Agrobacterium -mediated transformation of annatto ( Bixa orellana L.): protocol optimization and overexpression of microRNA 156 in transgenic plants

Annatto ( Bixa orellana L.) is a Neotropical woody plant and the main source of the natural dyes bixin and norbixin. To study the bixin biosynthesis pathway and genetically improve the species, a suitable transformation protocol is required. Here, an efficient method for Agrobacterium -mediated transformation of hypocotyl segments of annatto was developed. The effect of different factors on the efficiency of transformation was examined; these included three types of antibiotics (Timentin ® , cefotaxime, and meropenem) and three Agrobacterium strains (AGL1, EHA105, and GV3101) at different concentrations. The strains contained the plasmid pCAMBIA2301 harboring the gus reporter gene and npt II as a selection marker under the constitutive 35 S promoter. Strain AGL1 achieved significantly higher gus expression compared to the other candidates. The highest transformation efficiency (8.2% ± 3.8%) was obtained for cefotaxime or meropenem with strain AGL1 at OD 600 = 0.5, as revealed by a gus histochemical assay in hypocotyls and regenerated shoots. This is the highest transformation frequency ever reported for the target species. The optimized protocol was successfully validated by obtaining transgenic annatto plants containing the hpt II gene under constitutive expression and overexpressing miR156. Successful miR156 overexpression was confirmed by histochemical gus staining in regenerated plantlets, as well as by PCR amplification of the hpt II gene. Flow cytometry confirmed that all regenerated lines were diploid, guaranteeing the genetic stability of the transgenes.