Transgenic Research

, Volume 21, Issue 3, pp 619–632 | Cite as

Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene

  • S. Panter
  • P. G. Chu
  • E. Ludlow
  • R. Garrett
  • R. Kalla
  • M. Z. Z. Jahufer
  • A. de Lucas Arbiza
  • S. Rochfort
  • A. Mouradov
  • K. F. Smith
  • G. SpangenbergEmail author
Original Paper


Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T0 and T1 generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T4 generation in an agronomically elite ‘Grasslands Sustain’ genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T4 generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.


Agrobacterium-mediated plant transformation Viral coat protein Coat protein-mediated virus resistance Field evaluation Gene flow Trifolium repens 



Alfalfa mosaic virus


Cauliflower mosaic virus


Nopaline synthase


Neomycin phosphotransferase


Subgenomic RNA4



The research described in this paper was supported by Dairy Australia, the Molecular Plant Breeding Co-operative Research Centre and Heritage Seeds. The authors would like to thank all of the staff in DPI, CSIRO and Heritage Seeds who contributed to this project over many years.

Supplementary material

11248_2011_9557_MOESM1_ESM.pdf (37 kb)
Online Resource 1 Schematic representation of the pKYLX71:35S 2 AMV4 plasmid. LB - left border of T-DNA region; P35S2 – enhanced Cauliflower mosaic virus 35S promoter; AMV CP – Alfalfa mosaic virus CP gene coding region; rbcST – Pisum sativum RuBisCO small subunit E9 gene terminator; nosT – nopaline synthase gene terminator; npt2 – neomycin phosphotransferase 2 gene coding region; nosT – nopaline synthase gene promoter; RB – right border of T-DNA region. (PDF 29 kb)
11248_2011_9557_MOESM2_ESM.pdf (29 kb)
Online Resource 2 Summary of Alfalfa mosaic virus strains used for isolation of the AMV CP gene and for viral inoculation experiments. (PDF 30 kb)
11248_2011_9557_MOESM3_ESM.pdf (26 kb)
Online Resource 3 Summary of climatic data collected near the Hamilton, Victoria and Howlong, NSW sites for field evaluation of transgenic virus-resistant white clover plants. Although climatic data for Howlong NSW is not available, representative data from the three closest weather stations at Albury, Corowa and Rutherglen is provided. Summer, November–January; Winter, June–August. Source: (PDF 26 kb)
11248_2011_9557_MOESM4_ESM.pdf (62 kb)
Online Resource 4 Molecular and phenotypic characterization of the T0 generation of AMV CP transgenic white clover plants. a) Southern hybridization analysis of HindIII-digested genomic DNA from T0 AMV CP transgenic white clover plants using an nptII hybridization probe. b) Southern hybridization analysis as for a) using an AMV CP hybridization probe. Lanes in 1a and 1b: (1–4) T0 AMV CP lines H1-A, H1-B, H1-C and H6; (5) non-transgenic white clover control line; (6) positive control. c) Northern hybridization analysis of T0 AMV CP lines using an AMV CP hybridization probe. Lanes: (1–4) T0 AMV CP lines H1-A, H1-B, H1-C and H6; (5) non-transgenic white clover control line. d) Western immunoblot analysis of two representative T0 AMV CP lines using a polyclonal anti-AMV CP antibody. Lanes: (1) H1-A; (2) H2; (3) AMV CP extracted from an AMV-infected non-transgenic white clover plant; (4) non-transgenic white clover control plant. e) Field evaluation of the T0 generation of AMV CP transgenic white clover plants. Symptoms of viral infection were scored visually and confirmed using bioassays. The bar charts show the percentage of AMV-infected plants from non-transgenic control lines (cv Irrigation) and three transgenic lines (H6 and H1-B) over the 1998 growing season. 6 plants from each line were evaluated. (PDF 66 kb)
11248_2011_9557_MOESM5_ESM.pdf (40 kb)
Online Resource 5 Flow chart showing the breeding strategy used to generate transgenic ‘Sustain’-type Syn0 white clover plants with resistance to Alfalfa mosaic virus. (PDF 32 kb)
11248_2011_9557_MOESM6_ESM.pdf (91 kb)
Online Resource 6 Molecular characterization of the ‘Sustain’ type Syn0 generation of white clover plants containing the AMV CP transgene, H6 event. a) and b) Southern hybridization analysis of Syn0 plants containing the AMV CP transgene (H6 event), using AMV CP (A) and nptII (B) probes respectively. Lanes: (1)-(14) Syn0 AMV CP genotypes; (15) non-transgenic white clover plant. c) Northern hybridization analysis of Syn0 AMV CP genotypes using an AMV CP probe. Lanes: (1)-(12) Syn0 AMV CP genotypes; (13) non-transgenic white clover control plant; (14) AMV-infected non-transgenic white clover plant. d) Western immunoblot analysis of Syn0 AMV CP genotypes using an antibody against the AMV CP. Lanes: (1–12) Syn0 AMV CP genotypes; (13) non-transgenic white clover control plant; (14) AMV-infected non-transgenic white clover plant. (PDF 83 kb)
11248_2011_9557_MOESM7_ESM.pdf (44 kb)
Online Resource 7 Analysis of weediness-related agronomic traits in ‘Sustain’ type Syn0 generation white clover plants containing the AMV CP transgene, H6 event. Six agronomic traits related to potential weediness, namely plant height, plant width, internode length, stolon diameter and vigor, were measured in the field to compare the performance of 11 non-transgenic (cv Grasslands Sustain) parents of the cultivar (S2-S12) to the average performance of 600 transgenic lines (T). (PDF 32 kb)


  1. Ayres JF, Murison RD, Turner AD et al (2001) A rapid semi-quantitative procedure for screening hydrocyanidic acid in white clover. Aust J Exp Agric 41:515–521CrossRefGoogle Scholar
  2. Barnett OW, Gibson PB (1975) Identification and prevalence of white clover mosaic virus and the resistance of Trifolium species to these viruses. Crop Sci 15:32–37CrossRefGoogle Scholar
  3. Baulcombe DC (1996) Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8:1833–1844PubMedCrossRefGoogle Scholar
  4. Bennett D, Morley FHW, Axelen A (1976) Bioassay responses of ewes to legume swards. Aust J Agric Res 18:495–504CrossRefGoogle Scholar
  5. Berardo N (1997) Prediction of the chemical composition of white clover by near-infrared reflectance spectroscopy. Grass Forage Sci 52:27–32CrossRefGoogle Scholar
  6. Bol JF (2005) Replication of Alfamo- and Alfamoviruses: role of the coat protein. Annu Rev Phytopathol 43:39–62PubMedCrossRefGoogle Scholar
  7. Brunt AA, Crabtree K, Dallwitz MJ et al (1996) Viruses of plants. CAB International, WallingfordGoogle Scholar
  8. Campbell CL, Moyer JW (1984) Yield responses of six white clover clones to virus infection under field conditions. Plant Dis 68:1033–1035Google Scholar
  9. Carlsen S, Fomsgaard I (2008) Biologically active secondary metabolites in white clover (Trifolium repens L.)—a review focusing on contents in the plant, plant-pest interactions and transformation. Chemoecol 18:129–170CrossRefGoogle Scholar
  10. Chen CC, Gibson PB (1972) Barriers to hybridisation of Trifolium repens with related species. Can J Genet Cytol 14:381–389Google Scholar
  11. Crill P, Hanson EW, Hagedorn DJ (1971) Resistance and tolerance to alfalfa mosaic virus in alfalfa. Phytopathol 61:369–371CrossRefGoogle Scholar
  12. Davies WE (1971) Host/pollinator relationships in the evolution of herbage legumes in Britain. Sci Prog 59:573–589Google Scholar
  13. Ding Y-L, Aldao-Humble G, Ludlow E et al (2003) Efficient plant regeneration and Agrobacterium-mediated transformation in Medicago and Trifolium species. Plant Sci 165:1419–1427CrossRefGoogle Scholar
  14. Emmerling M, Chu P, Smith KF et al (2004) Field evaluation of transgenic white clover with AMV immunity and development of elite transgenic germplasm. In: Hopkins A, Wang ZU, Mian R et al (eds) Molecular breeding for the genetic improvement of forage and turf: proceedings of the 3rd international symposium, molecular breeding of forage and turf. Springer, New York, pp 359–366Google Scholar
  15. Fitchen JH, Beachy RN (1993) Genetically engineered protection against viruses in transgenic plants. Annu Rev Microbiol 47:739–763PubMedCrossRefGoogle Scholar
  16. Forster RLS, Musgrave DR (1985) Clover yellow vein virus in white clover (Trifolium repens) and sweet pea (Lathyrus odoratus) in the North Island of New Zealand. NZ J Agric Res 28:575–578CrossRefGoogle Scholar
  17. Forster RLS, Beck DL, Lough TJ (1997) Engineering for resistance to virus diseases. In: McKersie BD, Brown DCW (eds) Biotechnology and the improvement of forage legumes. CAB, WallingfordGoogle Scholar
  18. Franke AA, Custer LJ, Cerna CM et al (1994) Quantitation of phytoestrogens in legumes by HPLC. J Agric Food Chem 42:1095–1913CrossRefGoogle Scholar
  19. Fulkerson WJ, Slack K, Hennessy DW et al (1998) Nutrients in ryegrass (Lolium spp), white clover (Trifolium repens) and kikuyu (Pennisetum clandestinum) pastures in relation to season and stage of regrowth in a subtropical environment. Aust J Exp Agric 38:227–240CrossRefGoogle Scholar
  20. Garrett R (1991) Impact of viruses on pasture legume productivity. In: Proceedings of Department of Agriculture Victoria white clover conference, Victoria, pp 50–57Google Scholar
  21. George J, Dobrowolski MP, van Zijll de Jong E et al (2006) Assessment of genetic diversity in cultivars of white clover (Trifolium repens L.) detected by SSR polymorphisms. Genome 49:919–930PubMedCrossRefGoogle Scholar
  22. Gibson PB, Barnett OW, Skipper HD et al (1981) Effects of three viruses on growth of white clover. Plant Dis 65:50–51CrossRefGoogle Scholar
  23. Gibson PB, Barnett OW, Burrows PM et al (1982) Filtered-air enclosures exclude vectors and enable measurement of effects of viruses on white clover in the field. Plant Dis 66:142–144CrossRefGoogle Scholar
  24. Gibson PB, Barnett OW, Pederson MR et al (1989) Registration of southern regional virus resistant white clover germplasm. Crop Sci 29:241–242CrossRefGoogle Scholar
  25. Gilmour AR, Gogel BJ, Cullis BR et al (2002) ASReml user guide release 1. VSN, Hemel Hempstead, UKGoogle Scholar
  26. Guy P, Gibbs A, Harrower K (1980) The effect of white clover mosaic virus on nodulation of white clover (Trifolium repens L. cv. Ladino). Aust J Agric Res 31:307–311CrossRefGoogle Scholar
  27. Harris SL, Auldist MJ, Clark DA et al (1998) Effects of white clover content in the diet on herbage intake, milk, milk production and milk composition of New Zealand dairy cows housed indoors. J Dairy Res 65:389–400PubMedCrossRefGoogle Scholar
  28. Jayasena KW, Hajimorad MR, Law EG et al (2001) Resistance to alfalfa mosaic virus in transgenic barrel medic lines containing the virus coat protein gene. Aust J Agric Res 52:67–72CrossRefGoogle Scholar
  29. Kalantidis K, Psaradakis S, Tabler M et al (2002) The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol Plant Microbe Interact 15:826–833PubMedCrossRefGoogle Scholar
  30. Kapusta I, Janda B, Stochmal A et al (2005) Determination of saponins in aerial parts of barrel medic (Medicago truncatula) by liquid chromatography-electrospray ionization/mass spectrometry. J Agric Food Chem 53:7654–7660PubMedCrossRefGoogle Scholar
  31. Lechtenberg B, Schubert D, Forsbach A, Gils M, Schmidt R (2003) Neither inverted repeat T-DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing. Plant J 34:507–517PubMedCrossRefGoogle Scholar
  32. Lehmann J, Meister E, Gutzwiller A, Jans F, Charles JP, Blum J (1991) Should one use white clover (Trifolium repens L.) varieties rich in hydrogen cyanide? Rev Suisse d’Agric 23:107–112Google Scholar
  33. Martin PH, Coulman BE, Peterson JF (1997) Genetics of resistance to alfalfa mosaic virus in red clover. Can J Plant Sci 77:601–605CrossRefGoogle Scholar
  34. McLaughlin MR (1991) A greenhouse method for aphid inoculation of alfalfa mosaic virus in white clover by co-culture of virus, vector and clover. In: Peters DC, Webster JA, Chloubers CS (eds) Aphid plant interaction: populations to molecules. Okla. Agric. Exp. Stn., Stillwater, p 318Google Scholar
  35. Mendoza EMT, Laurena AC, Botella JR (2008) Recent advances in the development of transgenic papaya technology. Biotechnol Annu Rev 14:423–461CrossRefGoogle Scholar
  36. Norton MR, Johnstone GR (1998) Occurrence of alfalfa mosaic, clover yellow vein, subterranean clover red leaf, and white clover mosaic viruses in white clover throughout Australia. Aust J Agric Res 49:723–728CrossRefGoogle Scholar
  37. Parrella G, Lanave C, Marchoux G et al (2000) Evidence for two distinct subgroups of Alfalfa mosaic virus (AMV) from France and Italy and their relationships with other AMV strains. Arch Virol 145:2659–2667PubMedCrossRefGoogle Scholar
  38. Parrella G, Moretti A, Gognalons P et al (2004) The Am gene controlling resistance to alfalfa mosaic virus in tomato is located in the cluster of dominant resistance genes on chromosome 6. Phytopathology 94:345–350PubMedCrossRefGoogle Scholar
  39. Patterson HD, Thomson R (1971) Recovery of inter-block information when block sizes are unequal. Biometrika 58:545–554CrossRefGoogle Scholar
  40. Pedersen GA, McLaughlin MR (1994) Genetics of resistance to peanut stunt, clover yellow vein and alfalfa mosaic viruses in white clover. Crop Sci 34:896–900CrossRefGoogle Scholar
  41. Phillips GC, Collins GB, Taylor NL (1982) Interspecific hybridisation of red clover (Trifolium pratense L.) with T. sarosiense Hazsl. using in vitro embryo rescue. Theor Appl Genet 62:17–24Google Scholar
  42. Powell AP, Sanders PR, Tumer N et al (1990) Protection against tobacco mosaic virus infection in transgenic plants requires accumulation of coat protein rather than coat protein RNA sequences. Virology 175:124–130PubMedCrossRefGoogle Scholar
  43. Prins M, Laimer M, Noris E et al (2008) Strategies for antiviral resistance in transgenic plants. Mol Plant Pathol 9:73–83Google Scholar
  44. Schardl CL, Bryd AD, Benzion G et al (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61:1–11PubMedCrossRefGoogle Scholar
  45. Smith KF, Flinn PC (1991) Monitoring the performance of a broad-based calibration for measuring the nutritive value of two independent populations of pasture using infrared reflectance (NIRS) spectroscopy. Aust J Agric Res 31:205–210CrossRefGoogle Scholar
  46. Spangenberg G, Wang Z-Y, Wu X et al (1995) Transgenic perennial ryegrass (Lolium perenne) plants from microprojectile bombardment of embryogenic suspension cells. Plant Sci 108:209–217CrossRefGoogle Scholar
  47. Tenllado F, Bol JF (2000) Genetic detection of the multiple functions of alfalfa mosaic virus coat protein in viral RNA replication, encapsidation, and movement. Virology 268:29–40PubMedCrossRefGoogle Scholar
  48. Tepfer M (2002) Risk assessment of virus-resistant transgenic plants. Annu Rev Phytopathol 40:467–491PubMedCrossRefGoogle Scholar
  49. Thomas RG (1987) Reproductive development. In: Baker MJ, Williams WM (eds) White clover. CAB, UK, pp 63–123Google Scholar
  50. Timmerman-Vaughan GM, Pither-Joyce MD, Cooper PA et al (2001) Partial resistance of transgenic peas to alfalfa mosaic virus under greenhouse and field conditions. Crop Sci 41:846–853CrossRefGoogle Scholar
  51. Visschier PK, Seeley TD (1982) Foraging strategy of honeybee colonies in a temperate deciduous forest. Ecology 63:1790–1801CrossRefGoogle Scholar
  52. Williams WM (1987) White clover taxonomy and biosystematics. In: Baker MJ, Williams WM (eds) White clover. CAB, Wallingford, pp 323–342Google Scholar
  53. Woodfield DR, Clifford PTP, Baird IJ et al (1994) Gene flow and estimated isolation requirements for transgenic white clover. In: Jones DD (ed) Proceedings of the 3rd international symposium on the biosafety of field tests of genetically modified plants and microorganisms. University of California, Monterey, pp 509–514Google Scholar
  54. Xu DM, Collins GB, Hunt AG et al (1998) Resistance to alfalfa mosaic virus in transgenic burley tobacco expressing the AMV coat protein gene. Crop Sci 38:1661–1668CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • S. Panter
    • 1
    • 2
  • P. G. Chu
    • 3
  • E. Ludlow
    • 1
    • 2
  • R. Garrett
    • 1
  • R. Kalla
    • 1
  • M. Z. Z. Jahufer
    • 1
  • A. de Lucas Arbiza
    • 1
    • 2
    • 4
  • S. Rochfort
    • 2
    • 4
  • A. Mouradov
    • 1
    • 2
    • 4
  • K. F. Smith
    • 1
    • 2
  • G. Spangenberg
    • 1
    • 2
    • 4
    Email author
  1. 1.Molecular Plant Breeding CRCBundooraAustralia
  2. 2.Department of Primary Industries, Biosciences Research DivisionVictorian AgriBiosciences CentreBundooraAustralia
  3. 3.CSIRO, Plant IndustryCanberraAustralia
  4. 4.La Trobe UniversityBundooraAustralia

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