Abstract
A reliable and efficient genetic transformation protocol for various turfgrass species and elite cultivars has been achieved using Agrobacterium tumefaciens. We describe a general protocol for the establishment of embryogenic cell cultures, Agrobacterium tumefaciens-mediated transformation, selection, and regeneration of transgenic turfgrass plants. Embryogenic callus is initiated from mature seeds, maintained by visual selection, and infected with an Agrobacterium tumefaciens strain (LBA4404) that contains either an herbicide-resistant bar gene or an antibiotic-resistant hyg gene driven either by a rice ubiquitin or CaMV35S promoter. Stable transformation efficiencies up to 43.3% were achieved. Southern blot and genetic analysis was used to confirm transgene integration in the turfgrass genomes and normal transmission and stable expression of the transgene in the T1 generation. We demonstrate herein that five elite cultivars of bentgrass can be genetically transformed using this single tissue culture media regime. Additionally, we report the successful Agrobacterium-mediated transformation of an elite tall fescue variety using minor variations in the same transformation protocol.
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
Smith, R. and Hood, E.E. (1995) Agrobacterium tumefaciens transformation of monocotyledons. Crop Sci. 35, 301–309.
Schafer, W., Gorz, A., and Kahl, G. (1987) T-DNA integration and expression in a monocot crop plant after induction of Agrobacterium. Nature 327, 529–532.
Raineri, D.M., Bottino, P., Gordon, M.P., and Nester, E.W. (1990) Agrobacterium-mediated transformation of rice (Oryza sativa L.). Nat. Biotechnol. 8, 33–38.
Gould, J., Devey, M., Hasegawa, O., Ulian, E.C., Peterson, G., and Smith, R.H. (1991) Transformation of Zea mays L. using Agrobacterium tumefaciens and the shoot tip. Plant Physiol. 95, 426–434.
Li, X.-Q., Liu, C.-N., Ritchie, S.W., Peng, J.-Y., Gelvin, S.B., and Hodges, T.K. (1992) Factors influencing Agrobacterium-mediated transient expression of gusA in rice. Plant Mol. Biol. 20, 1037–1048.
Chan, M.-T., Lee, T.-M., and 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., and 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.
Ritchie, S.W., Liu, C.N., Sellmer, J.C., Kononowicz, H., Hodges, T.K., and Gelvin, S.B. (1993) Agrobacterium tumefaciens-mediated expression of gusA in maize tissues. Transgenic Res. 2, 252–265.
Hiei, Y., Ohta, S., Komari, T., and 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, 271–272.
Aldemita, R.R. and Hodges, T.K. (1996) Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties. Planta 199, 612–617.
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.
Cheng, M., Fry, J.E., Pang, S., et al. (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115, 971–980.
Tingay, S., McElroy, D., Kalla, R., et al. (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J. 11, 1369–1376.
Zhao, Z.-Y., Gu, W., Cai, T., et al. (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., et al. (2000) Agrobacterium-mediated sorghum transformation. Plant Mol. Biol. 44, 789–798.
Lupotto, E., Reali, A., Oassera, S., and Chan, M.T. (1999) Maize elite inbred lines are susceptible to Agrobacterium tumefaciens-mediated transformation. Maydica 44, 211–218.
Enriquez-Obregon, G.A., Prieto-Samsonov, D.L., de la Riva, G.A., Perez, M., Selman-Housein, G., and Vazquez-Padron, R.I. (1999) Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment. Plant Cell Tissue Organ Cult. 59, 159–168.
Frame, B.R., Shou, H., Chikwamba, R.K., et al. (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol. 129, 13–22.
Yu, T.T., Skinner, D.Z., Liang, G.H., Trick, H.N., Huang, B., and Muthukrishnan, S. (2000) Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene. Hereditas 133, 229–233.
Zilinskas, B.A. (1999) Agrobacterium-mediated transformation of turfgrass. USA PCT Patent application 98-0087PCT.
Bai, Y. and Qu, R. (2001) Genetic transformation of elite turf-type cultivars of tall fescue. Intl. Turf. Soci. Res. J. 9, 129–133.
Bai, Y. and Qu, R. (2001) Factors influencing tissue culture responses of mature seeds and immature embryos in turf-type tall fescue. Plant Breeding 120, 239–242.
Luo, H., Hu, Q., Nelson, K., et al. (2004) 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.
Zhong, H., Bolyard, M.G., Srinivasan, C., and Stricklen, M.B. (1993) Transgenic plants of turfgrass (Agrostis palustris Huds.) from microprojectile bombardment of embryogenic callus. Plant Cell Rep. 3, 1–6.
Hartman, C.L., Lee, L., Day, P.R., and Turner, N.E. (1994) Herbicide resistant turfgrass (Agrostis palustris Huds.) by biolistic transformation. Nat. Biotechnol. 12, 919–923.
Lee, L., Hartman, C., Laramore, C., Turner, N., and Day, P. (1995) Herbicide-resistant creeping bentgrass. USGA Green Section Record 33(3), 16–18.
Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J., and Schilperoort, R.A. (1983) A binary plant vector strategy based on separation of vir-and T region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303, 179–180.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Longo, C. et al. (2006). Turf Grasses. In: Wang, K. (eds) Agrobacterium Protocols Volume 2. Methods in Molecular Biology, vol 344. Humana Press. https://doi.org/10.1385/1-59745-131-2:83
Download citation
DOI: https://doi.org/10.1385/1-59745-131-2:83
Publisher Name: Humana Press
Print ISBN: 978-1-58829-843-0
Online ISBN: 978-1-59745-131-4
eBook Packages: Springer Protocols