Título:	Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canéfora.
Autoria:	CASARIN, T. FREITAS, N. C. PINTO, R. T. BREITLER, J. C. RODRIGUES, L. A. Z. MARRACCINI, P. ETIENNE, H. DINIZ, L. E. C. ANDRADE, A. C. PAIVA, L. V.
Afiliação:	TATIANE CASARIN, UNIVERSIDADE FEDERAL DE LAVRAS NATÁLIA CHAGAS FREITAS, UNIVERSIDADE FEDERAL DE LAVRAS RENAN TERASSI PINTO, UNIVERSIDADE FEDERAL DE LAVRAS JEAN‐CHRISTOPHE BREITLER, CENTRO DE COOPERAÇÃO INTERNACIONAL EM PESQUISA AGRÍCOLA PARA O DESENVOLVIMENTO LEONARDO AUGUSTO ZEBRAL RODRIGUES, UNIVERSIDADE FEDERAL DE LAVRAS PIERRE MARRACCINI, CENTRO DE COOPERAÇÃO INTERNACIONAL EM PESQUISA AGRÍCOLA PARA O DESENVOLVIMENTO HERVÉ ETIENNE, CENTRO DE COOPERAÇÃO INTERNACIONAL EM PESQUISA AGRÍCOLA PARA O DESENVOLVIMENTO LEANDRO EUGENIO CARDAMONE DINIZ, CNPSO ALAN CARVALHO ANDRADE, CNPCa LUCIANO VILELA PAIVA, UNIVERSIDADE FEDERAL DE LAVRAS.
Ano de publicação:	2022
Referência:	Scientific Reports, v. 12, 17270, 2022. 10 p.
Conteúdo:	Coffea canephora (2n=2x22 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 B-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.
Thesagro:	Biodiversidade Melhoramento Genético Vegetal Coffea Canephora Mutação Genótipo Café Robusta
Digital Object Identifier:	https://doi.org/10.1038/s41598-022-21566-w
Tipo do material:	Artigo de periódico
Acesso:	openAccess
Aparece nas coleções:	Artigo em periódico indexado (SAPC)