) In Vitro Regeneration and Genetic Transformation of Cucumis metuliferus via Cotyledon Organogenesis.

HortScience 04/2011; 46(4):616-621.


This study was undertaken to establish for the first time an efficient regeneration
and transformation system for Cucumis metuliferus line PI292190, which is
the source of a well-defined resistant gene, Wmv, that provides resistance against Papaya
ringspot virus type P (PRSV-P) and PRSV-W (formerly known as Watermelon mosaic
virus 1, WMV-1). Different combinations of growth regulators were evaluated for the
regeneration of cotyledon explants. Adventitious buds or shoot primordia were obtained
within 3 to 4 weeks on regeneration medium. After shoot development, adventitious buds
or shoot primordia were transferred to elongation medium for 3 to 4 weeks and these
shoots were subcultured onto rooting medium for another 1 to 2 weeks. Under optimal
culture conditions, a total of 7 to 10 weeks was necessary to obtain C. metuliferus plantlets
from cotyledons. Furthermore, transgenic plants were successfully obtained using an
Agrobacterium tumefaciens-mediated transformation method as shown by polymerase
chain reaction analysis and histochemical b-glucuronidase (GUS) assay. A total of nine
transgenic plants were

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    ABSTRACT: A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment length polymorphism (cDNA-AFLP) method. Transcript derived fragments (TDFs) identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future.
    Full-text · Article · Jul 2013 · PLoS ONE