Abstract
Plastid transformation is an attractive alternative to nuclear transformation enabling manipulation of native plastid genes and the insertion of foreign genes into plastids for applications in agriculture and industrial biotechnology. Transformation is achieved using dominant positive selection markers that confer resistance to antibiotics. The very high copy number of plastid DNA means that a prolonged selection step is required to obtain a uniform population of transgenic plastid genomes. Repair of mutant plastid genes with the corresponding functional allele allows selection based on restoration of the wild type phenotype. The use of deletion rather than point mutants avoids spontaneous reversion back to wild type. Combining antibiotic resistance markers with native plastid genes speeds up the attainment of homoplasmy and allows early transfer of transplastomic lines to soil where antibiotic selection is replaced by selection for photoautotrophic growth. Here we describe our method using the wild type rbcL gene as a plastid transformation marker to restore pigmentation and photosynthesis to a pale green heterotrophic rbcL mutant.
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
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
Ruf S, Hermann M, Berger IJ et al (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 19:870–875
Kanamoto H, Yamashita A, Asao H et al (2006) Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15:205–217
Lelivelt CLC, McCabe MS, Newell CA et al (2005) Stable plastid transformation in lettuce (Lactuca sativa L.). Plant Mol Biol 58:763–774
Schneider A, Stelljes C, Adams C et al (2015) Low frequency paternal transmission of plastid genes in Brassicaceae. Transgenic Res 24:267–277
Dufourmantel N, Pelissier B, Garcon F et al (2004) Generation of fertile transplastomic soybean. Plant Mol Biol 55:479–489
Avila EM, Day A (2014) Stable plastid transformation of petunia. Methods Mol Biol 1132:277–293
Zubko MK, Zubko EI, van Zuilen K et al (2004) Stable transformation of petunia plastids. Transgenic Res 13:523–530
Avila EM, Gisby MF, Day A (2016) Seamless editing of the chloroplast genome in plants. BMC Plant Biol 16(1):168. https://doi.org/10.1186/s12870-016-0857-6
Day A, Madesis P (2007) DNA replication, recombination, and repair in plastids. Top Curr Genet 19:65–119
Bock R (2015) Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Annu Rev Plant Biol 66:211–241
Iamtham S, Day A (2000) Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat Biotechnol 18:1172–1176
De Cosa B, Moar W, Lee SB et al (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74
Lu YH, Stegemann S, Agrawal S et al (2017) Horizontal transfer of a synthetic metabolic pathway between plant species. Curr Biol 27:3034–3041.e3. https://doi.org/10.1016/j.cub.2017.08.044
Madesis P, Osathanunkul M, Georgopoulou U et al (2010) A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera. J Biotechnol 145:377–386
Gisby MF, Mellors P, Madesis P et al (2011) A synthetic gene increases TGF beta 3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule. Plant Biotechnol J 9:618–628
Zoschke R, Liere K, Borner T (2007) From seedling to mature plant: Arabidopsis plastidial genome copy number, RNA accumulation and transcription are differentially regulated during leaf development. Plant J 50:710–722
Day A, Goldschmidt-Clermont M (2011) The chloroplast transformation toolbox: selectable markers and marker removal. Plant Biotechnol J 9:540–553
Tabatabaei I, Ruf S, Bock R (2017) A bifunctional aminoglycoside acetyltransferase/phosphotransferase conferring tobramycin resistance provides an efficient selectable marker for plastid transformation. Plant Mol Biol 93:269–281
Serino G, Maliga P (1997) A negative selection scheme based on the expression of cytosine deaminase in plastids. Plant J 12:697–701
Gisby MF, Mudd EA, Day A (2012) Growth of transplastomic cells expressing D-amino acid oxidase in chloroplasts is tolerant to D-alanine and inhibited by D-valine. Plant Physiol 160:2219–2226
Boynton JE, Gillham NW, Harris EH et al (1988) Chloroplast transformation in Chlamydomonas with high-velocity microprojectiles. Science 240:1534–1538
Klaus SMJ, Huang FC, Eibl C et al (2003) Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis. Plant J 35:811–821
Kode V, Mudd EA, Iamtham S et al (2006) Isolation of precise plastid deletion mutants by homology-based excision: a resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation. Plant J 46:901–909
Day A (2003) Antibiotic resistance genes in transgenic plants: their origins, undesirability and technologies for their elimination from genetically modified crops. In: Stewart CN (ed) Transgenic plants: current innovations and future trends. Horizon Scientific Press, Wymondham, pp 111–156
Day A, Kode V, Madesis P, Iamtham S (2004) Simple and efficient removal of marker genes from plastids by homologous recombination. In: Peña L (ed) Transgenic plants: methods and protocols. Humana Press, Totowa, NJ, pp 255–269
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Finer JJ, Vain P, Jones MW et al (1992) Development of the particle inflow gun for DNA delivery to plant-cells. Plant Cell Rep 11:323–328
Sugiura M, Shinozaki K, Zaita N et al (1986) Clone bank of the tobacco (Nicotiana tabacum) chloroplast genome as a set of overlapping restriction endonuclease fragments - mapping of 11 ribosomal-protein genes. Plant Sci 44:211–217
Twigg AJ, Sherratt D (1980) Trans-complementable copy-number mutants of plasmid Col E1. Nature 283:216–218
Acknowledgments
Supported in part by the Biotechnology and Biological Sciences Research Council (BB/I011552/1) and a University of Manchester (investment in success award). MEH was supported by a President’s Doctoral Scholar Award (University of Manchester), MFBH by the University Malaysia Terengganu and EMA by a Biotechnology and Biological Sciences Research Council PhD studentship.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
El Hajj, M., Hamdan, M.F.B., Avila, E.M., Day, A. (2018). Rescue of Deletion Mutants to Isolate Plastid Transformants in Higher Plants. In: Maréchal, E. (eds) Plastids. Methods in Molecular Biology, vol 1829. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8654-5_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8654-5_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8653-8
Online ISBN: 978-1-4939-8654-5
eBook Packages: Springer Protocols