水稻悬浮细胞培养.ppt

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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 50,1

2、51-158 (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

3、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 23 d

4、ays 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

5、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 m2 s1), plantlets were potted and grown in a growth chamber with 10 h light per day.Plant Molecular Biology 39: 3544, 1999.,NB Basic N6

6、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 and mino

7、r 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 6.0NBS NB B

8、asic + 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 hydrolysate,50 mg

9、/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 Skoog 1962), 50

10、 g/l sucrose, 2.6 g/l Phytagel, pH 5.8Theor Appl Genet (2003) 106:13961408,Rice (Oryza sativa L. cv. Nipponbare) was used in this study. Mature rice seeds were husked, sterilized with 70% ethanol for 5min and 1% NaClO for 30min. The seeds were washed with sterilized water and allowed to germinate on

11、 an agar plate of Murashige and Skoog (MS) medium 17 supplemented with 2mgL1 2,4-dichlorophenoxyacetic acid (2,4-D) and incubated at 25C in darkness. Cells were allowed to proliferate for 1month and were subcultured in MS medium supplemented with 1mgL1 2,4-D for rice cultured suspension cells. The m

12、edium was changed once every 2weeks during subculturing. To induce regeneration, the rice cultured suspension cells were spread on agar medium containing 0.01mgL1 naphthaleneacetic acid, 0.1mgL1 6-benzyladenine, 4% sucrose, 1% agarose and incubated at 25C under continuous light conditions.European J

13、ournal of BiochemistryVolume 267 Issue 3 Page 737-745, February 2000,Rice (Oryza sativa L. cv. Nipponbare) was used in this study.To induce regeneration, the rice cultured suspension cells were spread on agar medium containing 0.01 mg/L naphthaleneacetic acid, 0.1 mg/L 6-benzyladenine, 4% sucrose, 1

14、% agarose and incubated at 25 8C under continuous light conditions.Eur. J. Biochem. 267, 737745 (2000),In Vitro Binding of Agrobacterium tumefaciens to Plant Cellsfrom Suspension Culture. Plant Physiol. (1979) 63, 382-387Datura innoxia cells毛曼陀罗 Binding kinetics showed that adherence of bacteriato D

15、atura cells increased gradually during the first 60 minutes andattained a maximum level within 120 minutes of incubation. Maximumbinding occurred at pH 6.0. The presence of Ca2+ and Mg2e reducedbinding slightly and EDTA had little effect at concentrations of 0.1 to 10millimolar.,Stable Transformatio

16、n of Arabidopsis Cell CultureAgrobacteria carrying the pBlN plasmid were grown in YEB medium (0.5% w/v) beef extract, 0.5% w/v peptone, 0.1 % w/v yeast extract, 0.5% w/v sucrose, and 10 mM MgSO, pH 7.2) supplementedwith 250 mg/L streptomycin (Duchefa) and 50 mg/L kanamycin (Duchefa) at 28C to an OD,

17、oo of 1.5. Bacteria were collected by centrifugation (10 min at 4000 rpm) and resuspended in the same amount of cell culture medium. Four days after transfer to fresh medium, Arabidopsis cells (3 g fresh weight per 10 mL of medium) were incubated with 5 mL of an agrobacteria suspension in a Petri di

18、sh at 25C in the dark with gentle agitation (130 rpm). After 48 hr, the cells were loaded onto a nylon net and washed with excess of cell culture medium to remove most of the bacteria. Remaining cells were resuspended in 40 mL of culture media, vortexed vigorously for 20 sec, collected by a short ce

19、ntrifugation (1 min at 600g), and resuspended in fresh cell culture media. This procedure was repeated three times using 250 mg/L timenten (ticarcillin plus clavulanic acid, 151; Smith-Kline Beecham AG, Thorishaus, Switzerland) in the last solution. This antibiotic combination was highly effective i

20、n removing the remaining bacteria but had no effect on plant cell growth. Cells were plated on plant cell growth medium with 0.6% Gelrite (Merck), 250 mg/L timenten, and the indicated concentrations of kanamycin (see Results). The dishes were stored at 25C in the dark until calli formation was obser

21、ved, usually after 2 or 3 weeks.The Plant Cell, Vol. 9, 2171-2181, December 1997,Plant Physiol. (1979) 64, 374-378,路铁刚 Plant Science Volume 167, Issue 2, August 2004, Pages 281-288 T. Murashige and F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant

22、15 (1962), pp. 473479.21. L. Li, R. Qu, A. de Kochko, C. Fauquet and R.N. Beachy, An improved rice transformation system using the biolistic method. Plant Cell Rep. 12 (1993), pp. 250255. View Record in Scopus | Cited By in Scopus (96)22. C.C. Chu, C.C. Wang, C.S. Sun, C. Hsu, K.C. Yin, C.Y. Chu and

23、 F.Y. Bi, Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Scientia Sinica 5 (1975), pp. 659668.23. O.L. Gamborg, R.A. Miller and K. Ojima, Nutrient requirement suspension cultures of soybean root cells. Exp. Cell Res. 50 (1968), pp. 151158. Abstract | Article | PDF (629 K) | View Record in Scopus | Cited By in Scopus (2441),CC培养基配方,