Development of transgenic cucumber expressing TPSP gene and morphological alterations

Abstract

To develop transgenic cucumber tolerant to abiotic stress, a cotyledonary-node explants were co-cultivated with Agrobacterium tumefaciens (EHA101) carrying TPSP gene (pHC30-TPSP). After transfer to fresh medium every two week for eight weeks, putative transgenic plants were selected when shoots grown a length greater than 3 cm from the cotyledonary-node explants on selection medium supplemented with 5 mgl -1 phospinotricin as selectable agent. The confirmation of transgenic cucumber was based on the Northern blot analysis. Thirty four shoots (5.2%) with resistance to phospinotricin were obtained from 660 explants inoculated. Of them, transformants were only confirmed from 11 plants (1.7%). Transgenic cucumber expressing TPSP gene was more synthesized at 3.8 times amounts of trehalose (0.014 mg g fresh wt ). However, all of transgenic plants showed abnormal morphology, including stunted growth (< height 15 cm), shrunken leaves, and sterility as compared with non-transgenic plants (> height 150 cm) under the same growth environment. These results lead us to speculate that the overproduction of trehalose was toxic for cucumber, even though that had known for rice as non-toxic.

Current status of genetic transformation technology developed in cucumber (Cucumis sativus L.)

“…Cotyledonary node explants showed a very high regeneration frequency (94.3%) when incubated on MS medium supplementing with 6.66 μmol L -1 BA, and its transformation rate was approximately 4.8% (Wang et al 2013). However, the transformation efficiency of cotyledonary node explants was only 1.7% in another study (Kim et al 2010). Wang et al (2014) found the ratio of the transformation rate between cucumber cotyledon and hypocotyl explants was 2-fold.…”

“…In fact, Journal of Integrative Agriculture Advance Online Publication 2014 doi: 10.1016/S2095-3119(14)60899-6 target genes were introduced into cucumber genome mostly by Agrobacterium -mediated transformation approach. In the procedure of Agrobacterium-mediated transformation, the transformation frequency is depended on several main factors including plant regeneration frequency, plant genotype, Agrobacterium strains, bacterial concentration, length of exposure time to Agrobacterium, co-cultivation period, and selection system (Kose and Koç 2003;Yin et al 2005;Kim et al 2010).…”

“…In a word, cotyledon is a reliable explants used for cucumber transformation. Optimal sizes for different types of explants were variable, 4-6 mm for petiole, 0.5 cm 2 for leaf, and 2-3 cm for cotyledonary node, respectively (Sarmento et al 1992;Kim et al 2010). Rajagopalan and Perl-Treves (2005) demonstrated that dissecting cucumber cotyledons into proximal half by a V-shaped cut resulted in a higher transformation rate.…”

“…In a study using the Agrobacterium harboring npt II and bar genes for cucumber transformation, selection of transformed cucumber shoots with PPT led to successful results (Vengadesan et al 2005(Vengadesan et al , 2007Kim et al 2010). Selvaraj et al (2010) investigated the selection effects of kanamycin and PPT, and found that shoot regeneration from cotyledons was completely suppressed by adding kanamycin more than 100 mg L -1 and PPT greater than 2 mg L -1 , respectively, and no escapes were found in PPT selection at 2 mg L -1 level; a large percentage of regenerated shoots exhibited chimeric nature, compared to kanamycin selection.…”

“…With the development of transformation protocols of cucumber, lots of target genes have been transferred into cucumber to produce resistance cucumber plants to various diseases caused by viruses, bacteria and fungi, and environmental stresses. These genes include the coat protein of cucumber mosaic virus strain C gene (CMV-C) (Chee and Slightom 1991), CMV-O coat protein gene (Nishibayashi et al 1996b), chitinases genes (Kishimoto et al 2002(Kishimoto et al , 2004, cold-induced transcription activator CBF 3 gene (Deng et al 2004), abiotic resistant gene (Nit) (Jang et al 2013), trehalose-6-phosphate synthase and phosphatase (TPSP) gene (Kim et al 2010), and mitogen-activated protein kinase (MAPK) gene (Wang et al 2013).…”