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Molecular cloning and expression of a vacuolar Na+/H+ antiporter gene (AgNHX1) in fig (Ficus carica L.) under salt stress

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Abstract

Soil salinity can be a major limiting factor for productivity in agriculture and forestry and in order to fully utilize saline lands productively in plantation forestry for fig production, the genetic modification of tree species for salt tolerance may be required. Na+/H+ antiporters have been suggested to play important roles in salt tolerance in plants. Here, we isolated AgNHX1 a vacuolar Na+/H+ antiporter from a halophytic species Atriplex gmelini and introduced it into fig (Ficus carica L.) cv. Black Mission via Agrobacterium-mediated transformation. Leaf discs explants of fig were co-cultivated for 2 days with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the AgNHX1 gene and the hpt selectable marker gene which encodes hygromycin phosphotransferase. Explants were cultured on MS medium containing 30 mg L−1 hygromycin, 3 % sucrose, 0.2 mg L−1 kinetin and 2.0 mg L−1 2,4-dichlorophenoxyacetic acid solidified with 2.5 g L−1 phytagel in darkness for callus formation. The calli were cultured on MS medium containing 2.0 mg L−1 zeatin riboside in combination with 0.4 mg L−1 indole acetic acid in the light for plant regeneration. Putative regenerated transformant shoots were confirmed by polymerase chain reaction (PCR) and Southern hybridization for the AgNHX1 gene. Reverse transcriptase polymerase chain reaction analysis indicated that the gene was highly expressed in transgenic plants, but the degree of this expression varied among transformants. Overexpression of the AgNHX1 gene conferred high tolerance to salt stress and transgenic fig plants overexpressing AgNHX1 developed normally under salinity conditions compared to those of non-transgenic plants. Salt treated transgenic plants contained high proline and K+ but less Na+ compared to non-transgenic control plants.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

2ip:

N-6-(Δ2-isopentenyl) adenine

BAP:

Benzylaminopurine

CTAB:

Cetyltrimethylammonium bromide

Hpt :

Hygromycin phosphotransferase

IAA:

Indole acetic acid

IBA:

Indole-3-butaric Acid

LB:

Luria broth medium

NAA:

α-Naphthaleneacetic acid

OD:

Optical density

ORF:

Opening reading frame

PCR:

Polymerase chain reaction

RT-PCR:

Reverse transcriptase polymerase chain reaction

TDZ:

Thiadiazuron (N-phenyl-N-1,2,3,-thiadiasol-5-ylurea)

ZR:

Zeatin riboside

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Acknowledgments

This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant number (3-965-35-RG). The authors, therefore, acknowledge with thanks DSR technical and financial support. The authors are thankful to the Tissue culture and Biotechnology Labs., Marout Research Station, Desert Research Center, Egypt.

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Authors and Affiliations

  1. Biological Science Department, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia

    Ehab M. R. Metwali & H. S. Al-Zahrani

  2. Botany Department, Faculty of Agriculture, Suez Canal University, Ismailia, 41522, Egypt

    Ehab M. R. Metwali

  3. Plant Genetic Resources Department, Desert Research Center, El-Matariya, Cairo, 11753, Egypt

    Hemaid I. A. Soliman

  4. School of Biological Sciences, Faculty of Science and Environment, Plymouth University, Room 304, 3rd Floor Link Building, Plymouth, PL4 8AA, UK

    M. P. Fuller

  5. Biology Department, Faculty of Science, Albaha University, Albaha, Saudi Arabia

    S. M. Howladar

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  1. Ehab M. R. Metwali
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Correspondence to Ehab M. R. Metwali.

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Metwali, E.M.R., Soliman, H.I.A., Fuller, M.P. et al. Molecular cloning and expression of a vacuolar Na+/H+ antiporter gene (AgNHX1) in fig (Ficus carica L.) under salt stress. Plant Cell Tiss Organ Cult 123, 377–387 (2015). https://doi.org/10.1007/s11240-015-0842-z

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