The use of cotyledonary-node explants in Agrobacterium tumefaciensmediated transformation of cucumber (Cucumis sativus L.)

Abstract

Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic cucumber. Cotyledonary-node explants of cucumber (Cucumis sativus L. cv., Eunsung) were co-cultivated with Agrobacterium strains (EHA101) containing the binary vector (pPZP211) carrying with CaMV 35S promoter-nptII gene as selectable marker gene and 35S promoter-DQ gene (unpublished data) as target gene. The average of transformation efficiency (4.01%) was obtained from three times experiments and the maximum efficiency was shown at 5.97%. A total of 9 putative transgenic plants resistant to paromomycin were produced from the cultures of cotyledonary-node explants on selection medium. Among them, 6 transgenic plants showed that the nptII gene integrated into each genome of cucumber by Southern blot analysis.

Production of transgenic cucumber expressing phytoene synthase-2A carotene desaturase gene

“…The procedure of Agrobacterium-mediated transformation to develop transgenic cucumber (Cucumber sativus L. Cv. Eunsung), plants expressing phytoene synthase-2A carotene desaturase gene in this study basically followed the one described by Jang et al (2011). The estimate of transformation efficiency was based on the percentage of explants producing adventitious shoots growing on selection medium and the average number of plantlets regenerated per explant.…”

“…A subsequent vertical slice was made between the cotyledons, and then the embryonic axis was removed. The cotyledonary-node explants were used as a material for co-cultivation with Agrobacterium (Jang et al 2011).…”

“…The method of transformation and composition of the culture medium were performed according to Jang et al (2011). The cotyledonary-node explants were submerged suspension of Agrobacterium carrying pMBP-1 vector (GV3101) for 30 minutes, and then transferred to the co-cultivation medium without antibiotics for co-culture in the dark for three days ( Fig.…”

“…Up to now, the transformation protocols for cucumber have been optimized in various aspects involving in transformation methods, explants types, Agrobacterium strains, Agrobacterium concentration, length of exposure time to Agrobacterium, and selection markers, etc (Chee 1990;Kodama et al 1993;Kose and Koç 2003;Lin et al 2011;Kim et al 2008). Agrobacterium-mediated genetic transformation is proved to be a reliable way for transferring foreign genes into cucumber genome, and a stable transformation system has been improved without chimerical transgenic event (Jang et al 2011). Using the Agrobacteriummediated transformation technology, transgenic cucumber expressing TPSP gene or Nit gene related to abiotic stress resistance have recently been developed by our research team Jang et al 2013).…”

“…Using the Agrobacteriummediated transformation technology, transgenic cucumber expressing TPSP gene or Nit gene related to abiotic stress resistance have recently been developed by our research team Jang et al 2013). Using the stable transformation method (Jang et al 2011), we report here the development of a transgenic cucumber events expressing PAC gene to enhance beta-carotene content. The objectives of this study were to 1) evaluate the efficiency of the protocol of Agrobacterium-mediated transformation of cucumber to introduce PAC genes; 2) demonstrate the integration of PAC genes into the genome of putative transgenic cucumber based on analysis of Southern blot hybridization and gDNA-PCR; 3) evaluate the expression of PAC genes in transgenic cucumber based on analysis of RT-PCR and Northern blot hybridization.…”