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有斑百合多倍体诱导及鉴定

张锡庆 汪莲娟 曹钦政 贾桂霞

张锡庆, 汪莲娟, 曹钦政, 贾桂霞. 有斑百合多倍体诱导及鉴定[J]. 北京林业大学学报, 2017, 39(7): 96-102. doi: 10.13332/j.1000-1522.20170028
引用本文: 张锡庆, 汪莲娟, 曹钦政, 贾桂霞. 有斑百合多倍体诱导及鉴定[J]. 北京林业大学学报, 2017, 39(7): 96-102. doi: 10.13332/j.1000-1522.20170028
ZHANG Xi-qing, WANG Lian-juan, CAO Qin-zheng, JIA Gui-xia. Polyploidy induction and identification in Lilium concolor var. pulchellum[J]. Journal of Beijing Forestry University, 2017, 39(7): 96-102. doi: 10.13332/j.1000-1522.20170028
Citation: ZHANG Xi-qing, WANG Lian-juan, CAO Qin-zheng, JIA Gui-xia. Polyploidy induction and identification in Lilium concolor var. pulchellum[J]. Journal of Beijing Forestry University, 2017, 39(7): 96-102. doi: 10.13332/j.1000-1522.20170028

有斑百合多倍体诱导及鉴定

doi: 10.13332/j.1000-1522.20170028
基金项目: 

林业公益性行业科研专项 201204609

国家自然科学基金项目 31470106

详细信息
    作者简介:

    张锡庆,博士生。主要研究方向:园林植物种质资源与遗传育种。Email:zhangxiqing1983@126.com  地址:100083  北京市海淀区清华东路35号北京林业大学园林学院

    责任作者:

    贾桂霞,教授,博士生导师。主要研究方向:园林植物种质资源与遗传育种。Email:gxjia@bjfu.edu.cn  地址:同上

  • 中图分类号: S682.2+9

Polyploidy induction and identification in Lilium concolor var. pulchellum

  • 摘要: 为了建立野生有斑百合多基因型种子离体多倍体诱导方法及快速、准确和高效的多倍体鉴定技术,本研究在离体培养条件下,以野生有斑百合预培养7 d的无菌萌动种子(2n=2x=24)为材料,用不同含量的秋水仙素溶液进行不同时间的诱变处理。以种子下胚轴明显膨大作为变异植株早期鉴定的标准,通过流式细胞仪检测细胞核DNA含量,并结合染色体计数法对诱导后的植株进行倍性鉴定,探讨在相同的种子萌动状态下,不同含量、不同浸泡时间的秋水仙素诱变效果,并初步建立了种子离体多倍体诱导和鉴定技术流程。结果表明:0.10%的秋水仙素溶液可有效地诱导野生有斑百合萌动种子的染色体加倍,其中以浸泡处理36 h的诱导效果最好,诱导率达44.43%;与二倍体植株相比,四倍体植株表现出叶片肥厚且增宽、叶色深绿、叶表面轻微褶皱、叶柄粗壮、生长缓慢等特征。研究还发现,植物外部形态变化不仅在二倍体和四倍体之间存在,而且在不同基因型的四倍体之间也存在差异。本研究利用下胚轴的膨大作为早期筛选多倍体的有效指标,同时结合直接测定细胞中DNA含量和计数染色体数可快速有效地培育出野生有斑百合同源四倍体植株。本研究建立的离体多倍体诱导流程除可应用于有斑百合种子多倍体的诱导外,也可为其他野生百合种子离体染色体加倍提供参考。

     

  • 图  1  二倍体和四倍体倍性鉴定

    x轴和y轴分别代表荧光强度和细胞核数。图a、b为流式细胞分析,二倍体(图a)作内标,图b为四倍体;图c、d为根尖染色体数, 二倍体2n=2x=24(图c),四倍体2n=4x=48(图d); 比例尺=10 μm。

    Figure  1.  Ploidy confirmation of diploid and tetraploid plants

    The x-and y-axes represent the relative signal intensity and number of nuclei, respectively.Figures a, b are flow cytometry analysis of the relative DNA amounts of PI-stained nuclei from fresh young leaves. The diploid (a) was used as a control standard to compare the ploidy levels of tetraploid (b) plants; c, d are chromosomes of a root tip cell from diploid 2n = 2x = 24 (c) and induced tetraploid 2n = 4x = 48 (d); bars = 10 μm.

    表  1  不同秋水仙素含量和浸泡时间对有斑百合萌动种子的诱导结果

    Table  1.   Effects of colchicine concentrations and exposure times on tetraploid induction rate from germinating seeds of L. concolor var. pulchellum

    处理
    Treatment
    秋水仙素质量分数
    Colchicine mass fraction
    浸泡时间
    Exposure time/h
    处理种子粒数
    Number of seeds
    存活率
    Survival rate/%
    下胚轴膨大率
    Hypocotyl swollen rate/%
    四倍体诱导率
    Tetraploid induction rate/%
    T10.00%189097.77±2.23a0.00±0.00d0.00±0.00f
    T20.03%189094.43±1.13ab15.53±4.00c4.43±1.13ef
    T30.05%189094.47±4.00ab14.43±4.43c6.67±1.93ef
    T40.10%189091.10±4.02ab22.20±1.10c11.13±2.94e
    T50.00%249097.80±1.10a0.00±0.00d0.00± 0.00f
    T60.03%249084.43±6.78bc27.77±6.19c21.13±2.94d
    T70.05%249091.10±2.20ab43.33±1.93b35.53±2.23c
    T80.10%249087.80±1.10abc58.90±4.02a47.77±2.94ab
    T90.00%369092.20±1.10ab0.00±0.00d0.00±0.00f
    T100.03%369090.00±1.91abc67.77±2.94a55.53±2.23a
    T110.05%369091.10±4.85ab64.47±9.68a54.47±7.79ab
    T120.10%369080.00±1.91c65.57±4.43a44.43±5.57bc
    注:后3列数据为3个重复的均值±标准误。同列不同字母之间差异显著(P<0.05)。Notes: values represent means ±SE of three replicates. Different lowercase letters within a column indicate significant difference at P<0.05 level.
    下载: 导出CSV

    表  2  多变量方差分析主体间效应的检验

    Table  2.   Tests of among-subjects effects in multivariate


    Source
    因变量
    Dependent variable
    Ⅲ型平方和
    Sum of squares of type Ⅲ
    自由度
    df
    均方
    Mean square
    F
    F value
    显著性
    Sig.
    秋水仙素质量分数
    Colchicine mass fraction
    存活率Survival rate447.3833149.1284.7680.008
    下胚轴膨大率Hypocotyl swollen rate12 694.23634 231.41226.0600.000
    四倍体诱导率Tetraploid induction rate6 845.13032 281.71016.5980.000
    浸泡时间Exposure time存活率Survival rate234.1622117.0813.7430.035
    下胚轴膨大率Hypocotyl swollen rate7 965.98423 982.99224.5300.000
    四倍体诱导率Tetraploid induction rate6 683.42023 341.71024.3090.000
    误差Error存活率Survival rate938.3853031.279
    下胚轴膨大率Hypocotyl swollen rate4 871.24330162.375
    四倍体诱导率Tetraploid induction rate4 124.05830137.469
    总计Total存活率Survival rate299 845.14036
    下胚轴膨大率Hypocotyl swollen rate61 625.13036
    四倍体诱导率Tetraploid induction rate37 406.91036
    下载: 导出CSV

    表  3  多变量方差多重比较分析

    Table  3.   Multiple comparison analysis in ANOVA

    项目
    Item
    存活率
    Survival rate/%
    下胚轴膨大率
    Hypocotyl swollen rate/%
    四倍体诱导率
    Tetraploid induction rate/%
    秋水仙素Colchicine mass fraction0.00%95.92±1.22a0.00±0.00c0.00±0.00c
    0.03%89.62±2.52ab37.02±8.21ab27.03±7.60ab
    0.05%92.78±2.18ab28.88±6.81b21.10±6.59b
    0.10%87.50± 1.97b52.78±5.90a39.45±5.52a
    浸泡时间Exposure time18 h94.44±1.50a13.04±2.76b5.56±1.44b
    24 h90.28±2.15ab32.50±6.76a26.11±5.45a
    36 h88.32±1.90b49.45±8.93a38.61±7.16a
    注:同列不同字母之间差异显著(P<0.05)。Note :different lowercase letters within a column indicate significant difference at P<0.05.
    下载: 导出CSV

    表  4  下胚轴膨大植株数与四倍体植株数间相关分析

    Table  4.   Correlation analysis between number of swollen-hypocotyls plants and number of tetraploid plants

    下胚轴膨大植株数
    Number of swollen-hypocotyl plants
    四倍体植株数
    Number of tetraploid plants
    下胚轴膨大植株数Number of swollen-hypocotyl plants10.981**
    0.000
    3636
    四倍体植株数
    Number of tetraploid plants
    0.981**1
    0.000
    3636
    注:**表示在P<0.01水平(双侧)上相关性极显著;n=36。Notes: ** represents extremely significant correlation at P<0.01 level (bilateral);n=36.
    下载: 导出CSV
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