转基因相关药品配制

1、转基因相关药品配制stock solutionMSmax stock solution（10×）NH4NO3 16.5gKNO3 19gKH2PO4 1.7gMgSO4·7H2O 3.7gCaCl2·2H2O 4.4g or CaCl2 3.32gDissolve them one by one .Then add H2O to1000ml.MSmin stock solution (100×)KI 0.083gH3BO3 0.62gMnSO4·4H2O 2.23g or MnSO4·H2O 1.69gZnSO4·7H2O

2、0.86g Na2MoO4·2H2O 0.025gCuSO4·5H2O 0.0025gCoCl2·6H2O 0.0025gNote：Na2MoO4 must be dissolve independently and then mix with the other composition .Add H2O to 1000ml and store at room temperature.N6max stock solution（10×）KNO3 28.3gKH2PO4 4g(NH4)2SO4 4.63gMgSO4·7H2O 1.85gCaCl2&

3、#183;2H2O 1.66g or CaCl2 1.25gDissolve them one by one .Then add H2O to1000ml and store at room temperature.N6min stock solution (100×)KI 0.08gH3BO3 0.16gMnSO4·4H2O 0.44g or MnSO4·H2O 0.3335gZnSO4·7H2O 0.15gAdd H2O to1000ml and store at room temperature.Fe2+EDTA stock solution (1

4、00×)2.78g FeSO4·7H2O in a bottle containing 300ml H2O，3.73g Na2EDTA·H2O in anther bottle containing 300ml H2O and heat to 70，after they both dissolved，mix together and keep at 70 for 2 hours, then add H2O to1000ml and store at 4. Vitamin stock solution (100×)Nicotinic 0.1g Pyrido

5、xine HCl（VB6） 0.1gThiamine HC l（VB1） 0.1gGlycine 0.2gInositol 10gAdd H2O to1000ml and store at 4.AAmax stock solution（10×）KCl 29.5gNaH2PO4 1.5g or NaH2PO4·2H2O 1.95gMgSO4·7H2O 2.5gCaCl2·2H2O 1.5g or CaCl2 1.13gAdd H2O to1000ml and store at room temperature.AAmin stock solution (1

6、00×)MnSO4·H2O 1gH3BO3 0.3gZnSO4·7H2O 0.2gKI 0.075gNa2MoO4·2H2O 0.025gCuSO4·5H2O 0.0025gCoCl·6H2O 0.0025gNote：Na2MoO4 must be dissolve independently and then mix with the other composition add H2O to 1000ml and store at room temperature.A stock solution （10×）NH4NO3

7、16.5gKNO3 19gKH2PO4 1.7gMgSO4·7H2O 3.5gCaCl2 3gAdd H2O to1000ml and store at room temperature.B stock solution (100×)MnSO4·H2O 10gH3BO3 3gZnSO4·7H2O 2gKI 0.75gNa2MoO4·2H2O 0.25gCuSO4·5H2O 0.0389gCoCl·6H2O 0.025gNote：Na2MoO4 must be dissolve independently and then m

8、ix with the other composition add H2O to 1000ml and store at room temperature.2,4-D stock solution(1mgml)2,4-D 100mgAdd 1ml 1N KOH and shake for 5min ,and add 10ml H2O and shake till 2,4-D is dissolved ,then add H2O to100ml and store at 4.6-BA stock solution(1mgml)6-BA 100mgAdd 1ml 1N KOH and shake

9、till 6-BA is dissolved, then add H2O to100ml and store at 4.KT stock solution(1mgml)KT 100mgAdd 1ml 1N KOH and shake till KT is dissolved, then add H2O to100ml and store at 4.NAA stock solution(1mgml)NAA 100mgAdd 1ml 1N KOH and shake till NAA is dissolved, then add H2O to100ml and store at 4.IAA sto

10、ck solution(1mgml)IAA 100mgAdd 1ml 1N KOH and shake till IAA is dissolved, then add H2O to100ml and store at 4.1N KOH stock solutionKOH 5.6gDissolve with 100ml H2O and store at room temperature. 100mM AS stock solutionAS 0.196gDMSO 10mlAliquot to1.5ml eppendorf tube and store at 4. 1N KOH stock solu

11、tionKOH 5.6gDissolved with 100mL H2O and store at room temperature. 50% Glucose stock solutionGlucose 50g Dissolve with H2O to100ml，autoclave.MediumsNote：All mediums should be freshly prepared before use. Induction medium (for japonica rice)N6max stock solution（10×） 100mLN6min stock solution（10

12、0×） 10mLFe2+EDTA stock solution（100×） 10mLVitamin stock solution（100×） 10mL2,4-D stock solution 2.5mLProline 0.3gCasein Enzymatic Hydrolysate 0.6gSucrose 30gPhytagel 3gAdd H2O to 600-700mL and use 1N KOHadjust pH to 5.9. Boll and add H2O to 1000mL. Aliquot it to 50mL flask (25mL/flask

13、). Seal with sealfilm and autoclave them. Subculture medium (for japonica rice)N6max stock solution（10×） 100mLN6min stock solution（100×） 10mLFe2+-EDTA stock solution（100×） 10mLVitamin stock solution（100×） 10mL2,4-D stock solution 2.0mLProline 0.5gCasein Enzymatic Hydrolysate 0.6g

14、Sucrose 30gPhytagel 3gAdd H2O to 900mL and use 1N KOH,adjust pH to 5.9. Boll and add H2O to 1000mL. Aliquot it to 50mL flask (25mL/flask). Seal with sealfilm and autoclave them. Precultivation medium (for japonica rice)N6max stock solution（10×） 12.5mLN6min stock solution（100×） 1.25mLFe2+-E

15、DTA stock solution（100×） 2.5mLVitamin stock solution（100×） 2.5mL2,4-D stock solution 0.75mLCasein Enzymatic Hydrolysate 0.15gSucrose 5gAgarose 1.75gAdd H2O to 250mL andadjust pH to 5.6 using by 1N KOH,seal with sealfilm and autoclave it.Before use ,melt the medium and add 5mL Glucose stock

16、 solution（50% Glucose stock solution autoclaved）and 250L AS stock solution,then aliquot to plates（25mL medium per plate） Cocultivation medium (for japonica rice)N6max stock solution（10×） 12.5mLN6min stock solution（100×） 1.25mLFe2+-EDTA stock solution（100×） 2.5mLVitamin stock solution（

17、100×） 2.5mL2,4-D stock solution 0.75mLCasein Enzymatic Hydrolysate 0.2g Sucrose 5gAgarose 1.75gAdd H2O to 250mL adjust pH to 5.6 using by 1N KOH,seal with sealfilm and autoclave it. Before use ,melt the medium and add 5mL Glucose stock solution（50% Glucose stock solution autoclaved）and 250L AS

18、stock solution,then aliquot to plates（25mL medium per plate）.不用调 Agrobecterium suspension（100ml）N6max stock solution（10×） 5mLN6min stock solution（100×） 0.5mLFe2+-EDTA stock solution（100×） 0.5mLVitamin stock solution（100×） 1mL2,4-D stock solution 0.2mLCasein Enzymatic Hydrolysate

19、0.08gSucrose 2gAdd H2O to 100mL and adjust pH to 5.4 and aliquot to two 100mL bottle（50mL per bottle）.Seal with sealfilm and autoclave them.Before use add 1mL Glucose stock solution（50% Glucose stock solution autoclaved）（per 50mL）and 100L AS stock solution（per 50mL）. Selection medium (for japonica r

20、ice)N6max stock solution（10×） 25mLN6min stock solution（100×） 2.5mLFe2+-EDTA stock solution（100×） 2.5mLVitamin stock solution（100×） 2.5mL2,4-D stock solution 0.625mLCasein Enzymatic Hydrolysate 0.15gSucrose 7.5gAgarose 1.75gAdd H2O to 250mL andadjust pH to 6.0；Seal with sealfilm a

21、nd autoclave it. After autoclaved,add hygromycin 50g/mL.5.20phBefore use,melt the medium and add 250L Hn and add 500mg Cn per 1L，then aliquot to plates（25mL medium per plate）. Prepegeneration medium (for japonica rice)MSmax stock solution（10×） 25mLMSmin stock solution（100×） 2.5mLFe2+-EDTA

22、stock solution（100×） 2.5mLVitamin stock solution（100×） 2.5mL6-BA stock solution 0.5mLKT stock solution 0.5mLNAA stock solution 50LIAA stock solution 50LCasein Enzymatic Hydrolysate 0.15gSucrose 7.5gAgarose 1.75gAdd H2O to 250mL andadjust pH to 5.9，seal with sealfilm and autoclave it. Befor

23、e use,melt the medium and add 250L Hn and 200ppm Cn（国产），then aliquot to plates（25mL medium per plate）. Regeneration medium (for japonica rice)MSmax stock solution（10×） 100 mLMSmin stock solution（100×） 10 mLFe2+-EDTA stock solution（100×） 10 mLVitamin stock solution（100×） 10 mL6-BA

24、 stock solution 2 mLKT stock solution 2 mLNAA stock solution 0.2mLIAA stock solution 0.2mLCasein Enzymatic Hydrolysate 1 gSucrose 30 gPhytagel 3gAdd H2O to 1000mL and adjust pH to 6.0 using by 1N KOH，then boil them and aliquot them in 100mL flask（50mL medium per flask）.Seal with sealfilm and autocla

25、ve them. Add 500mg/L 头孢（国产）。5.4 Rooting medium (for japonica rice)MSmax stock solution（10×） 50 mLMSmin stock solution（100×） 5 mLFe2+-EDTA stock solution（100×） 10 mLVitamin stock solution（100×） 10 mLSucrose 20 gPhytagel 3gAdd H2O to 1000mL andadjust pH to 5.8 using by 1N KOH，then

26、boil them and aliquot them to cuboid vessel, then autoclave them.加NAA 0.4mg4.72proceduresCallus induction:1.Dehull the mature seeds and sterilize with 0.1% HgCl2粳稻 or 0.1% NaClO籼稻 30 min.2.Wash them with sterilize water 4-5 times.3.Incubate the seeds in callus induction medium.4.Put in the dark at 2

27、6 for about 4 weeks.Notice: choose the seeds which are good matured are relatively transparent and plump .and make sure that the sterile water is prepared before you sterilize the seeds .(so dose every step when the sterilized water ,bottles,fliter paper are needed)Callus subculture:Pick the light y

28、ellow compact and relatively dry embryogenitic and incubate on subculture medium then put it in dark at 26 for 2 weeks.Precultivation:Select the light yellow compact and relatively dry embryogenitic and incubate on precultivation medium then put it in dark at 26 for 4 days. Preparation of Agrobecter

29、ium culture 1. Preculture the Agrobecterium on YEB with appropriate selective agent at 26-28for 2 days 2. Transfer the Agrobecterium to suspension medium and culture for 2-3 hours with shaking in 26-28Agrobecterium infection (cocultivation)1. Transfer the calli precultured to a sterile tube.2. Adjus

30、t the Agrobecterium suspension to OD600=0.3.3. Immerse the calli with Agrobecterium suspension for 30 min.4. Blot the calli on a fliter paper and transfer the calli to the cocultivation medium. keep the cultures in the dark at 19-20 for 2-3 days.Notice：The temperature in the cocultivation process sh

31、ould be strictly controled and be no higher then 20.or otherwise it should be hard to wash effectively in the following step Wash and selection:1. Wash the calli several times with sterile water.2. Immerse the calli with sterile water with 500mg/1L Cn for 30 min.3. Blot the calli with the sterile fli