Abstract
Transgenic locus composition and T-DNA linkage configuration were assessed in a population of rice plants transformed using the dual-binary vector system pGreen (T-DNA containing the bar and gus genes)/pSoup (T-DNA containing the aphIV and gfp genes). Transgene structure, expression and inheritance were analysed in 62 independently transformed plant lines and in around 4,000 progeny plants. The plant lines exhibited a wide variety of transgenic locus number and composition. The most frequent form of integration was where both T-DNAs integrated at the same locus (56% of loci). When single-type T-DNA integration occurred (44% of loci), pGreen T-DNA was preferentially integrated. In around half of the plant lines (52%), the T-DNAs integrated at two independent loci or more. In these plants, both mixed and single-type T-DNA integration often occurred concurrently at different loci during the transformation process. Non-intact T-DNAs were present in 70–78% of the plant lines causing 14–21% of the loci to contain only the mid to right border part of a T-DNA. In 53–66% of the loci, T-DNA integrated with vector backbone sequences. Comparison of transgene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgene inheritance or T-DNA linkage, as only 60–80% of the transgenic loci were detected by the expression study. Co-expression (28% of lines) and backbone transfer (53–66% of loci) were generally a greater limitation to the production of marker-free T1 plants expressing the gene of interest than co-transformation (71% of lines) and unlinked integration (44% of loci).
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References
Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of T-DNA into wild-type and mutant lox sites in the plant genome. Plant J 7:649–659
Bec S, Chen L, Ferriere NM, Legave T, Fauquet C, Guideroni E (1998) Comparative histology of microprojectile-mediated gene transfer to embryonic calli in japonica rice (Oryza sativa L.): influence of the structural organization of target tissues on genotype transformation ability. Plant Sci 138:177–190
Bhattacharyya M, Stemer BA, Dixon RA (1994) Reduced variation in transgene expression from a binary vector with selectable markers at the right and left T-DNA borders. Plant J 6:957–968
Cluster PD, O’Dell M, Metzlaff M, Flavell RB (1996) Details of T-DNA structural organisation from a transgenic petunia population exhibiting co-suppression. Plant Mol Biol 32:1197–1203
Cotsaftis O, Sallaud C, Breitler JC, Meynard D, Greco R, Pereira A, Guiderdoni E (2002) Transposon-mediated generation of T-DNA and marker free rice plants expressing a Bt endotoxin gene. Mol Breed 10:165–180
Dale EC, Ow DE (1990) Intra and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase. Gene 91:79–85
Daley M, Knauf VC, Summerfelt KR, Turner JC (1998) Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants. Plant Cell Rep 19:489–496
De Block M, Debrouwer D (1991) Two T-DNAs co–transformed into Brassica napus by a double Agrobacterium infections are mainly integrated at the same locus. Theor Appl Genet 82:257–263
De Framond AJ, Back EW, Chilton WS, Kayes L, Chilton M-D (1986) Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation. Mol Gen Genet 202:125–131
De Neve M, De Buck S, Jacobs A (1997) T-DNA integration patterns in co-transformed plant cells suggests that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11:15–29
Dong J, Kharb P, Teng W, Hall TC (2001) Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach. Mol Breed 7:187–194
Ebinuma H, Sugita K, Matsunaga E, Endo S, Yamada K, Komamine A (2001) Systems for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep 20:383–392
Goldsbrough AP, Lastrella CN, Yoder J (1993) Transposition mediated repositioning and subsequent elimination of marker genes from transgenic tomato. BioTechnology 11:1286–1292
Hanson B, Engler D, Moy Y, Newman B (1999) A simple method to enrich an Agrobacterium-transformed population of plants containing only T-DNA sequences. Plant J 19:727–734
Hare P, Chua N-H (2002) Excision of selectable marker genes from transgenic plants. Nat Biotechnol 20:575–580
Hellens RP, Edwards A, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832
Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries T-DNA. Plant J 6:271–282
Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750
James VA, Avart C, Worland B, Snape JW, Vain P (2002) The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor Appl Genet 104:553–561
James VA, Worland B, Snape JW, Vain P (2004) Development of a standard operating procedure (SOP) for the precise quantification of transgene expression levels in rice. Physiol Plant 120:650–656
Jefferson RA, Kavanagh TA, Bevan MW (1987) β-glucuronidase as a sensitive and versatile fusion marker in higher plants. EMBO J 6:3901–3907
Jorgensen R, Snyder C, Jones JDG (1987) T-DNA is organized predominantly in inverted repeat structures in plants transformed with Agrobacterium tumefaciens C58 derivates. Mol Gen Genet 207:471–477
Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174
Koncz C, Nemeth K, Redei GP, Schell J (1994) Homology recognition during T-DNA integration into the plant genome. In: Paszkowski J (ed) Homologous recombination and gene silencing in plants. Kluwer, Dordrecht, pp 167–189
Lu HJ, Zhou X-R, Gong Z-X, Upadhyaya NM (2001) Generation of selectable marker-free transgenic rice using double right-border (DRB) binary vectors. Aust J Plant Physiol 28:241–248
Matthew PR, Wang M-B, Waterhouse PM, Thornton S, Fieg SJ, Gubler F, Jacobsen JV (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent “twin T-DNAs” on a standard Agrobacterium transformatiom vector. Mol Breed 7:195–202
McCormac AC, Elliot MC, Chen DF (1999) pBeCKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates “clean-gene” facilities. Mol Gen Genet 261:226–235
McKnight TD, Lillies MT, Simpson RB (1987) Segregation of genes transferred to one plant cell from two Agrobacterium strains. Plant Mol Biol 8:439–445
Miller M, Tagliani L, Wang N, Berka B, Bidney D (2002) High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Res 11:381–396
Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7:4021–4026
Sallaud C, Meynard D, Van Boxtel J, Gay C, Bes M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M, Ouwerkerk PBF, Rueb S, Delseny M, Guiderdoni E (2003) Highly efficient production and characterization of T-DNA plants for rice (Oriza sativa L.) functional genomics. Theor Appl Genet 106:1396–1408
Srivastava V, Anderson OD, Ow DV (1999) Single-copy transgenic wheat generated through the resolution of complex integration patterns. Proc Natl Acad Sci USA 96:11117–11121
Terada R, Urawa H, Inagaki Y, Tsugane K, Lida S (2002) Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20:1030–1034
Vain P, James VA, Worland B, Snape JW (2002) Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment. Theor Appl Genet 105:878–889
Vain P, Afolabi A, Worland B, Snape JW (2003) Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup. Theor Appl Genet 107:210–217
Xing A, Zhang Z, Sato S, Staswick P, Clement T (2000) The use of two T-DNA binary system to derive marker-free transgenic soybeans. In Vitro Cell Dev Plant 36:456–463
Yoder JI, Goldsbrough AP (1994) Transformation systems for generating marker-free transgenic plants. BioTechnology 12:263–267
Zubko E, Scutt C, Meyer P (2000) Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat Biotechnol 18:442–445
Acknowledgements
We gratefully acknowledge The Rockefeller Foundation for its support. This document is an output from projects (Plant Sciences Research Programme R8031) funded by the UK Department for International Development (DFID) and administered by the Centre for Arid Zone Studies (CAZS) for the benefit of developing countries. The views expressed are not necessarily those of the DFID.
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Afolabi, A.S., Worland, B., Snape, J.W. et al. A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations. Theor Appl Genet 109, 815–826 (2004). https://doi.org/10.1007/s00122-004-1692-y
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DOI: https://doi.org/10.1007/s00122-004-1692-y