The Complete Nucleotide Sequence of the Coffee (Coffea Arabica L.) Chloroplast Genome: Organization and Implications for Biotechnology and Phylogenetic Relationships Amongst Angiosperms

The chloroplast genome sequence of Coffea arabica L., the first sequenced member of the fourth largest family of angiosperms, Rubiaceae, is reported. The genome is 155 189 bp in length, including a pair of inverted repeats of 25 943 bp. Of the 130 genes present, 112 are distinct and 18 are duplicated in the inverted repeat. The coding region comprises 79 protein genes, 29 transfer RNA genes, four ribosomal RNA genes and 18 genes containing introns (three with three exons). Repeat analysis revealed five direct and three inverted repeats of 30 bp or longer with a sequence identity of 90% or more. Comparisons of the coffee chloroplast genome with sequenced genomes of the closely related family Solanaceae indicated that coffee has a portion of rps19 duplicated in the inverted repeat and an intact copy of infA. Furthermore, whole-genome comparisons identified large indels (\u3e 500 bp) in several intergenic spacer regions and introns in the Solanaceae, including trnE (UUC)–trnT (GGU) spacer, ycf4–cemA spacer, trnI (GAU) intron and rrn5–trnR (ACG) spacer. Phylogenetic analyses based on the DNA sequences of 61 protein-coding genes for 35 taxa, performed using both maximum parsimony and maximum likelihood methods, strongly supported the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids, asterids, eurosids II, and euasterids I and II. Coffea (Rubiaceae, Gentianales) is only the second order sampled from the euasterid I clade. The availability of the complete chloroplast genome of coffee provides regulatory and intergenic spacer sequences for utilization in chloroplast genetic engineering to improve this important crop

Transgenic coffee (Coffea Species)

Les essais de transformation génétique ont été conduits sur des embryons somatiques de Coffea canephora Pierre, Coffea arabica L. et de l'hybride interspécifique Arabusta, en utilisant Agrobacterium rhizogenes A4 ou Agrobacterium tumefaciens LBA4404. Deux plasmides ont été utilisés, portant l'un les gènes uidA (GUS) et NPTII ou l'autre, le gène csr 1-1 conférant la résistance à un herbicide (chlorsufuron) et le gène cryIA(c) codant l'endotoxine de Bacillus thuringiensis conférant la résistance à la mineuse des feuilles. Selon l'espèce Agrobacterium, on obtient des cals transformés, des racines ou directement des embryons somatiques qui peuvent être regénérés sur milieu sélectif ou non. Les embryons somatiques secondaires sont regénérés et se développent en plantules. L'intégration des gènes NPTII, uidA ou cryIA(c) est démontrée par PCR et le test d'activité de la bêta-glucuronidase a montré l'expression du gène GUS. Plusieurs de ces plantules transformées sont transférées en serre er apèrs floraison, il sera possible de suivre la transmission de l'ADN étranger dans la descendance. Les analyses "southern blot" ont confirmé l'intégration des gènes spécifiques uidA et Bt dans le génome des plantules. Les spécificités différentes ainsi que les limites des deux souches Agrobacterium rhizogenes et A. tumefaciens sont discutées. Des travaux sont en cours pour transformer Coffea arabic

Coffee (Coffea arabica L.)

Coffee (Coffea sp .) is a perennial plant widely cultivated in many tropical countries. It is a cash crop for millions of small farmers in these areas. As for other tree species, coffee has long breeding cycles, which makes conventional breeding programs time-consuming. For that matter, genetic transformation can be an effective way to introduce a desired trait in elite varieties or for functional genomics. In this chapter, we describe two highly efficient and reliable Agrobacterium -mediated transformation techniques developed for the C. arabica cultivated species: (1) A. tumefaciens to study and introduce genes conferring resistance/tolerance to biotic (coffee leaf rust, insects) and abiotic stress (drought, heat, seed desiccation) in fully transformed plants and (2) A. rhizogenes to study candidate gene expression for nematode resistance in transformed roots. (Résumé d'auteur

Transformation of coffee (Coffea Arabica L. cv. Catimor) with the cry1ac gene by biolistic, without the use of markers

The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible