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 plastids 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 plastids in Nicotiana tabacum using an optimized protoplast culture protocol. PEG-mediated plastid transformation has the potential to be developed into a high-throughput, automated pipeline.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Golds T, Maliga P, Koop HU (1993) Stable plastid transformation in PEG-treated protoplasts of Nicotiana tabacum. Biotechnology 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
Sporlein B, Streubel M, Dahlfeld G, Westhoff P, Koop HU (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(6):341–349
Nugent GD, Coyne S, Nguyen TT, Kavanagh TA, Dix PJ (2006) Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake into protoplasts. Plant Sci 170:135–142
Lelivelt C, McCabe M, Newell C, de Snoo B, Van Dunn K, Birch-Machin I, Gray JC, Mills K, Nugent JM (2005) Stable plastid transformation in lettuce (Lactuca sativa L). Plant Mol Biol 58:763–774. https://doi.org/10.1007/s11103-005-7704-8
Koop HU, Steinmüller K, Wagner H, Rössler C, Eibl C, Sacher L (1996) Integration of foreign sequences into the tobacco plastome via PEG-mediated protoplast tranformation. Planta 199:193–201
Kofer W, Eibl C, Steinmuller 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(1–2):114–118. https://doi.org/10.1007/Bf01282299
Gottschamel J, Lossl A (2016) Chloroplast-based expression of recombinant proteins by gateway(R) cloning technology. Methods Mol Biol 1385:3–27. https://doi.org/10.1007/978-1-4939-3289-4_1
Occhialini A, Piatek AA, Pfotenhauer AC, Frazier TP, Stewart CN Jr, Lenaghan SC (2019) MoChlo: a versatile, modular cloning toolbox for chloroplast biotechnology. Plant Physiol 179(3):943–957. https://doi.org/10.1104/pp.18.01220
Fuentes P, Armarego-Marriott T, Bock R (2018) Plastid transformation and its application in metabolic engineering. Curr Opin Biotechnol 49:10–15. https://doi.org/10.1016/j.copbio.2017.07.004
Daniell H, Chan HT, Pasoreck EK (2016) Vaccination via chloroplast genetics: affordable protein drugs for the prevention and treatment of inherited or infectious human diseases. Annu Rev Genet 50:595–618. https://doi.org/10.1146/annurev-genet-120215-035349
Ahmad N, Michoux F, Lossl AG, Nixon PJ (2016) Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot 67(21):5945–5960. https://doi.org/10.1093/jxb/erw360
Murashige T, Skoog F (1962) A revised medium for the growth and bioassay with tobacco tissue culture. Physiol Plant 15:473–497
Zou Z, Eibl C, Koop HU (2003) The stem-loop structure of the tobacco psbA 5′UTR is an important determinant of mRNA stability and translation efficiency. Mol Gen Genomics 269:340–349
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(1):114–118. https://doi.org/10.1007/bf01282299
Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci U S A 87(21):8526–8530
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
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Díaz, A.H., Koop, HU. (2021). Nicotiana tabacum: An Update on PEG-Mediated Plastid Transformation. In: Maliga, P. (eds) Chloroplast Biotechnology. Methods in Molecular Biology, vol 2317. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1472-3_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1472-3_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1471-6
Online ISBN: 978-1-0716-1472-3
eBook Packages: Springer Protocols