杜仲有性杂交与遗传作图群体的建立
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摘要
杜仲是一种以药用为主并具有多种用途的植物,具有极高的开发利用价值。遗传连锁图谱的构建使杜仲早期选择成为可能,可大大缩短选育时间、提高选择精度并降低选育成本,从而促进良种选育进程。建立合适的作图群体是构建遗传连锁图谱的前提并决定图谱的质量。因此,本文在开展杜仲人工杂交及苗木培育的基础上,进行了杂交子代的组织培养及无性系化研究,并进一步通过对F1家系田间表型性状和标志性功效成分总黄酮含量分化程度分析,初步确定了作图群体,从而为杜仲遗传连锁图谱构建和重要性状的分子标记研究奠定基础。主要研究结果如下:
1、初步探讨了杜仲杂交的花粉技术、授粉技术和育苗技术,结果表明:树上收集和室内水培收集两种不同方式所收集花粉的萌发特性没有明显的差异,说明在杜仲杂交育种中,可以通过水培花枝收集花粉。不同父本的散粉期和花粉生活力差异较大,秦仲2号和洛超3号是较好的杂交父本;不同母本的可授期相差较大。不同父本间杂交种子的发芽率差异不显著、而母本间差异显著;以杜仲优良无性系小叶做母本时种子发芽率最高,达99.6%。利用播种育苗方法对所获得的杂交种子进行繁殖,得到了7个F1家系。
2、将杜仲杂交种子剥去种皮,用清水反复冲洗干净,500ppm的赤霉素浸泡24h后,70%酒精消毒30s,1g/L HgCl2表面消毒12min,无菌水冲洗3~4次,切去少许胚芽和胚根端胚乳后胚根向下接入无激素的MS培养基中,30d后萌发成苗。组织培养培育无菌苗的方法可以显著地提高杜仲种子的成苗率。
3、子叶节不定芽诱导的最适宜培养基为MS+6-BA 2mg/L,诱导率85%;单节茎段快速增值的最适宜培养基为MS+6-BA 2mg/L,增值系数3.80;最适宜单节茎段生长的培养基为MS+ 6-BA 1 mg/L + NAA 0.1 mg/L,生长系数为3.71。1/2MS+IBA1.5 mg/L是最佳的生根培养基,生根率90%。以沙子、珍珠岩、腐殖质按1∶2∶1为基质移栽,成活率达80%。
4、通过对家系田间表型性状和标志性功效成分总黄酮含量分化程度分析,选出综合变异程度较高的家系6和家系3作为构建杜仲遗传连锁图谱的作图群体。它们的田间表型性状平均变异系数分别为24.02%和22.87%;标志性功效成分总黄酮含量的变异系数分别为31.89%和19.63%。
5、通过家系间性状差异性比较,结果表明各性状在家系间存在很大的差异。选出的优良家系5,其苗高生长量、地径生长量、叶片数、叶长、叶宽、叶面积和总黄酮含量都较群体均值有不同程度的提高,增幅在4.71%~15.85%之间。
Eucommia ulmoides is a multipurpose tree species, with high value for development and utilization. Genetic linkage map make it possible for early selection in Eucommia ulmoides breeding. It can promote the process of breeding by shortening the time and improving the accuracy as well as reducing the cost. Establishing a suitable mapping population is the first step in mapmaking and determine the quality of the map. For this purpose, the techniques of sexual cross and tissue culture for cloning hybrid seedlings of Eucommia ulmoides were studied, and the variation level of hybride families on field phenotypic characteristics and total flavones content were tested. The main results are as belows:
1. The techniques of pollen collection and pollination in hybridization, as well as culture of seedlings were studyed. The pollens collectd in two different ways (from trees and branches under hydroponic culture condition) has no obvious differences in germination characteristics. Branche hydroponic culture is a convenience way for pollen collecting in crossing. The activity and dispersal piriod of pollens are different among pollen parents. The best pollen parents are Qinzhong2 and Luochao3. Pollination period differed greatly among female parents. There were no obvious differences in seeds germination rates among pollen parents, but there were obvious differences among female parents. The seed germination rate of the clone Xiaoye is rather high, and the maximum level is 99.6%. By the way of sowing and culture of seedlings, seven F1 familes have been obtained.
2. With the process of striped the coats, rinsed repeatedly with water, immersioned in 500ppm gibberellin for 24h, disinfected with 70% alcohol for 30s and with 1g/L HgCl2 for 12min , washed with sterilized water for 3 to 4 times and truncated endosperms, the hybrid seeds chould germinate in a high ratio on the MS medium with hormonesfree in 30d and the seedlings grow well. The results showed that seeds germination ratio chould be improved greatly in tissue culture conditions .
3. The best medium for buds’initiation of cotyledonary nodes was MS+6-BA2mg/L. The inducing ratio could be up to 85%. The best medium for the propagation of stem with one node was MS+6-BA2mg/L. The multiplication coefficient was 3.80. The best medium for the growth of stem with one node was MS+6-BA1 mg/L+NAA0.1 mg/L. The growth ratio was 3.71. The best medium for rooting was 1/2MS+IBA1.5 mg/L, the rooting ratio reached 90%. The seedlings was transplanted in the substrate of sand and perlite mixed with humus for 1:2:1, the survival rate was 80%.
4. Based on the variation analysis of field phenotypic characteristics and total flavones content, family 6 and family 3 have been selected as the mapping populations, whose variation are higher than others. Their average variation coefficients of field phenotypic characteristics were 24.02% and 22.87%, and total flavones contens were 31.89% and 19.63%, respectively.
5. The variations is abundant among families, 5 of which was selected as the super one. Their gains of traits including seedling height, ground diameter, leaf number, leaf length, leaf width, leaf area and total flavones contents were between 4.71% and 15.85%.
1、初步探讨了杜仲杂交的花粉技术、授粉技术和育苗技术,结果表明:树上收集和室内水培收集两种不同方式所收集花粉的萌发特性没有明显的差异,说明在杜仲杂交育种中,可以通过水培花枝收集花粉。不同父本的散粉期和花粉生活力差异较大,秦仲2号和洛超3号是较好的杂交父本;不同母本的可授期相差较大。不同父本间杂交种子的发芽率差异不显著、而母本间差异显著;以杜仲优良无性系小叶做母本时种子发芽率最高,达99.6%。利用播种育苗方法对所获得的杂交种子进行繁殖,得到了7个F1家系。
2、将杜仲杂交种子剥去种皮,用清水反复冲洗干净,500ppm的赤霉素浸泡24h后,70%酒精消毒30s,1g/L HgCl2表面消毒12min,无菌水冲洗3~4次,切去少许胚芽和胚根端胚乳后胚根向下接入无激素的MS培养基中,30d后萌发成苗。组织培养培育无菌苗的方法可以显著地提高杜仲种子的成苗率。
3、子叶节不定芽诱导的最适宜培养基为MS+6-BA 2mg/L,诱导率85%;单节茎段快速增值的最适宜培养基为MS+6-BA 2mg/L,增值系数3.80;最适宜单节茎段生长的培养基为MS+ 6-BA 1 mg/L + NAA 0.1 mg/L,生长系数为3.71。1/2MS+IBA1.5 mg/L是最佳的生根培养基,生根率90%。以沙子、珍珠岩、腐殖质按1∶2∶1为基质移栽,成活率达80%。
4、通过对家系田间表型性状和标志性功效成分总黄酮含量分化程度分析,选出综合变异程度较高的家系6和家系3作为构建杜仲遗传连锁图谱的作图群体。它们的田间表型性状平均变异系数分别为24.02%和22.87%;标志性功效成分总黄酮含量的变异系数分别为31.89%和19.63%。
5、通过家系间性状差异性比较,结果表明各性状在家系间存在很大的差异。选出的优良家系5,其苗高生长量、地径生长量、叶片数、叶长、叶宽、叶面积和总黄酮含量都较群体均值有不同程度的提高,增幅在4.71%~15.85%之间。
Eucommia ulmoides is a multipurpose tree species, with high value for development and utilization. Genetic linkage map make it possible for early selection in Eucommia ulmoides breeding. It can promote the process of breeding by shortening the time and improving the accuracy as well as reducing the cost. Establishing a suitable mapping population is the first step in mapmaking and determine the quality of the map. For this purpose, the techniques of sexual cross and tissue culture for cloning hybrid seedlings of Eucommia ulmoides were studied, and the variation level of hybride families on field phenotypic characteristics and total flavones content were tested. The main results are as belows:
1. The techniques of pollen collection and pollination in hybridization, as well as culture of seedlings were studyed. The pollens collectd in two different ways (from trees and branches under hydroponic culture condition) has no obvious differences in germination characteristics. Branche hydroponic culture is a convenience way for pollen collecting in crossing. The activity and dispersal piriod of pollens are different among pollen parents. The best pollen parents are Qinzhong2 and Luochao3. Pollination period differed greatly among female parents. There were no obvious differences in seeds germination rates among pollen parents, but there were obvious differences among female parents. The seed germination rate of the clone Xiaoye is rather high, and the maximum level is 99.6%. By the way of sowing and culture of seedlings, seven F1 familes have been obtained.
2. With the process of striped the coats, rinsed repeatedly with water, immersioned in 500ppm gibberellin for 24h, disinfected with 70% alcohol for 30s and with 1g/L HgCl2 for 12min , washed with sterilized water for 3 to 4 times and truncated endosperms, the hybrid seeds chould germinate in a high ratio on the MS medium with hormonesfree in 30d and the seedlings grow well. The results showed that seeds germination ratio chould be improved greatly in tissue culture conditions .
3. The best medium for buds’initiation of cotyledonary nodes was MS+6-BA2mg/L. The inducing ratio could be up to 85%. The best medium for the propagation of stem with one node was MS+6-BA2mg/L. The multiplication coefficient was 3.80. The best medium for the growth of stem with one node was MS+6-BA1 mg/L+NAA0.1 mg/L. The growth ratio was 3.71. The best medium for rooting was 1/2MS+IBA1.5 mg/L, the rooting ratio reached 90%. The seedlings was transplanted in the substrate of sand and perlite mixed with humus for 1:2:1, the survival rate was 80%.
4. Based on the variation analysis of field phenotypic characteristics and total flavones content, family 6 and family 3 have been selected as the mapping populations, whose variation are higher than others. Their average variation coefficients of field phenotypic characteristics were 24.02% and 22.87%, and total flavones contens were 31.89% and 19.63%, respectively.
5. The variations is abundant among families, 5 of which was selected as the super one. Their gains of traits including seedling height, ground diameter, leaf number, leaf length, leaf width, leaf area and total flavones contents were between 4.71% and 15.85%.
引文
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