山东地区忍冬种质资源比较研究
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摘要
目的：经过长期的自然选择与人工定向培育,山东忍冬植株形态出现了分化变异,形成了数量众多的种质。对这些种质的遗传多样性进行评价,并对其产量与质量进行比较研究,为生产上高产、优质种质的筛选提供参考；研究光照等因素对药材质量的影响,为确定合理的人工栽培措施提供指导。
方法：从植株形态学,果皮、种皮、花粉粒超微形态,开花特性,种子蛋白、叶片过氧化物同工酶、药材红外光谱等方面对遗传多样性进行评价；采用于花期每日计数植株开花数目的方法对产量进行研究；采用人工遮阴法设置不同的光照强度,微量元素的施用采用人工叶面喷施法,活性成分含量测定采用高效液相色谱法。
结果：①不同种质忍冬植株形态存在差异,表现在株型、枝条长度及缠绕程度、叶片毛被情况、花蕾形态及着生方式、果实及种子大小等方面不同,以花蕾、果实、种子等繁殖器官的相关测量数据为指标进行聚类分析,结果与当地药农对品系的划分结果稍有偏差,表明不同种质间不仅其繁殖器官发生了变异,植株其他的表型特征亦发生了变异。②不同种质忍冬果皮、种皮超微形态均出现了变异,根据果皮表面角质纹理的形态,可以将其分为三类,结果与当地药农对品系的划分结果较一致。种质17花粉粒极面观为类方形,萌发孔为4个,与其他种质间差异较大；其他种质其花粉粒表现出较大一致性,表明亲缘关系较近,但其大小、表面刺突密度及尖锐程度、萌发孔大小等存在差异,可以此进行鉴别；根据花粉粒及萌发孔大小对所研究种质进行聚类分析,结果能较好的反应种质间的亲缘关系。③不同种质忍冬始花期除种质17(5月24日)外,其他均为5月23日,表现出较大一致性,但不同种质间其盛花期、末花期及开花振幅存在差异,表明了种质间的不同。④种子SDS-PAGE电泳分析结果表明,不同种质间谱带差异不大,不能作为种质划分的依据。叶片过氧化物酶分析结果表明,不同种质间谱带的数目、位置及强弱程度存在较大差异,可以作为金银花种质划分的依据。采用FTIR红外光谱三级鉴定技术对不同种质忍冬花蕾进行分析,结果表明,一维光谱有较大一致性,表明不同种质间其主体成分相似；不同种质间其二阶导数光谱存在较大差异,主要表现在吸收峰的位置上及部分吸收峰的强弱程度上存在差异；二维红外光谱上,所研究种质在1300～900cm-1波数范围内差异较大,表现在自动峰的数目及自动峰的强弱上各有不同,因此,傅立叶红外光谱三级鉴定技术可以作为不同种质金银花鉴定的辅助手段。⑤综合考虑产量与质量两大因素,认为种质7、种质8为优良种质；种质19产量较低,木犀草苷含量不符合药典规定,应将其淘汰；种质1、2、4、18活性成分含量均符合药典规定,但产量较低,应通过人工修剪、加强人工管理等措施增加其产量；种质5所测4种活性成分含量均较高,但因其植株长势较弱,产量低,应对其进行提纯、复壮；种质3、17木犀草苷含量不符合药典规定,产量中等,其是否可作为优良种质进行推广有待进一步研究。相关性分析结果表明,绿原酸含量与咖啡酸含量存在显著正相关(p<0.05),花蕾长度与花蕾宽度、花蕾重与花蕾长度存在极显著正相关(p<0.01),花蕾重与花蕾宽度间存在显著正相关(p<0.05)。多重线性回归分析结果表明,开花数目等因素对药材产量的影响大小为：开花数目>花蕾干重>花蕾长度>花蕾宽度。⑥光照有助于增加忍冬叶片叶绿体光合色素含量、药材可溶性糖含量及花蕾干重,木犀草苷和马钱苷含量亦以自然光条件下为最大,相关性分析结果亦表明了这一点；微量元素对金银花活性成分的影响是一个复杂过程,有些是正效应,有些是负效应,提示究竟施用哪种微量元素应经过具体实验后确定；蚜虫危害后,金银花可溶性糖含量和单花重量均降低,所测绿原酸等4种活性成分的含量均比正常植株低,提示忍冬人工栽培过程中蚜虫防治的重要性和必要性。
结论：不同种质忍冬在植株形态,果皮、种皮、花粉粒超微形态,开花特性,种子蛋白、叶片过氧化物同工酶、药材红外光谱等方面均存在差异,表明了种质的不同；不同种质间产量与质量不同,可以通过种质优选来提高药材的产量和质量。光照、微量元素、虫害胁迫等对药材质量均有影响,在人工栽培过程中应采用适宜的管理措施,以提高药材质量。
Objective:The plant morphology of Lonicera japonica Thunb.origined in ShanDong arised variation and formed different germplasms after long-time natural selection and artificial breeding during cultivation.The aim of this thesis is to evaluate genetic diversity and compare yield and quality of different germplasms so as to provide references for fine germplasm screening and to study the effects of some factors on quality so as to provide guidance for establishing reasonable cultivation measures.
Methods:The plant morphology, the micromorphology of fruit and seed coats and pollen grains,flowering habits,electophoresis of seed protein,peroxidase isoenzymes of leaves,infrared spectroscopy of buds were studied to evaluate genetic diversity;the flowering numbers were counted every day to appraise the yield;different lighting intensity were set up by shading; microelement were sprayed through leaves;the content of active constituent was determined by high performance liquid chromatography(HPLC).
Results:①There are some differences among different germplasms in many respects,such as plant shape,twining extent and length of branch,leaves hair, bud morphology and its manner of growth on branch,size of fruit and seed.Ten germplasms were clustered on the indexes of the measurment data of buds,fruits and seeds,the results showed that there had a little deviation from the results achieved by farmer,that showed that not only the reproductive organs but also other parts of the plant became difference among different germplasms.②There were some differences in micromorphology of fruit and seed coats.All germplasms could be divided into three categories on account of micromorphology of horny texture on the fruit,that was in agreement with the results achieved by farmer. There was very significant difference between germplasm17and others,with its pollen grain was square seen in polar axis and it had four apertures.Other germplasm had some similarity in pollen grain,that showed that the relationship of theirs was nearer.But we could distinguish them on account of their pollen size,aperture length and the density of spine.The result of cluster analysis with the indexes of pollen and aperture size showed their relationship.③All the germplasms showed great similarity in their initial time with the date May23except germplasm17(May24),but there was different in their blooming time,falling time and flowering amplitude.④Analysis of seed protein electophoresis showed that there was little differences in bands among different germplasms and it could not be used to diagnosis germplasm. Analysis of POD isoenzymes showed that there were much different in numbers and location of bands as well as the intensity of the bands,that it could be used to diagnosis germplasms.The IR spectra and2D-IR correlation analysis were used to analyze the buds of Lonicera japonica Thunb.from different germplasm,the result showed that the IR spectra have some similarity,showing that their main chemical compounds were similar;the second derivative sectra and2D-IR correlation spectra have significant differences,such as absorption strength frequency and form.The result showed that the FTIR analysis was effective for the identification of the buds of different germplasm resources.⑤Allowing for yield and quality,we thought that germplasm7and8were fine;the yield of germplasm19was lowest and its content of galuteolin did not conform to pharmacopoeia rules,so it should be reject;the content of active compounds of germplasm1,2,4,18met the pharmacopoeia rules,but they had poor yield,so we should increase their yield by means of strengthing field management;the content of active compounds in germplasm5was higer,but the yield was poor for its weak growing trend,so we should make it purify and rejuvenate;the content of galuteolin in germplasm3and17did not conform to pharmacopoeia rules and their yield were medium,so that they could or not be popularized as fine breeding should be through further studied.the results of correation analysis showed that the content of chlorogenic acid was positively correlated with that of caffeic acid(p<0.05), length of buds had a highly significant positive correlation with width and weight of buds (p<0.01),weight of buds was positively correlated with that of width (p<0.05).The result of linear regression showed that the extent of the factors such as flowering numbers affect the yield:flowering numbers>weight of buds>length of buds>width of buds.⑥Lighting was useful for increasing the content of the leaf photo synthetic pigment and soluble sugar in medicinal materials and weight of dried buds,the content of galuteolin and loganin was highest in the condition of natural light;the influences of microelement to the active compounds were complicated,some were positively and some were negatively,that prompted us what kind of microelement we used should be from experiments;the content of soluble sugar and weight of dried buds as well as4active compounds decreased after aphid occurring,that prompted us it was necessary and important to control aphid during Lonicera japonica Thunb. cultivation.
Conclusions:There were differences in such aspects as plant morphology,the micromorphology of fruit and seed coats and pollen grains,flowering habits,electophoresis of seed protein,peroxidase isoenzymes of leaves,infrared spectroscopy of buds among different germplasm;the yield and quality was also different,that prompted us we could increase the yield and quality by means of screening fine breeding.Some factors such as lighting,microelement and stress caused by pests had effect on quality of flos Lonicera japonica Thunb.,that told us we should take appropriate measures to improve quality of medicinal materials.
方法：从植株形态学,果皮、种皮、花粉粒超微形态,开花特性,种子蛋白、叶片过氧化物同工酶、药材红外光谱等方面对遗传多样性进行评价；采用于花期每日计数植株开花数目的方法对产量进行研究；采用人工遮阴法设置不同的光照强度,微量元素的施用采用人工叶面喷施法,活性成分含量测定采用高效液相色谱法。
结果：①不同种质忍冬植株形态存在差异,表现在株型、枝条长度及缠绕程度、叶片毛被情况、花蕾形态及着生方式、果实及种子大小等方面不同,以花蕾、果实、种子等繁殖器官的相关测量数据为指标进行聚类分析,结果与当地药农对品系的划分结果稍有偏差,表明不同种质间不仅其繁殖器官发生了变异,植株其他的表型特征亦发生了变异。②不同种质忍冬果皮、种皮超微形态均出现了变异,根据果皮表面角质纹理的形态,可以将其分为三类,结果与当地药农对品系的划分结果较一致。种质17花粉粒极面观为类方形,萌发孔为4个,与其他种质间差异较大；其他种质其花粉粒表现出较大一致性,表明亲缘关系较近,但其大小、表面刺突密度及尖锐程度、萌发孔大小等存在差异,可以此进行鉴别；根据花粉粒及萌发孔大小对所研究种质进行聚类分析,结果能较好的反应种质间的亲缘关系。③不同种质忍冬始花期除种质17(5月24日)外,其他均为5月23日,表现出较大一致性,但不同种质间其盛花期、末花期及开花振幅存在差异,表明了种质间的不同。④种子SDS-PAGE电泳分析结果表明,不同种质间谱带差异不大,不能作为种质划分的依据。叶片过氧化物酶分析结果表明,不同种质间谱带的数目、位置及强弱程度存在较大差异,可以作为金银花种质划分的依据。采用FTIR红外光谱三级鉴定技术对不同种质忍冬花蕾进行分析,结果表明,一维光谱有较大一致性,表明不同种质间其主体成分相似；不同种质间其二阶导数光谱存在较大差异,主要表现在吸收峰的位置上及部分吸收峰的强弱程度上存在差异；二维红外光谱上,所研究种质在1300～900cm-1波数范围内差异较大,表现在自动峰的数目及自动峰的强弱上各有不同,因此,傅立叶红外光谱三级鉴定技术可以作为不同种质金银花鉴定的辅助手段。⑤综合考虑产量与质量两大因素,认为种质7、种质8为优良种质；种质19产量较低,木犀草苷含量不符合药典规定,应将其淘汰；种质1、2、4、18活性成分含量均符合药典规定,但产量较低,应通过人工修剪、加强人工管理等措施增加其产量；种质5所测4种活性成分含量均较高,但因其植株长势较弱,产量低,应对其进行提纯、复壮；种质3、17木犀草苷含量不符合药典规定,产量中等,其是否可作为优良种质进行推广有待进一步研究。相关性分析结果表明,绿原酸含量与咖啡酸含量存在显著正相关(p<0.05),花蕾长度与花蕾宽度、花蕾重与花蕾长度存在极显著正相关(p<0.01),花蕾重与花蕾宽度间存在显著正相关(p<0.05)。多重线性回归分析结果表明,开花数目等因素对药材产量的影响大小为：开花数目>花蕾干重>花蕾长度>花蕾宽度。⑥光照有助于增加忍冬叶片叶绿体光合色素含量、药材可溶性糖含量及花蕾干重,木犀草苷和马钱苷含量亦以自然光条件下为最大,相关性分析结果亦表明了这一点；微量元素对金银花活性成分的影响是一个复杂过程,有些是正效应,有些是负效应,提示究竟施用哪种微量元素应经过具体实验后确定；蚜虫危害后,金银花可溶性糖含量和单花重量均降低,所测绿原酸等4种活性成分的含量均比正常植株低,提示忍冬人工栽培过程中蚜虫防治的重要性和必要性。
结论：不同种质忍冬在植株形态,果皮、种皮、花粉粒超微形态,开花特性,种子蛋白、叶片过氧化物同工酶、药材红外光谱等方面均存在差异,表明了种质的不同；不同种质间产量与质量不同,可以通过种质优选来提高药材的产量和质量。光照、微量元素、虫害胁迫等对药材质量均有影响,在人工栽培过程中应采用适宜的管理措施,以提高药材质量。
Objective:The plant morphology of Lonicera japonica Thunb.origined in ShanDong arised variation and formed different germplasms after long-time natural selection and artificial breeding during cultivation.The aim of this thesis is to evaluate genetic diversity and compare yield and quality of different germplasms so as to provide references for fine germplasm screening and to study the effects of some factors on quality so as to provide guidance for establishing reasonable cultivation measures.
Methods:The plant morphology, the micromorphology of fruit and seed coats and pollen grains,flowering habits,electophoresis of seed protein,peroxidase isoenzymes of leaves,infrared spectroscopy of buds were studied to evaluate genetic diversity;the flowering numbers were counted every day to appraise the yield;different lighting intensity were set up by shading; microelement were sprayed through leaves;the content of active constituent was determined by high performance liquid chromatography(HPLC).
Results:①There are some differences among different germplasms in many respects,such as plant shape,twining extent and length of branch,leaves hair, bud morphology and its manner of growth on branch,size of fruit and seed.Ten germplasms were clustered on the indexes of the measurment data of buds,fruits and seeds,the results showed that there had a little deviation from the results achieved by farmer,that showed that not only the reproductive organs but also other parts of the plant became difference among different germplasms.②There were some differences in micromorphology of fruit and seed coats.All germplasms could be divided into three categories on account of micromorphology of horny texture on the fruit,that was in agreement with the results achieved by farmer. There was very significant difference between germplasm17and others,with its pollen grain was square seen in polar axis and it had four apertures.Other germplasm had some similarity in pollen grain,that showed that the relationship of theirs was nearer.But we could distinguish them on account of their pollen size,aperture length and the density of spine.The result of cluster analysis with the indexes of pollen and aperture size showed their relationship.③All the germplasms showed great similarity in their initial time with the date May23except germplasm17(May24),but there was different in their blooming time,falling time and flowering amplitude.④Analysis of seed protein electophoresis showed that there was little differences in bands among different germplasms and it could not be used to diagnosis germplasm. Analysis of POD isoenzymes showed that there were much different in numbers and location of bands as well as the intensity of the bands,that it could be used to diagnosis germplasms.The IR spectra and2D-IR correlation analysis were used to analyze the buds of Lonicera japonica Thunb.from different germplasm,the result showed that the IR spectra have some similarity,showing that their main chemical compounds were similar;the second derivative sectra and2D-IR correlation spectra have significant differences,such as absorption strength frequency and form.The result showed that the FTIR analysis was effective for the identification of the buds of different germplasm resources.⑤Allowing for yield and quality,we thought that germplasm7and8were fine;the yield of germplasm19was lowest and its content of galuteolin did not conform to pharmacopoeia rules,so it should be reject;the content of active compounds of germplasm1,2,4,18met the pharmacopoeia rules,but they had poor yield,so we should increase their yield by means of strengthing field management;the content of active compounds in germplasm5was higer,but the yield was poor for its weak growing trend,so we should make it purify and rejuvenate;the content of galuteolin in germplasm3and17did not conform to pharmacopoeia rules and their yield were medium,so that they could or not be popularized as fine breeding should be through further studied.the results of correation analysis showed that the content of chlorogenic acid was positively correlated with that of caffeic acid(p<0.05), length of buds had a highly significant positive correlation with width and weight of buds (p<0.01),weight of buds was positively correlated with that of width (p<0.05).The result of linear regression showed that the extent of the factors such as flowering numbers affect the yield:flowering numbers>weight of buds>length of buds>width of buds.⑥Lighting was useful for increasing the content of the leaf photo synthetic pigment and soluble sugar in medicinal materials and weight of dried buds,the content of galuteolin and loganin was highest in the condition of natural light;the influences of microelement to the active compounds were complicated,some were positively and some were negatively,that prompted us what kind of microelement we used should be from experiments;the content of soluble sugar and weight of dried buds as well as4active compounds decreased after aphid occurring,that prompted us it was necessary and important to control aphid during Lonicera japonica Thunb. cultivation.
Conclusions:There were differences in such aspects as plant morphology,the micromorphology of fruit and seed coats and pollen grains,flowering habits,electophoresis of seed protein,peroxidase isoenzymes of leaves,infrared spectroscopy of buds among different germplasm;the yield and quality was also different,that prompted us we could increase the yield and quality by means of screening fine breeding.Some factors such as lighting,microelement and stress caused by pests had effect on quality of flos Lonicera japonica Thunb.,that told us we should take appropriate measures to improve quality of medicinal materials.
引文
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