High Efficiency Transgene Segregation in Co-Transformed Maize Plants using an Agrobacterium Tumefaciens 2 T-DNA Binary System
Tóm tắt
For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the β-glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R0 plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R0 events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R1 generation. Of these R0 events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R1 generation. A total of 64.4% of the R0 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R1 progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R0 plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R1 phenotypes were highly correlated to genetic segregation of the bar and GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.
Tài liệu tham khảo
An G, Mitra A, Choi HK, Costa MA, An K, Thornburg RW et al. (1989) Functional analysis of the 3′ control region of the potato wound-inducible proteinase inhibitor II gene. Plant Cell 1: 115–122.
Armstrong CL, Green CE and Phillips RL (1991) Development and availability of germplasm with high Type II culture formation response. Maize Genet Cooperat Newslett 65: 92–93.
Christensen AH, Sharrock RA and Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18: 675–689.
Daley M, Knauf V, Summerfelt K and Turner J (1998) Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants. Plant Cell Reports 17: 489–496.
De Block M and Debrouwer D (1991) 2 T-DNA' co-transformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus. Theor Appl Genet 82: 257–263.
De Framond AJ, Black EW, Chilton WS, Kayes L and 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.
Depicker A, Herman L, Jacobs A, Schell J and Van Montague M (1985) Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol Gen Genet 201: 477–484.
Feinberg AP and Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 659–668.
Feinberg AP and Vogelstein B (1984) Addendum: a technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137: 266–267.
Fromm M, Morrish F, Armstrong C, Williams R, Thomas J and Klein T (1990) Inheritance and expression of chimeric genes. in the progeny of transgenic maize plants. Bio/Technology 8: 833–839.
Goldsbrough A, Lastrella C and Yoder J (1993) Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Bio/Technology 11: 1286–1292.
Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG et al. (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2: 603–618.
Ishida Y, Saito H, Ohta S, Hiei Y, Komari T and Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14: 745–750.
Iyer LM, Kumpatla SP, Chandrasekharan MB and Hall TC (2000) Transgene silencing in monocots. Plant Mol Biol 43: 323–346.
Johnson DA, Gautsch JW, Sprotsman JR and Elder JH (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Tech 1: 3.
Komari T, Hiei Y, Saito Y, Murai N and 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.
Kramer MF, Coen DM (1997) Enzymatic amplification of DNA by PCR: standard procedures and optimization. In: Ausbel F, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA (eds), Short Protocols in Molecular Biology. 3rd edn, 15–3–15–5.
Labarca C and Paigen K (1980) A simple, rapid, sensitive DNA assay procedure. Anal Biochem 102: 344–352.
Lowe K, Abbit S, Glassman K, Gregory C, Hoerster G, Rasco-Gaunt S et al. (2000) Use of LEC1 to improve transformation. In Vitro Cell Deve Biol 36: 3 part 2 W-15.
Lu H, Zhou X, Gong Z and Upadhyaya N (2001) Generation of selectable marker-free transgenic rice using a double right-border (DRB) vector. Aust J Plant Physiol 28: 241–248.
Lyznik LA, Peterson D, Zhao ZY, Guan X, Bowen B, Drummond B et al. (2000) Gene transfer mediated by site-specific recombination systems. In: Gelvin SB and Schilperoort RA (eds), Plant Molecular Biology Manual N1. (pp. 1–26) Kluwer Academic Publishers, Dordrecht/Boston/London.
McCabe DE, Swain WF, Martinell BJ and Christou P (1988) Stable transformation of Soybean (Glycine max) by particle acceleration. Biotechnology 6: 923–926.
McKnight TD, Lillis MT and Simpson RB (1987) Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol Biol 8: 439–445.
Reed J, Chang Y, Dawson J, Dunder E, Hansen G, Launis K et al. (2000) The use of phosphomannose isomerase (pmi) as an efficient selectable marker for monocot and dicot transformation. In Vitro Plant Cell Dev Biol 36: 3 part 2 W-20.
Richards EJ (1997) Preparation of plant DNA using CTAB. In: Ausbel F, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds), Short Protocols in Molecular Biology. 3rd edn, Wiley, New York, pp. 2–10, 2–11.
Sidorenko LV, Li X, Cocciolone SM, Chopra S, Tagliani L, Bowen B et al. (2000) Complex structure of a maize Myb gene promoter: functional analysis in transgenic plants. Plant J 22: 471–482.
Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L and Rocha-Sosa M (1990). Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220: 245–250.
Walters D, Vetsch C, Potts D and Lundquist R (1992) Transformation and inheritance of a hygromycin phosphotransferase gene in maize plants. Plant Mol Biol 18: 189–200.
Xing A, Zhangyuan Z, Sato S, Staswick P and Clemente T (2000) The use of the two T-DNA binary system to obtain marker free transgenic soybeans. In Vitro Cell Dev Biol 36: 456–463.
Zhao Z, Gu W, Cai T, Hondred D, Bond D, Schroeder S et al. (2002) High throughput genetic transformation mediated by Agrobacterium tumfaciens in maize. Mol Breed (in press).