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Plant Molecular Biology

, Volume 76, Issue 3–5, pp 453–461 | Cite as

Visual spectinomycin resistance (aadA au ) gene for facile identification of transplastomic sectors in tobacco leaves

  • Tarinee Tungsuchat-Huang
  • Kristina Marie Slivinski
  • Sugey Ramona Sinagawa-Garcia
  • Pal Maliga
Article

Abstract

Identification of a genetically stable Nicotiana tabacum (tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea bar (bar au ) gene that confers a golden leaf phenotype and a spectinomycin resistance (aadA) gene that is necessary for the introduction of the bar au gene in the plastid genome. We developed a novel aadA gene that fulfills both functions: it is a conventional selectable aadA gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the clpP1 plastid gene by aurea aadA au transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new aadA au genes are useful to query rare events in large populations and for in planta manipulation of the plastid genome.

Keywords

Aurea aadA aadAau Leaf variegation Plastid transformation Spectinomycin resistance Nicotiana tabacum 

Notes

Acknowledgments

This work was supported by grants from the USDA National Institute of Food and Agriculture Biotechnology Risk Assessment Research Grant Program Award No. 2005-33120-16524 and 2008-03012. Kristina Marie Slivinski was supported by a fellowship from the Disabled American Veterans.

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Tarinee Tungsuchat-Huang
    • 1
  • Kristina Marie Slivinski
    • 1
  • Sugey Ramona Sinagawa-Garcia
    • 1
    • 2
  • Pal Maliga
    • 1
  1. 1.Waksman Institute of Microbiology, RutgersThe State University of New JerseyPiscatawayUSA
  2. 2.Universidad Autónoma de Nuevo LeónGral. EscobedoMexico

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