In Vitro Regeneration and Genetic Transformation of Cucumis metuliferus through Cotyledon Organogenesis

Abstract

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 β-glucuronidase (GUS) assay. A total of nine transgenic plants were developed from 360 cotyledon explants, giving a transformation frequency of 2.5%.