the genotype seems to have a strong influence on the success of gene silencing strategies in C. canephora, as well as the genes to be silenced, which can affect the development of edited plans.