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杉木体细胞胚胎发生胚性愈伤组织诱导条件的优化

吴夏雷 韩超 孙宇涵 曹森 胡瑞阳 徐金良 郑会全 李云

吴夏雷, 韩超, 孙宇涵, 曹森, 胡瑞阳, 徐金良, 郑会全, 李云. 杉木体细胞胚胎发生胚性愈伤组织诱导条件的优化[J]. 北京林业大学学报, 2020, 42(2): 79-86. doi: 10.12171/j.1000-1522.20190196
引用本文: 吴夏雷, 韩超, 孙宇涵, 曹森, 胡瑞阳, 徐金良, 郑会全, 李云. 杉木体细胞胚胎发生胚性愈伤组织诱导条件的优化[J]. 北京林业大学学报, 2020, 42(2): 79-86. doi: 10.12171/j.1000-1522.20190196
Wu Xialei, Han Chao, Sun Yuhan, Cao Sen, Hu Ruiyang, Xu Jinliang, Zheng Huiquan, Li Yun. Optimization of induction conditions for embryogenic callus of somatic embryogenesis in Cunninghamia lanceolata[J]. Journal of Beijing Forestry University, 2020, 42(2): 79-86. doi: 10.12171/j.1000-1522.20190196
Citation: Wu Xialei, Han Chao, Sun Yuhan, Cao Sen, Hu Ruiyang, Xu Jinliang, Zheng Huiquan, Li Yun. Optimization of induction conditions for embryogenic callus of somatic embryogenesis in Cunninghamia lanceolata[J]. Journal of Beijing Forestry University, 2020, 42(2): 79-86. doi: 10.12171/j.1000-1522.20190196

杉木体细胞胚胎发生胚性愈伤组织诱导条件的优化

doi: 10.12171/j.1000-1522.20190196
基金项目: 中央高校基本科研业务费专项资金资助(BJFUKF201918),中央高校基本科研业务费专项资金资助(2015ZCQ-SW-03),广东省省级科技计划项目(2016B020201002),北京林业大学科学技术研究项目(2018WS01)
详细信息
    作者简介:

    吴夏雷。主要研究方向:经济林木良种繁育。Email:wuxialei1993@163.com 地址:100083 北京市海淀区清华东路35号

    责任作者:

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

  • 中图分类号: S722.3

Optimization of induction conditions for embryogenic callus of somatic embryogenesis in Cunninghamia lanceolata

  • 摘要: 目的优化杉木体细胞胚胎发生过程中胚性愈伤组织的诱导条件,探究基本培养基、供体母株基因型和外源添加剂对杉木体胚诱导的影响。方法以3个供体母株基因型(Z1、Z2、Z3)的杉木雌配子体(其未成熟合子胚处于裂生多胚期)为外植体,采用6种基本培养基及不同浓度的外源添加剂塞苯隆(TDZ)和茉莉酸甲酯(MeJA)处理,并对诱导培养过程中的愈伤组织进行细胞学观察。结果以DCR为基本培养基,诱导培养效果最好,愈伤组织诱导率达70.74%,胚性愈伤组织的诱导率达17.36%;供体母株基因型对胚性愈伤组织诱导有较大影响,Z1基因型供体母株的雌配子体最适合用于诱导产生胚性愈伤组织,诱导率为14.73%;诱导培养时以DCR为基本培养基,添加蔗糖30 g/L、活性炭1 g/L、倍力凝5 g/L并附加植物生长调节剂2,4-D 1.5 mg/L、KT 0.4 mg/L、MeJA 1.2 μmol/L和TDZ 0.004 mg/L,杉木胚性愈伤组织的诱导率最高,达19.83%;仅需4 d就可诱导产生胚性愈伤组织,不同阶段原胚团的结构和极性特征有明显差异。结论基本培养基、供体母株基因型和外源添加剂都会明显影响杉木体胚诱导培养,以Z1供体母株基因型的杉木雌配子体为外植体、DCR为培养基并组合添加MeJA和TDZ可明显提高杉木体胚诱导率。

     

  • 图  1  诱导4 d时雌配子体体式镜及显微镜观察

    a. 以DCR为基本培养基,添加MeJA 1.2 μmol/L + TDZ 0.004 mg/L诱导培养4 d时,杉木雌配子体诱导产生的胚性愈伤组织形态结构,标尺 = 1 mm;b. 显微观察下处于原胚团I时期(PEM I)的胚性愈伤组织细胞团结构,标尺 = 500 μm。a, the morphological structure of embryogenic callus induced by female gametophyte of Cunninghamia lanceolata was induced by DCR as the basic medium and MeJA 1.2 μmol/L+ TDZ 0.004 mg/L for 4 days, bar = 1 mm; b, microscopically observed embryogenic callus cell mass structure in the proembryogenic masses I (PEM I), bar = 500 μm.

    Figure  1.  Stereoscopic and microscopic observation of female gametophyte at 4 days induction

    图  2  诱导28 d时雌配子体体式镜及显微镜观察

    a. 以DCR为基本培养基,添加MeJA 1.2 μmol/L + TDZ 0.004 mg/L诱导培养第28 d时,杉木雌配子体诱导产生的胚性愈伤组织形态结构,标尺 = 2 mm;b. 显微观察下处于原胚团II和原胚团III时期(PEM II和PEM III)的胚性愈伤组织细胞团结构,标尺 = 500 μm。a, the morphological structure of embryogenic callus induced by female gametophyte of Cunninghamia lanceolata was induced by DCR as the basic medium and MeJA 1.2 μmol/L + TDZ 0.004 mg/L. bar = 2 mm; b, microscopic observation of embryogenic callus cell cluster structure in proembryogenic masses II and proembryogenic masses III (PEM II and PEM III), bar = 500 μm.

    Figure  2.  Stereoscopic and microscopic observation of female gametophyte at 28 days induction

    图  3  LM培养基下杉木雌配子体的体胚诱导培养

    a. 以LM为基本培养基培养得到的非胚性愈伤组织形态结构,标尺 = 2 mm;b. 显微观察下非胚性愈伤组织细胞团结构,标尺 = 500 μm;c. 中心开始褐化的胚性愈伤组织形态结构,标尺 = 5 mm;d. 中心褐化状态下胚性愈伤组织细胞团显微结构,标尺 = 500 μm;e. 完全褐化条件下的愈伤组织形态结构,标尺 = 1 mm;f.完全褐化条件下愈伤组织细胞团显微结构,标尺 = 500 μm。a, non-embryonic callus morphological structure obtained by culturing LM as the basic medium, bar = 2 mm; b, microscopic observation of non-embryonic callus cell cluster structure, bar = 500 μm; c, the morphological structure of the embryogenic callus in which the center begins to brown, bar = 5 mm; d, microscopic structure of embryogenic callus cell clusters in a central browning state, bar = 500 μm; e, callus morphological structure under complete browning conditions, bar = 1 mm; f, microstructure of callus cell clusters under complete browning conditions, bar = 500 μm.

    Figure  3.  Somatic embryo induction culture of female gametophyte of Cunninghamia lanceolata under LM medium

    表  1  不同基本培养基下的杉木胚性愈伤组织诱导率

    Table  1.   Induction rate of embryogenic callus of Cunninghamia lanceolata under different basic media

    培养基类型Medium type 接种个数Inoculation number 愈伤组织诱导率
    Callus induction
    rate /%
    胚性愈伤组织诱导率Embryogenic callus induction rate /%
    MS 430 46.99 b 9.76 c
    DCR 428 70.74 a 17.36 a
    LM 432 29.79 c 6.09 d
    BM 430 72.72 a 16.14 ab
    SH 427 34.73 c 6.94 d
    GD 428 49.46 b 12.02 b
    注:同列不同小写字母表示差异显著(P < 0.05)。下同。Notes: different lowercase letters in the same column indicate significant differences (P < 0.05). Same as below.
    下载: 导出CSV

    表  2  不同供体母株基因型的胚性愈伤组织诱导率

    Table  2.   Induction rates of embryogenic callus of different maternal genotypes

    供体母株
    基因型
    Maternal genotype
    接种个数
    Inoculation number
    愈伤组织诱导率Callus induction rate/% 胚性愈伤组织诱导率Embryogenic callus induction rate/%
    Z1 576 62.87 a 14.73 a
    Z2 570 57.43 c 13.34 b
    Z3 571 59.14 b 13.39 b
    下载: 导出CSV

    表  3  MeJA和TDZ对杉木愈伤组织形成的影响

    Table  3.   Effects of MeJA and TDZ on callus formation of Cunninghamia lanceolata

    培养基编号
    Medium No.
    外源添加剂 Exogenous additive 首次产生天数
    First generation days
    愈伤组织诱导率
    Callus induction rate /%
    胚性愈伤组织诱导率
    Embryogenic callus induction rate /%
    茉莉酸甲酯 MeJA/(μmol·L− 1) 塞苯隆TDZ/(mg·L− 1)
    PM1 0 0 5 61.67 ± 1.03 d 12.33 ± 0.74 c
    PM2 0.4 0 6 63.33 ± 0.50 cd 12.33 ± 0.57 c
    PM3 0.4 0.002 5 64.17 ± 1.07 cd 13.17 ± 0.63 bc
    PM4 0.4 0.004 4 67.50 ± 0.82 bcd 16.50 ± 0.47 ab
    PM5 0.8 0 5 67.50 ± 0.67 bcd 13.17 ± 0.63 bc
    PM6 0.8 0.002 5 73.33 ± 0.63 ab 14.00 ± 0.47 b
    PM7 0.8 0.004 5 71.67 ± 0.68 abc 17.33 ± 0.92 ab
    PM8 1.2 0 5 74.16 ± 1.20 ab 13.17 ± 0.63 bc
    PM9 1.2 0.002 5 79.17 ± 0.83 a 14.83 ± 0.50 b
    PM10 1.2 0.004 4 75.83 ± 0.57 ab 19.83 ± 0.74 a
    下载: 导出CSV
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  • 收稿日期:  2019-04-26
  • 修回日期:  2019-08-29
  • 网络出版日期:  2019-12-02
  • 刊出日期:  2020-03-03

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