Article(id=1277240251089289332, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1277239982603502113, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2025.05.009, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1735488000000, receivedDateStr=2024-12-30, revisedDate=null, revisedDateStr=null, acceptedDate=1736524800000, acceptedDateStr=2025-01-11, onlineDate=1782447405835, onlineDateStr=2026-06-26, pubDate=1748102400000, pubDateStr=2025-05-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782447405835, onlineIssueDateStr=2026-06-26, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782447405835, creator=13701087609, updateTime=1782447405835, updator=13701087609, issue=Issue{id=1277239982603502113, tenantId=1146029695717560320, journalId=1235980609244409860, year='2025', volume='46', issue='5', pageStart='1025', pageEnd='1277', issueExtLink='null', onlineDate='null', pubDate='1748102400000', pubDateStr='2025-05-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782447341824, creator='13701087609', updateTime=1782447947315, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1277242522292319215, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1277239982603502113, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1277242522292319216, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1277239982603502113, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1107, endPage=1115, ext={EN=ArticleExt(id=1277240251634548854, articleId=1277240251089289332, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Effect of AgNO3 on Secondary Somatic Embryogenesis in Hevea brasiliensis, columnId=1236256430060261740, journalTitle=Chinese Journal of Tropical Crops, columnName=Germplasm Resources, Genetics & Breeding, runingTitle=null, highlight=null, articleAbstract=

The technology of secondary somatic embryo cyclic multiplication has realized the large-scale propagation of somatic embryo plantlets (self-rooted juvenile clones) of Hevea brasiliensis. This technology needs to cut the cotyledon embryo into 2 mm×2 mm pieces. A large amount of ethylene and reactive oxygen species (ROS) will be produced at the wound site, and much ethylene and ROS will also formed during culture process. Ethylene and ROS have adverse effects on somatic embryogenesis of H. brasiliensis. Silver nitrate (AgNO3) is an ethylene inhibitor and also affects enzyme activity. In this paper, the effects of different AgNO3 concentrations (0, 2.5, 5.0, 10.0, 15.0 mg/L) and different time (7, 14, 21, 28 days) on the embryo block browning and secondary somatic embryogenesis of H. brasiliensis were studied, and the phenotype and ploidy of regenerated plantlets were evaluated. AgNO3 significantly reduced the browning of embryo blocks and the production of ROS. The effect of 5-15 mg/L was better than that of 2.5 mg/L, and the effect of 21 days treatment was better than that of the other three treatments. 2.5-15.0 mg/L AgNO3 treated 7-28 days did not significantly increase the number of embryonic blocks, but the number of cotyledon embryos in 5.0 mg/L AgNO3 treated for 21 and 28 days, 10.0 mg/L AgNO3 treated for 21 days, and 15.0 mg/L AgNO3 treated for 7-21 days were significantly higher than those in the control. The number of embryonic blocks was negatively correlated with browning, but not significantly, the number of cotyledon embryos was significantly negatively correlated with browning. The phenotype of regenerated plantlets from AgNO3 treatments had no obvious difference compared with the control, and the chromosome ploidy was the same as the control. The results would provide technical support for improving the efficiency of secondary somatic embryogenesis in H. brasiliensis.

, authors=null, authorsList=Wen TIAN, Ziyu ZHANG, Zhengwei XU, Xiaoyi WANG, Quannan ZHOU, Xin JI, Rizhi WU, Wei XIA, Tiandai HUANG, authorCompany=null, correspAuthors=Wei XIA, Tiandai 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=1277240254469898366, articleId=1277240251089289332, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=硝酸银对橡胶树次生体胚发生的影响研究, columnId=1236256430219645304, journalTitle=热带作物学报, columnName=种质资源与遗传育种, runingTitle=null, highlight=null, articleAbstract=

橡胶树次生体胚循环增殖技术实现了橡胶树体胚苗(自根幼态无性系)规模化繁育。该技术需要将子叶胚切成2 mm×2 mm的胚块,伤口部位会产生大量的乙烯及活性氧,培养过程中也会形成较多乙烯和活性氧,乙烯及活性氧对橡胶树体胚发生产生不利影响。硝酸银(AgNO3)是乙烯抑制剂,同时影响酶的活性。本文研究了不同硝酸银浓度(0、2.5、5.0、10.0、15.0 mg/L)处理不同时间(7、14、21、28 d)对橡胶树胚块褐化及次生体胚发生的影响,并评估了再生植株的表型及倍性。结果表明:AgNO3可明显减轻胚块褐化情况,减少胚块活性氧的产生,5.0~15.0 mg/L AgNO3处理较2.5 mg/L效果更好,AgNO3处理21 d较其他3个处理时间效果好;2.5~15.0 mg/L AgNO3处理7~28 d未显著增加出胚胚块数,但5.0 mg/L AgNO3处理21、28 d,10.0 mg/L AgNO3处理21 d,15.0 mg/L AgNO3处理7~21 d子叶胚数显著高于对照;出胚胚块数与褐化呈负相关,但未达显著水平,子叶胚数与褐化呈显著负相关;AgNO3处理的再生植株表型与对照无明显差异,染色体倍性与对照相同。本研究结果为提高橡胶树次生体胚发生效率提供技术支撑。

, authors=

田雯(1997—),女,硕士,研究方向:农艺与种业。

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* 夏薇(XIA Wei),E-mail:;
黄天带(HUANG Tiandai),E-mail:
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2.School of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan 570228, China
3.Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences / Haikou Key Laboratory of Tropical Plant Seedling Innovation / Key Laboratory of Rubber Tree Biology and Genetic Resources Utilization, Ministry of Agriculture and Rural Areas / Hainan Provincial Key Laboratory of Tropical Crop Cultivation Physiology / Cultivation Base of State Key Laboratory Jointly Built by Province and Ministry, Haikou, Hainan 571101, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1277240258227994781, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1277240251089289332, authorId=1277240257741455512, language=CN, stringName=田雯, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, 3, address=1.海南大学三亚南繁研究院,海南三亚 572024
2.海南大学热带农林学院,海南海口 570228
3.中国热带农业科学院橡胶研究所/海口市热带植物种苗创新重点实验室/农业农村部橡胶树生物学与遗传资源利用重点实验室/海南省热带作物栽培生理学重点实验室/省部共建国家重点实验室培育基地,海南海口 571101, bio={"content":"

田雯(1997—),女,硕士,研究方向:农艺与种业。

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田雯(1997—),女,硕士,研究方向:农艺与种业。

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Laboratory of Rubber Tree Biology and Genetic Resources Utilization, Ministry of Agriculture and Rural Areas / Hainan Provincial Key Laboratory of Tropical Crop Cultivation Physiology / Cultivation Base of State Key Laboratory Jointly Built by Province and Ministry, Haikou, Hainan 571101, China
4.Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya, Hainan 572025, China
5.National Key Laboratory for Tropical Crop Breeding, Sanya, Hainan 572025, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1277240260723605673, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1277240251089289332, authorId=1277240259809247396, language=CN, stringName=徐正伟, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=3, 4, 5, address=3.中国热带农业科学院橡胶研究所/海口市热带植物种苗创新重点实验室/农业农村部橡胶树生物学与遗传资源利用重点实验室/海南省热带作物栽培生理学重点实验室/省部共建国家重点实验室培育基地,海南海口 571101
4.中国热带农业科学院三亚研究院,海南三亚 572025
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4.Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya, Hainan 572025, China
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4.中国热带农业科学院三亚研究院,海南三亚 572025
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(in Chinese), articleTitle=Effects of silver nitrate on phenolic content and related enzyme activities in rosa Roxburghii callus, refAbstract=null), Reference(id=1277240292323492124, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1277240251089289332, doi=null, pmid=null, pmcid=null, year=2003, volume=null, issue=6, pageStart=580, pageEnd=584, url=null, language=null, rfNumber=[42], rfOrder=54, authorNames=娄群峰, 陈劲枫, 任刚, 罗向东, journalName=植物生理学通讯, refType=null, unstructuredReference=娄群峰, 陈劲枫, 任刚, 罗向东. 硝酸银和乙烯利对黄瓜叶片中3种氧化还原酶同工酶和酶活性影响的比较[J]. 植物生理学通讯, 2003(6): 580-584., articleTitle=硝酸银和乙烯利对黄瓜叶片中3种氧化还原酶同工酶和酶活性影响的比较, refAbstract=null), Reference(id=1277240292415766813, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1277240251089289332, doi=null, pmid=null, pmcid=null, year=2003, volume=null, issue=6, pageStart=580, pageEnd=584, url=null, language=null, rfNumber=[42], rfOrder=55, authorNames=LOU Q F, CHEN J F, REN G, LUO X D, journalName=Plant Physiology Journal, refType=null, unstructuredReference=LOU Q F, CHEN J F, REN G, LUO X D. Comparison of effects of silver nitrate and ethephon on isozymes and enzyme activities of three oxidoreductases in cucumber leaves[J]. Plant Physiology Journal, 2003(6): 580-584. (in Chinese), articleTitle=Comparison of effects of silver nitrate and ethephon on isozymes and enzyme activities of three oxidoreductases in cucumber leaves, refAbstract=null), Reference(id=1277240292646453534, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1277240251089289332, doi=null, pmid=null, pmcid=null, year=1988, volume=39, issue=6, pageStart=787, pageEnd=794, url=null, language=null, rfNumber=[43], rfOrder=56, authorNames=ELTJO G M, SIMMONDS M J, journalName=Journal of Experimental Botany, refType=null, unstructuredReference=ELTJO G M, SIMMONDS M J. Polyamine levels in relation to growth and somatic embryogenesis in Medicago sativa L.[J]. 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A: Before DAB staining; B: After DAB staining.

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A:DAB染色前;B:DAB染色后。

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Effect of AgNO3 on secondary somatic embryogenesis in H. brasiliensis

, figureFileSmall=null, figureFileBig=null, tableContent=
处理时间Processing time/dAgNO3浓度AgNO3 concentration/(mg·L–1)出胚胚块数Number of embryonic blocks子叶胚数Number of cotyledon embryos胚块褐化程度Browning degree of embryo blocks
706.33±2.52ab68.00±2.00cd中等
2.56.33±1.53ab36.00±6.00d中等
5.04.50±2.89ab54.50±18.70d中等
10.04.75±2.99ab53.00±24.01d中等
15.06.33±2.08ab93.00±41.15bc中等
1404.00±2.16ab50.00±30.24d中等
2.54.75±2.36ab43.25±15.15d中等
5.04.67±2.31ab50.33±13.05d较轻
10.05.75±1.71ab65.00±17.15cd较轻
15.06.00±2.65ab122.00±2.00ab较轻
2105.33±0.58ab66.00±20.00cd中等
2.56.75±2.06ab45.25±7.32d中等
5.08.33±1.53a145.67±21.50a较轻
10.05.33±2.31ab75.33±6.43ab较轻
15.05.67±2.08ab127.00±2.00ab较轻
2803.67±1.53b35.00±1.00d中等
2.57.33±1.53ab56.00±14.00cd中等
5.07.50±4.20ab111.50±37.65ab较轻
10.08.00±1.00ab65.33±20.03cd较轻
15.04.50±1.73ab58.00±36.40cd中等
), ArticleFig(id=1277240276729069790, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1277240251089289332, language=CN, label=表1, caption=

AgNO3对橡胶树次生体胚发生的影响

, figureFileSmall=null, figureFileBig=null, tableContent=
处理时间Processing time/dAgNO3浓度AgNO3 concentration/(mg·L–1)出胚胚块数Number of embryonic blocks子叶胚数Number of cotyledon embryos胚块褐化程度Browning degree of embryo blocks
706.33±2.52ab68.00±2.00cd中等
2.56.33±1.53ab36.00±6.00d中等
5.04.50±2.89ab54.50±18.70d中等
10.04.75±2.99ab53.00±24.01d中等
15.06.33±2.08ab93.00±41.15bc中等
1404.00±2.16ab50.00±30.24d中等
2.54.75±2.36ab43.25±15.15d中等
5.04.67±2.31ab50.33±13.05d较轻
10.05.75±1.71ab65.00±17.15cd较轻
15.06.00±2.65ab122.00±2.00ab较轻
2105.33±0.58ab66.00±20.00cd中等
2.56.75±2.06ab45.25±7.32d中等
5.08.33±1.53a145.67±21.50a较轻
10.05.33±2.31ab75.33±6.43ab较轻
15.05.67±2.08ab127.00±2.00ab较轻
2803.67±1.53b35.00±1.00d中等
2.57.33±1.53ab56.00±14.00cd中等
5.07.50±4.20ab111.50±37.65ab较轻
10.08.00±1.00ab65.33±20.03cd较轻
15.04.50±1.73ab58.00±36.40cd中等
), ArticleFig(id=1277240277064614111, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1277240251089289332, language=EN, label=Tab. 2, caption=

Correlation between secondary somatic embryogenesis and browning

, figureFileSmall=null, figureFileBig=null, tableContent=
项目Item出胚胚块数Number of embryonic blocks子叶胚数Number of cotyledon embryos褐化等级Browning grade
出胚
胚块数
1.000
子叶
胚数
0.439**1.000
褐化
等级
–0.342–0.552**1.000
), ArticleFig(id=1277240277131722976, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1277240251089289332, language=CN, label=表2, caption=

次生体胚发生与褐化的相关性

, figureFileSmall=null, figureFileBig=null, tableContent=
项目Item出胚胚块数Number of embryonic blocks子叶胚数Number of cotyledon embryos褐化等级Browning grade
出胚
胚块数
1.000
子叶
胚数
0.439**1.000
褐化
等级
–0.342–0.552**1.000
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硝酸银对橡胶树次生体胚发生的影响研究
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田雯 1, 2, 3 , 张子愉 6 , 徐正伟 3, 4, 5 , 王笑一 3, 4, 5 , 周权男 3 , 吉辛 6 , 吴日智 3 , 夏薇 1, 2, * , 黄天带 3, 4, 5, *
热带作物学报 | 种质资源与遗传育种 2025,46(5): 1107-1115
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热带作物学报 |种质资源与遗传育种 2025 , 46 (5) : 1107 -1115
硝酸银对橡胶树次生体胚发生的影响研究
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田雯1, 2, 3, 张子愉6, 徐正伟3, 4, 5, 王笑一3, 4, 5, 周权男3, 吉辛6, 吴日智3, 夏薇1, 2, * , 黄天带3, 4, 5, *
作者信息
  • 1.海南大学三亚南繁研究院,海南三亚 572024
  • 2.海南大学热带农林学院,海南海口 570228
  • 3.中国热带农业科学院橡胶研究所/海口市热带植物种苗创新重点实验室/农业农村部橡胶树生物学与遗传资源利用重点实验室/海南省热带作物栽培生理学重点实验室/省部共建国家重点实验室培育基地,海南海口 571101
  • 4.中国热带农业科学院三亚研究院,海南三亚 572025
  • 5.热带作物生物育种全国重点实验室,海南三亚 572025
  • 6.云南农业大学热带作物学院,云南普洱 665099
通讯作者:
* 夏薇(XIA Wei),E-mail:;
黄天带(HUANG Tiandai),E-mail:
Effect of AgNO3 on Secondary Somatic Embryogenesis in Hevea brasiliensis
Wen TIAN1, 2, 3, Ziyu ZHANG6, Zhengwei XU3, 4, 5, Xiaoyi WANG3, 4, 5, Quannan ZHOU3, Xin JI6, Rizhi WU3, Wei XIA1, 2, * , Tiandai HUANG3, 4, 5, *
Affiliations
  • 1.Sanya Nanfan Research Institute, Hainan University, Sanya, Hainan 572024, China
  • 2.School of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan 570228, China
  • 3.Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences / Haikou Key Laboratory of Tropical Plant Seedling Innovation / Key Laboratory of Rubber Tree Biology and Genetic Resources Utilization, Ministry of Agriculture and Rural Areas / Hainan Provincial Key Laboratory of Tropical Crop Cultivation Physiology / Cultivation Base of State Key Laboratory Jointly Built by Province and Ministry, Haikou, Hainan 571101, China
  • 4.Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya, Hainan 572025, China
  • 5.National Key Laboratory for Tropical Crop Breeding, Sanya, Hainan 572025, China
  • 6.College of Tropical Crops, Yunnan Agricultural University, Pu’er, Yunnan 665099, China
出版时间: 2025-05-25 doi: 10.3969/j.issn.1000-2561.2025.05.009
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橡胶树次生体胚循环增殖技术实现了橡胶树体胚苗(自根幼态无性系)规模化繁育。该技术需要将子叶胚切成2 mm×2 mm的胚块,伤口部位会产生大量的乙烯及活性氧,培养过程中也会形成较多乙烯和活性氧,乙烯及活性氧对橡胶树体胚发生产生不利影响。硝酸银(AgNO3)是乙烯抑制剂,同时影响酶的活性。本文研究了不同硝酸银浓度(0、2.5、5.0、10.0、15.0 mg/L)处理不同时间(7、14、21、28 d)对橡胶树胚块褐化及次生体胚发生的影响,并评估了再生植株的表型及倍性。结果表明:AgNO3可明显减轻胚块褐化情况,减少胚块活性氧的产生,5.0~15.0 mg/L AgNO3处理较2.5 mg/L效果更好,AgNO3处理21 d较其他3个处理时间效果好;2.5~15.0 mg/L AgNO3处理7~28 d未显著增加出胚胚块数,但5.0 mg/L AgNO3处理21、28 d,10.0 mg/L AgNO3处理21 d,15.0 mg/L AgNO3处理7~21 d子叶胚数显著高于对照;出胚胚块数与褐化呈负相关,但未达显著水平,子叶胚数与褐化呈显著负相关;AgNO3处理的再生植株表型与对照无明显差异,染色体倍性与对照相同。本研究结果为提高橡胶树次生体胚发生效率提供技术支撑。

橡胶树  /  硝酸银  /  褐化  /  乙烯  /  体胚诱导

The technology of secondary somatic embryo cyclic multiplication has realized the large-scale propagation of somatic embryo plantlets (self-rooted juvenile clones) of Hevea brasiliensis. This technology needs to cut the cotyledon embryo into 2 mm×2 mm pieces. A large amount of ethylene and reactive oxygen species (ROS) will be produced at the wound site, and much ethylene and ROS will also formed during culture process. Ethylene and ROS have adverse effects on somatic embryogenesis of H. brasiliensis. Silver nitrate (AgNO3) is an ethylene inhibitor and also affects enzyme activity. In this paper, the effects of different AgNO3 concentrations (0, 2.5, 5.0, 10.0, 15.0 mg/L) and different time (7, 14, 21, 28 days) on the embryo block browning and secondary somatic embryogenesis of H. brasiliensis were studied, and the phenotype and ploidy of regenerated plantlets were evaluated. AgNO3 significantly reduced the browning of embryo blocks and the production of ROS. The effect of 5-15 mg/L was better than that of 2.5 mg/L, and the effect of 21 days treatment was better than that of the other three treatments. 2.5-15.0 mg/L AgNO3 treated 7-28 days did not significantly increase the number of embryonic blocks, but the number of cotyledon embryos in 5.0 mg/L AgNO3 treated for 21 and 28 days, 10.0 mg/L AgNO3 treated for 21 days, and 15.0 mg/L AgNO3 treated for 7-21 days were significantly higher than those in the control. The number of embryonic blocks was negatively correlated with browning, but not significantly, the number of cotyledon embryos was significantly negatively correlated with browning. The phenotype of regenerated plantlets from AgNO3 treatments had no obvious difference compared with the control, and the chromosome ploidy was the same as the control. The results would provide technical support for improving the efficiency of secondary somatic embryogenesis in H. brasiliensis.

Hevea brasiliensis  /  AgNO3  /  browning  /  ethylene  /  somatic embryogenesis
田雯, 张子愉, 徐正伟, 王笑一, 周权男, 吉辛, 吴日智, 夏薇, 黄天带. 硝酸银对橡胶树次生体胚发生的影响研究. 热带作物学报, 2025 , 46 (5) : 1107 -1115 . DOI: 10.3969/j.issn.1000-2561.2025.05.009
Wen TIAN, Ziyu ZHANG, Zhengwei XU, Xiaoyi WANG, Quannan ZHOU, Xin JI, Rizhi WU, Wei XIA, Tiandai HUANG. Effect of AgNO3 on Secondary Somatic Embryogenesis in Hevea brasiliensis[J]. Chinese Journal of Tropical Crops, 2025 , 46 (5) : 1107 -1115 . DOI: 10.3969/j.issn.1000-2561.2025.05.009
巴西橡胶树(Hevea brasiliensis)亦称“三叶橡胶”,为大戟科常绿乔木,原产于巴西亚马逊森林,是轮胎、绝缘材料、减震材料等的重要工业原料。橡胶树的生长周期长达35 a以上,因此种苗质量至关重要。体胚苗(自根幼态无性系)具有速生、高产等优异特性,是第三代新型种苗[1]。HUA等[1]建立了橡胶树次生体胚循环增殖技术,实现了橡胶树体胚苗规模化繁育。该技术以初(次)生胚状体为外植体,将其切成2 mm×2 mm的胚块,这些胚块经过体胚发生,产生新的胚状体,即完成胚状体的增殖。然而切胚产生伤口,造成的细胞膜降解使得原本分离开的酚类物质与多酚氧化酶(polyphenol oxidase,PPO)结合,被氧化成褐色的醌类物质[2-3]。此外,伤口部位的细胞同时产生大量的乙烯[4]及活性氧[5]。培养过程中添加的各类生长调节剂、蔗糖等成分也会对培养物造成非生物胁迫,进而促进乙烯的合成和活性氧的形成[6]
乙烯是植物的五大类激素之一,几乎所有的高等植物都能合成微量乙烯。乙烯对再生的影响因物种、基因型及外植体、培养阶段而异[6]。在白云杉和胡萝卜的非胚性愈伤组织中乙烯的含量比胚性愈伤组织高[7]。乙烯促进铁皮石斛胚性愈伤体积增大,但抑制体胚发生,抑制效果与浓度呈正相关,高浓度乙烯(5.6 mg/L)的体胚数比对照下降93.33%,几乎无法诱导体胚发生[8]。在苜蓿愈伤组织继代培养时,加入多胺生物合成抑制剂甲基乙二醛双脒基腙[methylglyoxal bis(guanylhydrazone),MGBG]促进乙烯的生成,抑制了愈伤组织及悬浮系的生长及体胚形成,在体胚诱导阶段加入影响更大,加入184 mg/L MGBG的体胚数为对照的60%(该浓度下乙烯释放量比对照增加21.1%),而368 mg/L MGBG则无法诱导体胚发生(该浓度乙烯释放量比对照增加36.8%)[9]。苜蓿无色松软的胚性愈伤组织转入将愈伤继代培养基[TDZ(thidiazuron)代替KT(kinetin)],发现愈伤组织变绿变硬,出现管状物和芽原基的分化,其体胚发生能力丧失,且乙烯合成量升高[10]。但部分植物体胚发生率与乙烯合成呈正相关,如大豆体胚数随着乙烯前体ACC(1-aminocyclopropane-1-carboxylic acid)增加而增加,随ACC合成抑制剂AVG(aminoethoxyvinylglycine)增加而下降[6]
硝酸银(AgNO3)是乙烯抑制剂,一定浓度一定处理时间对于体胚分化具有促进作用。15 mg/L硝酸银处理后的芦荟防褐化率高达90%;培养2周时,未发生褐化的外植体逐渐诱导出颗粒状的愈伤组织[11]。在花生幼叶芽再生[12]、油菜花梗植株再生[13]、石榴不定芽再生[14]中也表现出明显效果。而在有些物种中,与乙烯促进体胚发生效果相反。AgNO3在显著降低水曲柳外植体褐化指数和褐化率的同时也显著降低了体胚发生率[15]
切胚产生的活性氧爆发及组培胁迫过程产生的活性氧也抑制体胚发生。AgNO3对许多酶的活性有影响。2 mg/L AgNO3降低了凤丹牡丹愈伤的褐化程度,进一步研究表明,该处理降低了PPO、过氧化氢酶(CAT)活性,增加了过氧化物酶(POD)的活性[16]
本文研究AgNO3浓度及处理时间对橡胶树次生体胚胚块褐化及体胚发生的影响,并评估再生植株的表型及倍性,以期进一步提高橡胶树次生体胚发生效率。
选择处于成熟子叶胚阶段的热研73397次生体胚,由位于海南省儋州市的海南天然橡胶新型种植材料创新基地提供。切除子叶胚子叶外围卷曲、偏薄部位,剩余较厚的子叶在超净工作台用无菌手术刀切成2~3 mm的胚块作为外植体,分别将其均匀接种在添加不同浓度AgNO3的愈伤诱导培养基中,每皿接种12块外植体。
共设置AgNO3浓度(0、2.5、5.0、10.0、15.0 mg/L)、AgNO3处理时间(7、14、21、28 d)2个影响因素20个处理。将胚块接种于添加不同浓度(0、2.5、5.0、10.0、15.0 mg/L)AgNO3的M1[1]培养基中,室温培养不同时间(7、14、21、28 d),AgNO3处理结束后转入不添加AgNO3的M1培养基,室温继续培养至28 d后转入体胚诱导培养基M2[1],跟踪不同处理胚块体细胞胚形成及植株再生情况。每个处理4皿,每皿12个外植体。
每7 d对经不同AgNO3浓度处理后的胚块在培养基中的褐化情况进行观察,直至28 d。
对经不同AgNO3浓度及培养时间处理后的胚块随机取样进行二氨基联苯胺(diaminobenzidine,DAB)染色,观察并统计其显色情况,重复2次。
对转入M2培养基,60 d后统计子叶胚数、出胚胚块数。
每个处理取3株再生植株的叶片混合、切碎、解离,采用流式细胞仪进行倍性分析。
试验数据采用Excel 2023和IBM SPSS Statistics 20软件进行整理、统计和分析。
橡胶树次生体胚切胚后部分胚块切口褐化,部分对培养基反应敏感的胚块褐化不明显(图1A)。DAB在过氧化氢酶的催化下,可与过氧化氢(活性氧的一种)反应并迅速在组织表面形成棕红色化合物,进而确定活性氧含量。DAB染色明显看到对照(0 mg/L AgNO3)胚块比添加AgNO3的胚块染色深;2.5 mg/L AgNO3处理胚块较5.0~15.0 mg/L AgNO3处理同样时间的胚块染色深;AgNO3处理21 d的胚块较其他3个处理时间染色浅(图1B)。染色越深表明活性氧含量越高。可见,AgNO3可明显减少胚块培养中活性氧的产生,5.0~15.0 mg/L较2.5 mg/L效果好,处理21 d较其他3个处理时间效果好。
不同AgNO3浓度处理7 d的橡胶树出胚胚块数与对照组无显著差异;2.5~10.0 mg/L AgNO3处理的子叶型胚状体数低于对照,15.0 mg/L AgNO3处理的子叶型胚状体数高于对照,但均未达显著水平,15.0 mg/L AgNO3处理的子叶型胚状体数显著高于其他浓度;不同浓度间胚块在体胚诱导阶段褐化程度与对照无明显差异,浓度间也无明显差异(表1)。
处理14 d,不同AgNO3浓度处理后橡胶树出胚胚块数与对照组无显著差异;15.0 mg/L AgNO3处理的子叶型胚状体数显著高于对照及其他3个浓度,其他3个浓度处理的子叶型胚状体数与对照无显著差异;5.0~15.0 mg/L AgNO3处理的胚块在体胚诱导阶段褐化程度明显轻于2.5 mg/L AgNO3及对照,而2.5 mg/L AgNO3处理的褐化程度与对照相似(表1)。
处理21 d,5.0 mg/L AgNO3处理的橡胶树出胚胚块数高于其他浓度,但未达显著水平;而除2.5 mg/L AgNO3处理的子叶型胚状体数与对照无显著差异外,其余AgNO3浓度诱导的子叶型胚状体数均显著高于对照,其中5.0 mg/L AgNO3诱导的子叶型胚状体数量最多;2.5 mg/L AgNO3处理的胚块褐化程度与对照相似,其余AgNO3浓度处理的褐化程度均明显低于对照(表1)。
处理28 d,AgNO3处理的橡胶树次生体胚出胚胚块数均高于对照,特别是2.5~10.0 mg/L,但未达显著水平;5.0 mg/L AgNO3处理的子叶型胚状体数显著高于对照及其他浓度,其他3个浓度处理的子叶型胚状体数与对照无显著差异;5.0、10.0 mg/L AgNO3处理的胚块褐化程度较轻,2.5、15.0 mg/L AgNO3处理的胚块褐化程度中等,与对照相似(表1)。
AgNO3处理对出胚胚块数的影响不大,但对子叶型胚状体数的影响较大(表1)。低浓度AgNO3需要处理较长时间才能显著提升子叶胚数,2.5 mg/L AgNO3处理28 d子叶胚数才开始上升,5 mg/L AgNO3处理21 d达到峰值,28 d略有下降,但仍处于高值;而高浓度的AgNO3需要相对较短的处理时间就可显著提升子叶胚诱导率,如15 mg/L AgNO3处理的子叶胚诱导率的峰值出现在14~21 d,28 d开始显著下降。
综上所述,不同浓度AgNO3、不同处理时间未显著增加出胚胚块数,但5.0 mg/L AgNO3处理21、28 d,10.0 mg/L AgNO3处理21 d,15.0 mg/L AgNO3处理7~21 d子叶胚数显著高于对照(表1)。
相关性分析结果表明,出胚胚块数与褐化呈负相关,相关系数为–0.342,但未达到显著水平;子叶胚数与褐化呈负相关,相关系数为-0.552,达到极显著水平。此外,出胚胚块数与子叶胚数呈正相关,相关系数为0.439,达到极显著水平(表2)。
AgNO3处理的再生植株表型与对照植株相似(图2)。倍性分析结果表明,AgNO3处理再生植株与对照一样,均为二倍体,未产生倍性变异(图3)。
伤害会诱导乙烯大量合成[17],培养过程中添加的各类生长调节剂、蔗糖等成分也会对培养物造成逆境,进而促进乙烯的合成[6]。乙烯抑制白菜型油菜(Brassica campestris L.[18]、白菜[Brassica pekinensis(Lour.)Rupr.][19]、美洲狼尾草(Pennisetum glaucum[20]、白云杉(Picea glauca[21]、胡萝卜(Daucus carota L.)[22]、罗布斯塔咖啡(Coffea canephora[23-24]、拟南芥(Arabidopsis thaliana[25]、菠菜(Spinacia oleracea[26]、玉米(Zea mays L.)[27]、冰糖橙[28]等物种的体胚发生。AgNO3通过以银离子取代乙烯结合必须的铜离子,从而阻断乙烯信号传导,使培养物无法感受乙烯信号[29],达到抑制乙烯发挥作用的效果。添加AgNO3等乙烯抑制剂,可大幅提升体胚发生能力。如玉米幼胚胚性愈伤诱导率提高3~10倍,体胚发生能力提高3倍[27];添加1.70 mg/L AgNO3,每块菠菜愈伤体胚数增加3倍[26]
添加乙烯后,番茄体胚发育停留在球形胚阶段[30]。橡胶树体胚发生对乙烯也很敏感。内珠被培养需要将幼果切成薄片先诱导愈伤,再诱导体胚发生,此过程产生的乙烯会抑制胚性愈伤和体胚诱导[31]。橡胶树内珠被培养过程中添加0.44、4.40 mg/L乙烯抑制剂AOA及1.00、0.01 mg/L AgNO3均显著提高胚性愈伤诱导率和体胚诱导率,而添加ACC(乙烯合成前体)则抑制胚性愈伤诱导率和体胚诱导率,在IRCA144和PB260两个品种中趋势相同。IRCA144在9.98 mg/L、PB260在0.1 mg/L AgNO3处理分别获得了60个和7个原胚,而对照则无法诱导出原胚[31]。继代4 a以上胚性下降的橡胶树花药愈伤组织在体胚诱导第一个月添加5 mg/L AgNO3显著促进了体胚发生[32]。在其他物种中,如苜蓿[33-34]、大豆[35]、苏格兰松(Pinus sylvestris L.)[36]、夏雪片莲(Leucojum aestivum L.)[37]中乙烯却可以促进体胚发生。
在非胁迫环境中,细胞内的ROS主要由线粒体产生,ROS的产生和清除处于动态平衡,不会对细胞造成氧化损伤[38]。当生物体受到非生物胁迫时,呼吸作用加剧,释放出大量ROS,超出ROS清除系统的清除能力,使ROS在细胞内积累,从而对细胞造成氧化损伤[39]。组培对植物材料是一个非生物胁迫过程,期间会产生过量ROS。过氧化物酶(peroxidase,POD)脱除组织中的过氧化氢和超氧阴离子,保护细胞膜的通透性。研究表明,硝酸银可提高牡丹[16,40]、刺梨[41]愈伤组织POD的活性,减轻褐化。300 mg/L AgNO3提高了黄瓜叶片POD(过氧化物酶)和SOD(超氧化物歧化酶)的活性[42]。本研究也发现,AgNO3通过减少胚块组织中过氧化氢的积累,进而减少活性氧的含量,提高体胚发生率。
适宜浓度的AgNO3可提高正常胚状体诱导率,但长时间和高浓度AgNO3处理容易导致体胚畸形。体胚诱导第一个月添加5 mg/L AgNO3显著促进橡胶树花药愈伤组织体胚发生,但浓度提高到10 mg/L处理1个月或5 mg/L处理2个月,畸形胚率增加[32]。大量乙烯导致紫花苜蓿(Medicago sativa L.)植株畸形[43]。本研究表明,5.0~15.0 mg/L AgNO3在愈伤诱导阶段处理7~28 d提高了子叶胚诱导率,再生植株倍性正常,未发生变异。
  • 国家自然科学基金项目(32271915)
  • 国家天然橡胶产业技术体系(CARS33)
  • 热带作物育种国家重点实验室项目(PT2400008492)
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doi: 10.3969/j.issn.1000-2561.2025.05.009
  • 接收时间:2024-12-30
  • 首发时间:2026-06-26
  • 出版时间:2025-05-25
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  • 收稿日期:2024-12-30
  • 录用日期:2025-01-11
基金
国家自然科学基金项目(32271915)
国家天然橡胶产业技术体系(CARS33)
热带作物育种国家重点实验室项目(PT2400008492)
作者信息
    1.海南大学三亚南繁研究院,海南三亚 572024
    2.海南大学热带农林学院,海南海口 570228
    3.中国热带农业科学院橡胶研究所/海口市热带植物种苗创新重点实验室/农业农村部橡胶树生物学与遗传资源利用重点实验室/海南省热带作物栽培生理学重点实验室/省部共建国家重点实验室培育基地,海南海口 571101
    4.中国热带农业科学院三亚研究院,海南三亚 572025
    5.热带作物生物育种全国重点实验室,海南三亚 572025
    6.云南农业大学热带作物学院,云南普洱 665099

通讯作者:

* 夏薇(XIA Wei),E-mail:;
黄天带(HUANG Tiandai),E-mail:
参考文献
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https://castjournals.cast.org.cn/joweb/rdzwxb/CN/10.3969/j.issn.1000-2561.2025.05.009
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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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