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秋水仙碱处理诱导银灰杨2n花粉与杂种三倍体创制

周晴 吴剑 桑亚茹 赵征洋 刘美芹 张平冬

周晴, 吴剑, 桑亚茹, 赵征洋, 刘美芹, 张平冬. 秋水仙碱处理诱导银灰杨2n花粉与杂种三倍体创制[J]. 北京林业大学学报, 2020, 42(3): 119-126. doi: 10.12171/j.1000-1522.20190363
引用本文: 周晴, 吴剑, 桑亚茹, 赵征洋, 刘美芹, 张平冬. 秋水仙碱处理诱导银灰杨2n花粉与杂种三倍体创制[J]. 北京林业大学学报, 2020, 42(3): 119-126. doi: 10.12171/j.1000-1522.20190363
Zhou Qing, Wu Jian, Sang Yaru, Zhao Zhengyang, Liu Meiqin, Zhang Pingdong. Pollen chromosome doubling induced by colchicine treatment and creation of hybrid triploids in Populus canescens[J]. Journal of Beijing Forestry University, 2020, 42(3): 119-126. doi: 10.12171/j.1000-1522.20190363
Citation: Zhou Qing, Wu Jian, Sang Yaru, Zhao Zhengyang, Liu Meiqin, Zhang Pingdong. Pollen chromosome doubling induced by colchicine treatment and creation of hybrid triploids in Populus canescens[J]. Journal of Beijing Forestry University, 2020, 42(3): 119-126. doi: 10.12171/j.1000-1522.20190363

秋水仙碱处理诱导银灰杨2n花粉与杂种三倍体创制

doi: 10.12171/j.1000-1522.20190363
基金项目: 国家自然科学基金项目(31570646)
详细信息
    作者简介:

    周晴。主要研究方向:林木细胞遗传与细胞工程。Email:876034493@qq.com 地址:100083北京市海淀区学院路清华东路35号

    责任作者:

    张平冬,副教授,博士生导师。主要研究方向:林木遗传改良。Email:zhangpd@bjfu.edu.cn 地址:同上

  • 中图分类号: S722.3+5;S792.11

Pollen chromosome doubling induced by colchicine treatment and creation of hybrid triploids in Populus canescens

  • 摘要: 目的为创制银灰杨杂种三倍体新种质,研究完善银灰杨三倍体育种技术体系,筛选生长迅速、抗逆性强的银灰杨新品种。方法本研究以银灰杨、河北杨以及银腺杨为试验材料,在优化秋水仙碱诱导银灰杨花粉染色体加倍技术条件的基础上,开展了秋水仙碱注射处理对2n花粉粒表面结构的影响以及杂交创制杂种三倍体的研究。结果减数分裂时期、注射次数、减数分裂时期与注射次数的交互效应均对2n花粉诱导率具有极显著影响。利用0.5%的秋水仙碱溶液对发育至粗线期的花粉母细胞进行11次注射处理是秋水仙碱诱导银灰杨花粉染色体加倍的最佳处理组合,2n花粉平均诱导率最高可达(30.27 ± 8.69)%。与天然2n花粉粒相比,秋水仙碱诱导型2n花粉粒表面结构出现了明显的裂沟,说明秋水仙碱注射处理对2n花粉花粉壁形成具有一定的影响。选取含有一定2n花粉比例的花粉,分别与河北杨、银腺杨雌配子进行授粉杂交,共收获杂种苗木4 955株,检测出三倍体5株,其中,2株三倍体来源于河北杨 × 银灰杨的杂交组合;另3株三倍体来源于银腺杨 × 银灰杨的杂交组合。结论秋水仙碱能高效诱导银灰杨花粉染色体加倍并获得2n花粉,但是注射会影响2n花粉花粉壁的形成,使得2n花粉粒表面结构产生裂沟。秋水仙碱诱导的2n花粉可以用于授粉杂交,成功获得杂种多倍体植株。

     

  • 图  1  银灰杨天然2n花粉(a)与秋水仙碱诱导型2n花粉(b)

    a. 对照组中的天然2n花粉(箭头);b. 秋水仙碱诱导2n型花粉(箭头)。标尺 = 20 μm。a, natural 2n pollen in control group (arrow); b, colchicine-induced 2n pollen in treatments (arrow). Bar = 20 μm.

    Figure  1.  Natural 2n pollen (a) and colchicine-induced 2n pollen (b) in Populus canescens

    图  2  不同处理组合的银灰杨2n花粉诱导率

    Figure  2.  Induction rates of colchicine-induced 2n pollen in P. canescens under different treatments

    图  3  银灰杨单倍性花粉粒与2n花粉粒表面结构的电镜观察

    a. 对照组花粉粒形态;b. 粗线期注射5次花粉粒形态;c. 粗线期注射11次花粉粒形态;d. 对照组单倍性花粉粒表面结构;e. 粗线期注射5次花粉粒表面结构;f. 粗线期注射11次花粉粒表面结构;g. 对照组单倍性花粉粒表面沉积;h. 粗线期注射5次花粉粒表面沉积;i. 粗线期注射11次花粉粒表面沉积;j. 天然2n花粉粒表面结构;k. 粗线期注射5次2n花粉粒表面结构;l. 粗线期注射11次2n花粉粒表面结构;m. 天然2n花粉粒表面沉积;n. 粗线期注射5次2n花粉粒表面沉积;o. 粗线期注射11次2n花粉粒表面沉积。标尺 = 100 µm(a ~ c),10 µm(d ~ f),20 µm(j ~ l),5 µm(g ~ i;m ~ o)。a, morphology of pollen grains in control group; b, morphology of pollen grains derived from the treatment with 5 injections at pachytene; c, morphology of pollen grains derived from the treatment with 11 injections at pachytene; d, ectexine structure of haploid pollen grains in control group; e, ectexine structure of haploid pollen grains derived from the treatment with 5 injections at pachytene; f, ectexine structure of haploid pollen grains derived from the treatment with 11 injections at pachytene; g, ectexine deposition of haploid pollen grains in control group; h, ectexine deposition of haploid pollen grains derived from the treatment with 5 injections at pachytene; i, ectexine deposition of haploid pollen grains derived from the treatment with 11 injections at pachytene; j, ectexine structure of natural 2n pollen grains; k, ectexine structure of 2n pollen grains derived from the treatment with 5 injections at pachytene; l, ectexine structure of natural 2n pollen grains derived from the treatment with 11 injections at pachytene; m, ectexine deposition of natural 2n pollen grains; n, ectexine deposition of 2n pollen grains derived from the treatment with 5 injections at pachytene; o, ectexine deposition of 2n pollen grains derived from the treatment with 11 injections at pachytene; scale bar = 100 µm (a−c), 10 µm (d−f), 20 µm (j−l), 5 µm (g−i; m−o).

    Figure  3.  Scanning electron micrographs of haploid pollen grains and 2n pollen grains in P. canescens

    图  4  秋水仙碱诱导银灰杨花粉染色体加倍子代倍性分析

    a. 二倍体与三倍体混样流式分析;b. 三倍体流式分析;c. 二倍体染色体数目;d. 三倍体染色体数目。a, flow cytometric analysis of the mixed sample of diploid plant and triploid plant; b, flow cytometric analysis of the triploid plant; c, chromosome number of the diploid (2n = 2x = 38); d, chromosome number of the tripliod (2n = 3x = 57).

    Figure  4.  Ploidy analysis of the offsprings derived from pollen chromosome doubling induced by colchicine

    表  1  不同处理组合银灰杨2n花粉诱导率的方差分析

    Table  1.   Variance analysis on induction rates of colchicine-induced 2n pollen in P. canescens under different treatments

    变异来源 Source of variationdfMSFP
    减数分裂时期 Meiotic stage4251.6964.801**0.009
    注射次数 Injection time4652.65012.449**0.000
    减数分裂时期 × 注射次数 Meiotic stage × injection time1652.4273.006**0.002
    误差 Error5017.444
    注:** 代表差异极显著(P < 0.01)。Note: ** represents highly significant differences (P < 0.01).
    下载: 导出CSV

    表  2  不同减数分裂时期和不同注射次数银灰杨2n花粉诱导率的多重比较

    Table  2.   Multiple comparisons in induction rates of 2n pollen under different meiotic stages and different injection times in P. canescens

    减数分裂时期
    Meiotic stage
    平均2n花粉诱导率
    Average induction rate of 2n pollen/%
    注射次数
    Injection time
    平均2n花粉诱导率
    Average induction rate of 2n pollen/%
    细线期 Leptotene7.57 ± 2.51b34.83 ± 1.72d
    粗线期 Pachytene13.54 ± 10.30a56.03 ± 1.70cd
    双线期 Diplotene7.84 ± 2.921b77.94 ± 2.28bc
    终变期 Diakinesis10.77 ± 5.23ab911.18 ± 3.51b
    中期Ⅰ MetaphaseⅠ7.42 ± 5.39b1117.17 ± 8.94a
    注:不同小写字母代表差异显著(P < 0.05)。Note: different lowercase letters represent significant differences (P < 0.05).
    下载: 导出CSV

    表  3  银灰杨秋水仙碱诱导型2n花粉与河北杨、银腺杨雌配子杂交创制三倍体

    Table  3.   Crossing colchicine-induced 2n pollen in P. canescens with female gametes in P. hopeiensis and P. alba × P. glandulosa to produce triploids

    序号
    No.
    母本
    Female parent
    2n花粉比率
    Frequency of 2n pollen/%
    种子数
    Number of seeds
    成苗数
    Number of seedlings
    三倍体数
    Number of triploids
    三倍体得率
    Triploid production rate/%
    1河北杨
    P. hopeiensis
    30.27386116.67
    220.36339111.11
    316.70642100
    415.11311600
    511.03522000
    610.5327400
    7银腺杨
    P. alba × P. glandulosa
    9.0591261020.33
    8 8.761 02786410.12
    9 6.2270456300
    10 5.7997562300
    11 5.461 21796400
    12 4.3469455200
    对照 Control 2.0896770300
    总计 Total6 7414 9555
    下载: 导出CSV
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出版历程
  • 收稿日期:  2019-09-19
  • 修回日期:  2019-11-03
  • 网络出版日期:  2020-03-05
  • 刊出日期:  2020-03-31

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