Abstract
Stable plastid transformation in Nicotiana tabacum has been achieved by using two different methods, the biolistic method, using a particle gun, and the polyethylene glycol (PEG)-mediated transformation. PEG-mediated plastid transformation involves the treatment of isolated protoplasts (plant cells without cell wall) with PEG in the presence of DNA. We have previously shown that in Nicotiana tabacum both methods are equally efficient. The PEG-mediated transformation efficiencies range between 20 and 50 plastid transformants per experiment (106 viable treated protoplasts). One advantage of the PEG method is that no expensive equipment such as a particle gun is required. The only crucial points are the handling and the cultivation of protoplasts. Furthermore, markers for the selection of transformed chloroplasts are required. One of the most often used selection markers is the aadA gene which encodes for spectinomycin and streptomycin resistance. Here we describe a simplified and inexpensive protocol for the transformation of chloroplasts in Nicotiana tabacum using an optimized protoplast culture protocol.
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Acknowledgments
The authors wish to express their gratitude to Stefan Kirchner for his valuable technical assistance and the preparation of the pictures for this work. Areli Herrera Díaz was supported by a research scholarship from the DAAD (Deutscher Akademischer Austausch Dienst).
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Díaz, A.H., Koop, HU. (2014). Nicotiana tabacum : PEG-Mediated Plastid Transformation. 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_9
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DOI: https://doi.org/10.1007/978-1-62703-995-6_9
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Publisher Name: Humana Press, Totowa, NJ
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