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High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 50 and 30 regulatory sequences
Valkov, Vladimir T.; Gargano, Daniela; Mann, Carmela; Formisano, Gelsomina; Dix, Philip; Gray, John C.; Scotti, Nunzia
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 50-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 50-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 50-UTR construct, described above, and another containing the same terminator, but with the promoter and 50-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.
Keyword(s): Biology; Solanum tuberosum; Homologous and homeologous recombination; Chloroplasts; Amyloplasts
Publication Date:
2011
Type: Journal article
Peer-Reviewed: Yes
Institution: Maynooth University
Citation(s): Valkov, Vladimir T. and Gargano, Daniela and Mann, Carmela and Formisano, Gelsomina and Dix, Philip and Gray, John C. and Scotti, Nunzia (2011) High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 50 and 30 regulatory sequences. Transgenic Research, 20. pp. 137-151. ISSN 0962-8819
Publisher(s): Springer
File Format(s): other
Related Link(s): http://eprints.maynoothuniversity.ie/6280/1/PD-High-Efficiency%28Wait%29.pdf
First Indexed: 2015-07-23 05:12:58 Last Updated: 2018-09-04 06:14:53