银杏雄株资源多样性分析与评价
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摘要
银杏(Ginkgo biloba L.),雌雄异株,其雄株具有重要的经济、生态和观赏价值。我国银杏雄株资源丰富,研究其资源的多样性,评价其各类资源的孢粉学特性和黄酮类化合物等次生代谢物质含量水平,对于银杏雄株的分类、优良株系的选育及其花粉和叶片资源的综合开发利用具有重要的理论和实践意义。本研究在多年研究的基础上,选用江苏扬州、泰州、徐州等银杏主要产区的86棵银杏雄株为试材,采用孢粉学和ISSR分子标记等技术研究银杏雄株资源的多样性,并利用高效液相色谱(HPLC)技术检测雄株叶片和花粉中黄酮类化合物的含量,运用紫外分光光度法分析银杏叶片中银杏酸的含量,由此综合评价了银杏雄株不同类型的聚类关系,并根据设定的黄酮苷元和总黄酮含量的阈值筛选出银杏雄株优良株系。主要研究结果如下：
(1)通过光学显微镜、扫描电镜和透射电镜观察进行银杏花粉孢粉学研究。结果表明,银杏雄株的新鲜花粉呈球形或椭圆形,极轴长为19.93μm-25.63μm,赤道轴长为27.65gm-33.97gm,花粉形状指数(极轴长/赤道轴长,P/E)为0.64-0.86,其变异系数(CV)分别为4.87%、6.37%和6.72%；干燥花粉赤道面观呈银杏种核状,近极面萌发区呈沟状,其极轴长为12.05gm-20.29μm,赤道轴长为26.03μm-40.78gm,P/E值为0.43-0.56,其CV值分别为13.75%、13.26%和4.99%；银杏雄株花粉表面纹饰呈球珠镶嵌状、贝甲镶嵌状、线纹镶嵌状、弧纹镶嵌状等四种类型；银杏雄株花粉壁厚度为0.861μm-1.076gm,外壁厚度是内壁的2-6倍；供试雄株间上述各项性状指标存在显著或极显著差异。经采用花粉极轴长、赤道轴长及P/E值三元变量进行系统聚类分析,供试雄株可分为四种类型,其第Ⅰ、Ⅱ、Ⅲ、Ⅳ类分别覆盖4株、34株、44株和4株。
(2)采用改良CTAB法提取银杏雄株叶片基因组DNA,其条带清晰、完整,迁移率与λ-DNA相当。应用L16(44)正交试验设计筛选和优化ISSR-PCR分析体系,得到由模板DNA50ng、10×buffer2μl、Taq酶1.0U、Mg2+2.5mmol·L-1、dNTP0.20mmol·L-1和引物0.4μmol·L-1组成的适于银杏ISSR-PCR分析的优化体系。利用100个ISSR引物分别对试材进行扩增,筛选得到12个扩增条带信号清晰的引物,12个引物对供试银杏雄株扩增出94条DNA条带,其中多态性DNA条带为56条,多态位点百分数(P)为59.57%,每个引物扩增条带5-11条,片段大小为200-2000bp。供试雄株的平均有效等位基因数(Ne)为1.7149,平均基因多样度为(H)0.3966,平均Shannon's信息指数(Ⅰ)为0.5771,具丰富的遗传多样性。供试雄株个体间的Nei's距离在0.0443-0.9667之间,利用最长距离法进行系统聚类分析,取阈值为0.8234时,可分为Ⅰ、Ⅱ两类,取阈值为0.6342时,可分为五类。
(3)供试银杏雄株材料的总遗传变异中有10.48%的变异存在于群体间,而群体内的遗传变异为89.52%,明显高于银杏雄株群体间的遗传变异。三个银杏雄株群体的平均有效等位基因数(Ne)分别为1.7199、1.5520、1.5916,平均基因多样度(H)分别为0.3964、0.3066、0.3380,平均Shannon's信息指数(Ⅰ)分别为0.5760、0.4473、0.4964,其Ne、H、Ⅰ值的大小顺序完全一致,供试雄株的遗传多样性水平均为扬州株系>徐州株系>泰州株系。三个银杏雄株群体的基因流(Nm)为4.2710,且群体间的遗传一致度也较高,说明群体间存在广泛的基因交流。
(4)运用HPLC技术分析不同株系叶片主要黄酮苷元的含量,并采用三因子法计算总黄酮苷含量。研究结果表明,提取银杏雄株叶片黄酮类化合物的最佳提取组合为：料液比1：15,乙醇浓度70%,超声提取时间为40min,提取温度80℃,提取2次。供试银杏雄株间的各黄酮苷元含量和总黄酮含量存在显著差异,叶片的槲皮素、山奈黄素和异鼠李素三种黄酮苷元的平均含量分别为2.381mg·g-1DW、2.155mg·g-1DW和1.8515mg·g-1DW,总黄酮的平均含量为15.99mg·g-1DW.银杏雄株叶片的总黄酮含量与叶片厚度、比叶重(SLW)呈极显著正相关,其叶片厚度、SLW可作为评价银杏叶片中黄酮类化合物含量水平的重要指标。通过设定黄酮苷元和总黄酮含量的选择阈值,扬州有5个株系(04、05、12、39、49)、泰州有4个株系(59、66、68、74)、徐州有2个株系(80、85)符合叶用标准。
(5)运用HPLC技术分析不同株系花粉主要黄酮苷元的含量,并采用三因子法计算总黄酮苷含量。结果表明,供试银杏雄株花粉的槲皮素、山奈黄素和异鼠李素三种黄酮苷元的平均含量分别为0.327mg·g-1DW、7.891mg·g-1DW和0.254mg·g-1DW,其中山奈黄素含量相对较高,三种黄酮苷元的含量差异较大；总黄酮的平均含量为22.430mg·g-1DW,高于叶片。叶片和花粉两者间总黄酮含量的相关系数为0.9270*,呈显著正相关。通过设定黄酮苷元和总黄酮含量的选择阈值,扬州有14个株系(02、04、05、08、10、11、12、16、18、34、39、44、46、49)、泰州有5个株系(59、63、66、68、70)符合花粉用标准。
(6)本研究连续两年在不同时期采集扬州大学银杏种质资源圃生长条件一致的银杏雄株不同部位叶片,采用分光光度法分析了不同处理叶片的银杏酸的含量,旨在明确银杏酸的提取分离技术和检测方法及银杏雄株叶片银杏酸的变化规律,并优选出低酚酸成分的银杏雄株。结果显示：银杏雄株叶片银杏酸的含量9月30日、10月15日的较高,7月30日、8月15日的较低；长枝叶片中酚酸类物质的含量低于短枝叶片的含量；本地区银杏雄株采叶应在7月底至8月上、中旬,此时的叶片中银杏酸的平均含量为1.372%、1.361%；研究供试9株银杏雄株间叶片的银杏酸含量差异显著,58号的银杏酸类含量为1.404%,低于平均水平,可作为低酚酸银杏雄株供进一步试验研究,以决选出低酚酸银杏雄株。研究结果可为筛选低酚酸成分银杏叶用种质资源提供一定的理论依据和物质基础。
Ginkgo (Ginkgo biloba L.) is a dioecious, perennial tree. The male plant has important economic, ecological and ornamental values. Male plant resources is quite abundant in China, and it is important to study the diversity of male ginkgo resources and evaluate the palynology characterization and secondary metabolite levels of such as flavonoids. It may provide vital theoretical and practical significance for classification of ginkgo male plants, breeding of excellent male lines and exploitation of leaves and pollens Based on many years of research,86male plants of ginkgo distributed at Yangzhou, Xuzhou and Taizhou were used as materials in this study. The diversities of ginkgo male plant resources were investigated by techniques palynology and ISSR molecular marker techniques. The contents of flavonoids in leaves and pollens were determined by high performance liquid chromatography (HPLC). The ginkgolic acid levels were analzed with UV spectrophotometry. The comprehensive evaluation of clustering relations of different male ginkgo types was carried out. The excellent strains were screened on the basis of the set values of flavonoids aglycone total flavonoids. The main results were as followings:
(1) The pollens were observed with optical microscope, scanning electron microscope and transmission electron microscope. The fresh pollen was spherical or elliptical, its polar axis and equatorial axis were19.93μm-25.63μm and27.65μm-33.97μm in length, respectivly, the ratio of polar axis to equatorial axis (P/E) was0.64-0.86, their CV is4.87%,6.37%and6.72%respectivly; The dry pollen shows as the shape of a seed stone of ginkgo in equatorial view, its polar axis and equatorial axis were12.05μm-20.29μm and26.03μm-40.78μm in length, respectivly, the index of pollen shape (P/E) was0.43-0.56, their CV is13.75%,13.26%and4.99%, respectivly; The external veins of pollen could be classified into four types:mosaic spherical pearl-like, mosaic conch-like, mosaic lineolatus-like and mosaic curve-like. Thickness of pollen wall, in which the thickness of exine was2-6times as more as that of intine, was0.861μm-1.076μm. The differences of above characters were significant or much significant among the male plants. After3-variate cluster analysis on the indexes of polar axis, equatorial axis and P/E value, the experimental male plants could be divided into four types, in which the first, second, third and foruth type included4,34,44and4strains, respectivly.
(2) The genomic DNA was extracted from the leaves of male ginkgo plant by modified CTAB method, the bands were clear, complete and bright, and the mobilities of which were equaral to that of λDNA. The optimized system was selected for ISSR-PCR analysis by L16(44) orthogonal design. The results showed that the optimized system should consist of template DNA of50ng,10×buffer2μl, Taq DNA polymerase of1.0U, Mg2+of2.5mmol·L-1, dNTP of0.20mmol·L-1and primers of0.4μmol·L-1. Among100ISSR primers,12primers which can amplify more clear polymorphic bands were selected. Using these12selected primers,94bands were amplified, among which56were polymorphic bands, percentage of polymorphic loci was59.57%, with an average being5-11bands per primer and the segment size ranging from200to2000bp. The86male ginkgo plants used in this study showed abundant genetic diversities, with average effective number of alleles (Ne), average gene diversity (H), and average Shannon's information index (Ⅰ) of1.7149,0.3966and0.5771, respectively. The Nei's distance between individual male plant ranged from0.0443to0.9667. Clustering analysis by furthest-neighbor method showed the male plant of ginkgo were classified into two groups when based on the threshold value of0.8234, while five groups were gained when the threshold value equaled to0.6342.
(3) The results showed that10.48%out of the total genetic variation exists among populations, while the genetic variation within populations was89.52%, which was much higher than that among populations. For the three male ginkgo populations, the average effective numbers of alleles (Ne) was1.7199,1.5916and1.5520, respectively. The average gene diversity (H) was0.3964,0.3380and0.3066, respectively. The average Shannon's information index (Ⅰ) was0.5760,0.4964and0.4473, respectively. The values of Ne, H and I were in the same orders for these three ginkgo populations, that is, Yangzhou>Xuzhou> Taizhou. The gene flow (Nm) of the male plant of Ginkgo biloba L. were4.2710. Moreover, genetic identity was high. These indicated that extensive gene exchange exists in different populations.
(4) The flavonoid aglycone content in the leaves of different male strains were determined by HPLC. The flavonoid glycoside content of the samples were calculated according to the three-factor method. The results showed that the optimal extraction combinations of flavonoids in male ginkgo plant leaves were material liquid ratio1:15, ethanol concentration70%, extraction time60min, extraction temperature80℃, extraction2times.there were significant differences between contens of each flavonoid aglycone and the total flavonoid in the leaves of male ginkgo plants in this experiment. The average content of quercetin, kaempferol and isorhamnetin in leaf were2.381mg·g-1DW,2.155mg·g-1DW and1.8515mg·g-1DW, respectively. There was little difference in the content of individual flavonoid aglycones in leaves, with the total flavonoid of15.99mg·g-1DW. There was a significantly positive correlation between the content of flavonoid glycosides in leaf with leaf thickness and specific leaf weight (SLW). Therefore, the leaf thickness and SLW could be used to evaluate the content of flavonoids in leaf as an important index.Five strains from Yangzhou (04,05,12,39and49), four strains from Taizhou (59,66,68and74) and two strains from Xuzhou (80and85) met with the standard of leaf utilization by setting the choice threshold of flavonoid aglycone and total flavonoids content.
(5) The flavonoid aglycone content in the pollens of different male strains were determined by HPLC. The flavonoid glycoside content of the samples were calculated according to the three-factor method. The average content of quercetin, kaempferol and sorhamnetin in pollens was0.327mg·g-1DW,7.891mg·g-1DW and0.254mg·g-1DW, respectively. The content of kaempferol in pollens was higher than the other two flavone aglycones. The flavonoid glycoside content of pollens was22.240mg·g-1DW, which was higher than that of leaves. There was a positive relationship between the flavonoid glycoside content of leaves and pollens with correlation coefficient0.9270*. Fourteen strains from Yangzhou (02,04,05,08,10,11,12,16,18,34,39,44,46and49), five strains from Taizhou (59,63,66,68and70) met with the standard of pollen utilization by setting the choice threshold of flavonoid aglycone and total flavonoids content.
(6) In this study, the leaves on different part of branch, sampled from9male ginkgo plants growing uniformly in Ginkgo germplasm garden of Yangzhou University at different seasons in2008and2009, were used to determine the ginkgolic acid contents of ginkgo leaves with different treatments by UV spectrophotometric method, in order to clarify extraction technology and determination method of phenolic acids, and select male ginkgo plants which contain low phenolic acid. The results demonstrated that ginkgolic acid content in the leaf of male ginkgo plant varied from June15to November15. The average content of Ginkgo acid in Ginkgo leaves were1.372%.The ginkgolic acid contents on September30and October15were higher, and those on July30and August15were lower. The ginkgolic acid contents in the leaves of long branch was lower than that of short branch. The leave-collecting time at early in August or in mid-August in Yangzhou was recommended, at these time, the ginkgolic acid content was1.361%and1.384%, respectively. There existed significant difference between the ginkgolic acid contents in leaves of9tested male ginkgo plant. The ginkgolic acid content of58was lower than average level, with the value of1.404%, respectively. This male ginkgo plant can provide materials for further experiment research for low-acid male plant selection. The above results may provide important theoretical and material basis for screening low phenolic acid ginkgo male plant germplasm resources.
(1)通过光学显微镜、扫描电镜和透射电镜观察进行银杏花粉孢粉学研究。结果表明,银杏雄株的新鲜花粉呈球形或椭圆形,极轴长为19.93μm-25.63μm,赤道轴长为27.65gm-33.97gm,花粉形状指数(极轴长/赤道轴长,P/E)为0.64-0.86,其变异系数(CV)分别为4.87%、6.37%和6.72%；干燥花粉赤道面观呈银杏种核状,近极面萌发区呈沟状,其极轴长为12.05gm-20.29μm,赤道轴长为26.03μm-40.78gm,P/E值为0.43-0.56,其CV值分别为13.75%、13.26%和4.99%；银杏雄株花粉表面纹饰呈球珠镶嵌状、贝甲镶嵌状、线纹镶嵌状、弧纹镶嵌状等四种类型；银杏雄株花粉壁厚度为0.861μm-1.076gm,外壁厚度是内壁的2-6倍；供试雄株间上述各项性状指标存在显著或极显著差异。经采用花粉极轴长、赤道轴长及P/E值三元变量进行系统聚类分析,供试雄株可分为四种类型,其第Ⅰ、Ⅱ、Ⅲ、Ⅳ类分别覆盖4株、34株、44株和4株。
(2)采用改良CTAB法提取银杏雄株叶片基因组DNA,其条带清晰、完整,迁移率与λ-DNA相当。应用L16(44)正交试验设计筛选和优化ISSR-PCR分析体系,得到由模板DNA50ng、10×buffer2μl、Taq酶1.0U、Mg2+2.5mmol·L-1、dNTP0.20mmol·L-1和引物0.4μmol·L-1组成的适于银杏ISSR-PCR分析的优化体系。利用100个ISSR引物分别对试材进行扩增,筛选得到12个扩增条带信号清晰的引物,12个引物对供试银杏雄株扩增出94条DNA条带,其中多态性DNA条带为56条,多态位点百分数(P)为59.57%,每个引物扩增条带5-11条,片段大小为200-2000bp。供试雄株的平均有效等位基因数(Ne)为1.7149,平均基因多样度为(H)0.3966,平均Shannon's信息指数(Ⅰ)为0.5771,具丰富的遗传多样性。供试雄株个体间的Nei's距离在0.0443-0.9667之间,利用最长距离法进行系统聚类分析,取阈值为0.8234时,可分为Ⅰ、Ⅱ两类,取阈值为0.6342时,可分为五类。
(3)供试银杏雄株材料的总遗传变异中有10.48%的变异存在于群体间,而群体内的遗传变异为89.52%,明显高于银杏雄株群体间的遗传变异。三个银杏雄株群体的平均有效等位基因数(Ne)分别为1.7199、1.5520、1.5916,平均基因多样度(H)分别为0.3964、0.3066、0.3380,平均Shannon's信息指数(Ⅰ)分别为0.5760、0.4473、0.4964,其Ne、H、Ⅰ值的大小顺序完全一致,供试雄株的遗传多样性水平均为扬州株系>徐州株系>泰州株系。三个银杏雄株群体的基因流(Nm)为4.2710,且群体间的遗传一致度也较高,说明群体间存在广泛的基因交流。
(4)运用HPLC技术分析不同株系叶片主要黄酮苷元的含量,并采用三因子法计算总黄酮苷含量。研究结果表明,提取银杏雄株叶片黄酮类化合物的最佳提取组合为：料液比1：15,乙醇浓度70%,超声提取时间为40min,提取温度80℃,提取2次。供试银杏雄株间的各黄酮苷元含量和总黄酮含量存在显著差异,叶片的槲皮素、山奈黄素和异鼠李素三种黄酮苷元的平均含量分别为2.381mg·g-1DW、2.155mg·g-1DW和1.8515mg·g-1DW,总黄酮的平均含量为15.99mg·g-1DW.银杏雄株叶片的总黄酮含量与叶片厚度、比叶重(SLW)呈极显著正相关,其叶片厚度、SLW可作为评价银杏叶片中黄酮类化合物含量水平的重要指标。通过设定黄酮苷元和总黄酮含量的选择阈值,扬州有5个株系(04、05、12、39、49)、泰州有4个株系(59、66、68、74)、徐州有2个株系(80、85)符合叶用标准。
(5)运用HPLC技术分析不同株系花粉主要黄酮苷元的含量,并采用三因子法计算总黄酮苷含量。结果表明,供试银杏雄株花粉的槲皮素、山奈黄素和异鼠李素三种黄酮苷元的平均含量分别为0.327mg·g-1DW、7.891mg·g-1DW和0.254mg·g-1DW,其中山奈黄素含量相对较高,三种黄酮苷元的含量差异较大；总黄酮的平均含量为22.430mg·g-1DW,高于叶片。叶片和花粉两者间总黄酮含量的相关系数为0.9270*,呈显著正相关。通过设定黄酮苷元和总黄酮含量的选择阈值,扬州有14个株系(02、04、05、08、10、11、12、16、18、34、39、44、46、49)、泰州有5个株系(59、63、66、68、70)符合花粉用标准。
(6)本研究连续两年在不同时期采集扬州大学银杏种质资源圃生长条件一致的银杏雄株不同部位叶片,采用分光光度法分析了不同处理叶片的银杏酸的含量,旨在明确银杏酸的提取分离技术和检测方法及银杏雄株叶片银杏酸的变化规律,并优选出低酚酸成分的银杏雄株。结果显示：银杏雄株叶片银杏酸的含量9月30日、10月15日的较高,7月30日、8月15日的较低；长枝叶片中酚酸类物质的含量低于短枝叶片的含量；本地区银杏雄株采叶应在7月底至8月上、中旬,此时的叶片中银杏酸的平均含量为1.372%、1.361%；研究供试9株银杏雄株间叶片的银杏酸含量差异显著,58号的银杏酸类含量为1.404%,低于平均水平,可作为低酚酸银杏雄株供进一步试验研究,以决选出低酚酸银杏雄株。研究结果可为筛选低酚酸成分银杏叶用种质资源提供一定的理论依据和物质基础。
Ginkgo (Ginkgo biloba L.) is a dioecious, perennial tree. The male plant has important economic, ecological and ornamental values. Male plant resources is quite abundant in China, and it is important to study the diversity of male ginkgo resources and evaluate the palynology characterization and secondary metabolite levels of such as flavonoids. It may provide vital theoretical and practical significance for classification of ginkgo male plants, breeding of excellent male lines and exploitation of leaves and pollens Based on many years of research,86male plants of ginkgo distributed at Yangzhou, Xuzhou and Taizhou were used as materials in this study. The diversities of ginkgo male plant resources were investigated by techniques palynology and ISSR molecular marker techniques. The contents of flavonoids in leaves and pollens were determined by high performance liquid chromatography (HPLC). The ginkgolic acid levels were analzed with UV spectrophotometry. The comprehensive evaluation of clustering relations of different male ginkgo types was carried out. The excellent strains were screened on the basis of the set values of flavonoids aglycone total flavonoids. The main results were as followings:
(1) The pollens were observed with optical microscope, scanning electron microscope and transmission electron microscope. The fresh pollen was spherical or elliptical, its polar axis and equatorial axis were19.93μm-25.63μm and27.65μm-33.97μm in length, respectivly, the ratio of polar axis to equatorial axis (P/E) was0.64-0.86, their CV is4.87%,6.37%and6.72%respectivly; The dry pollen shows as the shape of a seed stone of ginkgo in equatorial view, its polar axis and equatorial axis were12.05μm-20.29μm and26.03μm-40.78μm in length, respectivly, the index of pollen shape (P/E) was0.43-0.56, their CV is13.75%,13.26%and4.99%, respectivly; The external veins of pollen could be classified into four types:mosaic spherical pearl-like, mosaic conch-like, mosaic lineolatus-like and mosaic curve-like. Thickness of pollen wall, in which the thickness of exine was2-6times as more as that of intine, was0.861μm-1.076μm. The differences of above characters were significant or much significant among the male plants. After3-variate cluster analysis on the indexes of polar axis, equatorial axis and P/E value, the experimental male plants could be divided into four types, in which the first, second, third and foruth type included4,34,44and4strains, respectivly.
(2) The genomic DNA was extracted from the leaves of male ginkgo plant by modified CTAB method, the bands were clear, complete and bright, and the mobilities of which were equaral to that of λDNA. The optimized system was selected for ISSR-PCR analysis by L16(44) orthogonal design. The results showed that the optimized system should consist of template DNA of50ng,10×buffer2μl, Taq DNA polymerase of1.0U, Mg2+of2.5mmol·L-1, dNTP of0.20mmol·L-1and primers of0.4μmol·L-1. Among100ISSR primers,12primers which can amplify more clear polymorphic bands were selected. Using these12selected primers,94bands were amplified, among which56were polymorphic bands, percentage of polymorphic loci was59.57%, with an average being5-11bands per primer and the segment size ranging from200to2000bp. The86male ginkgo plants used in this study showed abundant genetic diversities, with average effective number of alleles (Ne), average gene diversity (H), and average Shannon's information index (Ⅰ) of1.7149,0.3966and0.5771, respectively. The Nei's distance between individual male plant ranged from0.0443to0.9667. Clustering analysis by furthest-neighbor method showed the male plant of ginkgo were classified into two groups when based on the threshold value of0.8234, while five groups were gained when the threshold value equaled to0.6342.
(3) The results showed that10.48%out of the total genetic variation exists among populations, while the genetic variation within populations was89.52%, which was much higher than that among populations. For the three male ginkgo populations, the average effective numbers of alleles (Ne) was1.7199,1.5916and1.5520, respectively. The average gene diversity (H) was0.3964,0.3380and0.3066, respectively. The average Shannon's information index (Ⅰ) was0.5760,0.4964and0.4473, respectively. The values of Ne, H and I were in the same orders for these three ginkgo populations, that is, Yangzhou>Xuzhou> Taizhou. The gene flow (Nm) of the male plant of Ginkgo biloba L. were4.2710. Moreover, genetic identity was high. These indicated that extensive gene exchange exists in different populations.
(4) The flavonoid aglycone content in the leaves of different male strains were determined by HPLC. The flavonoid glycoside content of the samples were calculated according to the three-factor method. The results showed that the optimal extraction combinations of flavonoids in male ginkgo plant leaves were material liquid ratio1:15, ethanol concentration70%, extraction time60min, extraction temperature80℃, extraction2times.there were significant differences between contens of each flavonoid aglycone and the total flavonoid in the leaves of male ginkgo plants in this experiment. The average content of quercetin, kaempferol and isorhamnetin in leaf were2.381mg·g-1DW,2.155mg·g-1DW and1.8515mg·g-1DW, respectively. There was little difference in the content of individual flavonoid aglycones in leaves, with the total flavonoid of15.99mg·g-1DW. There was a significantly positive correlation between the content of flavonoid glycosides in leaf with leaf thickness and specific leaf weight (SLW). Therefore, the leaf thickness and SLW could be used to evaluate the content of flavonoids in leaf as an important index.Five strains from Yangzhou (04,05,12,39and49), four strains from Taizhou (59,66,68and74) and two strains from Xuzhou (80and85) met with the standard of leaf utilization by setting the choice threshold of flavonoid aglycone and total flavonoids content.
(5) The flavonoid aglycone content in the pollens of different male strains were determined by HPLC. The flavonoid glycoside content of the samples were calculated according to the three-factor method. The average content of quercetin, kaempferol and sorhamnetin in pollens was0.327mg·g-1DW,7.891mg·g-1DW and0.254mg·g-1DW, respectively. The content of kaempferol in pollens was higher than the other two flavone aglycones. The flavonoid glycoside content of pollens was22.240mg·g-1DW, which was higher than that of leaves. There was a positive relationship between the flavonoid glycoside content of leaves and pollens with correlation coefficient0.9270*. Fourteen strains from Yangzhou (02,04,05,08,10,11,12,16,18,34,39,44,46and49), five strains from Taizhou (59,63,66,68and70) met with the standard of pollen utilization by setting the choice threshold of flavonoid aglycone and total flavonoids content.
(6) In this study, the leaves on different part of branch, sampled from9male ginkgo plants growing uniformly in Ginkgo germplasm garden of Yangzhou University at different seasons in2008and2009, were used to determine the ginkgolic acid contents of ginkgo leaves with different treatments by UV spectrophotometric method, in order to clarify extraction technology and determination method of phenolic acids, and select male ginkgo plants which contain low phenolic acid. The results demonstrated that ginkgolic acid content in the leaf of male ginkgo plant varied from June15to November15. The average content of Ginkgo acid in Ginkgo leaves were1.372%.The ginkgolic acid contents on September30and October15were higher, and those on July30and August15were lower. The ginkgolic acid contents in the leaves of long branch was lower than that of short branch. The leave-collecting time at early in August or in mid-August in Yangzhou was recommended, at these time, the ginkgolic acid content was1.361%and1.384%, respectively. There existed significant difference between the ginkgolic acid contents in leaves of9tested male ginkgo plant. The ginkgolic acid content of58was lower than average level, with the value of1.404%, respectively. This male ginkgo plant can provide materials for further experiment research for low-acid male plant selection. The above results may provide important theoretical and material basis for screening low phenolic acid ginkgo male plant germplasm resources.
引文
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