鹅掌楸属内种间体细胞原生质体融合研究
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摘要
本实验采用的实验材料——中国马褂木和北美鹅掌楸,分别取自从中国及北美各地引种到南京林业大学下蜀林场的30多年种源实验林,以及南京林业大学校园内的纯种鹅掌楸优良单株。以新梢及花苞为材料进行原生质体分离和融合的实验研究。主要结果如下:
1.外植体的灭菌
中国马褂木和北美鹅掌楸优良单株春天抽芽后15天发出的幼嫩芽、嫩枝、嫩叶、叶柄为材料较好,花苞选取花瓣4~5cm长的较好,其适宜的灭菌处理为:将采摘的嫩枝用洗衣粉溶液洗15分钟,用清水冲洗2小时,用酒精处理30秒,再用升汞处理9分钟,用灭菌的清水冲洗4~5次,接种到诱导培养基上。
2.愈伤组织的诱导
中国马褂木和北美鹅掌楸初代培养基均以MS为佳。其中嫩芽、嫩枝、幼叶、幼叶叶柄适宜诱导培养基为MS+2,4-D2.0mg/L+6-BA0.2mg/L+SUC30g/L,其愈伤诱导率能达到100%;花药愈伤组织的诱导培养基为: 1/2MS+ 2,4D2mg/L+ 6-BA0.5mg/L+ NAA2mg/L+ KT0.2mg/L+ GA0.2mg/L+ Gln 200mg/L+ Asn200mg/L+ Pro100mg/L+ Arg200mg/L+ CH300mg/L。
3.悬浮体系的建立
愈伤组织经过培养基MS+ NAA0.5mg/L+ KT0.5mg/L+ 6-BA0.5mg/L+ Vc5mg/L+ LH500mg/L+ pH5.8+ SUC50g/L上培养,可诱导出胚性愈伤组织。
4.原生质体的游离和培养
确定叶肉原生质体分离的适用酶液组合为E组合:纤维素酶Cellulase2%+半纤维素酶Hemicellulase1%+果胶酶PectolyaseY-23 0.2%;确定悬浮细胞分离的适用酶液组合为F组合:纤维素酶Cellulase2%+半纤维素酶Hemicellulase1%+果胶酶PectolyaseY-23 0.1%。原生质体的培养基为KM8p,添加0.6mol/L的甘露醇,以及0.1%的MES。
5.原生质体的电融合处理
电融合的适宜参数为:AC强度110V/cm,时间30秒;直流脉冲,作用时间选择为40μs,作用强度为1000V/cm,作用次数为5次。原生质体融合率最高可达到30%以上。
6.外源DNA的转化
外源质粒DNA与原生质体的混合液,经过逐步添加PEG溶液(30%PEG-8000+ 0.3mol/L葡萄糖+10mmol CaCl2),可以使质粒DNA所携带的目的基因GFP瞬时表达。
In this experiment, explant materials were collected both from a 30years provenance testing of Liriodendron chinense and Liriodendron tulipifera on Xiashu Forest Farm of Nanjing Forestry University and on campus. New shoots, buds and flowers were taken as explants. The major results were as follows:
1. Sterilization of Explants
The materials were young shoots, twigs, leaves, petioles, flower bud petals in 4~5cm long. The new leaves from pure individual of Liriodendron chinense and Liriodendron tulipifera were collected 15 days after buds breaking in the spring. And the appropriate sterilization procedures are as fellows, for the shoot, wash with detergent for 15mins, rinse two hours, dealing with alcohol for 30 seconds, then mercuric chloride for 9 min, washed with sterile water for 4 to 5 times , inoculated into induction medium.
2. Callus Induction
The MS medium was suitable for initiation to Liriodendron Chinese and Liriodendron tulipifera basically. The rate of callus induction on medium MS+ 2,4-D2.0mg/L+ 6-BA0.2mg/L+ sucrose30g/L, for young shoots, twigs, leaves, petioles is up to 100%, but, the callus induction medium for anther was 1/2MS+ 2,4-D2mg/L+6-BA0.5mg/L+ NAA2mg/L+ KT0.2mg/L+ GA0.2mg/L+ Gln200mg/L+ Asn200mg/L+ Pro100mg/L+ Arg200mg/L+ CH_300mg/L.
3. Establishment of Suspension Culture System
The callus cultured in the medium of MS+NAA 0.5mg/L+KT 0.5mg/L+6-BA 0.5mg/L+Vc 5mg/L+LH 500mg/L+pH5.8+SUC50g/L can be induced into embryogenic callus.
4. Free and Culture of Protoplast
The optimal enzyme combination for the protoplast isolation from mesophyll tissue is combination E i.e.Cellulase2%+ Hemicellulase1%+ PectolyaseY-23 0.2%. And the most suitable enzyme combination for the protoplast free with suspension is combination F, i.e.Cellulase2%+ Hemicellulase1%+ PectolyaseY-23 0.1%. For protoplast culture was KM8p, add mannitol of 0.6mol/L, and MES of 0.1%.
5. Electro Fusion of Protoplast
The optimum parameters for electro fusion: alternating current (AC) 110V/cm, duration 30s; direct current (DC) 1000V/cm, duration 40μs, 5 times. The rate of protoplast fusion can be reached to 30%.
6. The Transformation of Exogenous DNA
The mixture of exogenous plasmid DNA and protoplasts, then adding the solution of PEG (30%PEG-8000+0.3mol/LGlucose+10mmolCaCl_2) steeply, the GFP gene carried by plasmid DNA can be expressed transiently.
1.外植体的灭菌
中国马褂木和北美鹅掌楸优良单株春天抽芽后15天发出的幼嫩芽、嫩枝、嫩叶、叶柄为材料较好,花苞选取花瓣4~5cm长的较好,其适宜的灭菌处理为:将采摘的嫩枝用洗衣粉溶液洗15分钟,用清水冲洗2小时,用酒精处理30秒,再用升汞处理9分钟,用灭菌的清水冲洗4~5次,接种到诱导培养基上。
2.愈伤组织的诱导
中国马褂木和北美鹅掌楸初代培养基均以MS为佳。其中嫩芽、嫩枝、幼叶、幼叶叶柄适宜诱导培养基为MS+2,4-D2.0mg/L+6-BA0.2mg/L+SUC30g/L,其愈伤诱导率能达到100%;花药愈伤组织的诱导培养基为: 1/2MS+ 2,4D2mg/L+ 6-BA0.5mg/L+ NAA2mg/L+ KT0.2mg/L+ GA0.2mg/L+ Gln 200mg/L+ Asn200mg/L+ Pro100mg/L+ Arg200mg/L+ CH300mg/L。
3.悬浮体系的建立
愈伤组织经过培养基MS+ NAA0.5mg/L+ KT0.5mg/L+ 6-BA0.5mg/L+ Vc5mg/L+ LH500mg/L+ pH5.8+ SUC50g/L上培养,可诱导出胚性愈伤组织。
4.原生质体的游离和培养
确定叶肉原生质体分离的适用酶液组合为E组合:纤维素酶Cellulase2%+半纤维素酶Hemicellulase1%+果胶酶PectolyaseY-23 0.2%;确定悬浮细胞分离的适用酶液组合为F组合:纤维素酶Cellulase2%+半纤维素酶Hemicellulase1%+果胶酶PectolyaseY-23 0.1%。原生质体的培养基为KM8p,添加0.6mol/L的甘露醇,以及0.1%的MES。
5.原生质体的电融合处理
电融合的适宜参数为:AC强度110V/cm,时间30秒;直流脉冲,作用时间选择为40μs,作用强度为1000V/cm,作用次数为5次。原生质体融合率最高可达到30%以上。
6.外源DNA的转化
外源质粒DNA与原生质体的混合液,经过逐步添加PEG溶液(30%PEG-8000+ 0.3mol/L葡萄糖+10mmol CaCl2),可以使质粒DNA所携带的目的基因GFP瞬时表达。
In this experiment, explant materials were collected both from a 30years provenance testing of Liriodendron chinense and Liriodendron tulipifera on Xiashu Forest Farm of Nanjing Forestry University and on campus. New shoots, buds and flowers were taken as explants. The major results were as follows:
1. Sterilization of Explants
The materials were young shoots, twigs, leaves, petioles, flower bud petals in 4~5cm long. The new leaves from pure individual of Liriodendron chinense and Liriodendron tulipifera were collected 15 days after buds breaking in the spring. And the appropriate sterilization procedures are as fellows, for the shoot, wash with detergent for 15mins, rinse two hours, dealing with alcohol for 30 seconds, then mercuric chloride for 9 min, washed with sterile water for 4 to 5 times , inoculated into induction medium.
2. Callus Induction
The MS medium was suitable for initiation to Liriodendron Chinese and Liriodendron tulipifera basically. The rate of callus induction on medium MS+ 2,4-D2.0mg/L+ 6-BA0.2mg/L+ sucrose30g/L, for young shoots, twigs, leaves, petioles is up to 100%, but, the callus induction medium for anther was 1/2MS+ 2,4-D2mg/L+6-BA0.5mg/L+ NAA2mg/L+ KT0.2mg/L+ GA0.2mg/L+ Gln200mg/L+ Asn200mg/L+ Pro100mg/L+ Arg200mg/L+ CH_300mg/L.
3. Establishment of Suspension Culture System
The callus cultured in the medium of MS+NAA 0.5mg/L+KT 0.5mg/L+6-BA 0.5mg/L+Vc 5mg/L+LH 500mg/L+pH5.8+SUC50g/L can be induced into embryogenic callus.
4. Free and Culture of Protoplast
The optimal enzyme combination for the protoplast isolation from mesophyll tissue is combination E i.e.Cellulase2%+ Hemicellulase1%+ PectolyaseY-23 0.2%. And the most suitable enzyme combination for the protoplast free with suspension is combination F, i.e.Cellulase2%+ Hemicellulase1%+ PectolyaseY-23 0.1%. For protoplast culture was KM8p, add mannitol of 0.6mol/L, and MES of 0.1%.
5. Electro Fusion of Protoplast
The optimum parameters for electro fusion: alternating current (AC) 110V/cm, duration 30s; direct current (DC) 1000V/cm, duration 40μs, 5 times. The rate of protoplast fusion can be reached to 30%.
6. The Transformation of Exogenous DNA
The mixture of exogenous plasmid DNA and protoplasts, then adding the solution of PEG (30%PEG-8000+0.3mol/LGlucose+10mmolCaCl_2) steeply, the GFP gene carried by plasmid DNA can be expressed transiently.
引文
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陈名红,陈学军,熊立,烟草原生质体再生植株的影响因子[J].种子, 2007, 26(5).
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