Rice Transgene Protocol

Solutions 1. MSmax stock solution(10X) NH4NO3 16.5 g KNO3 19.0 g KH2PO4 1.7 g use a bottle dissolve them one by one then add MgSO4·7H2O 3.7 g after it has been dissolved completely then slowly add CaCl2·2H2O 4.4 g add H2O to 1000 ml NOTE: If add too fast will bring deposition 2. MS min stock solution (100X) KI 0.083 g H3BO3 0.62 g MnSO4·4H2O 2.23 g ZnSO4·7H2O 0.86 g Na2MoSO4·2H2O 0.025 g CuSO4·5H2O 0.0025 g CoCl2·6H2O 0.0025 g add H2O to 1000 ml 3. N6 max stock solution (10X) KNO3 28.3 g KH2PO4 4.0 g (NH4)2SO4 4.63 g use a bottle dissolve them one by one then add MgSO4·7H2O 1.85 g after it has been dissolved completely then slowly add CaCl2·2H2O 1.66 g add H2O to 1000 ml NOTE: If add too fast will bring deposition 4. N6 min stock solution(100X) KI 0.08 g H3BO3 0.16 g MnSO4·4H2O 0.44 g ZnSO4·7H2O 0.15 g add H2O to 1000 ml Fe2EDTA (100X) use one bottle add 300 ml H2O add FeSO4·7H2O 2.78 g use another bottle add 300 ml H2O heat to 70℃ then add Na2·EDTA·2H2O 3.73 g After they both are dissolved then mix together and keep at 70℃ for about 45 minutes. 5. Vitamin (100X) Nicotinic acid 0.05 g Pyridoxine HCl 0.05 g Thiamine HCl 0.1 g Glycine 0.2 g add H2O to 1000 ml 6. AAmax stock solution (10X) CaCl2·2 H2O 150 g MgSO4·H2O 2.50 g NaH2PO4·H2O 1.50 g KCl 29.50 g add H2O to 1000 ml 7. AAmin stock solution (100X) KI 0.075 g H3BO3 0.3 g MnSO4·H2O 1.0 g ZnSO4·7 H2O 0.2 g Na2MoO4·2 H2O 0.025 g CuSO4·5 H2O 0.0025 g CoCl2·6 H2O 0.0025 g add H2O to 1000 ml 8. Hormone 2,4-D stock solution use a bottle with less than 100 ml water and add several drop of 1N KOH then add 2,4-D 100 mg add H2O to 100 ml KT, 6-BA, NAA and IAA stock solution’s prepare method are the same as 2,4-D. 9. AS stock solution AS 2 g DMSO 50 ml add H2O to 100 ml Filter-sterilized. Disperse to 1.5 ml centrifugetube and store at -20℃. 10. Gus staining solution X-gluc (dissolved in DMSO) 10 mg 100 mM PBS (pH 7.0) 9.8 ml 5 mM potassium ferricyanide 0.1 ml 5 mM potassium ferrocyanide 0.1 ml Triton X-100 (10%) 0.1 ml 11. Hygromycin stock solution Hygromycin 1 g PBS 20 ml Filter-sterilized. Disperse to 1.5 ml centrifugetube and store at -20℃. 3. Medium 1. Induction and subculture medium (N6I) N6max 100 ml N6min 10 ml Fe2EDTA 10 ml Vitamin 10 ml 2,4-D 2.0 ml Casein Enzymatic Hydrolysate 0.4 g Proline 1.0 g Sucrose 45 g add H2O to 900 ml use 1N KOH adjust pH to 5.9 then add Phytagel 3 g and add H2O to 1000 ml 2. Precultivation medium (N6P) N6max 100 ml N6min 10 ml Fe2EDTA 10 ml Vitamin 10 ml 2,4-D 2.0 ml Casein Enzymatic Hydrolysate 0.6 g Proline 0.6 g Sucrose (or Maltose) 30 g add H2O to 900 ml use 1N KOH adjust pH to 5.6 then add Phytagel 3 g and add H2O to 1000 ml 3. Suspend medium (N6A) N6max 10 ml N6min 1 ml Fe2EDTA 1 ml Vitamin 1 ml 2,4-D 0.2 ml Casein Enzymatic Hydrolysate 0.06 g Proline 0.06 g Sucrose (or Maltose) 3 g add H2O to 90 ml use 1N KOH adjust pH to 5.6 and add H2O to 100 ml Before use add AS 0.1 ml 4. Cocultivation medium (N6C) N6max 10 ml N6min 1 ml Fe2EDTA 1 ml Vitamin 1 ml 2,4-D 0.2 ml Casein Enzymatic Hydrolysate 0.06 g Proline 0.06 g Sucrose (or Maltose) 3 g add H2O to 90 ml use 1N KOH adjust pH to 5.6 then add Phytagel 0.3 g and add H2O to 100 ml Before use add AS 0.1 ml 5. Resting medium (N6R) N6max 100 ml N6min 10 ml Fe2EDTA 10 ml Vitamin 10 ml 2,4-D 2.5 ml Casein Enzymatic Hydrolysate 0.4 g Proline 1.2 g Sucrose 30 g add H2O to 900 ml use 1N KOH adjust pH to 5.8 then add Phytagel 3 g and add H2O to 1000 ml add 400 mg/l Cefotaxime before using 6. Selection medium (N6S) N6max 100 ml N6min 10 ml Fe2EDTA 10 ml Vitamin 10 ml 2,4-D 2.5 ml Casein Enzymatic Hydrolysate 0.4 g Proline 1.2 g Sucrose 30 g add H2O to 900 ml use 1N KOH adjust pH to 5.9 then add Phytagel 3 g and add H2O to 1000 ml add 250 mg/l Cefotaxime , 50 mg/l Hygromycin before using 7. Preregeneration medium (MSP) MSmax 100 ml MSmin 10 ml Fe2EDTA 10 ml Vitamin 10 ml (6-BA 2.0 ml) KT 2.0 ml (IAA 0.2 ml) NAA 0.2 ml Casein Enzymatic Hydrolysate 2.0 g Sucrose (or Maltose) 30 g add H2O to 900 ml use 1N KOH adjust pH to 5.8 then add Phytagel 3 g and add H2O to 1000 ml 8. Regeneration medium (MSR) MSmax 100 ml MSmin 10 ml Fe2EDTA 10 ml Vitamin 10 ml (6-BA 2.0 ml) KT 2.5 ml (IAA 0.2 ml) NAA 0.5 ml Sucrose (or Maltose) 30 g add H2O to 900 ml use 1N KOH adjust pH to 5.8 then add Phytagel 3 g and add H2O to 1000 ml 9. Root growth medium MSmax 50 ml MSmin 5 ml Fe2EDTA 10 ml Vitamin 5 ml NAA 0.2 ml Sucrose 10 g add H2O to 900 ml use 1N KOH adjust PH to 5.8 then add add H2O to 900 ml use 1N KOH adjust pH to 5.8 then add Phytagel 2.5 g and add H2O to 1000 ml (add 50 mg/l Hygromycin before using) 3. Procedures 1. Callus induction ⑴ Sterilize the seeds with 70% ethylalcoho 1 minute and 0.15%HgCl2 15 minutes. ⑵ Wash them with sterile water 4-5 times. ⑶ Inoculate the seeds in callus induction medium ⑷ Put in the dark at 27℃ for about 3 weeks. ⒉ Callus subculture ⑴ Separate the light yellow、compact and relatively dry embryogenic callus from the endosperm. ⑵ Inoculate on subculture medium ⑶ Put in dark at 27℃ for 2 weeks. ⒊ Precultivation ⑴ Select the compact and relatively dry callus and inoculate on precultivation medium ⑵ Put in dark at 27℃ for 4 days ⒋ Agrobacterium prepare ⑴ Inoculate the Agrobacterium on LA (with Karr or other antibiotic) at 28℃ for 2 days. ⑵ Transfer the agrobacterium to the suspend medium and culture for 2 hours with shaking in 28℃. ⒌ Agrobacterium cocultivation ⑴ Transfer the precultivation cellus to a sterile dish ⑵ Adjust the Agrobacterium concentration to OD600 1.0-1.2. ⑶ Immerse the callus with the Agrobacterium liquids for 15 minutes. ⑷ Dry it with the sterile filter paper. ⑸ Inoculate on cocultivation medium and keep the cultures in the dark at 24℃ for 3 days. 6. Washing, resting and selection ⑴ Wash the cellus several times with the sterile water until there is no Agrobacterium visable. ⑵ washed twice with sterile water containing 400 mg/l of cefotaxime, 15 min every time with gently shaking (80rpm) ⑶ Dry the callus with the sterile filter paper. (4) Transfer the callus to the resting medium (N6R) medium at 27 °C for one week (5) Transfer the callus to selection medium (N6S) and were sub-cultured every 2 weeks. The cultures were kept in the dark at 27 °C for 6-8 weeks 7. Regeneration ⑴ Transfer the selected callus to preregeneration medium plate at dark for 7-10 days. ⑵ Transfer the good callus to the regeneration medium. ⑶ Under 16 hours light and 8 hours dark for 2-4 week. ⒏ Root growth ⑴ Cut all the original root ⑵ Transfer the little plant to the root growth medium ⒐ Plant cultivation (1) Recover the cover for about 2-3 days. ⑴ Transfer the plants with vigorous root to greenhouse. ⑵ Clean the medium in the root and keep the moisture the first few days