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

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Abstract

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.

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Abbreviations

AMV:

Alfalfa mosaic virus

CaMV:

Cauliflower mosaic virus

Nos:

Nopaline synthase

nptII:

Neomycin phosphotransferase

sgRNA4:

Subgenomic RNA4

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Acknowledgments

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.

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Authors and Affiliations

  1. Molecular Plant Breeding CRC, 1 Park Drive, Bundoora, VIC, 3083, Australia

    S. Panter, E. Ludlow, R. Garrett, R. Kalla, M. Z. Z. Jahufer, A. de Lucas Arbiza, A. Mouradov, K. F. Smith & G. Spangenberg

  2. Department of Primary Industries, Biosciences Research Division, Victorian AgriBiosciences Centre, Bundoora, VIC, 3086, Australia

    S. Panter, E. Ludlow, A. de Lucas Arbiza, S. Rochfort, A. Mouradov, K. F. Smith & G. Spangenberg

  3. CSIRO, Plant Industry, Canberra, ACT, 2601, Australia

    P. G. Chu

  4. La Trobe University, Bundoora, VIC, 3086, Australia

    A. de Lucas Arbiza, S. Rochfort, A. Mouradov & G. Spangenberg

Authors
  1. S. Panter
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  2. P. G. Chu
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  6. M. Z. Z. Jahufer
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  7. A. de Lucas Arbiza
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  9. A. Mouradov
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  10. K. F. Smith
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Corresponding author

Correspondence to G. Spangenberg.

Electronic supplementary material

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11248_2011_9557_MOESM1_ESM.pdf

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

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

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: http://www.bom.gov.au. (PDF 26 kb)

11248_2011_9557_MOESM4_ESM.pdf

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

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

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

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)

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Panter, S., Chu, P.G., Ludlow, E. et al. Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene. Transgenic Res 21, 619–632 (2012). https://doi.org/10.1007/s11248-011-9557-z

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