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.

The ploidy level of transgenic plants in Agrobacterium-mediated transformation of tomato cotyledons ( Lycopersicon esculentum Mill.) is genotype and procedure dependent [corrected].

A protocol avoiding the feeder-layer cell system was optimized for Agrobacterium-mediated transformation of tomato cotyledonary explants. Over 500 transgenic plants from five tomato cultivars were regenerated in 15 independent experiments. Depending on both genotype and procedure, transformation frequencies ranged from 1.8% to 11.3%. The optimal transformation rate was obtained by inoculating explants with a bacterial suspension in exponential growth ( D(600) = 10(2)-10(3) cells/ml) and transferring cotyledon explants to fresh selective regeneration medium every 3 weeks. The ploidy level of both tomato genotypes used as explant source and primary transformants, was studied by flow cytometry. The inbred lines and cultivars were diploid but a polysomatic pattern in the cotyledon explant was confirmed. The rate of tetraploid transgenic plants ranged from 24.5% to 80% and depended on both the genotype and the transformation procedure. Surprisingly, the percentages of transformed plants with higher ploidy levels were not related to the proportion of 4C and 8C nuclei in the cotyledonary tissue. For some genotypes the optimisation of the transformation rate resulted in an increase of tetraploid transgenic plants. Results obtained in this work indicate the convenience of checking the ploidy level of the primary transformants before performing basic studies or introducing tomato transgenic material in a breeding program.

Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt tolerance.

An Agrobacterium-mediated gene transfer method for production of transgenic melon plants has been optimized. The HAL1 gene, an halotolerance gene isolated from yeast, was inserted in a chimaeric construct and joined to two marker genes: a selectable-neomycin phosphotransferase-II (nptII)-, and a reporter-beta-glucuronidase (gus)-. The entire construct was introduced into commercial cultivars of melon. Transformants were selected for their ability to grow on media containing kanamycin. Transformation was confirmed by GUS assays, PCR analysis and Southern hybridization. Transformation efficiency depended on the cultivar, selection scheme used and the induction of vir-genes by the addition of acetosyringone during the cocultivation period. The highest transformation frequency, 3% of the total number of explants cocultivated, was obtained with cotyledonary explants of cv. 'Pharo'. Although at a lower frequency (1.3%), we have also succeeded in the transformation of leaf explants. A loss of genetic material was detected in some plants, and results are in accordance with the directional model of T-DNA transfer. In vitro cultured shoots from transgenic populations carrying the HAL1 gene were evaluated for salt tolerance on shoot growth medium containing 10 gl-1 NaCl. Although root and vegetative growth were reduced, transgenic HAL1-positive plants consistently showed a higher level of tolerance than control HAL1-negative plants.

Plant regeneration and Agrobacterium-mediated transformation of cotyledon explants of Citrullus colocynthis (L.) Schrad.

The effect of 6-benzylaminopurine (6-BA) alone or in combination with naphthaleneacetic acid or indoleacetic acid on the morphogenetic response of cotyledon explants of Citrullus colocynthis (L.) Schrad. was tested. The best results were obtained with a medium containing 25 µM 6-BA, which yielded organogenic calli at a frequency of 81.8%. When these organogenic calli were transferred to elongation medium (basal medium supplemented with 0.5 µM 6-BA), 80% produced well-developed shoots. These shoots rooted normally when cultured on rooting medium containing indolebutyric acid at 2.5 or 5.0 µM. Plants grew to maturity under greenhouse conditions and gave normal fruits. Cotyledon explants were transformed by cocultivation with Agrobacterium tumefaciens LBA4404 carrying the binary vector pBI121 which bears the reporter gene ß-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII). Transformants were selected for growth capacity on medium with 100 mgl-1 of kanamycin. On the basis of ß-glucuronidase expression, the transformation frequency was 14.2%. Molecular characterization by polymerase chain reaction confirmed the presence of the two genes transferred (gus, nptII) in the transgenic plants. Sexual transmission of both genes was also confirmed by studying their expression in progenies from several transgenic plants.