Article(id=1276531613148246438, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276531538535781212, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2025.04.011, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1732636800000, receivedDateStr=2024-11-27, revisedDate=null, revisedDateStr=null, acceptedDate=1733328000000, acceptedDateStr=2024-12-05, onlineDate=1782278453384, onlineDateStr=2026-06-24, pubDate=1745510400000, pubDateStr=2025-04-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782278453384, onlineIssueDateStr=2026-06-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782278453384, creator=13701087609, updateTime=1782278453384, updator=13701087609, issue=Issue{id=1276531538535781212, tenantId=1146029695717560320, journalId=1235980609244409860, year='2025', volume='46', issue='4', pageStart='777', pageEnd='1024', issueExtLink='null', onlineDate='null', pubDate='1745510400000', pubDateStr='2025-04-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782278435595, creator='13701087609', updateTime=1782278607615, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276532260098675208, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276531538535781212, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276532260098675209, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276531538535781212, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=887, endPage=893, ext={EN=ArticleExt(id=1276531615459307944, articleId=1276531613148246438, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Polyploid Germplasm Induction of Bletilla ochracea Schltr., columnId=1236256430060261740, journalTitle=Chinese Journal of Tropical Crops, columnName=Germplasm Resources, Genetics & Breeding, runingTitle=null, highlight=null, articleAbstract=

To generate polyploid B. ochracea Schltr and find out the optimal induction condition, the seeds of diploid B. ochracea were treated with different concentration of colchicine solution under a range of treatment time. The results of the study suggested that the seeds treated with 0.25% colchicine solution for six days was effective in polyploid induction for B. ochracea, with a 35.66% survival rate of treated seeds and a 45% induction rate. The tetraploid gave a significant enhancement on various agronomic traits, including plant height, leaf number, leaf width, stem diameter, bulb length and root diameter. In addition, tetraploid B. ochracea showed increased stomatal aperture and decreased stomatal density. The tetraploid B. ochracea was generated in this study, which would provide technical support and basic material for improving the genetic traits of medicinal orchids.

, authors=null, authorsList=Jiamin HE, Sujin ZHENG, Xinhua ZENG, Shaofan LUO, Siren LAN, Weichang HUANG, authorCompany=null, correspAuthors=Weichang HUANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1276531616482718131, articleId=1276531613148246438, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=黄花白及多倍体种质创制研究, columnId=1236256430219645304, journalTitle=热带作物学报, columnName=种质资源与遗传育种, runingTitle=null, highlight=null, articleAbstract=

本研究以黄花白及(Bletilla ochracea Schltr.)种子为诱变材料,采用不同浓度秋水仙素溶液和不同处理时间进行多倍体诱导处理,以期找到诱导黄花白及多倍体植株的试验条件。结果表明:以0.25%秋水仙素溶液浸泡黄花白及种子6 d为诱导黄花白及四倍体的最佳方式,可获得较高的种子存活率和诱导率,其中种子存活率为35.66%,诱导率为45%。从形态学方面观察,四倍体植株平均高度、叶数、叶宽、茎粗、块茎、根长和根粗均大于二倍体植株,并且四倍体植株气孔明显变大,气孔密度降低。本研究创制的黄花白及四倍体新种质将为黄花白及等药用兰科植物的遗传性状的改良提供技术参考和研究材料。

, authors=

何佳敏(2000—),女,硕士研究生,研究方向:兰花育种。

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* 黄卫昌(HUANG Weichang),E-mail:
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何佳敏(2000—),女,硕士研究生,研究方向:兰花育种。

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A:二倍体黄花白及;B:经秋水仙素多倍体诱导黄花白及。

, figureFileSmall=IkIiWrAs0CwdsWJtcdpsig==, figureFileBig=yagE+1jXwhJ8ao4y0rg3Fw==, tableContent=null), ArticleFig(id=1276531628470039025, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=EN, label=Fig. 2, caption=Chromosome counting from root tip cells of B. ochracea, figureFileSmall=rZCKQAiZlgD/qcePfmVfIQ==, figureFileBig=hMNYc51j85L2hLosu3BUAg==, tableContent=null), ArticleFig(id=1276531628532953586, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=CN, label=图2, caption=黄花白及根尖细胞染色体计数

A:二倍体黄花白及;B:经秋水仙素多倍体诱导黄花白及。

, figureFileSmall=rZCKQAiZlgD/qcePfmVfIQ==, figureFileBig=hMNYc51j85L2hLosu3BUAg==, tableContent=null), ArticleFig(id=1276531628604256755, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=EN, label=Fig. 3, caption=Phenotypic comparisons between 210-day-old plants of diploid B. ochracea and polyploidized B. ochracea, figureFileSmall=mQkvTrQEdMZLLvx84+Zo/A==, figureFileBig=v2YEUW+i9Uf83GoDRRXXyg==, tableContent=null), ArticleFig(id=1276531628662977012, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=CN, label=图3, caption=二倍体黄花白及和诱导四倍体黄花白及苗龄210 d表型

A:二倍体黄花白及;B:多倍体诱导黄花白及。

, figureFileSmall=mQkvTrQEdMZLLvx84+Zo/A==, figureFileBig=v2YEUW+i9Uf83GoDRRXXyg==, tableContent=null), ArticleFig(id=1276531628730085877, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=EN, label=Fig. 4, caption=Leaf stomata and blade cross section in diploid and tetraploid plants of B. ochracea, figureFileSmall=g3+vskmqTZUHTa9eAmihNQ==, figureFileBig=VWe32Nnp7zRM/qqqEg2YWA==, tableContent=null), ArticleFig(id=1276531628793000438, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=CN, label=图4, caption=二倍体和四倍体黄花白及植株叶片气孔和叶横截面

A:二倍体上表皮气孔;B:四倍体上表皮气孔;C:二倍体叶片横截面;D:四倍体叶片横截面。

, figureFileSmall=g3+vskmqTZUHTa9eAmihNQ==, figureFileBig=VWe32Nnp7zRM/qqqEg2YWA==, tableContent=null), ArticleFig(id=1276531628851720695, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=EN, label=Tab. 1, caption=

Effects of colchicine concentration and treatment time on B. ochracea

, figureFileSmall=null, figureFileBig=null, tableContent=
秋水仙素浓度Colchicine concentration/%处理时间Treated time/d存活率Survival rate/%检测株数Test number四倍体株数Tetraploid number非整倍体株数Aneuploid number变异率Variation rate/%诱导率Induction rate/%
0.25356.852015305
635.6620925545
919.7020444020
0.50327.292012155
60.00
90.00
1.0038.692084040
60.00
90.00
CK072.15
), ArticleFig(id=1276531628931412472, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=CN, label=表1, caption=

不同浓度秋水仙素处理不同时间对黄花白及的影响

, figureFileSmall=null, figureFileBig=null, tableContent=
秋水仙素浓度Colchicine concentration/%处理时间Treated time/d存活率Survival rate/%检测株数Test number四倍体株数Tetraploid number非整倍体株数Aneuploid number变异率Variation rate/%诱导率Induction rate/%
0.25356.852015305
635.6620925545
919.7020444020
0.50327.292012155
60.00
90.00
1.0038.692084040
60.00
90.00
CK072.15
), ArticleFig(id=1276531629011104249, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=EN, label=Tab. 2, caption=

Morphological characteristics between diploid and tetraploid B. ochracea

, figureFileSmall=null, figureFileBig=null, tableContent=
指标Index二倍体Diploid四倍体Tetraploid
平均值Mean范围Range平均值Mean范围Range
株高/cm6.16±1.574.00~7.506.31±3.331.30~10.80
叶宽/cm0.28±0.02**0.24~0.320.49±0.13**0.30~0.80
叶片数量2.60±0.552~33.93±1.383~8
茎粗/cm0.16±0.040.12~0.220.26±0.110.15~0.50
块茎直径/cm0.21±0.03*0.13~0.290.32±0.08*0.23~0.50
根数量2.60±0.402~44.10±2.362~11
根长/cm1.23±0.930.30~3.501.50±0.710.30~3.70
根茎直径/cm0.11±0.01***0.10~0.130.16±0.02***0.14~0.20
), ArticleFig(id=1276531629082407418, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=CN, label=表2, caption=

黄花白及对照和四倍体植株的形态学特征比较

, figureFileSmall=null, figureFileBig=null, tableContent=
指标Index二倍体Diploid四倍体Tetraploid
平均值Mean范围Range平均值Mean范围Range
株高/cm6.16±1.574.00~7.506.31±3.331.30~10.80
叶宽/cm0.28±0.02**0.24~0.320.49±0.13**0.30~0.80
叶片数量2.60±0.552~33.93±1.383~8
茎粗/cm0.16±0.040.12~0.220.26±0.110.15~0.50
块茎直径/cm0.21±0.03*0.13~0.290.32±0.08*0.23~0.50
根数量2.60±0.402~44.10±2.362~11
根长/cm1.23±0.930.30~3.501.50±0.710.30~3.70
根茎直径/cm0.11±0.01***0.10~0.130.16±0.02***0.14~0.20
), ArticleFig(id=1276531629149516283, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=EN, label=Tab. 3, caption=

Leaf stomata density and area in diploid and tetraploid plants of B. ochracea

, figureFileSmall=null, figureFileBig=null, tableContent=
样本Sample保卫细胞长度Guard cell length/μm保卫细胞宽度Guard cell width/μm气孔面积大小Stomatal area/μm2气孔密度Stomatal density/mm2
二倍体36.5±5.14***13.0±2.21***1276.1±350.48**7.7±0.82***
四倍体45.6±3.09***17.7±1.76***1740.6±225.28**4.4±0.50***
), ArticleFig(id=1276531629220819452, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, language=CN, label=表3, caption=

黄花白及二倍体和四倍体植株叶片气孔大小和气孔密度

, figureFileSmall=null, figureFileBig=null, tableContent=
样本Sample保卫细胞长度Guard cell length/μm保卫细胞宽度Guard cell width/μm气孔面积大小Stomatal area/μm2气孔密度Stomatal density/mm2
二倍体36.5±5.14***13.0±2.21***1276.1±350.48**7.7±0.82***
四倍体45.6±3.09***17.7±1.76***1740.6±225.28**4.4±0.50***
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黄花白及多倍体种质创制研究
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何佳敏 1, 2 , 郑苏瑾 2, 3 , 曾歆花 2, 3 , 罗韶凡 2 , 兰思仁 1 , 黄卫昌 1, 2, 3, *
热带作物学报 | 种质资源与遗传育种 2025,46(4): 887-893
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热带作物学报 |种质资源与遗传育种 2025 , 46 (4) : 887 -893
黄花白及多倍体种质创制研究
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2.Shanghai Chenshan Botanical Garden / Eastern China Conservation Centre for Wild Endangered Plant Resources, Shanghai 201602, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1276531617678094789, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531613148246438, authorId=1276531617292218817, language=CN, stringName=何佳敏, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, address=1.福建农林大学风景园林与艺术学院,福建福州 350002
2.上海辰山植物园/华东野生濒危资源植物保育中心,上海 201602, bio={"content":"

何佳敏(2000—),女,硕士研究生,研究方向:兰花育种。

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何佳敏(2000—),女,硕士研究生,研究方向:兰花育种。

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nameInitials=null, affiliation=null, department=null, xref=1, 2, 3, *, address=1.College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
2.Shanghai Chenshan Botanical Garden / Eastern China Conservation Centre for Wild Endangered Plant Resources, Shanghai 201602, China
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何佳敏1, 2, 郑苏瑾2, 3, 曾歆花2, 3, 罗韶凡2, 兰思仁1, 黄卫昌1, 2, 3, *
作者信息
  • 1.福建农林大学风景园林与艺术学院,福建福州 350002
  • 2.上海辰山植物园/华东野生濒危资源植物保育中心,上海 201602
  • 3.上海辰山植物园/上海市资源植物功能基因组学重点实验室,上海 201602
通讯作者:
* 黄卫昌(HUANG Weichang),E-mail:
Polyploid Germplasm Induction of Bletilla ochracea Schltr.
Jiamin HE1, 2, Sujin ZHENG2, 3, Xinhua ZENG2, 3, Shaofan LUO2, Siren LAN1, Weichang HUANG1, 2, 3, *
Affiliations
  • 1.College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
  • 2.Shanghai Chenshan Botanical Garden / Eastern China Conservation Centre for Wild Endangered Plant Resources, Shanghai 201602, China
  • 3.Shanghai Chenshan Botanical Garden / Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai 201602, China
出版时间: 2025-04-25 doi: 10.3969/j.issn.1000-2561.2025.04.011
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本研究以黄花白及(Bletilla ochracea Schltr.)种子为诱变材料,采用不同浓度秋水仙素溶液和不同处理时间进行多倍体诱导处理,以期找到诱导黄花白及多倍体植株的试验条件。结果表明:以0.25%秋水仙素溶液浸泡黄花白及种子6 d为诱导黄花白及四倍体的最佳方式,可获得较高的种子存活率和诱导率,其中种子存活率为35.66%,诱导率为45%。从形态学方面观察,四倍体植株平均高度、叶数、叶宽、茎粗、块茎、根长和根粗均大于二倍体植株,并且四倍体植株气孔明显变大,气孔密度降低。本研究创制的黄花白及四倍体新种质将为黄花白及等药用兰科植物的遗传性状的改良提供技术参考和研究材料。

黄花白及  /  多倍体  /  秋水仙素  /  流式细胞术  /  染色体计数

To generate polyploid B. ochracea Schltr and find out the optimal induction condition, the seeds of diploid B. ochracea were treated with different concentration of colchicine solution under a range of treatment time. The results of the study suggested that the seeds treated with 0.25% colchicine solution for six days was effective in polyploid induction for B. ochracea, with a 35.66% survival rate of treated seeds and a 45% induction rate. The tetraploid gave a significant enhancement on various agronomic traits, including plant height, leaf number, leaf width, stem diameter, bulb length and root diameter. In addition, tetraploid B. ochracea showed increased stomatal aperture and decreased stomatal density. The tetraploid B. ochracea was generated in this study, which would provide technical support and basic material for improving the genetic traits of medicinal orchids.

Bletilla ochracea Schltr.  /  polyploidy  /  colchicine  /  flow cytometer  /  chromosome counting
何佳敏, 郑苏瑾, 曾歆花, 罗韶凡, 兰思仁, 黄卫昌. 黄花白及多倍体种质创制研究. 热带作物学报, 2025 , 46 (4) : 887 -893 . DOI: 10.3969/j.issn.1000-2561.2025.04.011
Jiamin HE, Sujin ZHENG, Xinhua ZENG, Shaofan LUO, Siren LAN, Weichang HUANG. Polyploid Germplasm Induction of Bletilla ochracea Schltr.[J]. Chinese Journal of Tropical Crops, 2025 , 46 (4) : 887 -893 . DOI: 10.3969/j.issn.1000-2561.2025.04.011
黄花白及(Bletilla ochracea Schltr.)是兰科(Orchidaceae)白及属(Bletilla)多年生草本植物,在陕西、重庆、湖北、湖南、江西、广西等地区分布。黄花白及花序具3~8朵,花朵常黄色,叶片为长形披针状,花朵淡雅,叶片秀丽,具有较高的园艺观赏价值。此外,黄花白及和小白及[B. formosana(Hayata)Schltr.]与白及[B. striata(Thunb.)Reichb. f.]为同属植物,均为我国传统中草药[1],其块茎具备药用价值,在我国常被用作白及的替代品。前人研究发现白及与黄花白及、小白及之间HPLC指纹图谱相似度较高,这表明白及与黄花白及所含化学成分类似[2]。近期研究表明黄花白及多糖与白及多糖具有相似的结构,具有抗肿瘤疗效[3]。此外,相关研究发现黄花白及还具有促凝血[4]和抗胃溃疡[5]等作用。然而,近年由于对植物源性药物市场需求的不断增加,黄花白及也面临野生资源被过度采集和生境退化或丧失而导致数量锐减等问题[6]。黄花白及已被列入《世界自然保护联盟濒危物种红色名录》(IUCN)和国际贸易公约(CITES)附录Ⅱ,在我国属于濒危(EN)保护植物。
目前黄花白及的研究多集中在其药用化学成分的分析和菌根共生等方面,对于黄花白及新种质创新研究鲜有报道。因此,加快培育出性状更优良的黄花白及新品种对于满足不断增长的药材市场需求和保护黄花白及野生资源显得更加迫切。多倍体诱导作为育种工具中的有效技术之一,对于优良物种培育、品种杂交、性状改良、生产和商业化等具有重要作用[7-8]。为创制表型和栽培性状更优良黄花白及新种质,本研究采用秋水仙素溶液浸种法对黄花白及进行倍性诱导,通过流式细胞术、染色体计数、气孔观察等方法鉴定黄花白及表型变异植株。黄花白及倍性新种质的培育可为药用兰科植物育种提供技术参考。
选取上海辰山植物园科研中心兰科植物资源圃的黄花白及自交蒴果为试验材料。
采用TTC改良染色法[9]对黄花白及种子活力进行检测(适用于新鲜采摘的种子),根据TTC染色结果计算种子活力率,选取活力良好的种子进行后续试验。将种子混样后加入1.0%TTC染液没过种子并摇匀,将其置于40 ℃环境下避光染色12 h后吸取种子置于载玻片上,在体视显微镜下观察染色情况。种子种胚颜色越红,说明种子活力越高;若种胚不变色,说明种子无活力。随机取3个视野,进行统计并计算种子活力率,活力率=胚染上红色种子数/有胚种子总数×100%,本试验所选取种子活力率为89.29%。
以黄花白及种子为诱变材料,采用秋水仙素浸泡法进行倍性诱导。在超净工作台对种子进行无菌处理,先用75%酒精浸泡3 min,再用3%次氯酸钠浸泡杀菌5 min,无菌水冲洗3次后用无菌滤纸吸干水分。接着将种子分别转移至0.25%、0.50%、1.00%秋水仙素溶液(诱导液添加不超过2.0% DMSO)中避光诱导,对照组则用无菌水浸泡处理,各处理组均置于120 r/min转速摇床暗处理3、6、9 d。每个处理种子数量约为100粒,每个处理重复5次。诱导处理结束后,用无菌水冲洗3次,利用无菌滤纸吸干水分后置于播种培养基使其恢复正常生长。30 d后统计种子存活率,并继代1次,90 d后再转至壮苗培养基,待植株生长到210 d后,筛选形态变异植株并对其进行倍性鉴定。
每个处理选取20株诱变株进行流式细胞检测,取0.50~1.00 cm2的样本叶片,置于冰上的培养皿中,加入预冷的细胞核提取缓冲液,用锋利的双面刀片快速垂直切碎叶片30~60 s,此过程尽量将材料浸没于裂解液中。吸取混合溶液经滤膜过滤,转移至测定管内,得到细胞核悬浮液,加入染色液(染色缓冲液+碘化丙啶+RNAseA储备液),混匀后置于4 ℃避光染色15~30 min,随后使用PARTEC CyFlow流式细胞仪进行检测。具体试验操作参照GALBRAITH等[10]的方法。
参考王乐乐等[11]和MORAES等[12]的方法。从植株上切取3~4 mm长势旺盛根尖,将根尖置于对二氯苯饱和溶液中,并在4 ℃条件下浸泡处理5 h,然后用Carnoy溶液[乙醇∶乙酸,3∶1(v/v)]在4 ℃条件下固定12 h。再将固定的根尖用无菌水冲洗3~5次,将水吸干后,置于2.0%纤维素酶和果胶酶溶液,并在37 ℃水中解离约15~20 min。根尖分生区细胞核用卡宝品红染液染色3~5 min,压片,最后置于显微镜下观察根尖细胞染色体。
参考前人研究方法[13-14],对植株株高、叶片大小、数量、茎秆、块茎大小、分蘖数等形态特征进行统计、测量,对比加倍植株与对照植株的形态变化。此外,气孔特征通常是检测倍性水平的重要指标。采用OMIDBAIGI等[15]的方法,取对照和诱变植株叶片,用速干透明指甲油进行处理之后,在光学显微镜观察气孔大小,并计算气孔密度。
采用Image J软件和IBM SPSS Statistics 27软件进行试验数据处理。样本间差异的显著性采用独立样本T检验。使用Microsoft Excel 2021软件和Adobe Photoshop 2024软件绘制图表。
通过不同浓度的秋水仙素对黄花白及进行处理,结果如表1所示,处理浓度越高且处理时间越长,植株存活率越低。在处理时间均为6 d的情况下,秋水仙素浓度为0.50%和1.00%的处理植株死亡率达到100%。黄花白及在0.25%秋水仙素条件下处理6 d的诱导率达到45%,其次是1.00%秋水仙素处理种子3 d,诱导率为40%,但该处理条件下存活率只有8.69%。结合诱导率和存活率结果得出,黄花白及种子经0.25%秋水仙素处理6 d是诱导出四倍体植株的最佳处理方式。
诱导处理210 d后,采用流式细胞术对诱变植株进行DNA含量检测和分析,以未经处理的二倍体黄花白及为对照,根据二倍体黄花白及和诱变株黄花白及荧光强度比值进行快速鉴定,如图1可见,二倍体黄花白及荧光强度大概在200处达到峰值,诱变株黄花白及荧光强度大概在400处达到峰值,约为对照组的2倍。由此可见该诱变株为四倍体黄花白及。
对经过流式基因组检测的加倍植株的根尖进行染色体压片处理,结果表明,未处理对照黄花白及根尖染色体数目为2n=2x=34,经流式鉴定的基因组加倍黄花白及根尖染色体数目为2n=4x=68(图2)。
在相同的培养条件下,对黄花白及四倍体和二倍体植株进行形态比较分析。如图3表2所示,黄花白及四倍体植株表型发生显著性变化,其植株株高、叶片数量、叶宽、茎粗、块茎直径、根长和根粗平均数值均高于二倍体植株。其中四倍体黄花白及的叶宽、块茎直径和根粗数值分别比二倍体黄花白及增加75.0%、52.4%和45.5%,具有显著性差异(P<0.05)。此外,四倍体植株表型同时具有矮化(最矮株高为对照最矮的0.325倍)和变大(最高株高为对照最高的1.44倍)趋势,并均与二倍体黄花白及表型差异明显。与二倍体黄花白及对照相比较,四倍体植株叶片更宽更厚,根增长加粗,这些都可作为四倍体黄花白及辅助鉴定的特征。
通过光学显微镜对二倍体黄花白及和四倍体黄花白及叶表皮气孔进行观察和拍照,使用ImageJ软件测量气孔长度、气孔宽度和气孔面积大小。结果显示(表3图4A图4B),四倍体黄花白及叶表皮气孔显著大于二倍体黄花白及,同时气孔密度变小。此外,叶片解剖结构也受到多倍体化的影响,与二倍体植株相比,四倍体植株叶片整体比二倍体叶片厚46.31%(图4C图4D)。
黄花白及兼具观赏与药用价值,随着药用植物市场对植物源性药物的需求增长,黄花白及面临过度采集和生境退化或丧失等挑战。并且黄花白及在我国属于濒危(EN)植物,已被列入《世界自然保护联盟濒危物种红色名录》(IUCN)。多倍体诱导能使植物获得改良性状,如更大的器官(叶、花等)[16],更高的生物量,耐旱性和抗病性增强[17-18],这些特性的改变甚至可以使多倍体植物占据新的生态位[19-20]。并且这些多倍性状的变化提高了它们在农艺用途的可能性[16]。因此,培育出产量更高,抗性更强等优良性状的黄花白及品种将成为黄花白及可持续健康发展的关键途径。
多倍体诱导成功的因素涉及物种、抗有丝分裂剂、外植体材料类型、处理浓度和时间等。种子、不定芽、从生芽、原球茎、类原球茎、茎尖、带节茎段等分裂旺盛组织常作为兰科植物多倍体诱导的材料[21]。同其他植物一样,秋水仙素也是兰科植物倍性育种最常用的抗有丝分裂剂[21-22]。例如,LI等[23]使用秋水仙素对白及种子进行多倍体诱导,得出经0.1%秋水仙素处理7 d后,四倍体诱导率高达(40.67±0.89)%。崔广荣等[24]利用0.2%~0.4%秋水仙素处理文心兰(Oncidium)类原球茎薄切片10~20 d,四倍体诱导率可达26.70%。由于植物具有表皮蜡、种皮,或较厚的角质层细胞等诸多物理屏障,因此抗有丝分裂剂的有效性会受到溶剂影响。DMSO可通过增加细胞渗透性从而提升化学试剂的吸收率,根据HAMILL等[25]研究,与溶于水的秋水仙素对照相比,用溶于2%或4% DMSO的秋水仙素处理降低了香蕉外植体的存活率,但诱导获得的四倍体植株数目增加。
本研究发现,秋水仙素浓度和处理时间与存活率呈负相关趋势,这与LI等[23]诱导白及种子结果相似,随着秋水仙素浓度和处理时间延长,存活率逐渐降低,在0.4%浓度处理9 d时死亡率最高。此外,在LIU等[26]诱导铁皮石斛(Dendrobium officinale)研究中,也是随着秋水仙素浓度提高和处理时间延长,各处理组原球茎存活率均降低,以0.4%浓度处理48 h死亡率最高。在本研究中,黄花白及在0.50%和1.00%秋水仙素条件下处理6 d的死亡率最高达到了100%,这可能是由于秋水仙素浓度太高、处理时间太长以及添加了DMSO对种子毒害太强导致。不同物种对抗有丝分裂剂浓度和处理时间所呈现的效果也不同,因此,找到诱导率和存活率都较高的处理条件是倍性育种的关键。本研究黄花白及在0.25%秋水仙素条件下处理6 d时诱导率和变异率分别高达45%和55%,存活率为35.66%,其次是1.00%秋水仙素处理种子3 d,诱导率和变异率均为40%,但该处理条件下存活率只有8.69%。对比各处理组的诱导率和存活率数据,0.25%秋水仙素处理时间6 d是创制黄花白及四倍体植株的最佳方式。
本研究通过流式细胞术和染色体计数检测出23株四倍体,观察发现大部分四倍体黄花白及呈现叶片变宽变厚、茎秆变粗、块茎变大、根系变粗、气孔变大和气孔密度变小等性状,与白及[23]、铁皮石斛[26-27]、荧光蝴蝶兰(Phalaenopsis bellina[14]、紫毛兜兰(Paphiopedilum villosum[28]、纹瓣兰[Cymbidium aloifolium(L.)Sw.][29]等多倍体植株器官粗大化特征相一致。在大多数情况下,物种基因组加倍后植株器官(叶、花)通常会呈现“巨体效应(gigas effect)”特征。然而,在本研究中,出现部分四倍体植株较二倍体植株叶片更厚更宽、叶表皮更粗糙、气孔更大,但整体植株更矮小现象。在前人的研究中也具有类似情况,如PODWYSZYNSKA等[30]诱导的四倍体郁金香(Tulipa gesneriana)出现了矮化现象,与二倍体郁金香相比,四倍体郁金香的叶、花以及整体植株均显著变小。除此之外,LI等[31]诱导的三倍体木槿(Hibiscus moscheutos)的花比二倍体小;REGALADO等[32]诱导的四倍体矮牵牛(Petunia axillaris)比二倍体矮牵牛矮,但叶片比二倍体矮牵牛紧凑(54%);FETOUH等[33]诱导的四倍体日本女贞(Ligustrum japonicum)平均数据比二倍体矮31.0%,冠幅小33.1%;以及PADOAN等[34]诱导的三倍体柑橘(Citrus clementine)的叶比二倍体亲本小。相关学者推测该现象可能是由于在高倍性水平下,细胞周期成本更高,细胞分裂延迟,导致细胞数量更少和器官更小[35-36]。由于培育时间不足,后续的试验还需进一步探究四倍体黄花白及与二倍体植株开花性状、块茎药用成分等方面的变化。总之,本研究诱导出的黄花白及四倍体植株与原二倍体植株形态变异显著,为药用兰花种质库增添了“gigas”化和矮化的表型性状的多倍体植物新材料,可为黄花白及遗传性状改良等研究和实践应用提供帮助。
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2025年第46卷第4期
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doi: 10.3969/j.issn.1000-2561.2025.04.011
  • 接收时间:2024-11-27
  • 首发时间:2026-06-24
  • 出版时间:2025-04-25
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  • 收稿日期:2024-11-27
  • 录用日期:2024-12-05
基金
中央财政林业科技推广示范资金项目(沪[2023]TG01)
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    1.福建农林大学风景园林与艺术学院,福建福州 350002
    2.上海辰山植物园/华东野生濒危资源植物保育中心,上海 201602
    3.上海辰山植物园/上海市资源植物功能基因组学重点实验室,上海 201602

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* 黄卫昌(HUANG Weichang),E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

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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