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Institut für Sonderkulturen und Produktionsphysiologie

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Browsing Institut für Sonderkulturen und Produktionsphysiologie by Person "Khidr, Yehia"

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    Development of a strategy to induce RNA-silencing in squash against virus diseases by genetic transformation
    (2007) Khidr, Yehia; Reustle, Götz
    Viral diseases of Cucurbits are an important limitation in the production of the crop. Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus-2 (WMV-2) are the most important squash (Cucurbita pepo L.) infecting viruses. Mixed infections with these viruses are deleterious for cucurbitaceous plants leading annually to significant yield losses world wide. All varieties of economical importance are susceptible for these viruses and classical breeding did not yield resistance. Therefore, a transgenic approach was chosen to induce resistance against both viruses by post-transcriptional gene silencing (PTGS). Highly conserved regions in the coat protein genes of ZYMV and WMV-2 were chosen to establish an inverted repeat construct. This construct was cloned into binary vector under control of the 35S promoter. Embryogenic callus was induced from different organs of three squash cultivars as target tissues for Agrobacterium transformation. The embryogenic callus was developed within 13-20 weeks incubation on MS medium containing different plant growth regulator combinations of auxin and cytokinin. Induction of embryogenesis in different explants ranged from 5 to 100 % depending on the organ and genotype used. Efficiency of embryo maturation, conversion and germination into entire plants from squash embryogenic callus was found to be callus age depended. Regeneration with young (2 months old) material was efficient, whereas regeneration of material maintained under in vitro conditions for more than 2 years was not possible. Agrobacterium-mediated transformation of squash embryogenic callus was established using transient GUS-gene expression. The highest transformation efficiency was obtained with the supervirulent ATHV strain, bacterial density of 0.85, washing procedure of the embryogenic material prior to Agrobacterium co-culture, application of 1 mM Acetosyringone in induction medium and sub-culturing of embryogenic callus on fresh MS medium 5-9 days prior co-culturing with the Agrobacterium. Selection strategy was optimized using GFP as reporter gene. For the genotype CX3006 300 mg/l Kanamycin showed the highest number of green areas but most efficient selection agent was Paromomycin 150 mg/l. For the genotype Dundoo 200 mg/l Paromomycin was the effective selection agent and showed the highest number of green areas. Selection of transformed calli could be efficient with the used selection agents but regeneration of transgenic plant was not possible because the old material was only one to be used for transformation experiments. It seems that these old materials may have lost their competency when they were maintained for long term in tissue culture. Therefore, the functionality of the inverted repeat construct was evaluated in Nicotiana benthamiana as a model plant. Transgenic lines were analyzed by PCR, Southern blot analysis and segregation analysis of T1 offspring. The transgene-induced PTGS in transgenic lines was confirmed by infiltration of GFP-sensor constructs containing viral derived sequences as silencing target and /or a construct containing the p19 silencing suppressor. In all transgenic lines tested, GFP fluorescence in infiltrated leaves was extinguished three days post-infection with GFP-sensor constructs. In contrast, all transgenic lines showed GFP fluorescence in infiltrated leaves when GFP-sensor constructs were co-infiltrated with a binary vector containing the viral silencing suppressor p19. With this work, tools have been developed to engineer virus resistance in squash. Using the optimized Agrobacterium-mediated transformation procedure together with the efficient RNA-silencing of the inverted inverted repeat construct and freshely induced embryogenic material it is quite possible to establish virus resistance in squash.