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Improved methods in Agrobacterium–mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols

  • Genetic Transformation and Hybridization
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Abstract

A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 μM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.

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Acknowledgements

This work was supported by an ARC (Australian Research Council) linkage grant and the Almond Board of Australia. We thank Dr. Ursula Langridge for care of the almond plants in the PC2 containment glasshouse. We thank SYNGENTA for providing the pNOV2819 manA plasmid

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Authors and Affiliations

  1. School of Agriculture and Wine, Waite Campus, University of Adelaide, Glen Osmond, SA, 5064, Australia

    Sunita A. Ramesh, Brent N. Kaiser, Tricia Franks & Graham Collins

  2. Faculty of Sciences, University of New England, Armidale, NSW 2351, Australia

    Margaret Sedgley

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  1. Sunita A. Ramesh
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  2. Brent N. Kaiser
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  3. Tricia Franks
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  4. Graham Collins
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  5. Margaret Sedgley
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Correspondence to Brent N. Kaiser.

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Communicated by L. Peña

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Ramesh, S.A., Kaiser, B.N., Franks, T. et al. Improved methods in Agrobacterium–mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols. Plant Cell Rep 25, 821–828 (2006). https://doi.org/10.1007/s00299-006-0139-0

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  • DOI: https://doi.org/10.1007/s00299-006-0139-0

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