Abstract
A number of systems to insert foreign DNA into a plant genome have been developed so far. However, only a small percentage of transgenic plants are obtained using any of these methods. Stable transgenic plants are selected by co-introduction of a selectable marker gene, which in most cases are genes that confer resistance against antibiotics or herbicides. In this chapter we describe a new method for selection of transgenic plants after transformation. The selection agent used is the nontoxic and common sugar glucose. Wild-type Arabidopsis thaliana plantlets that have been germinated on glucose have small white cotyledons and remain petite because the external sugar switches off the photosynthetic mechanism. The selectable marker gene encodes the essential trehalose-6-phophate synthase, AtTPS1, that catalyzes the first reaction of the two-step trehalose synthesis. Upon ectopic expression of AtTPS1 driven by the 35S promoter, transformed Arabidopsis thaliana plants became insensitive to glucose in comparison to wild-type plants. After transformation using AtTPS1 as a selection marker and 6% glucose as selection agent it is possible to single out the green and normal sized transgenic plants amid the nontransformed plantlets.
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
Roger, H., Philip, M., and Harry, K. (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451.
WHO (1993) Health aspects of marker genes in genetically modified plants. Report of a WHO workshop of the food safety unit.
Kunze, I., Ebneth, M., Heim, U., Geiger, M., Sonnewald, U., and Herbers, K. (2001) 2-Deoxyglucose resistance: a novel selection marker for plant transformation. Mol. Breeding 7, 221–227.
Malca, I., Endo, R. M., and Long, M. R. (1967) Mechanism of glucose counteraction of inhibition of root elongation by galactose, mannose, and glucosamine. Phytopathology 57, 272–278.
Lucca, P., Ye, X., and Potrykus, I. (2001) Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol. Breed. 7, 43–49.
Joersbo, M., Donaldson, I., Kreiberg, J., Petersen, S. G., Brunstedt, J., and Okkels, F. T. (1998) Analysis of mannose selection used for transformation of sugar beet. Mol. Breed. 4, 111–117.
Negrotto, D., Jolley, M., Beer, S., Wenck, A. R., and Hansen, G. (2000) The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (zea mays L.) via Agrobacterium transformation. Plant Cell Rep. 19, 798–803.
Haldrup, A., Petersen, S. G., and Okkels, F. T. (1998) The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent. Plant Mol. Biol. 37, 287–296.
Zuo, J. and Chua, N.-H. (2000) Chemical-inducible systems for regulated expression of plant genes. Curr. Opin. Biotechnol. 11, 146–151.
Kunkel, T., Niu, Q.-W., Chan, Y.-S., and Chua, N.-H. (1999) Inducible isopentenyl transferase as a high-efficiency marker for plant transformation. Nat. Biotechnol. 17, 916–919.
McCormac, A., Fowler, M. R., Chen, D. F., and Elliott, M. C. (2001) Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation. Transgenic Res 10, 143–155.
DeBuck, S., Jacobs, A., Van Montagu, M., and Depicker, A. (1989) Agrobacterium tumefaciens transformation and cotransformation frequencies of Arabidopsis thaliana root explants and tobacco protoplasts. Mol. Plant Microbe Interact. 11, 449–457.
Yoder, J. I. and Goldsbrough, A. P. (1994) Transformation systems for generating marker-free transgenic plants. Bio/Technology 12, 263–267.
Ebinuma, H., Sugita, K., Matsunaga, E., Endo, S., and Yamada, K. (2001) Systems for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep. 20, 383–392.
Hare, P. D. and Chua, N.-H. (2002) Excision of selectable marker genes from transgenic plants. Nature Biotechnol. 20, 575–580.
Russell, S. H., Hoopes, J. L., and Odell, J. T. (1992) Directed excision of a transgene from the plant genome. Mol. Gen. Genet. 234, 49–59.
Dale, E. C. and Ow, D. W. (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc. Natl. Acad. Sci. USA 88, 10558–10562.
Zuo, J., Niu, Q. W., Moller, S. G., and Chua, N.-H. (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nat. Biotechnol. 19, 157–161.
Corneille, S., Lutz, K., Svab, Z., and Maliga, P. (2001) Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant J. 27, 171–178.
Hajdukiewicz, P. T., Gilbertson, L., and Staub, J. M. (2001) Multiple pathways for Cre/loxmediated recombination in plastids. Plant J. 27, 161–170.
Zubko, E., Scutt, C., and Meyer, P. (2000) Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes Nat. Biotechnology 18, 442–445.
Iamtham, S. and Day, A. (2000) Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat. Biotechnol. 18, 1172–1176.
Daniell, H., Muthukumar, B., and Lee, S. B. (2001) Marker free transgenic plants: engineering the chloropast genome without the use of antibiotic selection. Curr. Gen. 39, 109–116.
Vogel, G., Fiehn, O., L, L. J.-R.-d.-B., Boller, T., Wiemken, A., Aeschbacher, R. A., and Wingler, A. (2001) Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues. J. Exp. Bot. 52, 1817–1826.
Leyman, B., Van Dijck, P., and Thevelein, J. M. (2001) An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana. Trends Plant Sci. 6, 510–513.
Blasquez, M. A., Santos, E., Flores, C. L., Martinez-Zapater, J. M., Salinas, J., and Gancedo, C. (1998) Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase. Plant J. 13, 685–689.
Van Dijck, P., Mascorro-Gallardo, J. O., Bus, M. D., Royackers, K., Iturriaga, G., and Thevelein, J. M. (2002) Truncation of Arabidopsis thaliana and Selaginella lepidophylla trehalose-6-phosphate synthase unlocks high catalytic activity and supports high trehalose levels on expression in yeast. Biochem. J. 15, 63–71.
Eastmond, P. J., Dijken, A. J. v., Spielman, M., Kerr, A., Tissier, A. F., Dickinson, H. G., et.al., (2002) Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation. Plant J. 29, 225–235.
Sheen, J., Zhou, L., and Jang, J. C. (1999) Sugars as signaling molecules. Curr. Opin. Plant. Biol. 2, 410–418.
Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Leyman, B., Avonce, N., Ramon, M., Van Dijck, P., Thevelein, J.M., Iturriaga, G. (2004). New Selection Marker for Plant Transformation. In: Balbás, P., Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 267. Humana Press. https://doi.org/10.1385/1-59259-774-2:385
Download citation
DOI: https://doi.org/10.1385/1-59259-774-2:385
Publisher Name: Humana Press
Print ISBN: 978-1-58829-262-9
Online ISBN: 978-1-59259-774-1
eBook Packages: Springer Protocols