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Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.)

  • Genetic Transformation and Hybridization
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Abstract

Transient expression studies using blueberry leaf explants and monitored by β-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 μM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 μM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l−1 thidiazuron, 0.5 mg l−1 α-naphthaleneacetic, 10 mg l−1 kanamycin (Km), and 250 mg l−1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 μE m−2 s−1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

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Acknowledgements

We thank Dr. S. Gelvin, Purdue University, for providing plasmid pBISN1, Drs. Jim Hancock and Peter Callow, Michigan State University for providing the cv. Aurora and technical assistance on micropropagation, respectively, and Dr. A. N. Kravchenko for statistical consultation. This research was supported by MSU Project GREEEN (Generating Research and Extension to Meet Economic and Environmental Needs).

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  1. Plant Transformation Center, Department of Horticulture, Michigan State University, East Lansing, MI, 48824, USA

    Guo-Qing Song & K. C. Sink

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  1. Guo-Qing Song
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  2. K. C. Sink
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Correspondence to K. C. Sink.

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Communicated by J.M. Widholm

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Song, GQ., Sink, K.C. Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.). Plant Cell Rep 23, 475–484 (2004). https://doi.org/10.1007/s00299-004-0842-7

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  • DOI: https://doi.org/10.1007/s00299-004-0842-7

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