一种禾本科植物叶片表皮层气孔密度的估测方法 - Google Patents

[0019] 本方法通过对显微镜采集到的样本叶片图像进行处理，得到视场内样本叶片的气孔数量，从而计算得到样本叶片的气孔密度，能够快速获取样本叶片的气孔密度，而且 ... CN107179268A-一种禾本科植物叶片表皮层气孔密度的估测方法 -GooglePatents 一种禾本科植物叶片表皮层气孔密度的估测方法 DownloadPDF Info Publicationnumber CN107179268A CN107179268A CN201710384484.8A CN201710384484A CN107179268A CN107179268A CN107179268A CN107179268A CN201710384484A CN201710384484A CN201710384484A CN107179268A CN107179268A CN107179268A Authority CN China Priorartkeywords epidermis leaf blade adhesivetape leafsamples Priorartdate 2017-05-26 Legalstatus(Thelegalstatusisanassumptionandisnotalegalconclusion.Googlehasnotperformedalegalanalysisandmakesnorepresentationastotheaccuracyofthestatuslisted.) Pending Applicationnumber CN201710384484.8A Otherlanguages English(en) Inventor 郑甲成 詹秋文 李杰勤 尤依锦 刘言龙 CurrentAssignee(Thelistedassigneesmaybeinaccurate.Googlehasnotperformedalegalanalysisandmakesnorepresentationorwarrantyastotheaccuracyofthelist.) AnhuiUniversityofScienceandTechnology OriginalAssignee AnhuiUniversityofScienceandTechnology Prioritydate(Theprioritydateisanassumptionandisnotalegalconclusion.Googlehasnotperformedalegalanalysisandmakesnorepresentationastotheaccuracyofthedatelisted.) 2017-05-26 Filingdate 2017-05-26 Publicationdate 2017-09-19 2017-05-26 ApplicationfiledbyAnhuiUniversityofScienceandTechnology filed Critical AnhuiUniversityofScienceandTechnology 2017-05-26 PrioritytoCN201710384484.8A priority Critical patent/CN107179268A/zh 2017-09-19 PublicationofCN107179268A publication Critical patent/CN107179268A/zh Links Espacenet GlobalDossier Discuss 210000002615 Epidermis Anatomy 0.000 title claims abstract description 49 241000209504 Poaceae Species 0.000 title claims abstract description 13 239000002390 adhesivetape Substances 0.000 claims abstract description 39 210000000282 Nails Anatomy 0.000 claims abstract description 15 210000001367 Arteries Anatomy 0.000 claims description 16 210000003462 Veins Anatomy 0.000 claims description 16 WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Chemicalcompound 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Substances 0.000 description 1 235000019322 gelatine Nutrition 0.000 description 1 235000011852 gelatinedesserts Nutrition 0.000 description 1 238000005259 measurement Methods 0.000 description 1 230000029553 photosynthesis Effects 0.000 description 1 238000010672 photosynthesis Methods 0.000 description 1 238000009738 saturating Methods 0.000 description 1 230000005068 transpiration Effects 0.000 description 1 Classifications G—PHYSICS G01—MEASURING;TESTING G01N—INVESTIGATINGORANALYSINGMATERIALSBYDETERMININGTHEIRCHEMICALORPHYSICALPROPERTIES G01N15/00—Investigatingcharacteristicsofparticles;Investigatingpermeability,pore-volume,orsurface-areaofporousmaterials G01N15/08—Investigatingpermeability,pore-volume,orsurfaceareaofporousmaterials G01N15/088—Investigatingvolume,surfacearea,sizeordistributionofpores;Porosimetry Abstract 一种禾本科植物叶片表皮层气孔密度的估测方法，首先准备样本叶片，并保存在FAA固定液中；然后取出样本叶片，在表面涂上指甲油或透明胶，干燥后粘上透明胶带，将透明胶带连同指甲油或透明胶一起撕下，分别制作样本叶片的上层表皮标本和下层表皮标本，用显微镜观察表皮标本的细胞并拍照，计数视野范围内的气孔总数，并利用气孔总数除以视野范围总面积，计算出样本叶片的气孔密度。

本方法能够快速获取样本叶片的气孔密度，结果准确、效率高。

Description 一种禾本科植物叶片表皮层气孔密度的估测方法 技术领域 [0001]本发明涉及植物学领域，具体是一种禾本科植物叶片表皮层气孔密度的估测方法。

背景技术 [0002]植物叶片的气孔密度会影响植物的光合作用和蒸腾作用，是表征植物叶片气孔特性的重要参数之一。

目前大部分研究都是通过人工计数进行气孔密度的测量，该方法不但浪费劳动力，而且当数据量很大时，人工很容易因为疲劳出现错误，还会受外界环境因素的影响，无法及时对采集的数据进行处理。

发明内容 [0003]本发明的目的在于克服现有技术的不足，提供一种禾本科植物叶片表皮层气孔密度的估测方法，通过对光学显微镜采集到的样本叶片表皮标本图像进行处理，得到视场内样本叶片的气孔数量，从而计算得到样本叶片的气孔密度。

[0004]为解决上述技术问题，本发明提供一种禾本科植物叶片表皮层气孔密度的估测方法，包括以下步骤： [0005](1)样本叶片的准备： [0006]选取株型健壮、发育正常的禾本科植物叶片为估测对象，量取叶片总长度，在距离叶片长度中间2〜3cm处分别横向剪断叶片，然后在距离主叶脉0.5〜2cm处纵向剪掉主叶脉一侧的边缘叶片，最后沿着主叶脉纵向剪掉包括主叶脉的另外一半叶片，作为样本叶片，标记叶片样品的上、下表皮后，迅速置于提前预冷的FAA固定液中保存备用； [0007](2)表皮标本的制作： [0008]①、用镊子轻轻夹取保存在FAA固定液中的样本叶片，放置在预先准备好的白色滤纸上，用两个载玻片左右横向压住样本叶片以保持叶片平整，室温放置晾干或用脱脂棉拭去叶片表面液体； [0009]②、蘸取无色指甲油或透明胶，均匀的涂抹在样本叶片表面，室温放置使指甲油或透明胶干燥； [0010]③、用透明胶带粘在涂有指甲油或透明胶的样本叶片表面，赶走透明胶带底部气泡，使透明胶带和样本叶片表层紧密粘在一起； [0011]④、迅速撕下透明胶带，使透明胶带连同指甲油或透明胶一起被撕下，将撕下的透明胶带粘在载玻片上，标注上表皮或下表皮标本； [0012]⑤、用镊子将样本叶片翻转过来，利用同样的办法，制作样本叶片另一面的表皮标本； [0013](3)气孔观察： [0014]利用光学显微镜观察表皮标本的气孔细胞分布，选择视场内没有杂质，且气孔数目分布尽可能多的观察视场，拍摄图片，并保存，每个表皮标本拍摄η张图片，n>5; [0015]⑷气孔密度计算： [0016]数出η张图片的气孔数目总和，按下式计算，分别获得叶片样品上表皮层和下表皮层的气孔密度： [0017] [0018]为简单说明问题起见，以下对本发明所述的禾本科植物叶片表皮层气孔密度的估测方法均简称为本方法。

[0019]本方法通过对显微镜采集到的样本叶片图像进行处理，得到视场内样本叶片的气孔数量，从而计算得到样本叶片的气孔密度，能够快速获取样本叶片的气孔密度，而且效率较高。

附图说明 [0020]图1是实施例1拍摄的高粱叶片下表皮的气孔图像。

[0021]图2是实施例2拍摄的小麦叶片下表皮的气孔图像 具体实施方式 [0022]实施例1 [0023]高粱叶片表皮层气孔密度的估测方法，包括以下步骤： [0024](1)样本叶片的准备： [0025]在高粱灌浆初期，选取株型健壮、发育正常、距离高粱植株顶端的第三片叶片为估测对象，量取叶片总长度，在距离叶片长度中间3cm处分别横向剪断叶片，然后在距离主叶脉2cm处纵向剪掉主叶脉一侧的边缘叶片，最后沿着主叶脉纵向剪掉包括主叶脉的另外一半叶片，作为样本叶片，计算样本叶片的面积为6cmX2cm=12cm2,标记叶片样品的上、下表皮后，迅速置于提前预冷的FAA固定液中保存备用； [0026](2)表皮标本的制作： [0027]①、用镊子轻轻夹取保存在FAA固定液中的样本叶片，放置在预先准备好的白色滤纸上(注意区别样本叶片上、下表皮），用两个载玻片左右横向压住样本叶片以保持样本叶片平整，室温放置并用脱脂棉擦拭，使叶片表面液体晾干； [0028]②、用微小的刷子蘸取透明胶，均匀的涂抹在样本叶片表面，室温放置并适当扇风使透明胶干燥； [0029]③、用4cmX2.5cm的透明胶带粘在涂有指甲油的样本叶片表面，赶走透明胶带底部气泡，使透明胶带和样本叶片表层紧密粘在一起； [0030]④、迅速撕下透明胶带，使透明胶带连同指甲油一起被撕下，并保证指甲油的完整性，将撕下的透明胶带粘在载玻片上，记号笔标注上表皮或下表皮标本； [0031]⑤、用镊子将样本叶片翻转过来，利用同样的办法，制作样本叶片另一面的表皮标本； [0032](3)气孔观察： [0033]打开光学显微镜和连接显微镜的电脑，利用放大倍数100X(目镜IOX，物镜10X)下观察，启动显微镜操作软件，矫正显微镜白平衡参数;将制作的表皮标本+载玻片放置在载物台上，推片器弹簧夹夹住，然后旋转推片器螺旋，将所要观察的部位调到通光孔的正中，调节细准焦螺旋至可以观察到清晰的图像为止;观察表皮标本的气孔细胞分布，选择视场内没有杂质，且气孔数目分布尽可能多的观察视场，拍摄图片，并保存，每个表皮标本拍摄10张图片，如图1所示； [0034]⑷气孔密度计算： [0035]数出10张图片的气孔数目总和，按下式计算，分别获得叶片样品上表皮层和下表皮层的气孔密度： [0036] [0037]其中FAA固定液（IOOmL)含有89mL60%的乙醇、5mL38%的甲醛和6mL冰醋酸。

60%的乙醇的配置方法是:量取无水乙醇60mL，加入40mLCldH2O(双蒸水）。

38%的甲醛的配制方法是：量取38mL甲醛，加入62mLddH20。

[0038]FAA固定液现配现用。

[0039]实施例2 [0040]小麦叶片表皮层气孔密度的估测方法，包括以下步骤： [0041](1)样本叶片的准备： [0042]在小麦灌浆初期，选取株型健壮、发育正常、从植株顶端数起的第四片叶片为估测对象，量取叶片总长度，在距离叶片长度中间2cm处分别横向剪断叶片，然后在距离主叶脉0.5cm处纵向剪掉主叶脉一侧的边缘叶片，最后沿着主叶脉纵向剪掉包括主叶脉的另外一半叶片，作为样本叶片，计算样本叶片的面积为4cmX0.5cm=2.0cm2,标记叶片样品的上、下表皮后，迅速置于提前预冷的FAA固定液中保存备用； [0043](2)表皮标本的制作： [0044]①、用镊子轻轻夹取保存在FAA固定液中的样本叶片，放置在预先准备好的白色滤纸上(注意区别样本叶片上、下表皮），用两个载玻片左右横向压住样本叶片以保持样本叶片平整，室温放置30s晾干； [0045]②、用微小的刷子蘸取无色指甲油，均匀的涂抹在样本叶片表面，室温放置使指甲油干燥； [0046]③、用3cmXI.Ocm的透明胶带粘在涂有透明胶的样本叶片表面，赶走透明胶带底部气泡，使透明胶带和样本叶片表层紧密粘在一起； [0047]④、迅速撕下透明胶带，使透明胶带连同透明胶一起被撕下，并保证透明胶的完整性，将撕下的透明胶带粘在载玻片上，记号笔标注上表皮或下表皮标本； [0048]⑤、用镊子将样本叶片翻转过来，利用同样的办法，制作样本叶片另一面的表皮标本； [0049](3)气孔观察： [0050]打开光学显微镜和连接显微镜的电脑，利用放大倍数100X(目镜IOX，物镜10X)下观察，启动显微镜操作软件，矫正显微镜白平衡参数;将制作的表皮标本+载玻片放置在载物台上，推片器弹簧夹夹住，然后旋转推片器螺旋，将所要观察的部位调到通光孔的正中，调节细准焦螺旋至可以观察到清晰的图像为止;观察表皮标本的气孔细胞分布，选择视场内没有杂质，且气孔数目分布尽可能多的观察视场，拍摄图片，并保存，每个表皮标本拍摄5张图片，如图2所示； [0051]⑷气孔密度计算： [0052]数出5张图片的气孔数目总和，按下式计算，分别获得叶片样品上表皮层和下表皮层的气孔密度： [0053] [0054]其中FAA固定液（IOOmL)含有89mL60%的乙醇、5mL38%的甲醛和6mL冰醋酸。

60%的乙醇的配置方法是:量取无水乙醇60mL，加入40mLCldH2O(双蒸水）。

38%的甲醛的配制方法是：量取38mL甲醛，加入62mLddH20。

配制好的FAA固定液4°C低温保存，且在一个月内使用。

Claims(4) 1.一种禾本科植物叶片表皮层气孔密度的估测方法，具体包括以下步骤：(1)样本叶片的准备：选取株型健壮、发育正常的禾本科植物叶片为估测对象，量取叶片总长度，在距离叶片长度中间部位两侧各2〜3cm处分别横向剪断叶片，然后在距离主叶脉0.5〜2cm处纵向剪掉主叶脉一侧的边缘叶片，最后沿着主叶脉纵向剪掉包括主叶脉的另外一半叶片，作为样本叶片，标记叶片样品的上、下表皮后，迅速置于提前预冷的FAA固定液中保存备用；⑵表皮标本的制作：①、用镊子轻轻夹取保存在FAA固定液中的样本叶片，放置在预先准备好的白色滤纸上，用两个载玻片左右横向压住样本叶片以保持叶片平整，室温放置晾干或用脱脂棉拭去样本叶片表面液体；②、蘸取无色指甲油或透明胶，均匀的涂抹在样本叶片表面，室温放置使指甲油或透明胶干燥；③、用透明胶带粘在涂有指甲油或透明胶的样本叶片表面，赶走透明胶带底部气泡，使透明胶带和样本叶片表层紧密粘在一起；④、迅速撕下透明胶带，使透明胶带连同指甲油或透明胶一起被撕下，将撕下的透明胶带粘在载玻片上，标注上表皮或下表皮标本；⑤、用镊子将样本叶片翻转过来，利用同样的办法，制作样本叶片另一面的表皮标本；⑶气孔观察：利用光学显微镜观察表皮标本的气孔细胞分布，选择视野内没有杂质，且气孔数目分布尽可能多的观察视野，拍摄图片，并保存，每个表皮标本拍摄η张图片，n>5;⑷气孔密度计算：数出η张图片的气孔数目总和，按下式计算，分别获得叶片样品上表皮层和下表皮层的气孔密度： 2.根据权利要求1所述的禾本科植物叶片表皮层气孔密度的估测方法，其特征在于：FAA固定液含有:89体积份60%的乙醇、5体积份38%的甲醛和6体积份冰醋酸。

3.根据权利要求2所述的禾本科植物叶片表皮层气孔密度的估测方法，其特征在于:所述的60%的乙醇和38%的甲醛都用双蒸水配制。

4.根据权利要求2所述的禾本科植物叶片表皮层气孔密度的估测方法，其特征在于:所述的FAA固定液现配现用。

CN201710384484.8A 2017-05-26 2017-05-26 一种禾本科植物叶片表皮层气孔密度的估测方法 CN107179268A (zh) PriorityApplications(1) ApplicationNumber PriorityDate FilingDate Title CN201710384484.8A CN107179268A (zh) 2017-05-26 2017-05-26 一种禾本科植物叶片表皮层气孔密度的估测方法 ApplicationsClaimingPriority(1) ApplicationNumber PriorityDate FilingDate Title CN201710384484.8A CN107179268A (zh) 2017-05-26 2017-05-26 一种禾本科植物叶片表皮层气孔密度的估测方法 Publications(1) PublicationNumber PublicationDate CN107179268A true CN107179268A (zh) 2017-09-19 Family ID=59835100 FamilyApplications(1) ApplicationNumber Title PriorityDate FilingDate CN201710384484.8A CN107179268A (zh) 2017-05-26 2017-05-26 一种禾本科植物叶片表皮层气孔密度的估测方法 CountryStatus(1) Country Link CN (1) CN107179268A (zh) CitedBy(2) *Citedbyexaminer,†Citedbythirdparty Publicationnumber Prioritydate Publicationdate Assignee Title CN109632431A (zh) * 2019-02-12 2019-04-16 北京林业大学 一种简单快速的透明方法 CN112014178A (zh) * 2020-08-14 2020-12-01 天津农学院 一种用于观测草坪草叶脉网络结构的装片制备方法 Citations(2) *Citedbyexaminer,†Citedbythirdparty Publicationnumber Prioritydate Publicationdate Assignee Title CN101556215A (zh) * 2009-05-13 2009-10-14 西北农林科技大学 一种田间试验无损伤定位观察植物气孔的取样方法 CN102721583A (zh) * 2012-06-01 2012-10-10 河南科技大学 一种夹竹桃叶的气孔窝叶表皮的制片方法 2017 2017-05-26 CN CN201710384484.8A patent/CN107179268A/zh active Pending PatentCitations(2) *Citedbyexaminer,†Citedbythirdparty Publicationnumber Prioritydate Publicationdate Assignee Title CN101556215A (zh) * 2009-05-13 2009-10-14 西北农林科技大学 一种田间试验无损伤定位观察植物气孔的取样方法 CN102721583A (zh) * 2012-06-01 2012-10-10 河南科技大学 一种夹竹桃叶的气孔窝叶表皮的制片方法 Non-PatentCitations(3) *Citedbyexaminer,†Citedbythirdparty Title 刘明智，等:"指甲油涂抹撕取法制取植物叶气孔装片",《生物学通报》 * 王晓涧，等:"樱桃试管苗叶片气孔观察初报",《延安大学学报(自然科学版)》 * 陈佰鸿，等:"一种用透明胶带粘取叶片表皮观察气孔的方法",《植物生理学通讯》 * CitedBy(2) *Citedbyexaminer,†Citedbythirdparty Publicationnumber Prioritydate Publicationdate Assignee Title CN109632431A (zh) * 2019-02-12 2019-04-16 北京林业大学 一种简单快速的透明方法 CN112014178A (zh) * 2020-08-14 2020-12-01 天津农学院 一种用于观测草坪草叶脉网络结构的装片制备方法 SimilarDocuments Publication PublicationDate Title CN107179268A (zh) 2017-09-19 一种禾本科植物叶片表皮层气孔密度的估测方法 Erickson 1976 Modelingofplantgrowth Maliketal. 2015 Dewharvestingefficiencyoffourspeciesofcacti Lund 1959 ASimpleCountingChamberforNannoplankton1 CN109855936B (zh) 2020-08-11 一种组织完整、利于显微观察的植物组织切片的制备方法 Crowderetal. 2012 Ribhistomorphometryforadultageestimation US20200163656A1 (en) 2020-05-28 Hydrogel-enabledmicrofluidicsweatsequesteringforwearablehuman-deviceinterfaces CN106093048B (zh) 2019-02-05 一种用于树木年轮定年的方法及实现该方法的装置 CN202329577U (zh) 2012-07-11 阔叶植物叶面积活体测定装置 CN102620960A (zh) 2012-08-01 一种用于观察水稻叶鞘组织胼胝质的叶鞘组织制片方法 CN102905515A (zh) 2013-01-30 用于估计液体的张力、势和活度的二面体传感器 Hackettetal. 1969 Amethodfordeterminingthepositionandsizeoflateralprimordiaintheaxesofrootswithoutsectioning Noyceetal. 2015 Apracticalmethodforstaginggrapevineinflorescenceprimordiainseason1,withimproveddescriptionofstages CN101929853A (zh) 2010-12-29 一种腹足纲动物腹足吸盘面积的测量方法 Drakeetal. 1956 Ultravioletscanner-cameraforpaperchromatography Pengetal. 2019 Effectofaltitudeonclimate–growthrelationshipsofChinesewhitepine(Pinusarmandii)inthenorthernFuniuMountain,centralChina Moore 2017 Preparationofcytologysamples:tricksofthetrade Rodriguezetal. 2008 Automatedlasercapturemicrodissectionfortissueproteomics Pinheiroetal. 2015 Indirectmethodsfordeterminingtheareaindexofforagecactuscladodes Haetal. 2021 Domino‐likewatertransportonTillandsiathroughflexibletrichomewings RU2013134056A (ru) 2015-01-27 Способсравнительнойиндикациизагрязненностивоздухапофлуктуирующейасимметриилистьевберезы Beatty 1937 Amethodforgrowingandformakingpermanentslidesofpollentubes JPH11299792A (ja) 1999-11-02 肌年齢の推定方法 WO2014123844A1 (en) 2014-08-14 Cytologicalstainingcompositionsandusesthereof CN103063491A (zh) 2013-04-24 一种孢子捕捉仪捕捉物样本的制作与显微镜观察方法 LegalEvents Date Code Title Description 2017-09-19 PB01 Publication 2017-09-19 PB01 Publication 2017-10-20 SE01 Entryintoforceofrequestforsubstantiveexamination 2017-10-20 SE01 Entryintoforceofrequestforsubstantiveexamination 2020-04-17 RJ01 Rejectionofinventionpatentapplicationafterpublication 2020-04-17 RJ01 Rejectionofinventionpatentapplicationafterpublication Applicationpublicationdate: 20170919