Journal
PLANT CELL TISSUE AND ORGAN CULTURE
Volume 101, Issue 3, Pages 339-347Publisher
SPRINGER
DOI: 10.1007/s11240-010-9694-8
Keywords
Coffea canephora; In vitro; Organogenesis; Rooting; Shoots; Somatic embryos
Funding
- Department of Biotechnology, Government of India, New Delhi
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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 mu M 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 mu M BA and 2.85 mu M 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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