Springer Protocols: Abstract: Perennial Ryegrass (Lolium perenne L.)

Perennial Ryegrass (Lolium perenne L.)

By: Fredy Altpeter³

Abstract

Full Text

Download PDF (471K)

A protocol that facilitates rapid establishment of Agrobacterium-mediated transformation for perennial ryegrass is described. The synthetic green fluorescent protein (sgfpS65T) reporter gene is introduced in combination with the nptII selectable marker gene into axillary bud derived embryogenic calli of perennial ryegrass (Lolium perenne L.) by co-cultivation with Agrobacterium tumefaciens strain AGL0 harboring binary vector pYF132. Following the co-cultivation calli are cultured for 48 h in liquid callus medium containing timentin at 10°C and 70 rpm, which reduces Agrobacterium overgrowth. Using green fluorescent protein (GFP) as a nondestructive visual marker allows identification of responsive genotypes and transgenic cell clusters at an early stage. GFP screening is combined with paromomycin selection to suppress wild type cells. Transgenic plantlets ready to transfer to soil are obtained within 4 mo of explant culture. Between 8 and 16% of the Agrobacterium-inoculated calli regenerate independent, Southern positive transgenic plants. Reproducibility and efficiency in this perennial ryegrass transformation protocols is controlled by multiple factors including genotype dependent tissue culture and gene transfer response, a short tissue culture-and-selection period and the efficient suppression of Agrobacterium following Agrobacterium-mediated gene transfer.

Affiliation(s): (3) Agronomy Department, PMCB, Genetics Institute, Laboratory of Molecular Plant Physiology, University of Florida — IFAS, Gainesville, FL

Book Title: Agrobacterium Protocols Volume 2

Series: Methods in Molecular Biology | Volume: 344 | Pub. Date: Sep-01-2006 | Page Range: 55-64 | DOI: 10.1385/1-59745-131-2:55

Subject: Plant Sciences

Key Words: Ryegrass - Lolium perenne L. - Agrobacterium-mediated gene transfer - nptII - gfp - grass transformation

References

Download References

1.	Watschke, T.L. and Schmidt, R.E. (1992) Ecological aspects of turfgrass communities. In: Waddington, D.V. et al. (eds.) Turfgrass Agron. Monogr. 32, pp 129–174. ASA, CSSA and SSSA Madison, Wi, USA.

2.	Xu, J., Schubert, J., and Altpeter, F. (2001) Dissection of RNA mediated virus resistance in fertile transgenic perennial ryegrass (Lolium perenne L.). Plant J. 26, 265–274.

3.	Spangenberg, G., Wang, Z.-Y., Wu, X.L., Nagel, J., and Potrykus, I. (1995) Transgenic perennial ryegrass (Lolium perenne) plants from microprojectile bombardment of embryogenic suspension cells. Plant Sci. 108, 209–217. <Occurrence Type="DOI"><Handle>10.1016/0168-9452(95)04135-H</Handle></Occurrence>

4.	Dalton, S.J., Bettany, A.J.E., Timms, E., and Morris, P. (1999) Co-transformed, diploid Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass) and Lolium temulentum (darnel) plants produced by microprojectile bombardment. Plant Cell Rep. 18, 721–726.

5.	Dalton, S.J., Bettany, A.J.E., Timms, E., and Morris, P. (1998) Transgenic plants of Lolium multiflorum, Lolium perenne, Festuca arundinacea and Agrostis stolonifera by silicon carbide fibre-mediated transformation of cell suspension cultures. Plant Sci. 132, 31–43. <Occurrence Type="DOI"><Handle>10.1016/S0168-9452(97)00259-8</Handle></Occurrence>

6.	Wang, G.R., Binding, H., and Posselt, U.K. (1997) Fertile transgenic plants from direct gene transfer to protoplasts of Lolium perenne L. and Lolium multiflorum Lam. J. Plant Physiol. 151, 83–90.

7.	Creemers-Molenaar, J. and Loeffen J.P.M. (1991) Regeneration from protoplasts of perennial ryegrass; progress and applications. In: Fodder Crops Breeding: Achievements, Novel Strategies and Biotechnology, Proceedings of the 16th Meeting of the Fodder Crops Section of Eucarpia. (Den Nijs, A.P.M. and Elgersma, A. eds.) November 16–22, 1990. Wageningen, The Netherlands, pp 123–128.

8.	Altpeter, F., Xu, J., and Ahmed, S. (2000) Generation of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage-and turf-type cultivars. Mol. Breeding 6, 519–528.

9.	Altpeter, F., Baisakh, N., Beachy, R., et al. (2005) Particle bombardment and the genetic enhancement of crops: Myths and realities. Mol. Breeding 15, 305–327.

10.	Czernilofsky, A.P., Hain, R., Baker, B., and Wirtz, U. (1986) Studies of the structure and functional organization of foreign DNA integrated into the genome of Nicotiana tabacum. DNA 5, 473–482.

11.	Koncz, C., Martini, N., Mayerhofer, R., et al. (1989) High-frequency T-DNA-mediated gene tagging in plants. Proc. Natl. Acad. Sci. USA 86, 8467–8471.

12.	McKnight, T.D., Lillis, M.T., and Simpson, R.B. (1987) Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol. Biol. 8, 439–445.

13.	Komari, T., Hiei, Y., Saito, Y., Murai, N., and Kumashiro, T. (1996) Vector 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.

14.	Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994) Efficient transformation of rice (Oriza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271–282.

15.	Cheng, M., Fry, J.E., Pang, S., et al. (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115, 971–980.

16.	Tingay, S., McElroy, D., Kalla, R., et al. (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J. 11, 1369–1376.

17.	Zhao, Z.-Y., Cai, T., Tagliani, L., et al. (2000) Agrobacterium-mediated sorghum transformation. Plant Mol. Biol. 44, 789–798.

18.	Yu, T.T., Skinner, D.Z., Liang, G.H., Trick, G.N., Huang, B., and Muthukrishnan, S. (2000) Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene. Hereditas 133, 229–233.

19.	Somleva, M.N., Tomaszewski, Z., and Conger, B.V. (2002) Agrobacterium-mediated genetic transformation of switchgrass. Crop Sci. 42, 2080–2087.

20.	Toyama, K., Bae C.-H., Kang J.-G., et al. (2003) Production of herbicide-tolerant zoysiagrass by Agrobacterium-mediated transformation. Mol. Cells 16, 19–27.

21.	Popelka, J.C. and Altpeter, F. (2003) Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.). Mol. Breeding 11, 203–211.

22.	Bettany, A.J.E., Dalton, S.J., Timms, E., Manderyck, B., Dhanoa, M.S., and Morris, P. (2003) Agrobacterium tumefaciens-mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.). Plant Cell. Rep. 21, 439–444.

23.	Hellens, R., Mullineaux, P., and Klee, H. (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451. <Occurrence Type="DOI"><Handle>10.1016/S1360-1385(00)01740-4</Handle></Occurrence>

24.	Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J., and Schilperpoort, 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.

25.	Alt-Mörbe, J., Kühmann, H., and Schröder, J. (1989) Differences in induction of Ti-plasmid virulence genes virG and virD and continued control of virD expression by four external factors. Mol. Plant-Microbe Interact. 2, 301–308.

26.	Usami, S., Okamoto, S., Takebe, I., and Machida, Y. (1988) Factor inducing Agrobacterium tumefaciens vir gene expression is present in monocotyledonous plants. Proc. Natl. Acad. Sci. USA 85, 3748–3752.

27.	Nauerby, B., Billing, K., and Wyndaele, R. (1997) Influence of the antibiotic timentin on plant regeneration compared to carbenicillin and cefotaxime in concentrations suitable for elimination of Agrobacterium tumefaciens. Plant Science 123, 169–177. <Occurrence Type="DOI"><Handle>10.1016/S0168-9452(96)04569-4</Handle></Occurrence>

28.	Sheen, J., Hwang, S., Niwa, Y., Kobayashi, H., and Galbraith, D.W. (1995) Green-flourescent protein as a new vital marker in plant cells. Plant J. 8, 777–784.

29.	Vain, P., Worland, B., Kohli, A., Snape, J.W., and Christou, P. (1998) The green fluorescent protein (GFP) as a vital screenable marker in rice transformation. Theor. Appl. Genet. 96, 164–169.

30.	Ahlandsberg, S., Sathish, P., Sun, C.X., and Jansson, C. (1999) Green fluorescent protein as a reporter system in the transformation of barley cultivars. Physiol. Plant. 107, 194–200.

31.	Jordan, M.C., (2000) Green fluorescent protein as a visual marker for wheat transformation. Plant Cell Rep. 19, 1069–1075.

32.	Mersereau, M., Pazour, G.J., and Das, A. (1990) Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90, 149–151. <Occurrence Type="DOI"><Handle>10.1016/0378-1119(90)90452-W</Handle></Occurrence>

33.	Lazo, G.R., Stein, P.A., and Ludwig, R.A. (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat. Biotechnol. 9, 963–967.

34.	Christensen, A.H. and Quail, P.H. (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgen. Res. 5, 213–218.

35.	Hajdukiewicz, P., Svab, Z., and Maliga, P. (1994). The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989–994.

36.	Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plantarum 15, 473–479.

Comments (Loading...)

Post comment/View all comments