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Plastid transformants of tomato selected using mutations affecting ribosome structure

  • Genetic Transformation and Hybridization
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Abstract

Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.

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Abbreviations

BAP:

Benzylaminopurine

HEPES:

N-2-Hydroxyehtylpiperazine-N′-2-ethanesulfonic acid

MES buffer:

2-(4-Morpholino)-ethane sulfonic acid

NAA:

α-Naphthaleneacetic acid

PEG:

Polyethylene glycol

ptDNA:

Plastid DNA

Spec:

Spectinomycin

Strep:

Streptomycin

2,4-D:

2,4-Dichlorophenoxyacetic acid

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Acknowledgements

The authors thank Peter Medgyesy (National University of Ireland, Maynooth) and Tony Kavanagh (Trinity College, Dublin) for making available their plasmids, and Roger Peeters for useful discussions. The work was carried out with the EU grant “Plastid transformation in crop plants” (BIO-97-2245)

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Author notes

  1. G. D. Nugent

    Present address: Department of Primary Industries, Primary Industries Research Victoria, Plant Biotechnology Centre, La Trobe University, 3086, Bundoora, Victoria, Australia

  2. A. P. Mordhorst

    Present address: BASF Plant Science GmbH, Li444, Carl-Bosch-Str., 64, 67117, Limburgerhof, Germany

Authors and Affiliations

  1. Biology Department, National University of Ireland, Maynooth, County Kildare, Ireland

    G. D. Nugent & P. J. Dix

  2. Nunhems Zaden BV, P.O. Box 4005, 6080 AA, Haelen, The Netherlands

    M. ten Have, A. van der Gulik, B. A. Uijtewaal & A. P. Mordhorst

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  1. G. D. Nugent
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  2. M. ten Have
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  3. A. van der Gulik
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  4. P. J. Dix
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  5. B. A. Uijtewaal
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  6. A. P. Mordhorst
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Corresponding author

Correspondence to A. P. Mordhorst.

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Communicated by H. Lörz

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Nugent, G.D., ten Have, M., van der Gulik, A. et al. Plastid transformants of tomato selected using mutations affecting ribosome structure. Plant Cell Rep 24, 341–349 (2005). https://doi.org/10.1007/s00299-005-0930-3

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  • DOI: https://doi.org/10.1007/s00299-005-0930-3

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