Abstract
This paper establishes that the isolated shoot meristem of monocotyledons can be infected and transformed using Agrobacterium. Since this explant from nearly any cereal cultivar can rapidly regenerate into a plant, using this explant effectively eliminates the genotype regeneration restrictions to cereal crop transformation allowing direct transformation of elite germplasm. Shoot apices of Oryza sativa L. Tropical Japonica, cv. Maybelle were explants used for cocultivation, and gene transfer was accomplished using Agrobacterium containing plasmids for the bar gene expression driven by the CaMV 35S promoter or by the rice actin 1 promoter. Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinate-ammonium (PPT), established that no shoot apices survived on 0.5 or 1.0 mg/l PPT. After shoot apices were cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT. Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells appeared to be most effective in promoting transformation. Integration, expression and transmission of the transferred foreign genes in primary, R1 and R2 generation plants were confirmed by molecular analyses and herbicide application tests. A germination test of R2 progeny from one of the transgenic plants (R1) established a phenotype segregation ratio showing a non-Mendelian inheritance pattern. Inactivation of the transferred foreign gene in R2 progeny appeared to result from transgene methylation.
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References
Battraw MJ, Hall TC: Histochemical analysis of CaMV 35S promoter-β-glucuronidase gene expression in transgenic rice plants. Plant Mol Biol 15: 527–538 (1990).
Bhaskaran S, Smith RH: Regeneration in cereal tissue culture: a review. Crop Sci 30(6): 1328–1337 (1990).
Cao J, Duan X, McElroy D, Wu R: Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Cell Rep 11: 536–591 (1992).
Chan MT, Chang HH, Ho SL, Tong WF, Yu SM: Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene. Plant Mol Biol 22: 491–506 (1993).
Christou P, Ford TL, Kofron M: Production of transgenic rice (Oryza sativa L.) plants from agronomically important Indica and Japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/technology 9: 957–962 (1991).
Cornejo M-J, Luth D, Blankenship KM, Anderson OD, Blechl AE: Activity of maize ubiquitin promoter in transgenic rice. Plant Mol Biol 23: 567–581 (1993).
Datta SK, Datta K, Soltanifar S, Donn G, Potrykus I: Herbicideresistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts. Plant Mol Biol 20: 619–629 (1992).
Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: Version II″. Plant Mol Biol Rep 1: 19–21 (1983).
Finnegan J, McElroy D: Transgene inactivation: Plants fight back!. Bio/technology 12: 883–888 (1994).
Gerber-Huber S, May FEB, Westley BR, Felber KB, Hosbach HA, Andres A-C, Ryffel G: In contrast to other Xenopus genes the estrogen-inducible vittellogenin genes are expressed when totally methylated. Cell 33: 43–51 (1983).
Gould J, Devey M, Hasegawa O, Ulian E, Peterson G, Smith RH: Transformation of Zea mays L. using Agrobacterium tumefaciens and the shoot apex. Plant Physiol 95: 426–434 (1991).
Hart CM, Fischer B, Neuhaus JM, Meins FJ: Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestris plants containing a defense-related tobacco.
Hauptmann RM, Ozias-Akins P, Vasil V, Tabaeizadeh Z, Rogers SG, Horsch RB, Vasil IK, Fraley RT: Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species. Plant Cell Rep 6: 265–272 (1987).
Hiei Y, Ohta S, Komari T, Kumashiro T: Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271–282 (1994).
Hobbs SLA, Warkentin TD, DeLong CMO: Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol Biol 21: 17–26 (1993).
Hood EE, Helmer GL, Fraley RT, Chilton MD: The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168: 1291–1301 (1986).
Hussey G, Johnson RD, Warren S: Transformation of meristematic cells in the shoot apex of cultured pea shoots by Agrobacterium tumefaciens and A. rhizogenes. Protoplasma 148: 101–105 (1989).
Klaas M, John MJ, Crowell DN, Amasino RM: Rapid induction of genomic demethylation and T-DNA gene expression in plant cells by 5-azacytidine derivatives. Plant Mol Biol 12: 413–423 (1989).
McElroy D, Blowers A, Jenes B, Wu R: Construction of expression vectors based on the rice actin 1 (Act 1) 5′ region for use in monocot transformation. Mol Gen Genet 231: 150–160 (1991).
Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).
Ottaviani MP, Smits T: Häenisch ten Cate CH: Differential methylation and expression of the b-glucuronidase and neomycin phosphotransferase genes in transgenic potato cv Bintje. Plant Sci 88: 73–81 (1993).
Peerbolte R, Leehouts K, Hooykaas-van Slogteren GMS, Wullems GJ, Schilperoort RA: Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization, T-DNA methylation and conditional expression of opine genes. Plant Mol Biol 7: 285–299 (1986).
Peng J, Wen F, Lister RL, Hodges TK: Inheritance of gusA and neo genes in transgenic rice. Plant Mol Biol 27: 97–104 (1995).
Potrykus I: Gene transfer to cereals: an assessment. Bio/technology 8: 535–542 (1990).
Raineri DM, Bottino P, Gordon MP, Nester EW: Agrobacterium-mediated transformation of rice (Oryza sativa L.). Bio/technology 8: 33–38 (1990).
Rathore KS, Chowdhury VK, Hodges TK: Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts. Plant Mol Biol 21: 871–884 (1993).
Renckens S, de Greve H, van Montague M, Hernalsteens JP: Petunia plants escape from negative selection against a transgene by silencing foreign DNA via methylation. Mol Gen Genet 233: 53–64 (1992).
Simpson RB, Speilmann A, Margossian L, McKnight TD: A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes. Plant Mol Biol 6: 403–415 (1986).
Smith RH, Hood EE: Agrobacterium tumefaciens transformation of monocotyledons. Crop Sci 35: 301–309 (1995).
Ulian E, Smith RH, Gould J, McKnight TD: Transformation of plants via the shoot apex. In Vitro Cell Dev Biol 24: 951–954 (1989).
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Park, S.H., Pinson, S.R.M. & Smith, R.H. T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices. Plant Mol Biol 32, 1135–1148 (1996). https://doi.org/10.1007/BF00041397
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DOI: https://doi.org/10.1007/BF00041397