Wounding after cocultivation improves somatic embryogenesis response and agrobacterium-mediated genetic transformation of Coffea canephora L.

Artículo

Traditional breeding can take a long time and effort to provide better-adapted crop varieties. Biotechnological breeding can be used to generate desired agronomic traits much faster, but improvements in transformation efficiency and in vitro regeneration are needed in non-models crops. Cocultivation of Coffea canephora embryogenic leaf explants with Agrobacterium tumefaciens led to 82 percent transformation efficiency in the somatic embryos that developed on the wounded edges of the explants, but both the application of vacuum during agroinfiltration and the cocultivation process diminished the embryogenic response to 36 percent, as compared with the non-transformed explants. Application of novel wounds to the already co-cultivated discs activated the embryogenic potential in the newly wounded edges of the former non-embryogenic areas, leading to 97 percent of the former embryogenic response, with 98 percent transformation efficiency, as evaluated by the GUS histochemical assay, PCR amplification of the uidA and nptII genes, and qPCR. Fully developed embryos at the cotyledonal stage were obtained 8 weeks after somatic embryogenesis was triggered by the addition of 5 mM benzyladenine. Rooting of the transformed cotyledonal embryos under Hygromycin selection, acclimatization of the rooted plantlets, and final greenhouse acclimatization to the soil conditions were accomplished within a 48-week period. The transformed plants were maintained in a biosafety greenhouse, and the wild plants were subsequently transplanted to soil. Application of new wounds to leaf explants of C. canephora after co-cultivation with A. tumefaciens recovers the original embryogenic capacity of the explants without affecting the extremely high transformation yields. The high efficiency and reproducibility, as well as the relative rapidity of this optimized protocol constitutes a valuable experimental model for analyzing gene function, studying cell differentiation, and improving the biotechnological breeding of coffee.