Somatic embryogenesis and Agrobacterium-mediated transformation in Bixa orellana L.

Establishment, maintenance, regeneration, and transformation of somatic embryos by both direct and indirect means (callus-mediated) was achieved for Bixa orellana, a tropical plant whose seeds produce commercially edible 'annatto pigment,' which mainly constitutes an apocarotenoid called bixin. Callus-mediated methodology was found to be efficient in producing a greater number of embryos in a short time. The maximum of 28 somatic embryos were produced in 16-18 weeks when immature zygotic embryonic stalks were inoculated onto Murashige and Skoog (MS) medium containing B5 vitamins supplemented with 0.44 mu M benzyladenine (BA), 0.054 mu M alpha-naphthaleneacetic acid (NAA), 2.89 mu M gibberellic acid (GA(3)), 0.02 mu M triiodobenzoic acid (TIBA), and 0.011 mu M triacontanol (TRIA). Callus initiation from hypocotyl explants was obtained on MS medium supplemented with 1.07-2.14 mu M NAA and 10.2 mu M BA. In 3 months, somatic embryos were produced when callus was inoculated onto MS medium supplemented with 4.44 mu M BA, 40 mu M AgNO3, and 0.011 mu M TRIA. Somatic embryos were efficiently regenerated on MS basal solid and liquid media supplemented with 0.44-4.4 mu M BA, 0.54-2.69 mu M NAA, 4.92 mu M 2iP, 2.1 mu M calcium d-pantothenate, 0.21 mu M biotin, 227.7 mu M cysteine HCl monohydrate, and 108.6 mu M adenine sulfate. Agrobacterium tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector-mediated stable transformation of somatic embryos exhibited a transformation frequency of 2.56%. As somatic embryogenesis in any perennial system is useful in terms of both commercial and scientific nature, this somatic embryo-based transformation protocol for the commercially important dye-yielding tropical plant B. orellana is useful for its improvement through genetic engineering.