Sistema CRISPR/Cas9 visando a edição genômica no alotetraploide Coffea arabica

Abstract

The incorporation of biotechnological techniques has become increasingly important for the conventional plant genetic breeding because after the association between these approaches faster advances have been achieved in obtaining superior genotypes. By using the CRISPR/Cas9 genome editing technology in Coffea arabica the gains in coffee genetic breeding programs could be still more significant. This species is the most important in economic terms within Coffea genus but has also the greater complexity at the genomic level, once C. arabica is an allotetraploid plant resulting from natural hybridization of C. canephora with C. eugenioides. The silencing of the phytoene desaturase (PDS) gene, which has involvement into carotenoid biosynthesis, stands out as a good alternative for CRISPR/Cas9 system validation since the mutant plants regenerated after the transformation procedure will likely present a visible phenotype well different compared to the normal plants. According to the context, the initial purpose of this work focused on verifying the efficiency of genome editing in C. arabica through CRISPR/Cas9 technology by using the sitedirected mutation strategy for the PDS gene as a proof-of-concept. Three binary vectors (pCOFEDIT-PDS3, pCOFEDIT-PDS5 e pCOFEDIT-PDS14) were used to the co-transformation of embryogenic calli with Agrobacterium tumefaciens. Transgenic somatic embryos were obtained until cotyledonay stage. However, these embryos exhibited phenotypic abnormalities and difficulty of growth and development. Based on the unsatisfactory development of the putatively transformed plantlets, three embryos that exhibited abnormal leaf morphogenesis, leaf chlorosis and T-DNA integration had their target regions sequenced. The small-scale sequencing performed for the transformed events was not capable to detect any mutations. In parallel, this work also aimed to obtain plants with low content of caffeine through multiple silencing of the genes xanthosine metyltransferase (XMT), theobromine synthase (MXMT) and caffeine synthase (DXMT), all of which encode enzymes involved in the caffeine synthesis. It was verified the presence of Cas9 transcripts in plants transformed with the binary vectors pCOFEDIT-XMT1, pCOFEDIT-XMT2 e pCOFEDIT-XMT3, showing a stable genetic transformation. These plants presented normal content of caffeine (1.1 to 1.4%) and associated with the sequencing data was possible to confirm the absence of mutation in all the alleles of target genes. The pla nts obtained in this study will be useful to help us better understand on limiting factors that impact on the CRISPR/Cas9 system functionality in C. arabica, allowing us to define which aspects must be optimized to conduct future works.