Direct shoot organogenesis on hypocotyl explants with collar region from in vitro seedlings of Coffea canephora Pierre ex. Frohner cv. C × R and Agrobacterium tumefaciens-mediated transformation
2010
Sridevi, V | Giridhar, P | Simmi, P. S | Ravishankar, G. A
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 μM 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 μM BA and 2.85 μM 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.
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