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Evaluation of a grapevine-derived reporter gene system for precision breeding of Vitis

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Abstract

A grapevine-derived VvMybA1 transcription factor was evaluated for its efficiency as a reporter gene by comparing it with existing reporter genes, the green fluorescent protein (GFP) and β-glucuronidase (GUS). Embryogenic cultures of Vitis cultivars Thompson Seedless and Bronx Seedless were initiated from leaves of in vitro grown micropropagation cultures and somatic embryos were used as explants for Agrobacterium-mediated transformation. Transient and stable expression of the MybA1 gene was characterized by intense red pigmentation in co-cultivated explants, callus tissues and secondary embryos lines compared to GFP and GUS that exhibited green fluorescence and blue coloration following a substrate assay. No differences were observed in transient gene expression frequencies between the MybA1 gene and GUS among the two compared reporter genes. Visual levels of stable gene expression were higher in GFP and GUS expressing cultures compared to MybA1 expressing cultures. The presence of the inserted genes and their expression in regenerated plant lines was confirmed by PCR and RT-PCR. Embryo and plant lines expressing the MybA1 gene accumulated varying levels of anthocyanin pigment in plant tissues and organs, and were characterized by slower growth compared to plant lines expressing GFP and GUS. Scanning electron microscopy analyses revealed a significant change in abaxial and adaxial leaf epidermal cells of MybA1-expressing plant lines compared to those expressing GFP and GUS, and non-transformed control plants. The study demonstrated the utility of the VvMybA1 transcription factor as a reliable reporter gene for identification of gene insertion events in cell culture and regeneration of modified plants.

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Acknowledgments

S. A. Dhekney holds the E. A. Whitney Endowed Professorship in the Department of Plant Sciences. The research was supported in part by the Wyoming Agricultural Experiment Station and grants from the USDA/USAID US-Egypt Science and Technology Development Funds program (Grant No. 0210-22310-005-24G). Raju Kandel was supported by a graduate assistantship from the Department of Plant Sciences.

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Authors and Affiliations

  1. Sheridan Research and Extension Center, University of Wyoming, 3401 Coffeen Avenue, Sheridan, WY, 82801, USA

    R. Kandel, D. R. Bergey & S. A. Dhekney

  2. Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA

    M. Dutt

  3. Department of Biology, Morgan State University, 1700 E. Cold Spring Lane, Baltimore, MD, 21251, USA

    V. Sitther

  4. Mid-Florida Research and Education Center, University of Florida, 2725 Binion Road, Apopka, FL, 32703, USA

    Z. T. Li & D. J. Gray

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  1. R. Kandel
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  2. D. R. Bergey
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  3. M. Dutt
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  4. V. Sitther
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  5. Z. T. Li
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  6. D. J. Gray
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  7. S. A. Dhekney
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Correspondence to S. A. Dhekney.

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Kandel, R., Bergey, D.R., Dutt, M. et al. Evaluation of a grapevine-derived reporter gene system for precision breeding of Vitis . Plant Cell Tiss Organ Cult 124, 599–609 (2016). https://doi.org/10.1007/s11240-015-0918-9

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  • DOI: https://doi.org/10.1007/s11240-015-0918-9

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