秋水仙素对金花菜多倍体的诱导效应

doi:10.11924/j.issn.1000-6850.casb2025-0045

摘要/Abstract

摘要：

以上海金花菜种子和试管苗为试验材料，通过设置不同的秋水仙素浓度(0%、0.01%、0.03%、0.05%、0.07%、0.09%、0.11%)，综合运用植物组织培养、植物生化生理和植物分子生物学技术，探讨秋水仙素处理对金花菜种子萌发、试管苗生长发育以及相关生理生化指标和基因表达的影响。结果表明，MS培养基添加秋水仙素和秋水仙素浸泡会抑制金花菜种子的萌发及其萌发苗的株高，减少其叶片数，提高其茎粗，促进其生根，促进其CAT活性、SOD活性、POD活性、MDA含量、可溶性糖含量、可溶性蛋白含量和脯氨酸含量增加，促进其WRKY转录因子、蛋白TIFY 8、转录因子MYB13、NAC转录因子47、ABC转运蛋白、晚期胚胎发生丰富蛋白2和乙烯反应转录因子ERF110等基因表达上调。MS培养基添加秋水仙素和秋水仙素浸泡会抑制金花菜试管苗的株高增长、叶片数增加、根数增多和根长增长，促进金花菜试管苗茎粗增粗以及其CAT活性、SOD活性、POD活性、MDA含量、可溶性糖含量、可溶性蛋白含量和脯氨酸含量的增加，促进其WRKY转录因子、蛋白TIFY 8、转录因子MYB13、NAC转录因子47、ABC转运蛋白、晚期胚胎发生丰富蛋白2和乙烯反应转录因子ERF110等基因表达上调。金花菜种子在添加秋水仙素培养基上的多倍体诱导效果低于金花菜种子经秋水仙素浸泡后的多倍体诱导效果，金花菜种子经秋水仙素浸泡后的多倍体诱导效果低于试管苗在添加秋水仙素培养基上的多倍体诱导效果，试管苗在添加秋水仙素培养基上的多倍体诱导效果低于试管苗经秋水仙素浸泡的多倍体诱导效果。本试验结果可为金花菜的多倍体诱导的信号途径研究以及秋水仙素在诱导金花菜多倍体方面的应用提供理论参考。

关键词: 秋水仙素, 金花菜, 种子萌发, 试管苗, 生理生化指标, 基因表达

Abstract:

Using the seeds and plantlets of Medicago polymorpha in Shanghai as experimental materials, different concentrations of colchicine were set (0%, 0.01%, 0.03%, 0.05%, 0.07%, 0.09%, 0.11%), and plant tissue culture, plant biochemical physiology, and plant molecular biology techniques were used to investigate the effects of colchicine treatment on the germination of seeds, the growth and development of plantlets, as well as related physiological and biochemical indicators and gene expression in M. polymorpha. The results showed that the addition of colchicine and colchicine soaking to MS medium could inhibit the germination rate of M. polymorpha seeds and the plant height of germinated seedlings, reduce their leaf number, increase their stem thickness, promote their rooting, increase their CAT activity, SOD activity, POD activity, MDA content, soluble sugar content, soluble protein content, and proline content, promote their upregulation of WRKY transcription factor, protein TIFY 8, transcription factor MYB13, NAC transcription factor 47, ABC transporter, late embryogenesis rich protein 2, and ethylene responsive transcription factor ERF110 gene expression. The addition of colchicine to MS medium and soaking in colchicine could inhibit the plant height, leaf number, stem diameter, root number, and root length of plantlets of M. polymorpha, promote their increase of stem diameter, CAT activity, SOD activity, POD activity, MDA content, soluble sugar content, soluble protein content, and proline content of test tube seedlings of M. polymorpha, and promote their upregulation of gene expression of WRKY transcription factor, protein TIFY 8, transcription factor MYB13, NAC transcription factor 47, ABC transporter, late embryogenesis enriched protein 2, and ethylene responsive transcription factor ERF110. The polyploidy induction effect of M. polymorpha seeds on colchicine added medium was lower than the polyploidy induction effect of M. polymorpha seeds soaked in colchicine, the polyploidy induction effect of M. polymorpha seeds soaked in colchicine was lower than the polyploidy induction effect of M. polymorpha plantlets on colchicine added medium, the polyploidy induction effect of M. polymorpha plantlets on colchicine added medium was lower than the polyploidy induction effect of M. polymorpha plantlets soaked in colchicine. The results of this experiment had practical significance for the induction of polyploidy in M. polymorpha, and could provide theoretical support for the study of signal pathways for polyploidy induction in M. polymorpha and the application of colchicine in inducing polyploidy in M. polymorpha.

Key words: colchicine, Medicago polymorpha, seed germination, plantlet, physiological and biochemical indexes, gene expression

洪森荣, 吉莉莉. 秋水仙素对金花菜多倍体的诱导效应[J]. 中国农学通报, 2025, 41(22): 13-26.

HONG Senrong, JI Lili. Induction Effect of Colchicine on Polyploidy of Medicago polymorpha[J]. Chinese Agricultural Science Bulletin, 2025, 41(22): 13-26.

图/表 18

参考文献 42

[1]	赵嫚, 陈仕勇, 李亚萍, 等. 外源GABA对盐胁迫下金花菜种子萌发及幼苗抗氧化能力的影响[J]. 江苏农业学报, 2021, 37(2):310-316.
[2]	董磊, 王栋麟, 王琳, 等. 外源水杨酸缓解金花菜高温胁迫的生理响应[J]. 扬州大学学报(农业与生命科学版), 2022, 43(4):129-136.
[3]	尹明华, 胡佳欣, 李瑶瑶, 等. 金花菜叶绿体基因组特征及密码子偏好性分析[J]. 草业科学, 2024, 41(4):884-898.
[4]	马伯艳, 张广庆, 刘长喜. 金花菜对营养性肥胖大鼠体重及血脂的影响[J]. 中医杂志, 2016, 57(17):1500-1503.
[5]	周勃, 任海龙, 张龑, 等. 金花菜与苜蓿属主要物种基因组SSR分布特征的比较分析[J]. 新疆农业科学, 2022, 59(9):2217-2223. doi: 10.6048/j.issn.1001-4330.2022.09.016
[6]	王小山, 魏臻武, 曹德明, 等. 播种期对金花菜生长及产量的影响[J]. 草业科学, 2017, 34(2):347-351.
[7]	任海龙, 魏臻武, 陈祥. 金花菜应用研究进展[J]. 中国野生植物资源, 2014, 33(5):33-36.
[8]	贾雪杰, 游明鸿, 李达旭, 等. 减量施肥对金花菜——水稻轮作系统中产量和土壤养分的影响[J]. 草地学报, 2023, 31(3):876-883. doi: 10.11733/j.issn.1007-0435.2023.03.030
[9]	陈莉敏, 李达旭, 白史且, 等. ⁶⁰Co-γ射线辐射对金花菜种子生物学效应的影响[J]. 核农学报, 2022, 36(9):1701-1706. doi: 10.11869/j.issn.100-8551.2022.09.1701
[10]	徐远东, 范彦, 何玮. 多倍体育种在牧草和草坪草中的应用[J]. 草业与畜牧, 2012(9):12-15.
[11]	王炜, 陈琛, 葛玉彬, 等. 高丹草多倍体的诱导与鉴定[J]. 西北农业学报, 2021, 30(9):1394-1401.
[12]	赵汗青, 王强, 庞晓明, 等. ‘京枣39’离体多倍体诱导及其增殖、生根培养体系优化[J]. 北京林业大学学报, 2024, 46(6):118-126.
[13]	徐远东, 何玮. 牧草植物多倍体诱导的研究进展[J]. 草业与畜牧, 2015(6):1-4.
[14]	LIONNETON E, BRURET W, DELAITRE C, et al. Improved microspore culture and doubled-haploid plant regeneration in the brown condiment mustard (Brassica juncea)[J]. Plant cell reports, 2001, 20(2):126-130. doi: 10.1007/s002990000292 pmid: 30759898
[15]	马小鸥, 张前, 吴利雪, 等. 匍茎百合再生体系的建立及多倍体诱导[J]. 东北林业大学学报, 2024, 52(4):52-58.
[16]	高俊凤. 植物生理学实验指导[M]. 北京: 高等教育出版社, 2006:140-144,228.
[17]	洪森荣, 朱盈盈, 李紫莹, 等. 盐胁迫下金花菜和紫花苜蓿试管苗的转录组分析及其耐盐基因筛选[J]. 中国农学通报, 2023, 39(3):111-118. doi: 10.11924/j.issn.1000-6850.casb2022-0235
[18]	刘倩. 6种苜蓿属植物多倍体诱导及倍性鉴定研究[D]. 扬州: 扬州大学, 2014:22.
[19]	李泽根, 任威蓁, 于淑漫, 等. 秋水仙素对豆梨的诱变效应及四倍体鉴定[J]. 林业与生态科学, 2024, 39(4):440-447. doi: 10.13320/j.cnki.hjfor.2024.0051
[20]	VISHNUPANDI S, GANGA M, RAJAMANI K, et al. Effect of varying concentrations of colchicine on polyploid induction in Jasminum sambac (L.) Aiton[J]. Electronic journal of plant breeding, 2023, 14(2):496-501.
[21]	孙利娜, 吴林巧, 李冰, 等. 秋水仙素对桃金娘种子的诱变效应[J]. 广西林业科学, 2022, 51(5):670-675. doi: 10.19692/j.issn.1006-1126.202205012
[22]	魏尊苗, 刘佳遥, 程艳, 等. 秋水仙素诱变对油莎豆出苗率及品质的影响[J]. 河南农业科学, 2022, 51(11):50-55.
[23]	张志慧, 王岩, 郑志兴, 等. 秋水仙素浓度及处理时间对黄芩发芽的影响[J]. 陕西农业科学, 2023, 69(11):42-45.
[24]	闫瑾, 邬海艳, 邹宜芯, 等. 秋水仙素处理对青花菜种子萌发及幼苗生长的影响[J]. 天津农学院学报, 2024, 31(2):18-24. doi: 10.19640/j.cnki.jtau.2024.02.004
[25]	倪成凤, 冷青云, 虞道耿, 等. 秋水仙素对猪屎豆的诱变及同源四倍体种质创制的研究[J]. 热带作物学报, 2022, 43(10):2047-2056. doi: 10.3969/j.issn.1000-2561.2022.10.010
[26]	HARING F, FARID M, RIDWAN I, et al. Polyploidization of three Chrysanthemum varieties in vitro at various levels of colchicine soaking length[J]. Biodiversitas, 2024, 25(8):3496.
[27]	李承秀, 程甜甜, 燕语, 等. 流苏多倍体诱导试验初报[J]. 浙江农业科学, 2022, 63(4):744-747. doi: 10.16178/j.issn.0528-9017.20212645
[28]	张丞慧, 孟冰超, 杨茂磊, 等. 刺槐多倍体诱导及叶片解剖结构分析[J]. 天津农学院学报, 2021, 28(3):18-21. doi: 10.19640/j.cnki.jtau.2021.03.004
[29]	HERNANDEZ R, LOPEZ A, VALENZUELA B, et al. Organogenesis of plant tissues in colchicine allows selecting in field trial blueberry (Vaccinium spp. cv Duke) clones with commercial potential[J]. Horticulturae, 2024, 10(3):283.
[30]	唐宁, 汪福源, 高山林. 白术同源四倍体农艺性状和抗氧化酶活性的研究[J]. 药物生物技术, 2006(6):442-445.
[31]	颉嘉丽, 李明, 石铭福, 等. 钾肥对干旱胁迫下马铃薯生理特性、产量及品质的影响[J]. 西北农业学报, 2024, 33(11):2059-2069.
[32]	闫瑾, 邬海艳, 任玉欣, 等. 秋水仙素处理对盐胁迫下青花菜生理生化特性的影响[J]. 湖南生态科学学报, 2023, 10(3):19-27.
[33]	MOHAMMADI H, ABDOLLAHI B S, AGHAEE A, et al. Foliar-applied silicate potassium modulates growth, phytochemical, and physiological traits in Cichorium intybus L. under salinity stress[J]. BMC plant biology, 2024, 24(1):288.
[34]	XU H N, HE X Z, CHEN X Q, et al. Molecular cloning and functional analysis of spinach SoSMT2 in response to excess nitrate stress[J]. Scientia horticulturae, 2015, 184:93-100.
[35]	ELIK S. Gene expression analysis of potato drought-responsive genes under drought stress in potato (Solanum tuberosum L.) cultivars[J]. Peer J, 2024, 12:e17116.
[36]	白龙飞, 况勇, 肖逸, 等. 茭白WRKY基因家族鉴定及菰黑粉菌侵染下的表达分析[J]. 安徽农业大学学报, 2024, 51(5):759-770.
[37]	杨锐佳, 张中保, 吴忠义. 植物转录因子TIFY家族蛋白结构和功能的研究进展[J]. 生物技术通报, 2020, 36(12):121-128. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0307
[38]	郭飞翔, 李春霞, 周爽, 等. 绿豆R2R3-MYB转录因子家族鉴定及其类黄酮合成调控基因的筛选[J]. 作物学报, 2025, 51(1):117-133. doi: 10.3724/SP.J.1006.2025.44077
[39]	刘惠玲, 江炳玉, 张静, 等. 植物NAC转录因子的研究进展[J]. 福建农林大学学报(自然科学版), 2024, 53(6):742-753.
[40]	蔡方阳, 赵懿琛, 李义, 等. 杜仲ABC转运蛋白基因家族成员鉴定与分析[J]. 浙江农业学报, 2021, 33(9):1581-1591. doi: 10.3969/j.issn.1004-1524.2021.09.02
[41]	郑洁旋, 张会, 简曙光, 等. 厚藤胚胎发生晚期丰富蛋白基因IpLEA的克隆与功能分析[J]. 分子植物育种, 2018, 16(13):4235-4244.
[42]	张秋平, 杨宇红, 茆振川, 等. 辣椒乙烯反应转录因子基因CaJERF1的克隆及诱导表达[J]. 园艺学报, 2012, 39(4):705-712.

秋水仙素浓度/%	萌发率/%	成苗率/%	黄化率/%	根数/条	根长/cm
CK	96.30±3.71Aa	40.12±1.53Aa	5.240±1.673Bb	8.33±1.04Aa	0.69±0.05Aa
0.01	98.81±2.06Aa	47.29±2.89Aa	6.680±1.682Bb	0.00±0.00Bb	0.00±0.00Bb
0.03	94.08±5.65Aa	21.27±3.62Bb	11.49±2.774Aa	0.00±0.00Bb	0.00±0.00Bb
0.05	93.80±10.43Aa	25.37±1.30Bb	17.36±1.362Aa	0.00±0.00Bb	0.00±0.00Bb
0.07	91.68±8.02Aa	28.97±2.50Bb	14.68±1.741Aa	0.00±0.00Bb	0.00±0.00Bb
0.09	88.83±3.05Aab	21.90±2.79Bb	18.41±2.784Aa	0.00±0.00Bb	0.00±0.00Bb
0.11	86.29±3.92Abc	22.15±4.11Bb	14.56±1.896Aa	0.00±0.00Bb	0.00±0.00Bb

秋水仙素浓度/%	萌发率/%	成苗率/%	黄化率/%	根数/条	根长/cm
CK	96.30±3.71Aa	40.12±1.53Aa	5.240±1.673Bb	8.33±1.04Aa	0.69±0.05Aa
0.01	98.81±2.06Aa	47.29±2.89Aa	6.680±1.682Bb	0.00±0.00Bb	0.00±0.00Bb
0.03	94.08±5.65Aa	21.27±3.62Bb	11.49±2.774Aa	0.00±0.00Bb	0.00±0.00Bb
0.05	93.80±10.43Aa	25.37±1.30Bb	17.36±1.362Aa	0.00±0.00Bb	0.00±0.00Bb
0.07	91.68±8.02Aa	28.97±2.50Bb	14.68±1.741Aa	0.00±0.00Bb	0.00±0.00Bb
0.09	88.83±3.05Aab	21.90±2.79Bb	18.41±2.784Aa	0.00±0.00Bb	0.00±0.00Bb
0.11	86.29±3.92Abc	22.15±4.11Bb	14.56±1.896Aa	0.00±0.00Bb	0.00±0.00Bb

秋水仙素浓度/%	株高/cm	叶片数/片	茎粗/cm	根数/条	根长/cm
CK	6.00±0.12Aa	11.67±1.66Aa	0.10±0.01Cc	8.67±0.67Aa	3.13±0.27Aa
0.01	0.93±0.03Bb	10.33±0.15Aa	0.12±0.01BCc	0.00±0.00Ee	0.00±0.00Cc
0.03	1.02±0.06Bb	6.50±0.50Bb	0.16±0.02ABb	1.00±0.13Bb	0.23±0.11Bb
0.05	0.97±0.05Bb	2.08±0.13Cc	0.28±0.01Aa	0.66±0.08Cc	0.33±0.12Bb
0.07	0.83±0.03Bb	2.12±0.34Cc	0.26±0.04Aa	0.33±0.02Dd	0.20±0.01Bb
0.09	0.80±0.08Bb	2.24±0.31Cc	0.26±0.05Aa	0.32±0.03Dd	0.31±0.04Bb
0.11	0.80±0.03Bb	2.31±0.24Cc	0.23±0.03Aa	0.33±0.01Dd	0.36±0.02Bb

秋水仙素浓度/%	株高/cm	叶片数/片	茎粗/cm	根数/条	根长/cm
CK	6.00±0.12Aa	11.67±1.66Aa	0.10±0.01Cc	8.67±0.67Aa	3.13±0.27Aa
0.01	0.93±0.03Bb	10.33±0.15Aa	0.12±0.01BCc	0.00±0.00Ee	0.00±0.00Cc
0.03	1.02±0.06Bb	6.50±0.50Bb	0.16±0.02ABb	1.00±0.13Bb	0.23±0.11Bb
0.05	0.97±0.05Bb	2.08±0.13Cc	0.28±0.01Aa	0.66±0.08Cc	0.33±0.12Bb
0.07	0.83±0.03Bb	2.12±0.34Cc	0.26±0.04Aa	0.33±0.02Dd	0.20±0.01Bb
0.09	0.80±0.08Bb	2.24±0.31Cc	0.26±0.05Aa	0.32±0.03Dd	0.31±0.04Bb
0.11	0.80±0.03Bb	2.31±0.24Cc	0.23±0.03Aa	0.33±0.01Dd	0.36±0.02Bb

秋水仙素浓度/ %	CAT活性/ [U/(min·g·FW)]	SOD活性/ [U/(min·g·FW)]	POD活性/ [U/(min·g·FW)]	MDA含量/ [µmol/(g·FW)]	可溶性糖含量/ [mg/(g·FW)]	可溶性蛋白含量/ [mg/(g·FW)]	脯氨酸含量/ [µg/(g·FW)]
CK	169.35±25.62Ff	238.12±48.21Ee	856.39±56.32Ee	5.59±0.69Cc	3.46±0.63Dd	26.39±2.36Ee	125.49±23.61Gg
0.01	234.96±54.21Dd	364.95±26.31Cc	981.06±52.42Dd	5.36±0.86Cc	5.96±0.85Cc	36.29±5.12Dd	234.81±52.13Ff
0.03	316.95±41.87Bb	425.96±28.91Bb	1325.42±48.75Cc	12.63±1.36Bb	8.65±1.26Bb	42.55±1.85Cc	352.64±49.87Cc
0.05	423.82±28.19Aa	498.67±47.26Aa	1624.82±62.13Aa	24.19±3.24Aa	12.84±1.64Aa	52.61±4.23Aa	428.51±51.22Aa
0.07	263.48±22.11Cc	413.54±31.54Bb	1482.44±82.13Bb	22.43±3.16Aa	13.96±2.85Aa	46.82±5.12Bb	382.19±61.23Bb
0.09	244.85±32.16Dd	342.68±24.16Cc	1023.52±77.39Dd	25.21±1.96Aa	7.86±1.03Bb	45.62±1.69Bb	316.74±31.85Dd
0.11	219.77±35.49Ee	318.49±19.85Dd	989.67±49.88Dd	26.78±2.88Aa	7.94±0.96Bb	41.84±2.51Cc	271.99±27.49Ee