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Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype.

Vinod, Kumar and Satyanarayana, K. V. and Sarala Itty, S. and Indu, E. P. and Giridhar, P. and Chandrashekar, A. and Ravishankar, G. A. (2006) Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype. Plant cell reports, 25 (3). pp. 214-222. ISSN 0721-7714

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Abstract

A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained with a transformation efficiency up to 3% depending on the medium adjuvant used. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene was used for sonication-assisted transformation of Coffea canephora. The use of hygromycin in the secondary embryo induction medium allowed the selection of transgenic secondary embryos having Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. In addition transgenic secondary embryos devoid of Ri-T-DNA but with stable integration of the T-DNA from the binary vector were obtained. The putative transformants were positive for the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of the analysed plants and indicated single and multiple locus integrations. The study clearly demonstrates that A. rhizogenes can be used for delivering transgenes into tree species like Coffea using binary vectors with Agrobacterium tumefaciens T-DNA borders.

Item Type: Article
Uncontrolled Keywords: PCR; Agrobacterium tumefaciens
Subjects: 500 Natural Sciences and Mathematics > 10 Plants > 05 Tissue Culture
Divisions: Plant Cell Biotechnology
Depositing User: Users 16 not found.
Date Deposited: 31 May 2011 06:01
Last Modified: 31 May 2011 06:01
URI: http://ir.cftri.com/id/eprint/1959

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