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High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

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Abstract

Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15–18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5′-UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5′-UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5′-UTR construct, described above, and another containing the same terminator, but with the promoter and 5′-UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.

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Acknowledgments

Research partially funded by the collaborative grant from the European Union (FP6 Plastomics project LSHG-CT-2003-503228). The technical assistance of Mr. Alfonso Piccolo and Ms. Lorenza Sannino (CNR-IGV, Portici) are gratefully acknowledged.

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Author notes

  1. Daniela Gargano

    Present address: Center for Organelle Research, University of Stavanger, 4036, Stavanger, Norway

Authors and Affiliations

  1. CNR-IGV, Institute of Plant Genetics, Res. Div. Portici, via Università 133, 80055, Portici, Italy

    Vladimir T. Valkov, Daniela Gargano, Carmela Manna, Gelsomina Formisano, Nunzia Scotti & Teodoro Cardi

  2. Biology Department, National Univerisity of Ireland-Maynooth, Maynooth, Co. Kildare, Ireland

    Philip J. Dix

  3. Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK

    John C. Gray

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  1. Vladimir T. Valkov
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Correspondence to Teodoro Cardi.

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IGV publication no. 347

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Valkov, V.T., Gargano, D., Manna, C. et al. High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences. Transgenic Res 20, 137–151 (2011). https://doi.org/10.1007/s11248-010-9402-9

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