Abstract
Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60–65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.
Similar content being viewed by others
Abbreviations
- 2,4-D::
-
2,4-Dichlorophenoxyacetic acid
- bar::
-
Bialaphos resistance gene
- GUS::
-
β-Glucuronidase
- PPT::
-
Phosphinothricin
- ubi::
-
Ubiquitin
References
Aldemita RR, Hodges TK (1996) Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties. Planta 199:612–617
Basu C, Luo H, Kausch AP, Chandlee JM (2003) Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris, L.). J Plant Physiol 160:1233–1239
Chan M-T, Lee T-M, Chang H-H (1992) Transformation of indica rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens. Plant Cell Physiol 33:577–583
Chan M-T, Chang H-H, Ho S-L, Tong W-F, Yu S-M (1993) Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α–amylase promoter/β-glucuronidase gene. Plant Mol Biol 22:491–506
Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980
De Buck S, Depicker A (2001) Disruption of their palindromic arrangement leads to selective loss of DNA methylation in inversely repeated gus transgenes in Arabidopsis. Mol Genet Genomics 265:1060–1068
De Buck S, Van Montagu M, Depicker A (2001) Transgene silencing of invertedly repeated transgenes is released upon deletion of one of the transgenes involved. Plant Mol Biol 46:433–445
Enríquez-Obregón GA, Prieto-Samsónov DL, de la Riva GA, Pérez M, Selman-Housein G, Vázquez-Padrón RI (1999) Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment. Plant Cell Tissue Organ Cult 59:159–168
Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SEK, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13–22
Gould J, Devey M, Hasegawa O, Ulian EC, Peterson G, Smith RH (1991) Transformation of Zea mays L. using Agrobacterium tumefaciens and the shoot tip. Plant Physiol 95:426–434
Hartley RW (1988) Barnase and barstar: expression of its cloned inhibitor permits expression of a cloned ribonuclease. J Mol Biol 202:913–915
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 of the T-DNA. Plant J 6:271–282
Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180
Huq E, Hirayama L, Hossain MA, Hodges TK (1997) Characterization of a cDNA encoding a polyubiquitin gene in rice (accession no. U37687)(PRG97-004). Plant Physiol 113:305
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
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405
Jin S, Komari T, Gordon MP, Nester EW (1987) Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J Bacteriol 169:4417–4425
Klöti A, He X, Potrykus I, Hohn T, Fütterer J (2002) Tissue-specific silencing of a transgene in rice. Proc Natl Acad Sci USA 99:10881–10886
Komari T (1989) Transformation of callus cultures of nine plant species mediated by Agrobacterium. Plant Sci 60:223–229
Lee J-Y, Aldemita RR, Hodges TK (1996) Isolation of a tapetum-specific gene and promoter from rice. Int Rice Res Newsl 21:2–3
Li X-Q, Liu C-N, Ritchie SW, Peng J-Y, Gelvin SB, Hodges TK (1992) Factors influencing Agrobacterium-mediated transient expression of gusA in rice. Plant Mol Biol 20:1037–1048
Luo H, Van Coppenolle B, Seguin M, Boutry M (1995) Mitochondrial DNA polymorphism and phylogenetic relationship in Hevea brasiliensis. Mol Breed 1:51–63
Luo H, Hu Q, Nelson K, Longo C, Kausch AP (2003) Controlling transgene escape in genetically modified grasses. In: Hopkins A, Wang ZY, Mian R, Sledge M, Barker R (eds) Molecular breeding of forage and turf. Kluwer, Dordrecht (in press)
Lupotto E, Reali A, Oassera S, Chan MT (1999) Maize elite inbred lines are susceptible to Agrobacterium tumefaciens-mediated transformation. Maydica 44:211–218
McElroy D, Zhang W, Cao J, Wu R (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2:163–171
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Peterhans A, Datta SK, Datta K, Goodall GJ, Potrykus I, Paszkowski J (1990) Recognition efficiency of Dicotyledoneae-specific promoter and RNA processing signals in rice. Mol Gen Genet 222:361–368
Potrykus I (1990) Gene transfer to cereals: an assessment. Biotechnology 8:535–542
Raineri DM, Bottino P, Gordon MP, Nester EW (1990) Agrobacterium-mediated transformation of rice (Oryza sativa L.). Biotechnology 8:33–38
Ritchie SW, Liu CN, Sellmer JC, Kononowicz H, Hodges TK, Gelvin SB (1993) Agrobacterium tumefaciens-mediated expression of gusA in maize tissues. Transgenic Res 2:252–265
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York
Schafer W, Gorz A, Kahl G (1987) T-DNA integration and expression in a monocot crop plant after induction of Agrobacterium. Nature 327:529–532
Smith R, Hood EE (1995) Agrobacterium tumefaciens transformation of monocotyledons. Crop Sci 35:301–309
Tereda R, Shimamoto K (1990) Expression of CaMV 35S-GUS gene in transgenic rice plants. Mol Gen Genet 220:389–392
Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376
Wang Z, Hopkins A, Mian R (2001) Forage and turf grass biotechnology. Crit Rev Plant Sci 20:573–619
Yu TT, Skinner DZ, Liang GH, Trick HN, Huang B, Muthukrishnan S (2000) Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene. Hereditas 133:229–233
Zhao Z-Y, Gu W, Cai T, Tagliani LA, Hondred D, Bond D, Krell S, Rudert ML, Bruce WB, Pierce DA (1998) Molecular analysis of T0 plants transformed by Agrobacterium and comparison of Agrobacterium-mediated transformation with bombardment transformation in maize. Maize Genet Coop Newsl 72:34–37
Zhao Z-Y, Cai T, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J, Pierce D (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44:789–798
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by J.M. Widholm
Rights and permissions
About this article
Cite this article
Luo, H., Hu, Q., Nelson, K. et al. Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration. Plant Cell Rep 22, 645–652 (2004). https://doi.org/10.1007/s00299-003-0734-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-003-0734-2