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Improving fruit quality in tomato (Lycopersicum esculentum Mill) under heat stress by silencing the vis 1 gene using small interfering RNA technology

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Abstract

Constantly elevated temperatures cause an array of physio-biochemical changes in tomato (Lycopersicum esculentum Mill.) which make the fruit ripen quickly and up to 50 % yield loss. The development of tomato cultivars, using genetic engineering approaches which delay ripening, offers a new way to keep tomatoes healthy under heat stress. Over-expression of small heat shock protein gene, viscosity 1 (vis 1) plays a role in increasing juice viscosity, early ripening and tissue soften which emphasizes the importance of this gene in premature ripening. The aim of this work was therefore to develop a useful system for silencing the vis1 gene using small interfering RNA strategy. Agrobacterium strain GV3101 harbouring the binary vector pICBV19 containing the gus and bar genes was used to adapt the transformation process in this study. The primers were designed to amplified the first exon of the vis 1 gene and the amplified fragment was used for cloning into the pFGC5941 at XhoI/NcoI site at sense orientation then additional fragment was subsequently cloned at BamHI/XbaI to form sense/antisense cloned fragment interrupted by the CHSA-intron Agrobacterium strain LBA4404 with the binary vector pFGC5941 which harbors vis1 gene under the control of the 35S promoter containing bar gene under the control of a mannopine synthase 2′(Mas2′) as selectable marker, was used to reduce the expression of vis1 gene in fruit. Polymerase chain reaction (PCR), RT–PCR and northern blotting analysis were applied to detect putative transgenic plants. Significantly, silencing of vis 1 gene was potently occur and new transgenic tomato cultivars were produced with enhanced ripening qualities for recommendation for growing under heat stress.

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Abbreviations

BAP 6:

Benzylaminopurine

dsRNA:

Double strand RNA

IAA:

Indole-3-acetic acid

IR:

Inverted repeat

LB:

Luria broth medium

MCS:

Multiple cloning site

NAA 1:

Naphthaleneacetic acid

nt:

Nucleotide

RISC:

RNA induced silencing complex

RNAi:

RNA interference

shRNA:

Short hairpin RNA

siRNA:

Short interfering RNA

ZEA:

Zeatin

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Acknowledgments

This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, Saudi Arabia under Grant Number (965-003-D1434). The authors, therefore, acknowledge with thanks DSR technical and financial support.

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Authors and Affiliations

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

    Ehab M. R. Metwali & Omar A. Almaghrabi

  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, Plymouth, PL4 8AA, UK

    Michael P. Fuller

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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. Improving fruit quality in tomato (Lycopersicum esculentum Mill) under heat stress by silencing the vis 1 gene using small interfering RNA technology. Plant Cell Tiss Organ Cult 121, 153–166 (2015). https://doi.org/10.1007/s11240-014-0691-1

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