杂花和紫花苜蓿原生质体分离培养条件的筛选
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青藏高原甘肃甘南社区生态畜牧业关键技术集成与模式示范(201203010);甘肃牧区优质饲草生产技术研究与示范(201003023);牧草种质资源保护与利用(NB2130135);国家牧草产业技术体系专项(CARS-35)


Selection for Protoplast Isolation and Culture Conditions of Alfalfa
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    摘要:

    以杂花苜蓿‘甘农1号’和紫花苜蓿‘甘农4号’、‘阿尔冈金’及‘清水’4个适宜西北内陆黄土高原地区栽培的苜蓿愈伤组织为材料,研究酶解时间、酶液组合、酶液渗透压、愈伤组织继代培养时间、预处理措施及不同培养方法等对原生质体分离和培养效果的影响,并对培养条件进行优化。结果表明:(1)适宜4个苜蓿品种愈伤组织酶解的最佳预处理措施为0.55 mol/L蔗糖或CPW溶液中预质壁分离1 h,最佳继代时间均为12 d。(2)‘甘农1号’、‘甘农4号’和‘清水’的最佳酶液组合均为2%纤维素酶+0.5%果胶酶+0.3%崩溃酶;‘阿尔冈金’的最佳酶液组合为2%纤维素酶+0.5%果胶酶+0.3%半纤维素酶+0.3%离析酶+0.3%崩溃酶;‘甘农1号’和‘阿尔冈金’的最佳酶解时间为12 h,‘甘农4号’和‘清水’分别为14 h和10 h。(3)适宜4个品种酶解的甘露醇浓度分别为‘甘农1号’0.75 mol/L,‘甘农4号’0.65 mol/L,‘阿尔冈金’0.6 mol/L,‘清水’0.55~0.6 mol/L。(4)经液体浅层培养和固液培养方式均可获得4个苜蓿品种的再生愈伤组织,且固液培养法较液体浅层培养法更有利于苜蓿原生质体早期的培养和再生。

    Abstract:

    The protoplasts were isolated by enzymolysis method from calli of 4 cultivars of Medicago varia (‘Gannong 1’) and Medicago sativa (‘Gannong 4’,‘Algonquin’ and ‘Qingshui’) which have an excellent growth adaptability in Loess Plateau of northwest of China,and the yield and viability of these protoplasts were measured under the different enzymolysis conditions,and their influence factors,such as enzymolysis time,enzyme combination,mannitol concentration,pretreatment measures and subculture time were discussed.Results show that:(1)The optimum pretreatment measure was 0.55 mol/L sucrose or CPW solutions plasmolysis for 1 h and the optimum subculture time was 12 d for 4 cultivars.(2)The best enzyme solution combination for ‘Gannong 1’,‘Gannong 4’ and ‘Qingshui’is 2% cellulose onozuka R-10+0.5% pectinase Y-23+0.3% driselase and for ‘Algonquin’ is 2% cellulose onozuka R-10+0.5% pectinase Y-23+0.3% hemicellulose+0.3% macerozyme+0.3% driselase.Enzymolysis 12 h is best time for ‘Gannong 1’ and ‘Algonquin’,14 h is for‘Gannong 4’and 10 h is for‘Qingshui’.(3)The suitable mannitol concentration was 0.75 mol/L for ‘Gannong 1’,0.65 mol/L for ‘Gannong 4’,0.6 mol/L for ‘Algonquin’ and 0.55~0.6 mol/L for ‘Qingshui’.(4)The regenerated calli can be formed from protoplasts of 4 alfalfa cultivars both by liquid medium and solid-liquid medium,and solid-liquid double layer culture is more helpful for division and regeneration of 4 cultivars than thin layer liquid culture.

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李玉珠,师尚礼.杂花和紫花苜蓿原生质体分离培养条件的筛选[J].西北植物学报,2014,34(1):184-192

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  • 在线发布日期: 2014-02-13
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