Direct shoot organogenesis on hypocotyl explants with collar region from in vitro seedlings of Coffea canephora Pierre ex. Frohner cv. C 3 R and Agrobacterium tumefaciens-mediated transformation

Direct differentiation of shoot buds from the
collar region of hypocotyl segments of Coffea canephora
was obtained on Murashige and Skoog (MS) medium
supplemented with 40 lM silver nitrate (AgNO3) and
growth regulators indole-3-acetic acid (IAA) and N6 benzyladenine
(BA). The highest response to shoot differentiation
of 60% frequency and the maximum number of
multiple shoots (2–3) per explant were obtained on MS
medium containing 8.87 lM BA and 2.85 lM IAA. Apart
from this, 70% of hypocotyl explants produced yellow
friable embryogenic callus and also globular primary
somatic embryos. Subsequent transfer onto the same
medium induced secondary somatic embryogenesis. The
micro-shoots, upon transfer to the same medium, in the
following 6 weeks developed into 4-cm-long shoots with a
single root. Further subculturing onto the same medium
induced 4–5 roots in a 4-week period. The resulting
plantlets were hardened and transferred to micro-pots
containing sand:compost mixture (1:2), where 65% of them
survived and resumed growth. By using optimal levels of
AgNO3, it was possible to obtain effective direct organogenesis
and embryogenesis. This system was used for
genetic transformation using Agrobacterium tumefaciens.
A stable transformation frequency of 2–5% was obtained
when both types of explants, i.e., hypocotyl explants with
collar region or hypocotyl explants without collar region,
were co-cultivated with A. tumefaciens GV 3101 harboring
pCAMBIA 1305.2 binary vector. This is the first report of a
hypocotyl collar region-based Agrobacterium-mediated
transformation protocol for the economically important
tropical plant C. canephora.