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Creation of borer pests resistance genetically engineering peach (Prunus persica L.) plants by constitutively overexpressing the cry1Ab gene

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Abstract

The plasmid pBI121 cry1Ab used to transform peach explants to produce insect resistant plants was constructed by cloning the synthetic cryIAb gene with the intron of the castor bean catalase-1 gene into the pBI121 binary vector, under the control of the CaMV35S promoter. Leaf discs of peach (Prunus persica L.) were co-cultivated for two days with this construct in A. tumefaciens strain LBA 4404. Explants were plated on WPM medium supplemented with 125 mg L−1 kanamycin, 3% sucrose, 2.5 mg L−1 2,4-Dichlorophenoxyacetic acid, and 0.5 mg L−1 6-benzyladenine in darkness for callus formation. The calli were then selected on WPM medium supplemented with 125 mg L−1 kanamycin, 3% sucrose, 3.00 mg L−1 Thidiazuron, 1.0 mg L−1 kinetin, and 0.5 mg L−1 indoleacetic acid in the light for at least five subcultures (with a regeneration efficiency of 91.8%). The integration of the cry1Ab gene into the peach genome was confirmed by polymerase chain reaction (PCR) and northern blot analysis. A transformation efficiency of 28% was obtained when leaves were incubated for 15 min with A. tumefaciens. The cry1Ab gene expression was confirmed using RT-PCR, northern blot hybridization, immune-strip test, and insect bioassays, respectively. For insect bioassay, it was evident that the protein cryIAb expressed in transformed peach plants showed 100% mortality at 1000 ppm against Synanthedon exitiosa larvae after 96 h. The obtained results demonstrated a significant improvement of cryIAb toxin protein against lepidopteron larvae in peach.

Key message

In vitro peach regeneration from in vitro leaves is difficult, as well as regeneration using the method of genetic transfer such as agrobacterium to impart the desired characteristics of the plants with the aim of improvement is very difficult. This paper is the first to transgenic peach plants carrying cry1Ab gene for insect resistance particularly the peach tree borer.

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Abbreviations

BA:

6-Benzyladenine

bp:

Base pairs

Cry:

Crystal protein

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

2iP:

N-6-(Δ2-isopentenyl) adenine

Kn:

Kinetin

TDZ:

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

2,4-D:

2,4-Dichlorophenoxyacetic acid

WPM:

Woody plant medium

MS:

Murashige and Skoog’s medium

nptII:

Neomycin phosphotransferase

PCR:

Polymerase chain reaction;

RT-PCR:

Reverse transcriptase polymerase chain reaction

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Acknowledgements

The authors are thankful to the Tissue culture and Biotechnology Labs., Mariout Research Station, Desert Research Center, Egypt.

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Authors and Affiliations

  1. Department of Biology, College of Science, University of Jeddah, Jeddah, Saudi Arabia

    Naif M. Kadasa & Wafa A. Alshehri

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

    Ehab M. R. Metwali

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

    Hemaid I. A. Soliman

  4. Plant Tissue Culture and Biotechnology Labs., Mariout Research Station, Desert Research Center, Alexandria, Egypt

    Hemaid I. A. Soliman

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  1. Naif M. Kadasa
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  4. Wafa A. Alshehri
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Contributions

NK planned the whole work, performed the tissue culture experiments, helped write the draft and designed the figures and tables. EM analyzed data, helped in the write, participated in the discussion and results, contributed in the statistical analysis, and reviewed several drafts of the manuscript. HS conceived the research, performed the experiments, provided lab facilities, performed molecular analysis, and submitted the MS. WA helped in experimental accomplishment, reviewed and edited the manuscript. All authors contributed to the final manuscript. All authors read and approved the manuscript.

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Correspondence to Ehab M. R. Metwali.

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The authors declare that they have no conflict of interests. The authors do not have any competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.

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Kadasa, N.M., Metwali, E.M.R., Soliman, H.I.A. et al. Creation of borer pests resistance genetically engineering peach (Prunus persica L.) plants by constitutively overexpressing the cry1Ab gene. Plant Cell Tiss Organ Cult 148, 465–477 (2022). https://doi.org/10.1007/s11240-021-02198-w

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