1、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 27C. Cell growth in the R-2 medium was superior tothat obtained in B5, Heller, Murashige-Skoog and White media. Plant & CellPhysiol. 14: 1113-1121 (1973)Experimental cell research 50
2、,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 conta
3、ining 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 fo
4、r 23 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 supplem
5、ented 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 23 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: 3544, 1999.NB Ba
6、sic N6 major salts and iron source (Chu 1975), B5 minor salts and vitamins (Gamborg et al. 1968), 30 g/l sucroseNB 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 a
7、nd minor salts, vitamins and iron source (Ohira et al. 1973), 2.5 mg/l 2,4-DCCL R2 Basic + 10 g/l glucose, 100 mM acetosyringone, pH 5.2 liquid co-culture mediumCCS CCL + 7 g/l agaroseR2S R2 Basic + 30 g/l sucrose, 50 mg/l hygromycin, 400 mg/l cefotaxime, 100 mg/l vancomycine, 7 g/l agarose, pH 6.0N
8、BS NB Basic + 2.5 mg/l 2,4-D, 500 mg/l proline, 500 mg/l glutamine, 300 mg/l casein hydrolysate, 50 mg/l hygromycin,400 mg/l cefotaxime, 100 mg/l vancomycine,万古霉素 7 g/l agarose, pH 6.0PRAG NB Basic + 2 mg/l BAP, 1 mg/l NAA, 5 mg/l ABA, 500 mg/l proline, 500 mg/l glutamine, 300 mg/l casein hyd
9、rolysate,50 mg/l hygromycin, 100 mg/l cefotaxime, 100 mg/l vancomycine, 7 g/l agarose, pH 5.8预分化RN NB Basic + 3 mg/l benzylaminopurine, 0.5 mg/l a-naphthaleneacetic acid, 30 g/l sucrose, 50 mg/l hygromycina,4.5 g/l Phytagel, pH 5.8P MS major and minor salts, vitamins and iron source (Murashige and S
10、koog 1962), 50 g/l sucrose, 2.6 g/l Phytagel, pH 5.8Theor Appl Genet (2003) 106:13961408Rice (Oryza sativa L. cv. Nipponbare) was used in this study. Mature rice seeds were husked, sterilized with 70% ethanol for 5 min and 1% NaClO for 30 min. The seeds were washed with sterilized water and allowed
11、to germinate on an agar plate of Murashige and Skoog (MS) medium 17 supplemented with 2 mgL1 2,4-dichlorophenoxyacetic acid (2,4-D) and incubated at 25 C in darkness. Cells were allowed to proliferate for 1 month and were subcultured in MS medium supplemented with 1 mgL1 2,4-D for rice cultured susp
12、ension cells. The medium was changed once every 2 weeks during subculturing. To induce regeneration, the rice cultured suspension cells were spread on agar medium containing 0.01 mgL1 naphthaleneacetic acid, 0.1 mgL1 6-benzyladenine, 4% sucrose, 1% agarose and incubated at 25 C under continuous light conditions.European Journal of BiochemistryVolume 267 Issue 3 Page 737-745, February 2000
