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Article(id=1321785496964018270, tenantId=1146029695717560320, journalId=1316781310003478668, issueId=1321785454333121105, articleNumber=null, orderNo=null, doi=10.11983/CBB24022, pmid=null, cstr=32102.14.CBB24022, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1708185600000, receivedDateStr=2024-02-18, revisedDate=null, revisedDateStr=null, acceptedDate=1711728000000, acceptedDateStr=2024-03-30, onlineDate=1793067820062, onlineDateStr=2026-10-27, pubDate=1736438400000, pubDateStr=2025-01-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793067820062, onlineIssueDateStr=2026-10-27, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793067820062, creator=13701087609, updateTime=1793067820062, updator=13701087609, issue=Issue{id=1321785454333121105, tenantId=1146029695717560320, journalId=1316781310003478668, year='2025', volume='60', issue='1', pageStart='1', pageEnd='143', issueExtLink='null', onlineDate='null', pubDate='1736438400000', pubDateStr='2025-01-10', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1793067809899, creator='13701087609', updateTime=1793068026855, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1321786364417397082, tenantId=1146029695717560320, journalId=1316781310003478668, issueId=1321785454333121105, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1321786364417397083, tenantId=1146029695717560320, journalId=1316781310003478668, issueId=1321785454333121105, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=81, endPage=89, ext={EN=ArticleExt(id=1321785498482356319, articleId=1321785496964018270, tenantId=1146029695717560320, journalId=1316781310003478668, language=EN, title=A New Cereal Seed Treatment Method for Displaying Endosperm Cell Structures Under Micro CT Scanning, columnId=1317485704066748799, journalTitle=Chinese Bulletin of Botany, columnName=TECHNIQUES AND METHODS, runingTitle=null, highlight=null, articleAbstract=

Cereal starch endosperm is the main source of human staple food, but the methods for observing its mature cell structure are not well developed. Micro CT technology, a non-destructive three-dimensional imaging technique, is a powerful tool for studying plant morphology. However, due to the uniform density of cereal starch endosperm, whose cell structures cannot be distinguished clearly by conventional micro CT techniques. In this study, we used phosphotungstic acid to treat ten different kinds of cereal seeds of seven crops, and then dried them by CO2 critical point dryer. We found that the cell structure of the treated endosperms can be clearly displayed by micro CT. Our method provides a new way for studying the structures and functions of cereal starch endosperm cells.

, authors=Xiuping Xu1, 2, Xiaoyu Yang3, 4, Min Feng1, 2, *, authorsList=Xiuping Xu, Xiaoyu Yang, Min Feng, authorCompany=null, correspAuthors=Min Feng, authorNote=null, correspAuthorsNote=
* E-mail:
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禾谷类种子淀粉胚乳是人类主食的重要来源, 但观察其成熟细胞形态的方法非常有限。显微CT是一种非破坏性的三维成像技术, 是研究植物形态的有力工具。由于成熟禾谷类种子淀粉胚乳密度均匀, 常规显微CT技术无法应用。在前期研究的基础上, 用磷钨酸处理10种不同禾谷类作物种子, 经CO2临界点干燥后, 再用显微CT扫描, 可以清晰地分辨出禾谷类作物淀粉胚乳的细胞结构, 为研究此类细胞结构提供了一种新方法。

, authors=徐秀苹1, 2, 杨小雨3, 4, 冯旻1, 2, *, authorsList=徐秀苹, 杨小雨, 冯旻, authorCompany=null, correspAuthors=冯旻, authorNote=

徐秀苹, 中国科学院植物研究所正高级工程师, 植物形态与结构平台主管, 负责显微CT扫描系统和冷冻扫描电镜等大型仪器的运行、管理及功能开发。第一完成人授权发明专利3项、实用新型专利1项。以第一作者发表技术方法类文章4篇, 出版仪器图书1册。完成2项中国科学院仪器设备功能开发项目。入选2022年度中国科学院技术支撑人才项目

, correspAuthorsNote=
* E-mail:
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2国家植物园, 北京 100093, bio={"content":"

徐秀苹, 中国科学院植物研究所正高级工程师, 植物形态与结构平台主管, 负责显微CT扫描系统和冷冻扫描电镜等大型仪器的运行、管理及功能开发。第一完成人授权发明专利3项、实用新型专利1项。以第一作者发表技术方法类文章4篇, 出版仪器图书1册。完成2项中国科学院仪器设备功能开发项目。入选2022年度中国科学院技术支撑人才项目

"}, bioImg=null, bioContent=

徐秀苹, 中国科学院植物研究所正高级工程师, 植物形态与结构平台主管, 负责显微CT扫描系统和冷冻扫描电镜等大型仪器的运行、管理及功能开发。第一完成人授权发明专利3项、实用新型专利1项。以第一作者发表技术方法类文章4篇, 出版仪器图书1册。完成2项中国科学院仪器设备功能开发项目。入选2022年度中国科学院技术支撑人才项目

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Insights from in vivo micro-CT analysis: testing the hydraulic vulnerability segmentation in Acer pseudoplatanus and Fagus sylvatica seedlings. New Phytol 221, 1831-1842., articleTitle=Insights from in vivo micro-CT analysis: testing the hydraulic vulnerability segmentation in Acer pseudoplatanus and Fagus sylvatica seedlings, refAbstract=null), Reference(id=1321785509031030975, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, doi=null, pmid=null, pmcid=null, year=2007, volume=39, issue=null, pageStart=391, pageEnd=399, url=null, language=null, rfNumber=[10], rfOrder=11, authorNames=Perret JS, Al-Belushi ME, Deadman M, journalName=Soil Biol Biochem, refType=null, unstructuredReference=Perret JS, Al-Belushi ME, Deadman M (2007). Non-destructive visualization and quantification of roots using computed tomography. 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Crop Seed Science, 2nd edn. Beijing: China Agriculture Press. pp. 16-18. 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High-efficiency procedure to characterize, segment, and quantify complex multicellularity in raw micrographs in plants. 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(A) Rice seed treated by FAA; (B) Rice seed treated by 10% cesium iodide; (C) Rice seed treated by 10% phosphotungstic acid; (D) Enlarged view of rice seed treated by 10% phosphotungstic acid. Bars=500 µm

, figureFileSmall=dn1eryIEPbLTlRVCOBrHcA==, figureFileBig=Nfw1VLkDwqVvSAR3Nw7PSg==, tableContent=null), ArticleFig(id=1321785504467628186, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=CN, label=图1, caption=不同处理下大米显微CT扫描

(A) FAA处理后的大米; (B) 10%碘化铯处理后的大米; (C) 10%磷钨酸处理后的大米; (D) 10%磷钨酸处理后的大米局部放大。Bars=500 µm

, figureFileSmall=dn1eryIEPbLTlRVCOBrHcA==, figureFileBig=Nfw1VLkDwqVvSAR3Nw7PSg==, tableContent=null), ArticleFig(id=1321785504681537691, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=EN, label=Figure 2, caption=Micro CT scanning of purple rice seeds by different treatments

(A) Purple rice seed treated by FAA; (B) Purple rice seed treated by 10% cesium iodide; (C) Purple rice seed treated by Lugols solution; (D) Purple rice seed treated by 10% phosphotungstic acid; (E) Enlarged view of purple rice seed treated by 10% phosphotungstic acid. Bars=400 µm

, figureFileSmall=3zjUlOuIWcX3SDlHSX7GRA==, figureFileBig=SAfBfdseugNHc8dWFitTlA==, tableContent=null), ArticleFig(id=1321785504757035164, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=CN, label=图2, caption=不同处理下紫米显微CT扫描

(A) FAA处理后的紫米; (B) 10%碘化铯处理后的紫米; (C) Lugols溶液处理后的紫米; (D) 10%磷钨酸处理后的紫米; (E) 10%磷钨酸处理后的紫米局部放大。Bars=400 µm

, figureFileSmall=3zjUlOuIWcX3SDlHSX7GRA==, figureFileBig=SAfBfdseugNHc8dWFitTlA==, tableContent=null), ArticleFig(id=1321785504828338333, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=EN, label=Figure 3, caption=Micro CT scanning of red rice seeds by different treatments

(A) Red rice seed treated by FAA; (B) Red rice seed treated by 10% phosphotungstic acid. Bars=400 µm

, figureFileSmall=cQg5akR75Qqt7rbuKdxTAA==, figureFileBig=/K4VB/2nEr/X5OWWRVry4g==, tableContent=null), ArticleFig(id=1321785504903835806, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=CN, label=图3, caption=不同处理下红米显微CT扫描

(A) FAA处理后的红米; (B) 10%磷钨酸处理后的红米。Bars=400 µm

, figureFileSmall=cQg5akR75Qqt7rbuKdxTAA==, figureFileBig=/K4VB/2nEr/X5OWWRVry4g==, tableContent=null), ArticleFig(id=1321785504987721887, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=EN, label=Figure 4, caption=Micro CT scanning of polished glutinous rice seeds by different treatments

(A) Polished glutinous rice seed treated by FAA; (B) Polished glutinous rice seed treated by 10% phosphotungstic acid. Bars=400 µm

, figureFileSmall=+KlOq3BT8tvHI9kaYUJ01A==, figureFileBig=CQibFmhDP8zmoerRN5FZsQ==, tableContent=null), ArticleFig(id=1321785505050636448, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=CN, label=图4, caption=不同处理下糯米显微CT扫描

(A) FAA处理后的糯米; (B) 10%磷钨酸处理后的糯米。Bars=400 µm

, figureFileSmall=+KlOq3BT8tvHI9kaYUJ01A==, figureFileBig=CQibFmhDP8zmoerRN5FZsQ==, tableContent=null), ArticleFig(id=1321785505126133921, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=EN, label=Figure 5, caption=Micro CT scanning of millet seeds by different treatments

(A) Millet seed treated by FAA; (B) Millet seed treated by 10% phosphotungstic acid. Bars=300 µm

, figureFileSmall=0Q8ZsOtLx9oggzL/a+SaOQ==, figureFileBig=CCh5xJJL50RsqjfYNrHKuw==, tableContent=null), ArticleFig(id=1321785505193242786, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=CN, label=图5, caption=不同处理下谷子种子显微CT扫描

(A) FAA处理后的谷子种子; (B) 10%磷钨酸处理后的谷子种子。Bars=300 µm

, figureFileSmall=0Q8ZsOtLx9oggzL/a+SaOQ==, figureFileBig=CCh5xJJL50RsqjfYNrHKuw==, tableContent=null), ArticleFig(id=1321785505293906083, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=EN, label=Figure 6, caption=Micro CT scanning of oat seeds by different treatments

(A) Oat seed treated by FAA; (B) Oat seed treated by 10% phosphotungstic acid. Bars=500 µm

, figureFileSmall=SMGODLSjYNciex6bUCHeYg==, figureFileBig=Viz+tgPbmPgXpgpS6RhcMQ==, tableContent=null), ArticleFig(id=1321785505361014948, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=CN, label=图6, caption=不同处理下燕麦种子显微CT扫描

(A) FAA处理后的燕麦种子; (B) 10%磷钨酸处理后的燕麦种子。Bars=500 µm

, figureFileSmall=SMGODLSjYNciex6bUCHeYg==, figureFileBig=Viz+tgPbmPgXpgpS6RhcMQ==, tableContent=null), ArticleFig(id=1321785505453289637, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=EN, label=Figure 7, caption=Micro CT scanning of sorghum seeds by different treatments

(A) Sorghum seed treated by FAA; (B) Sorghum seed treated by 10% phosphotungstic acid. Bars=500 µm

, figureFileSmall=D6GGCfxDWW2ZKY8QE1bq3g==, figureFileBig=MR6xxCO0OvkMcD1dE7sIBw==, tableContent=null), ArticleFig(id=1321785505528787110, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=CN, label=图7, caption=不同处理下高粱种子显微CT扫描

(A) FAA处理后的高粱种子; (B) 10%磷钨酸处理后的高粱种子。Bars=500 µm

, figureFileSmall=D6GGCfxDWW2ZKY8QE1bq3g==, figureFileBig=MR6xxCO0OvkMcD1dE7sIBw==, tableContent=null), ArticleFig(id=1321785506787078311, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=EN, label=Figure 8, caption=Micro CT scanning of the seeds of Job’s tears by different treatments

(A) The seed of Job’s tears treated by FAA; (B) The seed of Job’s tears treated by 10% phosphotungstic acid. Bars=500 µm

, figureFileSmall=InJr22xJjhH5kPsJGbtDQw==, figureFileBig=JMdauCkqSFBER8KjTJqDXA==, tableContent=null), ArticleFig(id=1321785506887741608, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=CN, label=图8, caption=不同处理下薏米种子显微CT扫描

(A) FAA处理后的薏米种子; (B) 10%磷钨酸处理后的薏米种子。Bars=500 µm

, figureFileSmall=InJr22xJjhH5kPsJGbtDQw==, figureFileBig=JMdauCkqSFBER8KjTJqDXA==, tableContent=null), ArticleFig(id=1321785506950656169, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=EN, label=Figure 9, caption=Micro CT scanning of maize seeds by different treatments

(A) Maize seed treated by FAA; (B) Maize seed treated by 10% phosphotungstic acid; (C) Part of maize seed treated by 10% phosphotungstic acid; (D) Enlarged view of part of maize seed treated by 10% phosphotungstic acid. Bars=400 µm

, figureFileSmall=3d9FtL7C5FpZwJVePjt59Q==, figureFileBig=6AqDYRj7o81H/nujVLyQXQ==, tableContent=null), ArticleFig(id=1321785507021959338, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1321785496964018270, language=CN, label=图9, caption=不同处理下玉米种子显微CT扫描

(A) FAA处理后的玉米种子; (B) 10%磷钨酸处理后的玉米种子; (C) 10%磷钨酸处理后的玉米种子小块; (D) 10%磷钨酸处理后的玉米种子小块局部放大。Bars=400 µm

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(A) Wheat seed treated by FAA; (B) Wheat seed treated by 10% phosphotungstic acid. Bars=500 µm

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(A) FAA处理后的小麦种子; (B) 10%磷钨酸处理后的小麦种子。Bars=500 µm

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Scanning voltage and resolution of ripe seeds

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Seed Voltage (kV) Resolution (µm)
Rice 100 0.88
Rice (partially enlarged) 50 0.56
Purple rice 100 0.95
Red rice 100 0.74
Glutinous rice 100 0.88
Millet 41 0.68
Oats 100 0.88
Sorghum 100 1.15
Job’s tears 100 2.60
Corn 100 1.48
Corn (partially enlarged) 49 0.56
Wheat 100 1.15
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成熟种子扫描电压和分辨率

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Seed Voltage (kV) Resolution (µm)
Rice 100 0.88
Rice (partially enlarged) 50 0.56
Purple rice 100 0.95
Red rice 100 0.74
Glutinous rice 100 0.88
Millet 41 0.68
Oats 100 0.88
Sorghum 100 1.15
Job’s tears 100 2.60
Corn 100 1.48
Corn (partially enlarged) 49 0.56
Wheat 100 1.15
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Effect of cereal seed endosperm by phosphotungstic acid treatment

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Kind of
seeds		 Rice Purple rice Red
rice		 Glutinous rice Millet
(remove seed coat)		 Oats Sorghum
(remove seed coat)		 Job’s tears Corn
(remove seed coat)		 Wheat
Treatment time (day) 40 40 40 40 30 60 70 90 90 180
Valid or not Valid Valid Valid Valid Valid Valid Valid Valid Valid Invalid
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磷钨酸处理禾谷类种子胚乳效果

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Kind of
seeds		 Rice Purple rice Red
rice		 Glutinous rice Millet
(remove seed coat)		 Oats Sorghum
(remove seed coat)		 Job’s tears Corn
(remove seed coat)		 Wheat
Treatment time (day) 40 40 40 40 30 60 70 90 90 180
Valid or not Valid Valid Valid Valid Valid Valid Valid Valid Valid Invalid
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成熟禾谷类作物种子胚乳细胞的显微CT扫描方法
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徐秀苹 1, 2 , 杨小雨 3, 4 , 冯旻 1, 2, *
植物学报 | 技术方法 2025,60(1): 81-89
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植物学报 |技术方法 2025 , 60 (1) : 81 -89
成熟禾谷类作物种子胚乳细胞的显微CT扫描方法
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徐秀苹, 中国科学院植物研究所正高级工程师, 植物形态与结构平台主管, 负责显微CT扫描系统和冷冻扫描电镜等大型仪器的运行、管理及功能开发。第一完成人授权发明专利3项、实用新型专利1项。以第一作者发表技术方法类文章4篇, 出版仪器图书1册。完成2项中国科学院仪器设备功能开发项目。入选2022年度中国科学院技术支撑人才项目

"}, bioImg=null, bioContent=

徐秀苹, 中国科学院植物研究所正高级工程师, 植物形态与结构平台主管, 负责显微CT扫描系统和冷冻扫描电镜等大型仪器的运行、管理及功能开发。第一完成人授权发明专利3项、实用新型专利1项。以第一作者发表技术方法类文章4篇, 出版仪器图书1册。完成2项中国科学院仪器设备功能开发项目。入选2022年度中国科学院技术支撑人才项目

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徐秀苹1, 2, 杨小雨3, 4, 冯旻1, 2, *
作者信息
  • 1中国科学院植物研究所, 系统与进化植物学重点实验室, 北京 100093
  • 2国家植物园, 北京 100093
  • 3中国农业科学院作物科学研究所, 北京 100081
  • 4贵州省农业科学院, 贵州省亚热带作物研究所, 兴义 562400
通讯作者:
作者简介:

徐秀苹, 中国科学院植物研究所正高级工程师, 植物形态与结构平台主管, 负责显微CT扫描系统和冷冻扫描电镜等大型仪器的运行、管理及功能开发。第一完成人授权发明专利3项、实用新型专利1项。以第一作者发表技术方法类文章4篇, 出版仪器图书1册。完成2项中国科学院仪器设备功能开发项目。入选2022年度中国科学院技术支撑人才项目

A New Cereal Seed Treatment Method for Displaying Endosperm Cell Structures Under Micro CT Scanning
Xiuping Xu1, 2, Xiaoyu Yang3, 4, Min Feng1, 2, *
Affiliations
  • 1Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
  • 2China National Botanical Garden, Beijing 100093, China
  • 3Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • 4Guizhou Institute of Subtropical Crops, Guizhou Academy of Agricultural Sciences, Xingyi 562400, China
    • Corresponding Author:
    * E-mail:
出版时间: 2025-01-10 doi: 10.11983/CBB24022
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摘要
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禾谷类种子淀粉胚乳是人类主食的重要来源, 但观察其成熟细胞形态的方法非常有限。显微CT是一种非破坏性的三维成像技术, 是研究植物形态的有力工具。由于成熟禾谷类种子淀粉胚乳密度均匀, 常规显微CT技术无法应用。在前期研究的基础上, 用磷钨酸处理10种不同禾谷类作物种子, 经CO2临界点干燥后, 再用显微CT扫描, 可以清晰地分辨出禾谷类作物淀粉胚乳的细胞结构, 为研究此类细胞结构提供了一种新方法。

显微CT  /  禾谷类淀粉胚乳  /  磷钨酸

Cereal starch endosperm is the main source of human staple food, but the methods for observing its mature cell structure are not well developed. Micro CT technology, a non-destructive three-dimensional imaging technique, is a powerful tool for studying plant morphology. However, due to the uniform density of cereal starch endosperm, whose cell structures cannot be distinguished clearly by conventional micro CT techniques. In this study, we used phosphotungstic acid to treat ten different kinds of cereal seeds of seven crops, and then dried them by CO2 critical point dryer. We found that the cell structure of the treated endosperms can be clearly displayed by micro CT. Our method provides a new way for studying the structures and functions of cereal starch endosperm cells.

micro CT  /  cereal starch endosperm  /  phosphotungstic acid
徐秀苹, 杨小雨, 冯旻. 成熟禾谷类作物种子胚乳细胞的显微CT扫描方法. 植物学报, 2025 , 60 (1) : 81 -89 . DOI: 10.11983/CBB24022
Xiuping Xu, Xiaoyu Yang, Min Feng. A New Cereal Seed Treatment Method for Displaying Endosperm Cell Structures Under Micro CT Scanning[J]. Chinese Bulletin of Botany, 2025 , 60 (1) : 81 -89 . DOI: 10.11983/CBB24022
正文
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胚乳是植物种子的营养贮藏组织。禾谷类作物种子含有丰富的胚乳, 胚乳最外层为糊粉层, 向内的胚乳细胞含有大量淀粉粒, 称为淀粉胚乳(张春庆和李岩, 2019)。禾谷类胚乳是人类文明的基础, 是目前世界上绝大多数人的主食。禾谷类在工业上也有重要应用价值, 如制成生物燃料和塑料(Becraft and Gutierrez-Marcos, 2012)。淀粉胚乳占据胚乳的中心区域, 决定胚乳的质量, 是谷物胚乳中最重要的细胞类型(Becraft and Gutierrez-Marcos, 2012)。淀粉是一种不溶于水的葡萄糖聚合物, 其颗粒具有高度复杂的层次结构。淀粉广泛分布于植物种子中, 以淀粉粒的形式贮存于胚乳细胞, 是禾谷类种子中最主要的贮藏物质, 在种子的其它部位极少存在(胡晋和关亚静, 2022)。
研究种子淀粉胚乳的常用方法为扫描电子显微镜观察, 掰开种子后露出淀粉粒结构, 胚乳细胞的形态较难分辨。灌浆期种子可以制作成切片, 在光学显微镜或透射电镜下观察胚乳细胞。但成熟种子硬度大, 制作切片极其困难。因此, 观察成熟禾谷类种子淀粉胚乳细胞形态的方法极少。
自显微CT技术诞生以来, 在医学、地质学及材料学等领域发挥了巨大作用, 并越来越多地应用于植物研究(Kaestner et al., 2006; Perret et al., 2007; Korte and Porembski, 2012; Staedler et al., 2013; Knipfer et al., 2019; Losso et al., 2019; Zhang et al., 2020; Liao et al., 2020; Huang and Fu, 2021; Snell et al., 2022)。对于高淀粉含量的禾谷类种子, 由于其胚乳内部密度均匀, 导致射线透过率无差别, 重构后的图片淀粉部分为均一质地, 无法区分细胞的结构特征。
本研究将不同禾谷类成熟种子用10%磷钨酸溶液长时间浸泡处理, 然后进行CO2临界点干燥和显微CT扫描观察。结果显示, 使用该处理方法能清晰地观察到淀粉胚乳的细胞形态且能在不破坏成熟禾谷类种子结构的情况下, 直接观察胚乳细胞的排布状态, 为研究此类细胞结构提供了一种新方法。
1 材料与方法
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1.1 实验材料
实验材料为禾谷类作物的成熟种子: 水稻(Oryza sativa L.), 包括大米、糯米、紫米和红米; 谷子(Setaria italica var. germanica (Mill.) Schred); 高粱(Sorghum bicolor Moench); 薏米(Coix lacryma-jobi var. ma-yuen (Rom. Caill.) Stapf); 燕麦(Avena sativa L.); 玉米(爆裂型) (Zea mays L.); 小麦(Triticum aestivum L.)。
1.2 实验方法
1.2.1 固定与染色
磷钨酸(H3PW12O40)溶液的配制: 将磷钨酸溶于FAA固定液中, 得到质量浓度为10%的磷钨酸溶液。
碘化铯(CsI)溶液的配制: 将碘化铯溶于FAA固定液中, 得到质量浓度为10%的碘化铯溶液。
Lugols溶液的配制: 将4 g碘化钾(KI)、2 g碘(I2)加入50 mL FAA固定液中, 得到Lugols溶液。
FAA固定液(100 mL): 90 mL 50%乙醇, 5 mL冰乙酸, 5 mL甲醛。
选取不同禾谷类作物成熟种子, 将大样品(如玉米种子)破碎成小块, 投入磷钨酸溶液中, 上下颠倒数次以去除气泡, 直至样品沉入溶液中。
4°C条件下染色固定30-180天, 体积大或密度高的样品需要时间较长, 每2周更换1次染色固定液。
1.2.2 CO2临界点干燥
取染色固定后的样品进行梯度乙醇(70%、80%、90%和100%)脱水, 各处理20分钟。将脱水后的样品放入CO2临界点干燥仪(徕卡, CPD300)内进行干燥。
1.2.3 显微CT扫描与重构
取干燥样品进行显微CT扫描(Skyscan1172, 布鲁克公司), 扫描电压为41-100 kV, 分辨率为0.56-2.60 µm。样品扫描后用NRecon软件重构, 得到横切面图。各样品扫描电压和分辨率见表1。
2 结果与讨论
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本课题组前期研究表明, 磷钨酸处理可提高植物样品显微CT图像的对比度(徐秀苹等, 2021), 但未对高淀粉含量的种子进行尝试。林金星等(2019)证实碘化铯染色可提高拟南芥(Arabidopsis thaliana)种子的显微CT成像质量。而Lugols溶液中含有碘, 常用于检测样品中的淀粉。为提高禾谷类成熟种子胚乳部分显微CT图像的对比度, 本研究分别采用以上3种溶液进行实验。
2.1 水稻种子显微CT扫描
2.1.1 大米
为比较不同染色液对大米的染色效果, 分别使用碘化铯溶液(图1B)和磷钨酸溶液(图1C, D)进行处理, 对照仅用FAA溶液(图1A), 染色40天后进行CO2临界点干燥。显微CT扫描后经NRecon软件重构, 得到种子横切面图。结果显示, 经10%碘化铯溶液处理后的大米, 胚乳部分亮度较高, 但形态非常均一, 不能分辨出任何细胞结构, 与经FAA处理的对照类似。而经10%磷钨酸处理后的大米, 胚乳细胞的形态分明, 胚的形态结构也更清晰, 对比度显著提高。
2.1.2 紫米
为比较不同染色液对紫米的染色效果, 分别用碘化铯溶液(图2B)、Lugols溶液(图2C)和磷钨酸溶液(图2D, E)进行处理, 以FAA溶液(图2A)处理作为对照, 染色40天后经CO2临界点干燥。显微CT扫描后经NRecon软件重构, 得到横切面图(图2)。结果显示, 经10%碘化铯溶液和Lugols溶液处理的紫米, 虽然胚乳部分亮度增高, 但质地均一, 不能分辨出细胞结构; 而经10%磷钨酸处理的紫米, 对比度显著提高, 胚乳细胞的形态清晰, 与大米(图1C)类似。
2.1.3 红米
红米染色处理40天后进行CO2临界点干燥, 显微CT扫描后得到横切面图。FAA处理(图3A)的胚乳部分形态结构不清晰, 而经10%磷钨酸处理(图3B)后, 胚乳细胞形态清晰, 胚的对比度也显著提高。
2.1.4 糯米
糯米经磷钨酸染色处理40天、CO2临界点干燥后进行显微CT扫描。与对照(图4A)相比, 处理后(图4B)的胚乳细胞形态清晰, 胚的对比度也显著提高。
2.2 谷子种子显微CT扫描
谷子种子去皮后的小米经10%磷钨酸溶液染色, 仅用FAA溶液处理作为对照, 染色后进行CO2临界点干燥。显微CT扫描后得到横切面图。由于小米胚乳淀粉较大米、紫米、红米和糯米稀疏, 经FAA处理后(图5A), 淀粉粒形态模糊, 胚乳细胞结构不清晰。而经10%磷钨酸处理后(图5B), 小米胚乳细胞的结构清晰。由于小米较小, 染色处理用时较其它种子短, 只需30天。但如果小米最外层的果种皮(禾谷类种子种皮和果皮愈合, 简称果种皮)和糊粉层未脱掉, 染液不易渗透, 染色时间需延长几倍。
2.3 燕麦种子显微CT扫描
燕麦种子用磷钨酸处理60天后, 经CO2临界点干燥, 显微CT扫描后得到横切面图。燕麦种子淀粉粒排列疏松, 与小米相似, 经FAA固定液处理(图6A)的燕麦种子显微CT扫描后, 可以看到淀粉粒的排布, 胚乳细胞形态不清晰。经10%磷钨酸溶液处理(图6B)燕麦种子显微CT扫描后, 胚乳细胞的形态清晰。
2.4 高粱种子显微CT扫描
高粱种子较大, 虽已脱掉果种皮, 但仍需用磷钨酸处理70天。经FAA固定液处理(图7A)的高粱种子显微CT扫描后, 胚乳部分形态非常均一。经10%磷钨酸溶液处理(图7B)高粱种子显微CT扫描后, 胚乳细胞的结构清晰。
2.5 薏米种子显微CT扫描
薏米种子经磷钨酸处理90天, CO2临界点干燥后进行显微CT扫描。经FAA固定液处理(图8A)的薏米种子胚乳部分形态结构不清晰。经10%磷钨酸溶液处理(图8B)后, 胚乳细胞的形态清晰。
2.6 玉米种子显微CT扫描
本实验用爆裂型玉米, 其内部淀粉结构非常致密, 导致染色6个月仍未染透, 仅种皮边缘和胚的部分染色较亮, 胚乳细胞结构不十分清晰(图9B), 与对照(图9A)差别不大。由于种皮过于致密, 导致染液不易渗透, 故将种子破碎, 取小块用水浸泡过夜后再固定染色90天, 胚乳细胞结构清晰(图9C), 局部放大提高分辨率扫描后, 可见清晰的胚乳细胞淀粉粒形态(图9D)。
2.7 小麦种子显微CT扫描
小麦种子经磷钨酸处理6个月(图10B), 显微CT扫描后糊粉层细胞比对照(图10A)更为清晰, 但胚乳部分细胞形态不清晰。将其切成小块染色, 并用水浸泡膨胀后再染色, 效果均不理想。
2.8 讨论
磷钨酸分子量大, 能提高样品的对比度。在透射电子显微镜下观察时, 可用磷钨酸进行样品的负染色, 染色后背景呈黑色, 样品相对透明, 从而衬托出样品结构。本课题组前期研究表明, 磷钨酸可用于提高花芽、花柄、茎、 幼叶、根及种子等植物样品的显微CT图像对比度(徐秀苹等, 2021), 种子使用了绿豆, 仅提高了种皮及胚的对比度, 其余部分未见明显变化。本研究尝试提高禾谷类高淀粉含量种子胚乳部分的对比度。使用本研究方法可在不破坏禾谷类种子结构的前提下, 直接观察胚乳细胞的二维结构, 生成3D图像后, 还可观察到完整种子或者单个胚乳细胞的三维结构。
本研究中, 用磷钨酸处理后, 与对照相比, 大米、糯米、紫米、红米、谷子、高粱、薏米、燕麦和爆裂型玉米的胚乳细胞结构清晰。但对于小麦种子, 笔者经过长期多次尝试, 均未获得满意的效果(表2)。可能由于小麦种皮致密或其它原因, 染色6个月也未能观察到清晰的胚乳细胞结构(图10), 将其切成小块, 染色并用水浸泡膨胀后再染色, 效果也均不理想。这可能是由于所用硬质小麦角质化程度较高, 导致磷钨酸不易渗透。具体原因有待进一步探索。
由于禾本科作物种子较致密, 染色时间比其它植物长几倍至几十倍。但进行CO2临界点干燥时, 受技术所限, CO2交换的时间不能无限延长, 部分样品(如小米和高粱种子)有开裂现象。如何避免上述问题有待进一步研究。
作者贡献声明
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徐秀苹: 设计并完成大部分实验, 撰写论文; 杨小雨: 提供实验样品, 构思实验, 完成部分实验; 冯旻: 分析数据, 修改文章。
基金
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  • 中国科学院技术支撑人才项目(2022)
  • 黔农科院青年科技基金(2021-20)
  • 黔农科院国基后补助(2021-12)
文献
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2025年第60卷第1期
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doi: 10.11983/CBB24022
  • 接收时间：2024-02-18
  • 首发时间：2026-10-27
  • 出版时间：2025-01-10
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  • 收稿日期：2024-02-18
  • 录用日期：2024-03-30
基金
中国科学院技术支撑人才项目(2022)
黔农科院青年科技基金(2021-20)
黔农科院国基后补助(2021-12)
作者信息
    1中国科学院植物研究所, 系统与进化植物学重点实验室, 北京 100093
    2国家植物园, 北京 100093
    3中国农业科学院作物科学研究所, 北京 100081
    4贵州省农业科学院, 贵州省亚热带作物研究所, 兴义 562400

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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