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Genetic engineering of sunflower (Helianthus annuus L.) for resistance to necrosis disease through deployment of the TSV coat protein gene

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Abstract

Necrosis disease incited by tobacco streak virus (TSV) causes significant yield losses in sunflower in the tropics and sub-tropics. Genetic engineering of sunflower through deployment of the coat protein gene of TSV (TSV-CP) was attempted along with neomycin phosphotransferase gene (nptII) for selection of putative transformants on kanamycin. Split cotyledons from decoated mature seeds were used as target tissues for Agrobacterium tumefaciens-mediated transformation and putative transformed shoots were obtained with an average frequency of 3.3%. Presence of the introduced transgene was identified by PCR; expression was determined through RT-PCR, northern blot analysis and quantitative Real-time PCR (qRT-PCR); stable integration was ascertained through Southern blot analysis of plants in T0 to T4 generations. Out of 102 positive T0 events, 20 events were carried to T1 generation from which five events (CP-S-237, CP-S-247, CP-S-481, CP-S-648 and CP-S-753) were advanced till T4 generation. Challenge inoculations of plants of event No 481 showed resistance to necrosis disease and plants grew to maturity in TSV-CP transgenics while control plants (untransformed) showed mortality within 1–2 weeks following inoculation. Expression analysis of the TSV-CP and nptII genes in different tissues at flowering and seed setting stages revealed constitutive expression of the transgene till seed maturation. One event (No 481) was selected for transfer of the TSV-CP gene into agronomically superior genotypes.

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Abbreviations

BAP:

6-Benzylaminopurine

CP-MR:

Coat protein-mediated resistance

2-iP:

N6-[2-Isopentenyl] adenine

IAA:

Indole-3-acetic acid

MS:

Murashige and Skoog

NAA:

α-Naphthaleneacetic acid

NptII:

Neomycin phosphotransferase gene

PCR:

Polymerase chain reaction

SND:

Sunflower necrosis disease

TDZ:

1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)

TSV:

Tobacco streak virus

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Acknowledgements

The authors thank the Director, ICAR-Indian Institute of Oilseeds Research, Hyderabad for providing necessary facilities for carrying out the work and Dr. K.K. Sharma, ICRISAT, Hyderabad for extending the facilities for Real-time PCR analysis.

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Author notes

  1. Vasavi Singareddy and Vijay Reddy Sheri contributed equally.

Authors and Affiliations

  1. ICAR-Indian Institute of Oilseeds Research, Rajendranagar, Hyderabad, 500 030, India

    Vasavi Singareddy, Vijay Reddy Sheri, Tarakeswari Muddanuru, Revathi Tatineni, Chander Rao Sankaraneni, Varaprasad S. Kodeboyina & Sujatha Mulpuri

  2. ICAR-Indian Agricultural Research Institute, Pusa Campus, New Delhi, 110 012, India

    Rakesh Kumar Jain

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  1. Vasavi Singareddy
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Contributions

SV, SV, and TR assisted in the transformation work; MT helped in molecular analysis, RKJ provided the gene construct, SCR helped in virus challenge assays, KSV edited the manuscript, MS conceived and designed the research, analysed the results and prepared the manuscript. All authors read and approved the manuscript.

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Correspondence to Sujatha Mulpuri.

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The authors declare that they have no conflict of interest.

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Communicated by Yu-Jin Hao.

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Singareddy, V., Sheri, V.R., Muddanuru, T. et al. Genetic engineering of sunflower (Helianthus annuus L.) for resistance to necrosis disease through deployment of the TSV coat protein gene. Plant Cell Tiss Organ Cult 135, 263–277 (2018). https://doi.org/10.1007/s11240-018-1461-2

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