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Gladiolus plants transformed with single-chain variable fragment antibodies to Cucumber mosaic virus

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Abstract

Plants of Gladiolus ‘Peter Pears’ or ‘Jenny Lee’ were transformed with single-chain variable fragments (scFv) to Cucumber mosaic virus (CMV) subgroup I or II. The CMV subgroup I heavy and light chain scFv fragments were placed under control of either the duplicated CaMV 35S or sugarcane Ubi9 promoters. Integration of the transgenes was verified by Southern hybridization. Plants were challenged in vitro by inoculating with purified CMV using the Helios hand-held gene gun. An initial pathogen challenge was performed using six plants/plant line for the 51 lines containing the CMV subgroup I and 37 lines with the CMV subgroup II scFv fragments. Four plant lines with the CMV subgroup I scFv and four lines with the CMV subgroup II scFv were selected for further challenging. Less than 30% of the plants challenged from two plant lines with the CMV subgroup I scFv and three plant lines with the CMV subgroup II scFv contained CMV as compared to 70% for the non-transformed control plants. Less than 40% of the plants from two other plant lines with the CMV subgroup I scFv contained CMV 1–2 months after challenge. These eight plant lines that showed resistance and two susceptible lines expressed the transgene as determined by Northern hybridization and reverse transcriptase PCR.

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Abbreviations

scFv:

Single-chain variable fragment

CMV:

Cucumber mosaic virus

TSWV:

Tomato spotted wilt virus

Mab:

Monoclonal antibody

EDTA:

Ethylenediamine tetraacetic acid

SSC:

Citric acid trisodium salt

SDS:

Sodium dodecyl sulfate

PVP:

Polyvinylpyrrolidone

MS:

Murashige and Skoog’s medium

2,4-D:

2,4-Dichorophenoxyacetic acid

PPT:

Phosphinothricin

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Acknowledgments

Mention of a trademark, proprietary product or vendor does not imply its approval to the exclusion of other products or vendors that may also be suitable. Anne O’Connor is thanked for technical assistance with the DNA and RNA blots. Margaret Dienelt is thanked for using transmission electron microscopy to view our purified virus.

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Authors and Affiliations

  1. Floral and Nursery Plants Research Unit, U.S. National Arboretum, U.S. Department of Agriculture, Beltsville, MD, 20705, USA

    Kathryn Kamo, Mary Ann Guaragna, Charity James, Hei-ti Hsu & Ramon Jordan

  2. National Institute of Health/NIAID/MVDU, Rockville, MD, 20850, USA

    Joan Aebig

Authors
  1. Kathryn Kamo
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  2. Joan Aebig
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  3. Mary Ann Guaragna
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  4. Charity James
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  5. Hei-ti Hsu
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  6. Ramon Jordan
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Correspondence to Kathryn Kamo.

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Kamo, K., Aebig, J., Guaragna, M.A. et al. Gladiolus plants transformed with single-chain variable fragment antibodies to Cucumber mosaic virus . Plant Cell Tiss Organ Cult 110, 13–21 (2012). https://doi.org/10.1007/s11240-012-0124-y

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  • DOI: https://doi.org/10.1007/s11240-012-0124-y

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