欧洲七叶树体细胞胚发生和植株再生研究
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摘要
欧洲七叶树是重要的园林绿化树种，并具有重要的医药价值，国内引种栽培有限，种苗供不应求，通过细胞工程技术快速繁殖性状优良的再生植株，具有重要的应用价值和理论意义。本文就欧洲七叶树的组织培养和体细胞胚发生以及植株再生进行了系统研究，主要结果如下：
以植物细胞具有全能性的理论为依据，以欧洲七叶树幼嫩叶片为外植体，进行体细胞胚胎发生研究，研究结果表明，诱导愈伤组织的适宜培养基是MS+2，4-D 2mg／L+KT0.2mg／L，MS+BA 8mg／L+NAA 1mg／L有利于胚性愈伤组织的诱导和增殖，添加ZT 2mg／L或BA 5mg／L和IAA 0.2mg／L的MS培养基有利于体细胞胚发育和成熟，体细胞胚可直接诱导次生胚发生，MS+KT 0.1mg／L+NAA 0.01 mg／L或MS+ZT 0.1mg／L+NAA 0.01 mg／L培养基诱导效果最好，增殖频率分别为214％、256％。
以欧洲七叶树体细胞胚的子叶为外植体，诱导次生胚发生或分化不定芽。结果表明，MS+ZT 0.5～2mg／L+BA 1mg／L+IAA 0.2mg／L培养基可直接诱导体胚发生，诱导率超过80％；MS+BA 2mg／L+NAA 0.2 mg／L或MS+TDZ 0.01 mg／L+NAA 0.2 mg／L培养基可直接分化不定芽，出芽诱导率分别为88％、100％。
以欧洲七叶树成熟种子的胚芽进行离体培养和快速繁殖，结果表明：高约2cm的无菌苗在MS+0.6mg／L 6-BA+0.1 mg／L NAA或MS+0.4～0.6mg／L ZT+0.1 mg／L NAA培养基上培养15 d左右可诱导出不定芽，分化频率为100％，平均每株产芽35.7个；MS+0.2mg／L 6-BA+0.1mg／L NAA+10mg／L AD培养基有利于芽伸长；生根培养基为1／2MS+0.4 mg／L NAA+0.2mg／L IBA时，生根率可达75％。
通过组织细胞学方法研究体细胞胚的起源与发生部位，发现欧洲七叶树体细胞胚为单细胞起源，外植体的表面及内部都可以发生体细胞胚。胚性细胞多为椭圆形，细胞质浓厚，核仁明显，代谢旺盛；体细胞胚经过球形胚、心形胚、鱼雷形胚和子叶胚而发育成再生植株，子叶期体细胞胚可观察到明显的V型维管束、茎尖分生组织以及根尖分生组织。
综合上述研究结果，为大规模生产欧洲七叶树，以完整的体细胞胚诱导次生胚效果最好，合适的培养基为MS+KT 0.1mg／L+NAA 0.01 mg／L或MS+ZT 0.1mg／L+NAA 0.01 mg／L。
It was of great value to propagate Aesculus hippocastanum L rapidly, which was an important landscape tree and with great value for medical treatment, by the means of cell engineering.
Somatic embryogenesis systems were investigated by means of vitro culture from young leaf of Aesculus hippocastanum L. The results were showed that embryogenic calli were induced from young leaves, which were cultured on MS medium supplemented with 2,4-D 2mg/L and KT 0.2mg/L. For the proliferation of embryogenic calli, the suitable culture medium was MS+BA 8mg/L +NAA 1mg/L. The development and maturation of somatic embryo could be much improved by using the medium of MS+ZT 2mg/L or BA 5mg/L +IAA 0.2mg/L. For the induction of secondary somatic embryo from integral somatic embryo, the suitable culture medium was MS+KT 0.1mg/L+NAA 0.01 mg/L or MS+ZT 0.1mg/L+NAA 0.01 mg/L, the proliferation frequency is 214%, 256% respectively.
The cotyledonary generated from somatic embryos of Aesculus hippocastanum L. can be used to induce secondary somatic embryogenesis or bud differentiation in vitro culture also. The results were showed that somatic embryos can be directly re-induced from cotyledonary, which was cultured on MS medium supplemented with ZTO.5-2 mg/L, BA 1mg/L and NAA 0.2mg/L, and the induction rate was over 80%. For the bud directly differentiation , the suitable culture medium was MS+BA 2mg/L +NAA 0.2mg/L or MS+TDZ0.01mg/L +NAA0.2 mg/L, of which the induction rate was 88% and 100% respectively.
An efficient tissue culture system has been developed with the bud of mature seed of Aesculus hippocastanum L. as explants . Buds were induced from 2cm high young plantlet cultured on MS medium supplemented with 0.6 mg/L 6-BA plus 0.1mg/L or 0.4~0.6mg/L ZT plus 0.1 mg/L NAA for 15 d, and the induction rate was 100%, the mean No. of buds was 35.7; The combination of MS+0.2mg/L 6-BA +0.1mg/L NAA + 10mg/L AD was the suitable culture medium for elongation of the buds. The medium of 1/2MS+ 0.4mg/L NAA +0,2mg/L IBA was used for rooting, and the rooting rate was 75%.
The origin and development of somatic embryos was observed by histological method. It was found that the somatic embryos of Aesculus hippocastanum L were developed from single cell. Somatic embryos can be produced both from the surface and the inner of the calli. Embryogenic cell has normal shape, dense cytoplasm , clear nucleolus, hearty metabolize; Plantlet regenerations were developed from global embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledon embryo, in which the V-shaped vessel vascular bundles and shoot tip meristem and root tip meristem were observed.
Inconclution, for produce Aesculus hippocastanum L in a mass, we should induce secondary somatic embryos with integral somatic embryos, the suitable culture medium is MS+KT 0.1mg/L+NAA 0.01 mg/L or MS+ZT 0.1mg/L+NAA 0.01 mg/L.
以植物细胞具有全能性的理论为依据，以欧洲七叶树幼嫩叶片为外植体，进行体细胞胚胎发生研究，研究结果表明，诱导愈伤组织的适宜培养基是MS+2，4-D 2mg／L+KT0.2mg／L，MS+BA 8mg／L+NAA 1mg／L有利于胚性愈伤组织的诱导和增殖，添加ZT 2mg／L或BA 5mg／L和IAA 0.2mg／L的MS培养基有利于体细胞胚发育和成熟，体细胞胚可直接诱导次生胚发生，MS+KT 0.1mg／L+NAA 0.01 mg／L或MS+ZT 0.1mg／L+NAA 0.01 mg／L培养基诱导效果最好，增殖频率分别为214％、256％。
以欧洲七叶树体细胞胚的子叶为外植体，诱导次生胚发生或分化不定芽。结果表明，MS+ZT 0.5～2mg／L+BA 1mg／L+IAA 0.2mg／L培养基可直接诱导体胚发生，诱导率超过80％；MS+BA 2mg／L+NAA 0.2 mg／L或MS+TDZ 0.01 mg／L+NAA 0.2 mg／L培养基可直接分化不定芽，出芽诱导率分别为88％、100％。
以欧洲七叶树成熟种子的胚芽进行离体培养和快速繁殖，结果表明：高约2cm的无菌苗在MS+0.6mg／L 6-BA+0.1 mg／L NAA或MS+0.4～0.6mg／L ZT+0.1 mg／L NAA培养基上培养15 d左右可诱导出不定芽，分化频率为100％，平均每株产芽35.7个；MS+0.2mg／L 6-BA+0.1mg／L NAA+10mg／L AD培养基有利于芽伸长；生根培养基为1／2MS+0.4 mg／L NAA+0.2mg／L IBA时，生根率可达75％。
通过组织细胞学方法研究体细胞胚的起源与发生部位，发现欧洲七叶树体细胞胚为单细胞起源，外植体的表面及内部都可以发生体细胞胚。胚性细胞多为椭圆形，细胞质浓厚，核仁明显，代谢旺盛；体细胞胚经过球形胚、心形胚、鱼雷形胚和子叶胚而发育成再生植株，子叶期体细胞胚可观察到明显的V型维管束、茎尖分生组织以及根尖分生组织。
综合上述研究结果，为大规模生产欧洲七叶树，以完整的体细胞胚诱导次生胚效果最好，合适的培养基为MS+KT 0.1mg／L+NAA 0.01 mg／L或MS+ZT 0.1mg／L+NAA 0.01 mg／L。
It was of great value to propagate Aesculus hippocastanum L rapidly, which was an important landscape tree and with great value for medical treatment, by the means of cell engineering.
Somatic embryogenesis systems were investigated by means of vitro culture from young leaf of Aesculus hippocastanum L. The results were showed that embryogenic calli were induced from young leaves, which were cultured on MS medium supplemented with 2,4-D 2mg/L and KT 0.2mg/L. For the proliferation of embryogenic calli, the suitable culture medium was MS+BA 8mg/L +NAA 1mg/L. The development and maturation of somatic embryo could be much improved by using the medium of MS+ZT 2mg/L or BA 5mg/L +IAA 0.2mg/L. For the induction of secondary somatic embryo from integral somatic embryo, the suitable culture medium was MS+KT 0.1mg/L+NAA 0.01 mg/L or MS+ZT 0.1mg/L+NAA 0.01 mg/L, the proliferation frequency is 214%, 256% respectively.
The cotyledonary generated from somatic embryos of Aesculus hippocastanum L. can be used to induce secondary somatic embryogenesis or bud differentiation in vitro culture also. The results were showed that somatic embryos can be directly re-induced from cotyledonary, which was cultured on MS medium supplemented with ZTO.5-2 mg/L, BA 1mg/L and NAA 0.2mg/L, and the induction rate was over 80%. For the bud directly differentiation , the suitable culture medium was MS+BA 2mg/L +NAA 0.2mg/L or MS+TDZ0.01mg/L +NAA0.2 mg/L, of which the induction rate was 88% and 100% respectively.
An efficient tissue culture system has been developed with the bud of mature seed of Aesculus hippocastanum L. as explants . Buds were induced from 2cm high young plantlet cultured on MS medium supplemented with 0.6 mg/L 6-BA plus 0.1mg/L or 0.4~0.6mg/L ZT plus 0.1 mg/L NAA for 15 d, and the induction rate was 100%, the mean No. of buds was 35.7; The combination of MS+0.2mg/L 6-BA +0.1mg/L NAA + 10mg/L AD was the suitable culture medium for elongation of the buds. The medium of 1/2MS+ 0.4mg/L NAA +0,2mg/L IBA was used for rooting, and the rooting rate was 75%.
The origin and development of somatic embryos was observed by histological method. It was found that the somatic embryos of Aesculus hippocastanum L were developed from single cell. Somatic embryos can be produced both from the surface and the inner of the calli. Embryogenic cell has normal shape, dense cytoplasm , clear nucleolus, hearty metabolize; Plantlet regenerations were developed from global embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledon embryo, in which the V-shaped vessel vascular bundles and shoot tip meristem and root tip meristem were observed.
Inconclution, for produce Aesculus hippocastanum L in a mass, we should induce secondary somatic embryos with integral somatic embryos, the suitable culture medium is MS+KT 0.1mg/L+NAA 0.01 mg/L or MS+ZT 0.1mg/L+NAA 0.01 mg/L.
引文
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