The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].
An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.
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Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-C.S.I.C. Avenida de los Naranjos s/n. 46022 Valencia, Spain. pellul@upvnet.upv.es
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Atarés
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Roig
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Serrano
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Moreno
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D015152:Blotting, Northern; D015139:Blotting, Southern; D029773:Citrullus / Q000235:genetics* / Q000502:physiology; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D047908:Intracellular Signaling Peptides and Proteins; D019868:Kanamycin Kinase / Q000235:genetics; D030821:Plants, Genetically Modified / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D012965:Sodium Chloride
D004269:DNA, Bacterial; C076232:HAL1 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; C036483:T-DNA; D012965:Sodium Chloride; D019868:Kanamycin Kinase