Abstract
A detailed protocol for PEG-mediated plastid transformation of Lactuca sativa cv. Flora, using leaf protoplasts, is described. Successful plastid transformation using protoplasts requires a large number of viable cells, high plating densities, and an efficient regeneration system. Transformation was achieved using a vector that targets genes to the trnI/trnA intergenic region of the lettuce plastid genome. The aadA gene, encoding an adenylyltransferase enzyme that confers spectinomycin resistance, was used as a selectable marker. With the current method, the expected transformation frequency is 1–2 spectinomycin-resistant cell lines per 106 viable protoplasts. Fertile, diploid, homoplasmic, plastid-transformed lines were obtained. Transmission of the plastid-encoded transgene to the T1 generation was demonstrated.
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Acknowledgements
This work was initially funded under the EU Fifth Framework initiative, grant number QLK-CT-1999-00692.
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Lelivelt, C.L.C., van Dun, K.M.P., de Snoo, C.B., McCabe, M.S., Hogg, B.V., Nugent, J.M. (2014). Plastid Transformation in Lettuce (Lactuca sativa L.) by Polyethylene Glycol Treatment of Protoplasts. In: Maliga, P. (eds) Chloroplast Biotechnology. Methods in Molecular Biology, vol 1132. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-995-6_20
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DOI: https://doi.org/10.1007/978-1-62703-995-6_20
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