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Marker-free transgenic cucumber expressing Arabidopsis cbf1 gene confers chilling stress tolerance

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Biologia Plantarum

Abstract

Marker-free transgenic cucumber (Cucumis sativus L.) cv. Poinsett 76 SR plants were produced by Agrobacterium mediated transformation. A transformation efficiency of 1.62 was observed on using Agrobacterium tumefaciens strain LBA4404 harbouring Arabidopsis cbf1 gene driven by the inducible promoter RD29A in a binary vector system pCAMBIA. Transgene integration and single copy insert in transgenic cucumber was confirmed by polymerase chain reaction (PCR) and Southern blot analysis in T0 lines and also confirmed marker-free status in T1 generation. Transgene expression was confirmed by reverse transcription (RT)-PCR in T1 generation transgenic cucumber and advanced to T2 generation. Upon exposure to chilling stress (4 °C), the T2 generation transgenic plants survived up to 36 h; however, wild-type plants could not survive and gradually died. A significant decrease in membrane injury index (MII), increase in activities of antioxidant enzymes (SOD and CAT), free proline content and relative water content (RWC) in the leaves were observed in transgenic cucumber as compared to wild-type under chilling stress. Thus, the transgenic cucumber plants expressing Arabidopsis cbf1 gene conferred protection against chilling stress.

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Abbreviations

BAP:

6-benzylaminopurine

CAT:

catalase

CBF1:

c-repeat binding factor-1

CTAB:

cetyltrimethylammonium bromide

IBA:

indole-3-butyric acid

MII:

membrane injury index

MS:

Murashige and Skoog’s medium

NBT:

nitroblue tetrazolium chloride

nptII:

neomycin phosphotransferase-II

RT-PCR:

reverse transcription polymerase chain reaction

RWC:

relative water content

SOD:

superoxide dismutase

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Acknowledgements

Authors are grateful to DRDO HQ, Ministry of Defence, Govt. of India for the financial assistance. The generous gift of the co-transformation constructs by Dr. A.K. Sharma (Centre for Plant Molecular Biology, University of Delhi, South Campus, India) is gratefully acknowledged.

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Authors and Affiliations

  1. Defence Institute of Bio-Energy Research, Ministry of Defence, Haldwani, 263139, Uttarakhand, India

    N. Gupta, M. Rathore, D. Goyary, N. Khare, S. Anandhan & Z. Ahmed

  2. Department of Biotechnology, Kumaon University, Nainital, 263 001, Uttarakhand, India

    V. Pande

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  1. N. Gupta
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  2. M. Rathore
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  7. Z. Ahmed
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Correspondence to D. Goyary.

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Gupta, N., Rathore, M., Goyary, D. et al. Marker-free transgenic cucumber expressing Arabidopsis cbf1 gene confers chilling stress tolerance. Biol Plant 56, 57–63 (2012). https://doi.org/10.1007/s10535-012-0016-3

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  • DOI: https://doi.org/10.1007/s10535-012-0016-3

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