水稻悬浮细胞培养方法.pptVIP

水稻悬浮细胞培养的方法 * The flasks were set on a gyratory shaker agitated at 120 rpm under fluorescent light of 200-300 lux at 27°C. Cell growth in the R-2 medium was superior to that obtained in B5, Heller, Murashige-Skoog and White media. Plant CellPhysiol. 14: 1113-1121 (1973) Experimental cell research 50,151-158 (1968) Molec gen Genet 161 , 67 一 76 ( 1978 ) A japonica rice variety, Nackdong, was used for transformation by the Agrobacterium cocultivation method as described previously with the following modifications [14]. Calli were induced from the scutellum of mature seeds on an N6 medium containing 2 mg/l 2,4-D. An A. tumefaciens strain LBA4404 carrying the pGA1647 plasmid was grown for 3 days in an AB liquid medium supplemented with 30 mg/l hygromycin B and 3 mg/l tetracycline. Three-week-old calli were cocultivated with Agrobacterium on a 2N6-As medium supplemented with 1 mM betaine for 2–3 days in darkness at 25 C. The cocultivated calli were washed with sterile water containing 100 mg/l cefotaxime, and incubated on an N6 medium containing 40 mg/l hygromycin B and 250 mg/l cefotaxime for 3 weeks. Actively growing calli were transferred onto a regeneration medium, MS media supplemented with 0.1 mg/l NAA, 2 mg/l kinetin, 2% sorbitol, 1.6% phytagar (Gibco), 50 mg/l hygromycin B, and 250 mg/l cefotaxime. After 2–3 weeks under continuous light (40 mol m??2 s??1), plantlets were potted and grown in a growth chamber with 10 h light per day. Plant Molecular Biology 39: 35–44, 1999. NB Basic N6 major salts and iron source (Chu 1975), B5 minor salts and vitamins (Gamborg et al. 1968), 30 g/l sucrose NB NB Basic + 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 500 mg/l proline, 500 mg/l glutamine, 300 mg/l casein hydrolysate, 2.6 g/l Phytagel, pH 5.8 (Li et al. 1993) R2 Basic R2 major and minor salts, vitamins and iron source (Ohira et al. 1973), 2.5 mg/l 2,4-D CCL R2 Basic + 10 g/l glucose, 100 mM acetosyringone, pH 5.2 liquid co-culture medium CCS CCL + 7 g/l agarose