方法1NB基本培养基（右边的为N6,大量相同，加glycine甘氨酸2 mg/L）KNO32830 mg/L（NH4）2SO4463 mg/LKH2pO4400 mg/LMgSO4.7H2O185 mg/LCaCl2.2H2O166 mg/LFeSO4.7H2O27.8mg/L5.6Na2EDTA37. 5 mg/L7.5MnSO4.4H2O10 mg/L27.8H3BO33 mg/L1.6ZnSO4.7H2O2 mg/L1.5Na2MoO4.2H2O0.25 mg/L0.25CuSO4.5H2O0.025 mg/L0.025CoCl2.6H2O0.025 mg/L0.025KI0.75 mg/L0.8盐酸硫胺素 thiamine CHL VB110 mg/L0.1盐酸吡哆醇 pyridoxine-CHL VB61 mg/L0.5烟酸 nicotinic acid1 mg/L0.5肌醇 myo-inositol100 mg/L100水解酪蛋白300 mg/L谷氨酰胺500 mg/L脯氨酸500 mg/L蔗糖30，000 mg/LpHytagel2.6 mg/LpHBasic培养基B N6 （大量）50ml （ 20 倍）5.8A Ms-Fe 盐10-20ml（ 100 倍）CH0.3g/LS B5 macro10ml （ 100 倍）phytogel4g/L 或agar 8g/LI B5 vita10ml （ 100 倍）sucrose30g/LC proline0.5g/Lglutamine0.5g/LN6 （大量梗稻种子）终浓度母液（20倍） 终浓度母液（20倍）培 kno32830mg/L56.6g/LMgSO4.7H2O185 mg/L3.7 g/L养 （NH4）2SO4463 mg/L9.26 g/LCaCl2.2H2O1663.32mg/Lg/L基 KH2pO4400 mg/L8.00 g/L制备1 KNO3, （NH4）2SO4, kh2po4同时倒入烧杯，加水搅拌，使之完全溶解.2 MgSO4.7H2O 先溶于加了 100ML 水的烧杯中溶解后再缓慢倒入 1 中,边 倒边搅, 不能有沉淀.3 CaCl2.2H2O 的配制同 MgSO4.7H2O4配好后放于棕色瓶4°C保存MS（大量籼稻种子）终浓度 母液（20倍）母液（20 倍）终浓度培 kno31900 mg/L38g/LMgSO4.7H2O370 mg/L7.4 g/L 养nh4no31650 mg/L33 g/LCaCl2.2H2O440 mg/L8.8 g/L基 KH2PO4 170 mg/L 3.4 g/L制备1 KNO3, NH4NO3, MgSO4.7H2O同时倒入烧杯，加水搅拌2 KH2PO4先溶于加了 100ML水的烧杯中溶解后再缓慢倒入1中,边倒边 搅,3 CaCl2.2H2O 同 KH2PO44配好后放于棕色瓶4C保存Ms（大量花药用） 终浓度母液（20 倍）终浓度母液元素 KNO3 2830mP/L56.6g/L MgSO4.7H2O370 mg/L 7.4g/L培养(NH〉SO4 231 mg/L4.62 g/L CaCl2.2H2O160 mg/L3.2 g/L基 KH2PO4 641 mg/L12.82 g/L制备1 kno3, （nh4）2so4, kh2po4同时倒入烧杯，加水搅拌2 MgSO47H2O先溶于加了 100ML水的烧杯中溶解后再缓慢倒入1中，边 倒边搅,不能有沉淀.CaCl2.2H2O的配制同MgSO4.7H2O 3配好后放于棕色瓶4C保存YM脓杆菌培养基KH2PO42g/L0.5g/LMannitol10g/LL-GlutamineNaCl0.2g/LMgSO40.2g/LYeast extract0.3g/LAgar15g/LPH=7.0诱导愈伤、继代培养基basic+2,4-D(2mg/L) PH=5.8共培养培养基液体:N6(大)+Fe+B5(micro)+B5(vita)+ sucrose(前四项与 basic 相同)+ 肌醇 2g/L+ CH500mg/L+phytogel+0.1%AS固体:N6(大)+Fe+B5(micro)+B5(vita)+ sucrose(前四项与 basic 相同) + 肌醇 2g/L+ CH500mg/L+2,4-D (2mg/L)+ phytogel +0.1%AS PH=5.5加2,4-D前后都要调PH值.灭完菌，温度降下来后加AS筛选培养基诱导愈伤+cef(300mg/L)+hyg(50mg/ml) PH=5.5一筛,二筛的cef,hyg的浓度依次逐渐下降分化 1: basic+KT(4mg/L)+NAA(0.25mg/L)+cef(300mg/L)+hyg(50mg/L)培养 2: basic+KT(0.5mg/L)+NAA(0.25mg/L)+6-BA(2mg/L)+cef(300mg/L)+hyg(50mg/L)基 PH=5.8分化培养基 NB 培养基：6BA,2.5mg,NAA,0.25mg,KT,0.5mg壮苗培养基(生根)sucrose 30g/L N6 25ml/L MS-Fe 盐 10ml/LB5vita 10ml/L agar 7g/LMS-Fe盐(必须单独配制，否则会沉淀，用鳌合铁)终浓度 母液(100倍)FeSO4.7H2O 27.8mg/L 2.78g/LNa-EDTA 37.5mg/L 3.75g/L制备1 FeSO4.7H2O溶解于水,Na-EDTA溶解于热水，将两者混合定容，于微波炉中加热，煮沸，颜色变深，冷却到室温，补水，棕色瓶4°C保存2,4-D母液(1g/ml)用无水乙醇溶解2,4-D后缓慢加入到H2O中，搅拌，如产生沉 淀，则重配.配好后4 C保存NAA母液(1g/ml)用1N KOH溶解NAA,用水稀释定容,4C保存PAA母液(1g/ml)用无水乙醇溶解加H2O搅拌，定容,4C保存6-BA母液(1g/ml)用HCl溶解，加少量HCl后，用玻棒研磨成糊状，再加HC1使 之完全溶解AS (乙酰丁香酮)用DMSO直接溶解定容19.62mg/ml,分装到无菌小管KT母液(5mg/ml)用1NKOH溶解，用水稀释定容到10ml,过滤灭菌后分装到 EP管中，冰冻保存B5 vitamin终浓度母液(100倍)终浓度母液(100倍)肌醇100mg/L10g/lNico-acid1mg/l0.1g/lpyrido(B6)1 mg/L0.1 g/lthiamin(B1)10 mg/L1 g/l棕色瓶4C保存(每次配100ml为好)，肌醇在配培养基时，加入到培养基中B5(micro)KI 0.75mg/L H3BO3 3.0mg/L MnSO4 10mg/L ZnSO4 2.0mg/LNa2MnO4.2H2O 0.25mg/LCuSO4 0.025mg/L CoCl2 0.025mg/L程序1. 种子去皮，挑成熟完整健康的种子2. 用70%乙醇清洗浸泡2-3分钟，洗3-4次，用0.1%升汞浸泡20-40分钟3. 无菌水洗 3 至 4 次4. 置于虑纸上吹干，吹3小时以上5. 摆放到诱导愈伤培养基上，黑暗中培养15-20天，直到长出黄色较大的愈伤6. 剥下愈伤，转至继代培养基上，黑暗培养2周7. 共培养。
脓杆菌与愈伤在液体共培养基中轻轻摇培 30 分钟，转至共培养基(用 虑纸盖住培养基)上，吹风3-4 小时黑暗培养 3 天8. 转至筛选培养基上，吹风吹干黑暗培养2 周转至二筛培养基上，黑暗培养 2 周，直到长出新的愈伤9. 转至分化培养基上，黑暗培养一周然后明暗交替(16 小时：8 小时)10. 剪苗后，转至生根壮苗培养基上11. 移苗到温床，田间方法2 水稻转化 (来自文献 rice transformation, mature rice seeds were de-husked and sterilised with 3% sodium hypochlorite (30 min). The seeds were thoroughly washed three times with sterile distilled water and transferred onto MS basal medium (Murashige and Skoog 1962) containing 500 mg/l proline, 300 mg/l casein hydrolyte, 2 mg/l 2,4-dichlorphenoxy acidic acid (2,4-D) and 30 g sucrose, for 1 week. The freshly induced calli were dipped in Agrobacterium tumefaciens (AGL1 strain) and transferred onto NB medium (N6 macro-elements, B5 micro-elements) containing 500 mg/l proline, 300 mg/l casein hydrolysate, and 30 g sucrose. After co-cultivation with Agrobacterium (no dim light, 28 °C, 2 days), the calli were transferred onto NB medium containing 2 mg/l 2,4-D, 120 mg/l G418, and 500 mg/l Cefotaxime. The resistant calli were subcultured on fresh plates at 2-week intervals for 4 weeks, and then transferred onto MS medium containing 0.2 mg/l anaphthalene acetic acid (NAA), 3 mg/l 6-benzylaminopurine (6-BA) and 150 mg/l G418 until shoots regenerated. Shoots were transfe。