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茶壶枣离体多倍体诱导关键技术研究

郭烨 崔艳红 孔德仓 曹明 庞晓明 李颖岳

郭烨, 崔艳红, 孔德仓, 曹明, 庞晓明, 李颖岳. 茶壶枣离体多倍体诱导关键技术研究[J]. 北京林业大学学报, 2019, 41(7): 49-56. doi: 10.13332/j.1000-1522.20190230
引用本文: 郭烨, 崔艳红, 孔德仓, 曹明, 庞晓明, 李颖岳. 茶壶枣离体多倍体诱导关键技术研究[J]. 北京林业大学学报, 2019, 41(7): 49-56. doi: 10.13332/j.1000-1522.20190230
Guo Ye, Cui Yanhong, Kong Decang, Cao Ming, Pang Xiaoming, Li Yingyue. Study on key techniques of polyploid induction in Ziziphus jujuba Mill. cv. ‘Teapot’[J]. Journal of Beijing Forestry University, 2019, 41(7): 49-56. doi: 10.13332/j.1000-1522.20190230
Citation: Guo Ye, Cui Yanhong, Kong Decang, Cao Ming, Pang Xiaoming, Li Yingyue. Study on key techniques of polyploid induction in Ziziphus jujuba Mill. cv. ‘Teapot’[J]. Journal of Beijing Forestry University, 2019, 41(7): 49-56. doi: 10.13332/j.1000-1522.20190230

茶壶枣离体多倍体诱导关键技术研究

doi: 10.13332/j.1000-1522.20190230
基金项目: 国家重点研发计划项目(2018YFD1000607)
详细信息
    作者简介:

    郭烨。主要研究方向:经济林木良种繁育。Email:guoye1630@163.com 地址:100083 北京市海淀区清华东路35号北京林业大学生物学院

    责任作者:

    李颖岳,博士,教授。主要研究方向:经济林木良种繁育。Email:yingyueli@bjfu.edu.cn 地址:同上

  • 中图分类号: S722.3

Study on key techniques of polyploid induction in Ziziphus jujuba Mill. cv. ‘Teapot’

  • 摘要: 目的离体多倍体育种是枣树育种的重要方法,对其关键技术的研究有利于建立和完善枣树离体多倍体育种体系,加快枣树多倍体种质的创制。方法以茶壶枣无菌叶片为材料,将预培养不同天数(7 ~ 20 d)的叶片固定后制成石蜡切片,进行细胞学观察以确定适宜的预培养时间。在此基础上,将叶片不定芽再生技术与秋水仙素染色体加倍技术相结合,在叶片预培养8、9、10、11 d后,分别用70和90 mg/L的秋水仙素处理48和72 h,并对处理后得到的不定芽进行流式细胞术倍性检测。结果通过对叶片最佳预培养时间的细胞学研究可以得出,茶壶枣无菌叶片在预培养8 ~ 9 d时,分生细胞的分裂能力最强,且未出现分化现象,在此时期用秋水仙素对叶片进行处理,可大大提高诱导率,并且可以有效避免嵌合体和混倍体的出现。染色体加倍试验发现,在预培养8 d后,用70 mg/L的秋水仙素处理72 h,其诱导率最高,达4.11%,并成功获得8株多倍体植株。通过流式细胞术确定这8株多倍体均为纯合四倍体。结论本研究探索出了将茶壶枣叶片不定芽再生体系与秋水仙素染色体加倍技术相结合,人工诱导茶壶枣多倍体的技术方法。

     

  • 图  1  茶壶枣离体多倍体诱导过程中不同预培养时间的叶片

    a. 预培养7 d;b. 预培养9 d;c. 预培养11 d;d. 预培养13 d;e. 预培养15 d;f. 预培养20 d。a, 7 days pre-culture time; b, 9 days pre-culture time; c, 11 days pre-culture time; d, 13 days pre-culture; e, 15 days pre-culture; f, 20 days pre-culture.

    Figure  1.  Leaves of different pre-cultivation time during in vitro polyploid induction of ‘Teapot’ jujube

    图  2  茶壶枣叶片不同预培养时间的组织学观察

    a. 预培养7 d;b、c. 预培养8、9 d;d、e、f. 预培养10、11、12 d;g、h. 预培养13、14 d;i. 预培养20 d。a, 7 days pre-culture time; b, c, 8, 9 days pre-culture time, respectively; d, e, f, 10, 11, 12 days pre-culture time; g, h, 13, 14 days pre-culture time; i, 20 days pre-culture time.

    Figure  2.  Histological observation on different pre-culture time of ‘Teapot’ jujube leaves

    图  3  秋水仙素浓度及处理时间对外植体存活率的影响

    Figure  3.  Effects of colchicine concentration and colchicine treatment time on the explant survival rate

    图  4  秋水仙素浓度及处理时间对不定芽再生数的影响

    Figure  4.  Effects of colchicine concentration and colchicine treatment time on the number of adventitious bud regeneration

    图  5  流式细胞仪倍性分析图

    Figure  5.  Analysis diagram of ploidy by FCM

    表  1  秋水仙素处理茶壶枣组培苗叶片诱导多倍体处理设计表

    Table  1.   Design table of ‘Teapot’ jujube leaves with colchicine treatments

    处理
    Treatment
    预培养时间
    Pre-culture
    time/d
    秋水仙素浓度
    Colchicine concentration/
    (mg·L− 1)
    秋水仙素处理时间
    Colchicine treatment
    time/h
    1 8 70 48
    2 8 70 72
    3 8 90 48
    4 8 90 72
    5 9 70 48
    6 9 70 72
    7 9 90 48
    8 9 90 72
    9 10 70 48
    10 10 70 72
    11 10 90 48
    12 10 90 72
    13 11 70 48
    14 11 70 72
    15 11 90 48
    16 11 90 72
    下载: 导出CSV

    表  2  秋水仙素处理对茶壶枣叶片存活率、再生芽数以及四倍体诱导率的影响

    Table  2.   Effects of colchicine treatment on leaf survival rate, number of regenerated buds and tetraploid induction rate in ‘Teapot’ jujube

    处理
    Treatment
    外植体存活率
    Explant survival rate/%
    再生芽数
    Regenerated
    buds
    四倍体诱导率
    Tetraploid induction rate/%
    1 83.33 ± 1.25a 8.40 ± 0.41df 0
    2 69.33 ± 1.46ab 5.60 ± 0.48bcd 4.11
    3 62.33 ± 1.79bc 6.30 ± 0.31df 0
    4 52.00 ± 2.14cde 5.10 ± 0.53a 0
    5 80.60 ± 2.31a 7.98 ± 0.21df 0
    6 62.67 ± 3.21bc 6.21 ± 0.55cdf 1.37
    7 56.67 ± 7.27bcde 6.10 ± 0.54cdf 0
    8 40.67 ± 6.26e 5.40 ± 0.32a 0
    9 70.67 ± 3.73ab 8.40 ± 0.31f 0
    10 59 ± 11.88cdef 6.50 ± 0.81df 0
    11 55.33 ± 4.945bcd 6.30 ± 0.67df 0
    12 45.00 ± 0.91de 5.50 ± 0.62ab 0
    13 79.40 ± 1.57a 7.88 ± 0.29df 0
    14 65.33 ± 7.83bcd 6.45 ± 0.24df 0
    15 60.33 ± 5.51bcd 6.70 ± 0.33df 0
    16 45.23 ± 2.61de 5.70 ± 0.3abc 0
    注:不同的小写字母表示差异显著,P < 0.05。下同。Notes: different lowercase letters indicate a significant difference at P < 0.05 level. The same below.
    下载: 导出CSV

    表  3  秋水仙素处理茶壶枣叶片外植体存活率的方差分析

    Table  3.   Variance analysis of explant survival rate on colchicines treatment of ‘Teapot’ jujube

    变异来源 Source of variation自由度 Degree of freedom (df)均方 Mean square (MS)F
    预处理时间 Pre-culture time 3 102.702 2.498
    秋水仙素浓度 Colchicine concentration 1 2 238.692 54.443**
    秋水仙素处理时间 Colchicine treatment time 1 1 279.600 31.119**
    随机误差 Random error 31 41.120
    总计 Total 36
    注:**表示达到极显著差异,P < 0.01。下同。Notes: ** indicates extremely significant difference at P < 0.01 level. The same below.
    下载: 导出CSV

    表  4  秋水仙素处理茶壶枣叶片外植体再生芽数的方差分析

    Table  4.   Variance analysis of explant survival rate on colchicine treatment of ‘Teapot’ jujube

    变异来源 Source of variation自由度 Degree of freedom (df)均方 Mean square (MS)F
    预处理时期 Pre-culture time 3 0.318 0.527
    秋水仙素浓度 Colchicine concentration 1 20.358 20.358**
    秋水仙素处理时间 Colchicine treatment time 1 24.225 40.111**
    随机误差 Random error 32 0.645
    总计 Total 37
    下载: 导出CSV
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出版历程
  • 收稿日期:  2019-05-18
  • 修回日期:  2019-06-04
  • 网络出版日期:  2019-07-03
  • 刊出日期:  2019-07-01

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