作者：上海路阳生物时间：2022-01-04 09:22:45浏览981 次
近日北京大学先进农业科学研究所发表文献《Efficient transformation and genome editing of watermelon assisted by genes that encode developmental regulators》，文献中提及利用LUYOR-3415RG便携式荧光蛋白激发光源快速筛选dsred在西瓜上的表达。
Establishment of a watermelon transformation system
To improve the transformation efficiency of watermelon, we first established a fast and simple detection system for watermelon transformation. Both the neomycin phosphotransferase II (nptII) gene, which confers kanamycin (Kan) resistance in transgenic plants, and the hygromycin phosphotransferase II (hptII) gene, which confers hygromycin (Hyg) resistance, have been used as selectable markers in watermelon tissue culture. Although both Kan and Hyg are aminoglycoside antibiotics that act as protein synthesis inhibitors, Hyg causes more severe damage than Kan during watermelon tissue culture (Park et al., 2005), and the time required for tissue culture in Hyg medium is usually longer than required for Kan; thus, nptII was chosen as the selectable marker. In addition, given the high frequency of false positive plants obtained after Kan selection (Park et al., 2005), the DsRed2 fluorescent protein, which can be easily observed using the LUYOR-3415RG hand-held lamp (Luyor Corporation, Shanghai, China), was introduced into the binary vector as another marker to visually identify stable transgenic events (Figure 1a). The reporter vector was named pW501.
The genetic transformation was conducted as previously described with slight modifications (Tian et al., 2017). In brief, surface-sterilized watermelon seeds were sown on Murashige and Skoog (MS) solid medium for 3 days in the dark. Then the middle parts of the cotyledons without embryo were cut into 1.5 × 1.5 mm pieces as explants. A. tumefaciens strains harboring the indicated binary vectors were co-cultivated with the cotyledon fragments in the dark for 3 days on MS solid medium containing 1.5 mg/L 6-BA. Then the cotyledon fragments were transferred onto selective induction medium containing 2 mg/L 6-BA, 0.2 mg/L IAA, 50 mg/L Kan, and 100 mg/L Timentin. The regenerated adventitious buds were excised and transferred onto elongation medium containing 0.1 mg/L 6-BA, 0.01 mg/L NAA, 100 mg/L Timentin, and 50 mg/L Kan. Plants with full leaves and stems were transferredto rooting medium containing 1mg/L IBA and 100 mg/L Timentin. Positive transgenic events were detected using the hand-held dual-wavelength fluorescent protein excitation light source LUYOR-3415RG (Luyor Corporation, Shanghai, China) or Zeiss 357 SteREO Discovery.V20.