三种豆科牧草的原生质体的培养及骆驼刺和鹰嘴紫云英的体细胞杂交
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摘要
以草木樨状黄芪发根农杆菌A_4转化系再生植株幼叶为材料，通过酶法分离出大量有活力的原生质体。原生质体经持续分裂形成愈伤组织，并高频率分化出再生苗。比较了不同叶龄、预处理时间、不同酶解时间和培养密度及不同激素组合对原生质体分裂和再生的影响。结果表明，原生质体以3×10~5个／ml的植板密度，采用液体浅层培养在附加有2.0mg/L2,4-D，0.2mg／L6BA，0.3 M甘露醇，2％(w／v)蔗糖和500mg／L水解酪蛋白的DPD培养基中，可获得最佳培养效果，其细胞分裂频率达38％左右。由原生质体获得的愈伤组织可再生植株。
对骆驼刺发根农杆菌A_4转化系愈伤组织分离的原生质体进行了培养，并获得了愈伤组织和分化的幼苗。结果表明，新转代8d的黄色颗粒状愈伤组织，为获得大量有活力(80％)的原生质体的最佳状态。渗透压为540±3mmol／L时，原生质体以4×10~5个／ml的植板密度培养于附加有1.5mg/L2,4-D，0.2mg/L6BA，0.3M甘露醇，2％(w／v)蔗糖和500mg/L水解酪蛋白的DPD培养基中，分裂频率可达41.7％。由原生质体获得的愈伤组织有再生植株的能力。
用甲硫氨酸抗性系鹰嘴紫云英的愈伤组织分离原生质体，发现该愈伤组织在转代12d时，游离的原生质体活力最高。原生质体以2×10~5个／ml的植板密度，培养于附加有2.0mg／L 2,4-D，0.2mg/L6BA,，0.3M甘露醇，2％(w／v)蔗糖和500mg／L水解酪蛋白的DPD培养基中，分裂频率可达到38％。原生质体形成的愈伤组织在附加有高浓度KT的MS基本培养基上，可诱导出丛生芽。
Mesophyll protoplasts of Astragalus melilotoides Pall, transfromed by Agrobact-erium rhizogene were obtained enzymatically. Protoplasts of sustainable divisions developed into calli, and high frequency of shoot differentiation was obtained on differentiated medium. We have compared the effect of cotyledon age, pretreatment time and enzyme digestion time on protoplast division frequency. Liquid culture method was appropriate for the protoplast division. The highest division frequency was 38% in DPD medium supplemented with 2.0 mg/L 2,4-D, 0.2mg/L 6-BA, 0.3M mannitol, 2%(w/v) sucrose and 500mg/L casein hydroIysate(CH) at a plating density of 3×105/ml. Protocalli could regenerate hairy roots and roots.
Calli were induced from hairy root segments of Agrobacterium rhizogene A4-transformed Alhagi pseudal hagi. The highest yield of protoplasts (80%) was obtained from 8-day-old friable calli after subcultured on fresh medium. Protoplasts were induced to undergo sustained divisions in DPD medium aupplemented with 1.5mg/L 2,4-D, 0.2mg/L 6BA, 0.3 M mannitol, 2%(w/v) sucrose and 500mg/L casein hydrolysate(CH) at a plating density of 4×105/ml on the condition that osmotic pressure was 540±3mmol/L. The division frequency was about 42 %. Numerous buds were induced from protocalli on MS medium supplemented with Phytohormone 6-BA.
Protoplasts were isolated from the methionine resistant cell line of Astragalus cicer L. .The highest yield of protoplasts was obtained from 12-day-old friable calli after subcultured on fresh medium. The viability of protoplasts reached over 80%. Protolasts underwent sustained divisions when cultured in DPD medium supplemented with 2.0mg/L 2,4-D, 0.2mg/L 6BA, 0.3 M mannitol, 2%(w/v) sucrose and 500mg/L casein hydrolysate(CH) at a plating density of 2×105/ml.
对骆驼刺发根农杆菌A_4转化系愈伤组织分离的原生质体进行了培养，并获得了愈伤组织和分化的幼苗。结果表明，新转代8d的黄色颗粒状愈伤组织，为获得大量有活力(80％)的原生质体的最佳状态。渗透压为540±3mmol／L时，原生质体以4×10~5个／ml的植板密度培养于附加有1.5mg/L2,4-D，0.2mg/L6BA，0.3M甘露醇，2％(w／v)蔗糖和500mg/L水解酪蛋白的DPD培养基中，分裂频率可达41.7％。由原生质体获得的愈伤组织有再生植株的能力。
用甲硫氨酸抗性系鹰嘴紫云英的愈伤组织分离原生质体，发现该愈伤组织在转代12d时，游离的原生质体活力最高。原生质体以2×10~5个／ml的植板密度，培养于附加有2.0mg／L 2,4-D，0.2mg/L6BA,，0.3M甘露醇，2％(w／v)蔗糖和500mg／L水解酪蛋白的DPD培养基中，分裂频率可达到38％。原生质体形成的愈伤组织在附加有高浓度KT的MS基本培养基上，可诱导出丛生芽。
Mesophyll protoplasts of Astragalus melilotoides Pall, transfromed by Agrobact-erium rhizogene were obtained enzymatically. Protoplasts of sustainable divisions developed into calli, and high frequency of shoot differentiation was obtained on differentiated medium. We have compared the effect of cotyledon age, pretreatment time and enzyme digestion time on protoplast division frequency. Liquid culture method was appropriate for the protoplast division. The highest division frequency was 38% in DPD medium supplemented with 2.0 mg/L 2,4-D, 0.2mg/L 6-BA, 0.3M mannitol, 2%(w/v) sucrose and 500mg/L casein hydroIysate(CH) at a plating density of 3×105/ml. Protocalli could regenerate hairy roots and roots.
Calli were induced from hairy root segments of Agrobacterium rhizogene A4-transformed Alhagi pseudal hagi. The highest yield of protoplasts (80%) was obtained from 8-day-old friable calli after subcultured on fresh medium. Protoplasts were induced to undergo sustained divisions in DPD medium aupplemented with 1.5mg/L 2,4-D, 0.2mg/L 6BA, 0.3 M mannitol, 2%(w/v) sucrose and 500mg/L casein hydrolysate(CH) at a plating density of 4×105/ml on the condition that osmotic pressure was 540±3mmol/L. The division frequency was about 42 %. Numerous buds were induced from protocalli on MS medium supplemented with Phytohormone 6-BA.
Protoplasts were isolated from the methionine resistant cell line of Astragalus cicer L. .The highest yield of protoplasts was obtained from 12-day-old friable calli after subcultured on fresh medium. The viability of protoplasts reached over 80%. Protolasts underwent sustained divisions when cultured in DPD medium supplemented with 2.0mg/L 2,4-D, 0.2mg/L 6BA, 0.3 M mannitol, 2%(w/v) sucrose and 500mg/L casein hydrolysate(CH) at a plating density of 2×105/ml.
引文
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