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Fusion gene construct preparation with lectin and protease inhibitor genes against aphids and efficient genetic transformation of Brassica juncea using cotyledons explants

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Abstract

Globally, Brassica juncea is one of the significant oilseed crops, which is commercially important and cultivated under diverse agro-climatic region preferably for its high oil content as well as superior oil quality. Biotic stress is one of the dominant factors which hinders plant growth and yield globally. Though substantial progress has been made in breeding and genetic manipulation of plants for imposing resistance, the task remains as a challenge even today. Here, in this study, two major defense genes of plant origin, lectin from lentil and protease inhibitors from chickpea, which have insecticidal activity against phytophagous insect pests have been isolated and stacked into one ORF by overlapping extension PCR and a fusion gene construct is prepared. For tissue specific expression of this fusion gene, phloem specific promoter rolC was utilized and the construct was mobilized into Agrobacterium tumefaciens strain GV3101. Further, genetic transformation of B. juncea cv. Varuna was performed with fusion gene using Agrobacterium-mediated genetic transformation. The regeneration of the transformed putative plants was screened with hygromycin selection. Fusion gene integration in randomly selected transformed plants gives 17% overall transformation efficiency by PCR and Southern hybridization. A quantitative real-time PCR analysis showed that the transcript level of the fusion gene was within 1.8–2.8-fold. The expression of the fusion gene was significantly higher in the third line (2.8-fold). The aphid resistance test demonstrated that inhibition of larval survivability is about 40%, and the extent of leaf damage measured was also diminished in the transgenic plants compared to non transformed control plants. This study provides insight the multi-toxin approach developed through overlap extension PCR in B. juncea and may facilitate genetic engineering for the potential improvement of resistance against aphids.

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Abbreviations

LL:

Lentil lectin gene

CPPI:

Chickpea protease inhibitor gene

ORF:

Open reading frame

CCM:

Co-cultivation medium

PCM:

Pre-cultivation medium

SRM:

Shoot regeneration and growth medium

SEM:

Shoot elongation and growth medium

SM:

Selection culture medium

RM:

Rooting medium

MS:

Murashige and Skoog culture medium

NTC:

Non-transgenic control

WT:

Wild type

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Acknowledgements

We are grateful to Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing the financial assistance to Prof. K.R. Koundal under Emeritus Scientist scheme to perform this research work. The authors are sincerely thankful to Dr. P. Anand Kumar, former Project Director of National Research Centre on Plant Biotechnology (NRCPB), New Delhi, India for his kind support.

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Authors and Affiliations

  1. NRC on Plant Biotechnology, Pusa Campus, New Delhi, 110012, India

    Sushma Rani, Alkesh Hada, R. C. Bhattacharya & K. R. Koundal

  2. Head of Department of Biosciences and Biotechnology, Banasthali University, Banasthali, Jaipur, India

    Vinay Sharma

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  1. Sushma Rani
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  2. Vinay Sharma
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  3. Alkesh Hada
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  4. R. C. Bhattacharya
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  5. K. R. Koundal
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Contributions

RK and RCB, conceived and designed the research. SR, performed experiments and wrote the manuscript. AH, analyzed the experimental data and helped in experimental and writing part. VS, contributed to part of conception and finalized the revised manuscript. All authors read and approved the manuscript.

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Correspondence to Sushma Rani.

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The author(s) declare that they have no competing interests.

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Communicated by J.-H. Liu.

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Rani, S., Sharma, V., Hada, A. et al. Fusion gene construct preparation with lectin and protease inhibitor genes against aphids and efficient genetic transformation of Brassica juncea using cotyledons explants. Acta Physiol Plant 39, 115 (2017). https://doi.org/10.1007/s11738-017-2415-8

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  • DOI: https://doi.org/10.1007/s11738-017-2415-8

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