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Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites

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Abstract

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Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing.

Abstract

An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3′ terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean®, containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2–8 μg L−1 Glean®, whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 μg L−1 Glean®, respectively. Glean®-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 μg L−1 Glean®. The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

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Abbreviations

BAP:

6-Benzylaminopurine

CaMV:

Cauliflower mosaic virus

IBA:

Indole-3-butyric acid

MS:

Murashige and Skoog

TDZ:

Thidiazuron

NAA:

α-Naphthylacetic acid

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Acknowledgments

We would like to acknowledge Sakuntala Karunairetnam for providing plasmid pSAK778, Bhawana Nain for sequencing ALS mutants, Nihal de Silva for advice in statistical analysis, Tim Holmes for photographing apple transgenic plants and Charles Ampomah-Dwamena, Mary Christey, Sue Gardiner and Anne Gunson for helpful comments on the manuscript.

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Authors and Affiliations

  1. The New Zealand Institute for Plant & Food Research Ltd., Private Bag 92169, Auckland, New Zealand

    Jia-Long Yao, Sumathi Tomes & Andrew P. Gleave

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  1. Jia-Long Yao
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  2. Sumathi Tomes
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  3. Andrew P. Gleave
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Correspondence to Jia-Long Yao.

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Communicated by S. Merkle.

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Yao, JL., Tomes, S. & Gleave, A.P. Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites. Plant Cell Rep 32, 703–714 (2013). https://doi.org/10.1007/s00299-013-1404-7

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  • DOI: https://doi.org/10.1007/s00299-013-1404-7

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