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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Golds T, Maliga P, Koop H (1993) Stable plastid transformation in PEG-treated protoplasts of Nicotiana tabacum. Bio/Technology 11:95–97
O‘Neill C, Horvath GV, Horvath E, Dix PJ, Medgyesy P (1993) Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. Plant J 3:729–738
Spörlein B, Streubel M, Dahlfeld G, Westhoff P, Koop H (1991) PEG-mediated plastid transformation: a new system for transient gene expression assays in chloroplasts. Theor Appl Genet 82:717–722
Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci U S A 87:8526–8530
Nugent GD, Ten Have M, van der Gulik A, Dix PJ, Uijtewaal BA, Mordhorst AP (2005) Plastid transformants of tomato selected using mutations affecting ribosome structure. Plant Cell Rep 24:341–349
Nugent GD, Coyne S, Nguyen TH, Kavanagh TA, Dix PJ (2005) Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts. Plant Sci 170:135–142
Lelivelt CL, McCabe MS, Newell CA, Desnoo CB, van Dun KM, Birch-Machin I, Gray JC, Mills KH, Nugent JM (2005) Stable plastid transformation in lettuce (Lactuca sativa L.). Plant Mol Biol 58:763–774
Koop HU, Steinmuller K, Wagner H, Rossler C, Eibl C, Sacher L (1996) Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta 199:193–201
Kofer W, Eibl C, Steinmüller K, Koop H-U (1998) PEG-mediated plastid transformation in higher plants. In Vitro Cell Dev Biol Plant 34:303–309
Dovzhenko A, Bergen U, Koop HU (1998) Thin-alginate-layer technique for protoplast culture of tobacco leaf protoplasts: shoot formation in less than two weeks. Protoplasma 204:114–118
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Koop HU, Schweiger HG (1985) Regeneration of plants from individually cultivated protoplasts using and improved microculture system. J Plant Physiol 121:245–257
Zou Z, Eibl C, Koop HU (2003) The stem-loop region of the tobacco psbA 5′-UTR is an important determinant of mRNA stability and translation efficiency. Mol Genet Genomics 269:340–349
Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci U S A 90:913–917
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).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-62703-995-6_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-994-9
Online ISBN: 978-1-62703-995-6
eBook Packages: Springer Protocols