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

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 27degC Cell growth in the R-2 medium was superior tothat obtained in B5 Heller Murashige-Skoog and White media Plant amp CellPhysiol 14 1113-1121 (1973)

Experimental cell research 50151-158 (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 mgl 24-D An A tumefaciens strain LBA4404 carrying the pGA1647 plasmid was grown for 3 days in an AB liquid medium supplemented with 30 mgl hygromycin B and 3 mgl tetracycline Three-week-old calli were cocultivated with Agrobacterium on a 2N6-As medium supplemented with 1 mM betaine for 2ndash3 days in darkness at 25 C The cocultivated calli were washed with sterile water containing 100 mgl cefotaxime and incubated on an N6 medium containing 40 mgl hygromycin B and 250 mgl cefotaxime for 3 weeks Actively growing calli were transferred onto a regeneration medium MS media supplemented with 01 mgl NAA 2 mgl kinetin 2 sorbitol 16 phytagar (Gibco) 50 mgl hygromycin B and 250 mgl cefotaxime After 2ndash3 weeks under continuous light (40 mol m10485762 s10485761) plantlets were potted and grown in a growth chamber with 10 h light per dayPlant Molecular Biology 39 35ndash44 1999

NB Basic N6 major salts and iron source (Chu 1975) B5 minor salts and vitamins (Gamborg et al 1968) 30 gl sucrose

NB NB Basic + 2 mgl 24-dichlorophenoxyacetic acid (24-D) 500 mgl proline 500 mgl glutamine 300 mgl casein hydrolysate 26 gl Phytagel pH 58 (Li et al 1993)

R2 Basic R2 major and minor salts vitamins and iron source (Ohira et al 1973) 25 mgl 24-D

CCL R2 Basic + 10 gl glucose 100 mM acetosyringone pH 52 liquid co-culture medium

CCS CCL + 7 gl agarose

R2S R2 Basic + 30 gl sucrose 50 mgl hygromycin 400 mgl cefotaxime 100 mgl vancomycine 7 gl agarose pH 60

NBS NB Basic + 25 mgl 24-D 500 mgl proline 500 mgl glutamine 300 mgl casein hydrolysate 50 mgl hygromycin400 mgl cefotaxime 100 mgl vancomycine 万古霉素 7 gl agarose pH 60

PRAG NB Basic + 2 mgl BAP 1 mgl NAA 5 mgl ABA 500 mgl proline 500 mgl glutamine 300 mgl casein hydrolysate50 mgl hygromycin 100 mgl cefotaxime 100 mgl vancomycine 7 gl agarose pH 58 预分化

RN NB Basic + 3 mgl benzylaminopurine 05 mgl a-naphthaleneacetic acid 30 gl sucrose 50 mgl hygromycina45 gl Phytagel pH 58

P MS major and minor salts vitamins and iron source (Murashige and Skoog 1962) 50 gl sucrose 26 gl Phytagel pH 58

Theor Appl Genet (2003) 1061396ndash1408

Rice (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 to germinate on an agar plate of Murashige and Skoog (MS) medium [17] supplemented with 2 mgmiddotLminus1 24-dichlorophenoxyacetic acid (24-D) and incubated at 25 degC in darkness Cells were allowed to proliferate for asymp 1 month and were subcultured in MS medium supplemented with 1 mgmiddotLndash1 24-D for rice cultured suspension 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 001 mgmiddotLminus1 naphthaleneacetic acid 01 mgmiddotLminus1 6-benzyladenine 4 sucrose 1 agarose and incubated at 25 degC under continuous light conditionsEuropean Journal of BiochemistryVolume 267 Issue 3 Page 737-745 February 2000

Rice (Oryza sativa L cv Nipponbare) was used in this studyTo induce regeneration the rice cultured suspension cells were spread on agar medium containing 001 mgL naphthaleneacetic acid 01 mgL 6-benzyladenine 4 sucrose 1 agarose and incubated at 25 8C under continuous light conditionsEur J Biochem 267 737plusmn745 (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 Datura cells increased gradually during the first 60 minutes andattained a maximum level within 120 minutes of incubation Maximumbinding occurred at pH 60 The presence of Ca2+ and Mg2e reducedbinding slightly and EDTA had little effect at concentrations of 01 to 10millimolar

Stable Transformation of Arabidopsis Cell Culture

Agrobacteria carrying the pBlN plasmid were grown in YEB medium (05 [wv]) beef extract 05 [w

v] peptone 01 [wv] yeast extract 05 [wv] sucrose and 10 mM MgSO pH 72) supplemented

with 250 mgL streptomycin (Duchefa) and 50 mgL kanamycin (Duchefa) at 28degC to an ODoo of 15

Bacteria were collected by centrifugation (10 min at 4000 rpm) and resuspended in the same amount of c

ell 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 dish at 25degC in the da

rk with gentle agitation (130 rpm) After 48 hr the cells were loaded onto a nylon net and washed with e

xcess of cell culture medium to remove most of the bacteria Remaining cells were resuspended in 40 m

L of culture media vortexed vigorously for 20 sec collected by a short centrifugation (1 min at 600g) a

nd resuspended in fresh cell culture media This procedure was repeated three times using 250 mgL time

nten (ticarcillin plus clavulanic acid 151 Smith-Kline Beecham AG Thorishaus Switzerland) in the las

t solution This antibiotic combination was highly effective in removing the remaining bacteria but had n

o effect on plant cell growth Cells were plated on plant cell growth medium with 06 Gelrite (Merck)

250 mgL timenten and the indicated concentrations of kanamycin (see Results) The dishes were stored

at 25degC in the dark until calli formation was observed usually after 2 or 3 weeks

The Plant Cell Vol 9 2171-2181 December 1997