Abstract
Uniform transformation of the thousands of plastid genome (ptDNA) copies in a cell is driven by selection for plastid markers. When each of the plastid genome copies is uniformly altered, the marker gene is no longer needed. Plastid markers have been efficiently excised by site-specific recombinases expressed from nuclear genes either by transforming tissue culture cells or introducing the genes by pollination. Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the bar au gene, confers a visual aurea leaf phenotype; thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The bar au marker-free plastids are transmitted through seed to the progeny. PCR and DNA gel blot (Southern) protocols to confirm transgene integration and plastid marker excision are also provided herein.
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
Iamtham S, Day A (2000) Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat Biotechnol 18:1172–1176
Kode V, Mudd E, Iamtham S, Day A (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
Klaus SMJ, Huang FC, Eibl C, Koop HU, Golds TJ (2003) Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis. Plant J 35: 811–821
Klaus SMJ, Huang FC, Golds TJ, Koop H-U (2004) Generation of marker-free plastid transformants using a transiently cointegrated selection gene. Nat Biotechnol 22:225–229
Ye GN, Colburn S, Xu CW, Hajdukiewicz PTJ, Staub JM (2003) Persistence of unselected transgenic DNA during a plastid transformation and segregation approach to herbicide resistance. Plant Physiol 133:402–410
Corneille S, Lutz K, Svab Z, Maliga P (2001) Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant J 72:171–178
Hajdukiewicz PTJ, Gilbertson L, Staub JM (2001) Multiple pathways for Cre/lox-mediated recombination in plastids. Plant J 27:161–170
Kittiwongwattana C, Lutz KA, Clark M, Maliga P (2007) Plastid marker gene excision by the phiC31 phage site-specific recombinase. Plant Mol Biol 64:137–143
Lutz KA, Bosacchi MH, Maliga P (2006) Plastid marker gene excision by transiently expressed CRE recombinase. Plant J 45:447–456
Lutz KA, Maliga P (2007) Construction of marker-free transplastomic plants. Curr Opin Biotechnol 18:107–114
Lutz KA, Azhagiri AK, Tungsuchat-Huang T, Maliga P (2007) A guide to choosing vectors for transformation of the plastid genome of higher plants. Plant Physiol 145:1201–1210
Maliga P, Bock R (2011) Plastid biotechnology: food, fuel and medicine for the 21st century. Plant Physiol 155:1501–1510
Maliga P (2012) Plastid transformation in flowering plants. In: Bock R, Knoop V (eds) Genomics of chloroplasts and mitochondria. Springer, Berlin, pp 393–414
Lutz KA, Svab Z, Maliga P (2006) Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system. Nat Protocols 1:900–910
Lutz KA, Knapp JE, Maliga P (2001) Expression of bar in the plastid genome confers herbicide resistance. Plant Physiol 125:1585–1590
Tungsuchat-Huang T, Slivinski KM, Sinagawa-Garcia SR, Maliga P (2011) Visual spectinomycin resistance gene for facile identification of transplastomic sectors in tobacco leaves. Plant Mol Biol 76:453–461
Tungsuchat-Huang T, Maliga P (2012) Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants. Plant J 70:717–725
Kapila J, De Rycke R, Van Montagu M, Angenon G (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–108
Murashige T, Skoog F (1962) A revised medium for the growth and bioassay with tobacco tissue culture. Physiol Plant 15:473–497
Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci U S A 81: 1991–1995
Dillen W, De Clercq J, Kapila J, Zambre J, Van Montagu M, Angenon G (1997) The effect of temperature on Agrobacterium tumefaciens-mediated gene transfer to plants. Plant J 12: 1459–1463
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325
Acknowledgments
This work was supported by grants from the USDA National Institute of Food and Agriculture Biotechnology Risk Assessment Research Grant Program Award No. 2005-33120-16524, 2008-03012, and 2010-2716.
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
Tungsuchat-Huang, T., Maliga, P. (2014). Plastid Marker Gene Excision in Greenhouse-Grown Tobacco by Agrobacterium-Delivered Cre Recombinase. 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_12
Download citation
DOI: https://doi.org/10.1007/978-1-62703-995-6_12
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