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RESEARCH ARTICLE

Development and validation of protocols for product stewardship in transgenic white clover (Trifolium repens L.): detection of the AMV CP and npt2 transgenes in seeds, herbage and hay

S. Panter A , A. Mouradov B , K. F. Smith C and G. Spangenberg A D E
+ Author Affiliations
- Author Affiliations

A Department of Environment and Primary Industries, Biosciences Research Division, AgriBio, La Trobe University, Bundoora, Vic. 3083, Australia.

B School of Applied Sciences, RMIT University, Bundoora, Vic., 3083, Australia.

C Melbourne School of Land and Environment, The University of Melbourne, Private Bag 105, Hamilton, Vic. 3300, Australia.

D Faculty of Science, Technology and Engineering, La Trobe University, Bundoora, Vic. 3083, Australia.

E Corresponding author. Email: german.spangenberg@ecodev.vic.gov.au

Crop and Pasture Science 66(10) 1039-1048 https://doi.org/10.1071/CP14337
Submitted: 1 December 2014  Accepted: 7 June 2015   Published: 8 September 2015

Abstract

White clover (Trifolium repens L.) is an important pasture legume in temperate areas throughout the world, providing fodder for grazing animals and improving soil fertility via symbiotic nitrogen fixation. However, the persistence and stress tolerance of white clover is affected by several viruses, chiefly Alfalfa mosaic virus (AMV), Clover yellow vein virus (ClYVV) and White clover mosaic virus (WClMV). Efforts to introgress natural forms of virus resistance from other Trifolium spp. into white clover and lucerne (alfalfa) have had only limited success. This has been addressed by developing white clover germplasm exhibiting viral-coat-protein-mediated resistance to AMV and non-transgenic resistance to ClYVV. This report describes PCR-based assays for detecting the transgenes associated with the H6 transformation event in seeds, fresh leaves, air-dried leaves and mixtures of air-dried herbage of white clover and perennial ryegrass (hay). Although further development is required to convert these assays for use in the field, this paper demonstrates the ability to detect these transgenes in a range of agricultural products associated with the commercial use of white clover.

Additional keywords: Alfalfa mosaic virus, GMO, transgenic, tracing, Trifolium repens, white clover.


References

Abberton MT, Marshall AH (2010) White clover. In ‘Handbook of plant breeding. Vol. 5: Fodder crops and amenity grasses’. (Eds UK Posselt, F Veronesi) (Springer: New York)

Barnett OW, Gibson PB (1975) Identification and prevalence of white clover viruses and the resistance of Trifolium species to these viruses. Crop Science 15, 32–37.
Identification and prevalence of white clover viruses and the resistance of Trifolium species to these viruses.Crossref | GoogleScholarGoogle Scholar |

Battistini E, Noli E (2009) Real-time quantification of wild-type contaminants in glyphosate tolerant soybean. BMC Biotechnology 9, 16
Real-time quantification of wild-type contaminants in glyphosate tolerant soybean.Crossref | GoogleScholarGoogle Scholar | 19267904PubMed |

Campbell CL, Moyer JW (1984) Yield responses of 6 white clover clones to virus infection under field condition. Plant Disease 68, 1033–1035.
Yield responses of 6 white clover clones to virus infection under field condition.Crossref | GoogleScholarGoogle Scholar |

Crill P, Hanson EW, Hagedorn DJ (1971) Resistance and tolerance to Alfalfa mosaic virus in alfalfa. Phytopathology 61, 369–379.
Resistance and tolerance to Alfalfa mosaic virus in alfalfa.Crossref | GoogleScholarGoogle Scholar |

Devic M, Guilleminot J, Debeaujon I, Bechtold N, Bensaude E, Koornneef M, Pelletier G, Delseny M (1999) The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development. The Plant Journal 19, 387–398.
The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXms1Srtbc%3D&md5=2d33a4b0ad1e07d2984619f0944f2896CAS | 10504561PubMed |

Dudas B, Woodfield DR, Tong PM, Nicholls MF, Cousins GR, Burgess R, White DWR, Beck DL, Lough TJ, Forster RLS (1998) Estimating the agronomic impact of white clover mosaic virus on white clover performance in the North Island of New Zealand. New Zealand Journal of Agricultural Research 41, 171–178.
Estimating the agronomic impact of white clover mosaic virus on white clover performance in the North Island of New Zealand.Crossref | GoogleScholarGoogle Scholar |

Forster JW, Panter S, Mouradov A, Mason J, Spangenberg GC (2013) Transgenic technologies for enhanced molecular breeding of white clover (Trifolium repens L.). Crop & Pasture Science 64, 26–38.
Transgenic technologies for enhanced molecular breeding of white clover (Trifolium repens L.).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXmvFyrs70%3D&md5=827f44281b401de3b96fd8eed5b52fc2CAS |

Gerdes L, Busch U, Pecoraro S (2012) Parallelised real-time PCR for identification of maize GMO events. European Food Research and Technology 234, 315–322.
Parallelised real-time PCR for identification of maize GMO events.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xos1aisg%3D%3D&md5=f4a54a430e1ecc40d5dc3576d7082dd9CAS |

Gibson PB, Barnett OW, Skipper HD, McLaughlin MR (1981) Effects of three viruses on growth of white clover. Plant Disease 65, 50–51.
Effects of three viruses on growth of white clover.Crossref | GoogleScholarGoogle Scholar |

Hancock K, Collette V, Fraser K, Grieg M, Xue H, Richardson K, Jones C, Rasmussen S (2012) Expression of the R2-R3-MYB transcription factor TaMYB14 from Trifolium arvense activates proanthocyanidin biosynthesis in the legumes Trifolium repens and Medicago sativa. Plant Physiology 159, 1204–1220.
Expression of the R2-R3-MYB transcription factor TaMYB14 from Trifolium arvense activates proanthocyanidin biosynthesis in the legumes Trifolium repens and Medicago sativa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhtVGlsbnN&md5=81a8917ae25e3a9f613213651eb22880CAS | 22566493PubMed |

Hancock K, Collette V, Chapman E, Hanson K, Temple S, Moraga R, Caradus J (2014) Progress towards developing bloat safe legumes for the farming industry. Crop & Pasture Science 65, 1107–1113.
Progress towards developing bloat safe legumes for the farming industry.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXhvVOnsrvE&md5=bf42dc726b18b5f8ff6682fe92aad228CAS |

Jones RAC (2013) Virus diseases of perennial legumes in Australia: incidences, losses, epidemiology, and management. Crop & Pasture Science 64, 199–215.
Virus diseases of perennial legumes in Australia: incidences, losses, epidemiology, and management.Crossref | GoogleScholarGoogle Scholar |

Lamb EG, Booker HM (2011) Quantification of low-level genetically modified (GM) seed presence in large seed lots: a case study of GM seed in Canadian flax breeder seed lots. Seed Science Research 21, 315–321.
Quantification of low-level genetically modified (GM) seed presence in large seed lots: a case study of GM seed in Canadian flax breeder seed lots.Crossref | GoogleScholarGoogle Scholar |

Lane LA, Ayres JF, Lovett JV (1997) A review of the introduction and use of white clover (Trifolium repens L.) in Australia—significance for breeding objectives. Australian Journal of Experimental Agriculture 37, 831–839.
A review of the introduction and use of white clover (Trifolium repens L.) in Australia—significance for breeding objectives.Crossref | GoogleScholarGoogle Scholar |

Lane LA, Ayres JF, Lovett JV (2000) The pastoral significance, adaptive characteristics, and grazing value of white clover (Trifolium repens L.) in dryland environments in Australia: a review. Australian Journal of Experimental Agriculture 40, 1033–1046.
The pastoral significance, adaptive characteristics, and grazing value of white clover (Trifolium repens L.) in dryland environments in Australia: a review.Crossref | GoogleScholarGoogle Scholar |

Latch GCM, Skipp RA (1987) Diseases. In ‘White clover’. (Eds MJ Baker, WM Williams) pp. 421–446. (CAB International: Wallingford, UK)

Lewis CD, Malcolm B, Jacobs JL, Spangenberg G, Smith KF (2013) A method to estimate the potential net benefits of trait improvements in pasture species: Transgenic white clover for livestock grazing systems. AFBM Journal 10, 30–45.

Mather RDJ, Melhuish DT, Herlihy M (1995) Trends in the global marketing of white clover cultivars. In ‘White clover: New Zealand’s competitive edge’. Special Publication No. 11. pp. 7–14. (Agronomy Society of New Zealand)

Moar NT (1985) Pollen analysis of New Zealand honey. New Zealand Journal of Agricultural Research 28, 39–70.
Pollen analysis of New Zealand honey.Crossref | GoogleScholarGoogle Scholar |

Nageswara-Rao M, Kwit C, Agarwal S, Patton MT, Skeen JA, Yuan JS, Manshardt RM, Stewart CN (2013) Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.). BMC Biotechnology 13, 69
Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhsFSqsrjL&md5=7094444fb4ce658ee5069aba49419da4CAS | 24004548PubMed |

Panter S, Chu PG, Ludlow E, Garrett R, Kalla R, Jahufer MZZ, deLucas Arbiza A, Mouradov A, Smith KF, Spangenberg G (2012) Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of the AMV coat protein. Transgenic Research 21, 619–632.
Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of the AMV coat protein.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xms12gtrs%3D&md5=81dd19960bf8702359513bc93829bf0cCAS | 21947755PubMed |

Panter S, Mouradov A, Smith KF, Spangenberg G (2015) Development and validation of protocols for product stewardship in transgenic white clover (Trifolium repens L.): Detection of the AMV CP and npt2 transgenes in pollen, honey and honey bees. Crop & Pasture Science 66, 474–480.
Development and validation of protocols for product stewardship in transgenic white clover (Trifolium repens L.): Detection of the AMV CP and npt2 transgenes in pollen, honey and honey bees.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXns1Ggu7g%3D&md5=d80a7a4dede22909db268e115511f624CAS |

Potrykus G, Spangenberg G (Eds) (1995) ‘Gene transfer to plants 1.’ (Springer-Verlag: Berlin)

Sambrook J, Fritsch EF, Maniatis T (1989) ‘Molecular cloning: a laboratory manual.’ (Cold Spring Harbour Press: New York)

Smith KF, Forster JW, Spangenberg GC (2007) Converting genomic discoveries into genetic solutions for dairy pastures. Australian Journal of Experimental Agriculture 47, 1032–1038.
Converting genomic discoveries into genetic solutions for dairy pastures.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXos1Kgur4%3D&md5=1f112e9485625bcb4c0382a0a445d594CAS |

Xie DY, Sharma SB, Paiva NL, Ferreira D, Dixon RA (2003) Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 299, 396–399.
Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXjsF2rsw%3D%3D&md5=a60954e78ff53d1dc5ba4c45e0b1b964CAS | 12532018PubMed |