Transgenic watermelon lines expressing the nucleocapsid gene of <Emphasis Type="Italic">Watermelon silver mottle virus</Emphasis> and the role of thiamine in reducing hyperhydricity in regenerated shoots

Viral diseases are very detrimental to watermelon production. Watermelon silver mottle virus (WSMoV) is a major limiting factor for the production of watermelon and other cucurbit fruits. There are no effective natural sources of resistance to WSMoV, making transgenic resistance an appropriate solution for attenuating virus infection. Hyperhydricity is an important problem in watermelon culture in vitro, resulting from lower multiplication rates, poor quality shoots and tissue necrosis. In this study, we report an Agrobacterium-mediated genetic transfer protocol for commercial watermelon cultivars expressing the nucleocapsid (N) gene of WSMoV and a suitable approach to overcome hyperhydricity in watermelon culture in vitro. Murashige and Skoog (MS) salts containing Schenk and Hildebrandt (SH) vitamins + 50 mg l⁻¹ thiamine HCl could diminish the hyperhydric phenotype. The proximal halves of cotyledons from 3-day-old seedlings were cut into 1.5 × 1.5 mm segments as explants. Four days after co-cultivation, the explants were transferred to a selection medium for shoot regeneration. The putative transgenic shoots developed within 6 weeks of culture and were then transferred to stringent medium for 8 weeks to eliminate ‘escape type’ shoots. Fifty putative transgenic watermelon lines were obtained from three cultivars. PCR and Southern blot analysis confirmed that the foreign gene was incorporated into the genomic DNA of the transgenic lines.