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气动生物反应器油松胚性愈伤增殖体系的建立研究

李亦轩 赵健 付双彬 董明亮 杨硕 李珊珊 KongLisheng 张金凤

李亦轩, 赵健, 付双彬, 董明亮, 杨硕, 李珊珊, KongLisheng, 张金凤. 气动生物反应器油松胚性愈伤增殖体系的建立研究[J]. 北京林业大学学报, 2019, 41(11): 37-43. doi: 10.13332/j.1000-1522.20190221
引用本文: 李亦轩, 赵健, 付双彬, 董明亮, 杨硕, 李珊珊, KongLisheng, 张金凤. 气动生物反应器油松胚性愈伤增殖体系的建立研究[J]. 北京林业大学学报, 2019, 41(11): 37-43. doi: 10.13332/j.1000-1522.20190221
Li Yixuan, Zhao Jian, Fu Shuangbin, Dong Mingliang, Yang Shuo, Li Shanshan, Kong Lisheng, Zhang Jinfeng. Enhancement of embryogenic callus proliferation in Chinese pine (Pinus tabuliformis) by airlift bioreactor[J]. Journal of Beijing Forestry University, 2019, 41(11): 37-43. doi: 10.13332/j.1000-1522.20190221
Citation: Li Yixuan, Zhao Jian, Fu Shuangbin, Dong Mingliang, Yang Shuo, Li Shanshan, Kong Lisheng, Zhang Jinfeng. Enhancement of embryogenic callus proliferation in Chinese pine (Pinus tabuliformis) by airlift bioreactor[J]. Journal of Beijing Forestry University, 2019, 41(11): 37-43. doi: 10.13332/j.1000-1522.20190221

气动生物反应器油松胚性愈伤增殖体系的建立研究

doi: 10.13332/j.1000-1522.20190221
基金项目: 国家重点研发计划(2017YFD0600501),国家“948”项目(2014-4-59)
详细信息
    作者简介:

    李亦轩。主要研究方向:油松的体胚发生。Email:yxli0913@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科学与技术学院

    责任作者:

    赵健,博士,讲师。主要研究方向:林木体细胞胚胎发生及其分子调控机制。Email:zhaojian0703@bjfu.edu.cn 地址:同上

    张金凤,教授,博士生导师。主要研究方向:树木体胚胎发生与倍性育种。Email:zjf@bjfu.edu.cn 地址:同上

  • 中图分类号: S722.3

Enhancement of embryogenic callus proliferation in Chinese pine (Pinus tabuliformis) by airlift bioreactor

  • 摘要: 目的大量具有活性的胚性愈伤组织的增殖可为油松体细胞胚胎发生研究和植株再生提供充足的材料,锥形瓶悬浮培养体系下胚性愈伤组织的增殖率低且易酸化坏死,建立气动生物反应器油松胚性愈伤增殖体系可促进油松胚性愈伤组织的增殖。方法本研究以油松愈伤组织为材料,利用L9(34)正交试验设计,探究了生物反应器中愈伤接种量、新旧培养基的配比和激素浓度3个因素对愈伤组织增殖的影响,建立了增殖体系。而后在相同条件下与锥形瓶悬浮培养进行对比。结果结果表明:气动生物反应器中每100 mL液体培养基内接种胚性愈伤10 g,2,4-D 0.2 mg/L,保留20%的旧培养基时能使胚性愈伤增殖率达到最高,可达216.18%;结论与锥形瓶悬浮培养体系相比,ALB系统在一周内胚性愈伤生长速度更快,为锥形瓶悬浮培养体系的3.15倍;显微观察显示增殖的胚性愈伤组织稳定且优质。该研究为基于油松体胚体系的大规模扩繁提供技术支持。

     

  • 图  1  油松胚性愈伤组织

    Figure  1.  Embryogenic callus of Pinus tabuliformis

    图  2  锥形瓶悬浮培养和ALB培养的愈伤组织增殖对比

    不同的字母代表存在差异显著。Different letters indicate significant differences.

    Figure  2.  Comparison of callus proliferation in flask suspension culture and ALB culture

    图  3  锥形瓶悬浮培养和ALB培养的愈伤组织pH值对比图

    Figure  3.  Comparison of pH values of callus in flask suspension culture and ALB culture

    图  4  增殖继代年龄与增殖率的关系

    Figure  4.  Correlation between subculture time and proliferation rate

    图  5  锥形瓶悬浮培养和生物反应器培养的愈伤组织形态对比

    A. ALB培养的胚性愈伤组织;B. 锥形瓶悬浮培养的胚性愈伤组织;C. 体式显微镜下观察到的ALB培养的胚性愈伤组织结构,黄色和红色箭头分别表示胚柄和胚头;D. 体式显微镜下观察到锥形瓶悬浮培养的胚性愈伤组织结构。A, embryogenic callus cultured by ALB; B, embryogenic callus suspension culture in conical flask; C, embryogenic callus structure in ALB observed under the stereo microscope; yellow and red arrows represent the suspensor and the blastocephalon, respectively; D, embryogenic callus in suspension culture observed under the stereo microscope.

    Figure  5.  Comparison of callus morphology between flask suspension culture and bioreactor culture

    表  1  正交设计表L9(34

    Table  1.   Orthogonal design L9 (34)

    编号
    No.
    接种量
    Inoculum size/g
    2,4-D质量浓度
    2,4-D mass concentration/
    (mg·L− 1)
    旧培养基保留量
    Retention of old medium/mL
    1 5 0.1 20
    2 5 0.2 50
    3 5 0.3 80
    4 10 0.1 50
    5 10 0.2 80
    6 10 0.3 20
    7 15 0.1 80
    8 15 0.2 20
    9 15 0.3 50
    下载: 导出CSV

    表  2  接种量、激素浓度、旧培养基保留量对愈伤组织增殖量的影响

    Table  2.   Effects of inoculation quantity, hormone concentrations and the ratio of used medium on growth rate

    编号
    No.
    接种量
    Inoculum size/g
    2,4-D质量浓度
    2,4-D mass concentration/(mg·L− 1)
    旧培养基保留量
    Retention of old medium/mL
    增殖量
    Proliferation/g
    1 5 0.1 20 139.67 ± 18.21
    2 5 0.2 50 143.33 ± 14.70
    3 5 0.3 80 58.93 ± 6.52
    4 10 0.1 50 111.0 ± 10.91
    5 10 0.2 80 96.3 ± 4.03
    6 10 0.3 20 206.87 ± 8.29
    7 15 0.1 80 11.80 ± 4.75
    8 15 0.2 20 216.18 ± 5.00
    9 15 0.3 50 169.56 ± 7.16
    k1 113.98 87.49 187.57
    k2 138.06 151.94 141.30
    k3 132.51 145.12 55.68
    R 24.08 64.45 131.89
    最优组合 Optimal combination 10 0.2 20
    注:表中数据为平均值 ± 标准差。Note: the data in the table are mean ± standard deviation.
    下载: 导出CSV

    表  3  ALB试验方差分析

    Table  3.   Analysis of variance of ALB test

    变异来源 Source of variation自由度 df均方 MSFF value显著性 Sig.
    接种量 Inoculum size 2 1 427.125 153.380 0.042*
    旧培养基保留量 Retention of old medium 2 318.063 34.184 0.031*
    2,4-D质量浓度 2,4-D mass concentration 2 553.212 59.456 0.027*
    误差 Error 20 9.304
    总变异 Total variation 26
    注:*表示在P < 0.05水平差异显著。Note: * represents significant difference at P < 0.05 level.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2019-05-10
  • 修回日期:  2019-06-03
  • 网络出版日期:  2019-11-02
  • 刊出日期:  2019-11-01

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