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Abstract Coffea canephora (2n = 2x = 22 chromosomes) is a species with extensive genetic diversity and desirable agronomic traits for coffee breeding programs. However, obtaining a new coffee cultivar through conventional breeding techniques may require more than 30 years of crossing cycles and selection, which hampers the effort of keeping up with market demands and rapidly proposing more resilient to climate change varieties. Although, the application of modern biotechnology tools such as precision genetic engineering technologies may enable a faster cultivar development process. Therefore, we aimed to validate the CRISPR/Cas9 system to generate mutations on a selected genotype of C. canephora, the clone 14. Embryogenic calli and a multiplex binary vector containing two sgRNAs targeting different exons of the CcPDS gene were used. The sgRNAs were under the C. canephora U6 promoter regulation. The target gene encodes phytoene desaturase, an enzyme essential for photosynthesis involved in β-carotene biosynthesis. Somatic seedlings and embryos with albino, variegated and green phenotypes regenerated after Agrobacterium tumefaciens-mediated genetic transformation were analyzed by verifying the insertion of the Cas9 gene and later by sequencing the sgRNAs target regions in the genome of Robusta modified seedlings. Among them, 77% had the expected mutations, and of which, 50% of them had at least one target with a homozygous mutation. The genotype, temperature of co-cultivation with the bacteria, and light intensity used for subsequent embryo regeneration appeared to strongly influence the successful regeneration of plants with a mutated CcPDS gene in the Coffea genus.

Abstract The aim of this study was to identify the correlation between photochemical efficiency and candidate genes expression to elucidate the drought tolerance mechanisms in coffee progenies (Icatu Vermelho IAC 3851-2 × Catimor UFV 1602-215) previously identified as tolerant in field conditions. Four progenies (2, 5, 12 and 15) were evaluated under water-deficit conditions (water deficit imposed 8 months after transplanting seedlings to the pots) and under irrigated system. Evaluations of physiological parameters and expression of candidate genes for drought tolerance were performed. Progeny 5 showed capacity to maintain water potential, which contributed to lower qP variation between irrigated and deficit conditions. However, the increases of qN and NPQ in response to stress indicate that this progeny is photochemically responsive to small variations of Ψam protecting the photosystem and maintaining qP. Data obtained for progeny 12 indicated a lower water status maintenance capacity, but with increased qN and NPQ providing maintenance of the ɸPSII and ETR parameters. A PCA analysis revealed that the genes coding regulatory proteins, ABA-synthesis, cellular protectors, isoforms of ascorbate peroxidase clearly displayed a major response to drought stress and discriminated the progenies 5 and 12 which showed a better photochemical response. The genes CaMYB1, CaERF017, CaEDR2, CaNCED, CaAPX1, CaAPX5, CaGolS3, CaDHN1 and CaPYL8a were up-regulated in the arabica coffee progenies with greater photochemical efficiency under deficit and therefore contributing to efficiency of the photosynthesis in drought tolerant progenies.