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 lM benzyladenine (BA),
0.054 lM a-naphthaleneacetic acid (NAA), 2.89 lM gibberellic
acid (GA3), 0.02 lM triiodobenzoic acid (TIBA),
and 0.011 lM triacontanol (TRIA). Callus initiation from
hypocotyl explants was obtained on MS medium supplemented
with 1.07–2.14 lM NAA and 10.2 lM BA. In
3 months, somatic embryos were produced when callus
was inoculated onto MS medium supplemented with
4.44 lM BA, 40 lM AgNO3, and 0.011 lM TRIA.
Somatic embryos were efficiently regenerated on MS basal
solid and liquid media supplemented with 0.44–4.4 lM
BA, 0.54–2.69 lM NAA, 4.92 lM 2iP, 2.1 lM calcium
D-pantothenate, 0.21 lM biotin, 227.7 lM cysteine HCl
monohydrate, and 108.6 lM 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.