笋用竹的组织培养研究

The Research on Bamboo Shoots Tissue Culture

作者: 专业:林木遗传育种 导师:陈建华 年度:2009 学位:硕士  院校: 中南林业科技大学

Keywords

Bamboo Shoots, Tissue culture, Browing

        本研究选取笋用竹中的红哺鸡竹和白哺鸡竹为研究材料,分别选择对成年红哺鸡竹和白哺鸡竹的节芽、地下茎和嫩枝条的嫩茎段三种外植体进行组织培养。对基本培养基、激素组合、培养条件以及防褐变措施进行了研究,确立了笋用竹节芽萌芽、愈伤组织形成和增殖培养的最适培养基,找到了减轻褐变的有效方法。主要研究结果如下:(1)外植体的消毒处理:采集3月初地下茎和5月健康的嫩枝条,洗涤剂溶液浸泡15~30min左右,半胱氨酸浸泡30min,流动的清水冲洗30~60min。然后在超净工作台上用75%酒精和0.1%的生汞分别消毒30~45s和6~9min,消毒效果最好,诱导率高,污染率低,可有效控制污染的发生(2)培养基:MS培养基适合笋用竹的节芽诱导和愈伤组织的诱导,以及继代培养。均附加蔗糖25g/L,琼脂7g/L,pH值5.8。(3)以节芽诱导萌芽:以笋用竹红哺鸡竹和白哺鸡竹的饱满的节芽作为外植体,红哺鸡竹节芽诱导的最佳培养基为MS+TDZ0.1mg/L+BA5mg/L,白哺鸡竹节芽诱导的最佳培养基为MS+TDZ0.01mg/L+BA5 mg/L(4)选取笋用竹红哺鸡竹和白哺鸡竹的地下茎和嫩枝条的嫩茎段为外植体诱导愈伤组织:红哺鸡竹地下茎的最佳培养基是MS+2,4-D2.0mg/L+KT1.0 mg/L,红哺鸡竹嫩枝条嫩茎段的最佳培养基是MS+2,4-D3.0mg/L+KT1.0mg/L。白哺鸡竹地下茎的最佳培养基是MS+2,4-D3.0mg/L+KT1.0mg/L,白哺鸡竹嫩枝条嫩茎段的最佳培养基是MS+2,4-D2.0mg/L+KT1.0 mg/L(5)不定芽的增殖:以初代培养获得的红哺鸡竹和白哺鸡竹无菌芽诱导丛生芽,BA和NAA影响笋用竹增殖的主要因子。红哺鸡竹和白哺鸡竹继代培养的最佳培养MS+NAA1mg/L+BA5mg/L(6)愈伤组织的增殖:在愈伤组织增殖试验过程中,愈伤组织生长比较的慢,并且褐变现象越来越严重,大多数的愈伤组织在继代第二次之后,逐渐发黑直至死亡。只有极少数的存活,但增殖率非常的低,也没有出现分化的现象。(7)防褐变措施:外植体在半胱氨酸溶液(100 mg/L)中浸泡30min,培养基中添加一定量的聚乙烯吡哆烷酮和10mg/L AgNO3,以及在20℃的温度下进行暗培养,能有效抑制外植体的褐变。
    The study selected bamboo shoots in Phyllostachys iridenscens C. Y. Yaoet S. Y chen and Phyllostachys dulcis Mc.Clure for the study. Three different kind of organs were used as explants, i.e., nodal bud, undergound stem and stem from shoot. Various basic culture media, combinations of hormones, culture condition and Measures to prevent browning were tested. It is recealed that the optium basic medium of bamboo shoots’nodal bud induction, callus induction and multiplication. During subculture and redifferentiation, the callus growth ability is the most important. Found out an effective way to reduce the browning.The main results are shown as follows:(1) Explant sterilization:Underground stems collected from Marth. Stem which growing this year collected from May. These materials were immersed in ordinary detergent for 15-30min, and were then immersed in cysteine 30min. Afer that, washed with flowing water for 30~60min. On the super clean benchm, the explant was dipped into 70% alcohol for 30~45s, and then treated with 0.1% HgCl2 for 6~9min. This procedure proved very good in raising the callus induction percentage and decreasing the pollution and browning.(2) Culture medium:MS culture medium was fit for callus induction and bud induction of Phyllostachys iridenscens C. Y. Yaoet S. Y chen and Phyllostachys dulcis Mc.Clure, and sucrose 25g/L, ager7.5 g/L, pH5.8. so as the multiplication.(3) Bud bud inducement budding:Buds were used as explant. Results showed that the best medium for Phyllostachys iridenscens C. Y. Yaoet S. Y chen was MS added with 0.1mg/L of TDZ, and 5mg/L of BA. The best medium for Phyllostachys dulcis Mc.Clure was MS added with 0.01mg/L of TDZ, and 5mg/L of BA.(4) Callus induction:Stems of underground and shoot were used as explant. Results showed that the best medium for Phyllostachys iridenscens C. Y. Yaoet S. Y chen’ underground stem was MS added with 2.0mg/L of 2,4-D, and 1.0mg/L of KT. The best medium for Phyllostachys iridenscens C. Y. Yaoet S. Y chen’ shoot stem was MS added with 3.0mg/L of 2,4-D, and 1.0mg/L of KT. The best medium for Phyllostachys dulcis Mc.Clure’ underground stem was MS added with 3.0mg/L of 2,4-D, and 1.0mg/L of KT. the best medium for Phyllostachys dulcis Mc.Clure’ shoot stem was MS added with 2.0mg/L of 2,4-D, and 1.0mg/L of KT(5) Cluster bud multipication:The bud cluster was multiplicated rapidly on the MS medium with 1.0mg/L of NAA, and 5.0mg/L of BA. NAA and BA were the main factor about bamboo shoots multiplication.(6) Callus multiplication:During the callus multiplication experiment. Callus were growing slowly and browning became more and more worse. Most callus dead after the multiplication of second time. There were no differentiation.(7) Measures to prevent browning:Explant were then immersed in cysteine 30min, added PVP and 10mg/L of AgNO3, the temperture controled at 20℃and darking were inhibit browning.
        

笋用竹的组织培养研究

摘要4-6
ABSTRACT6-7
缩写词表8-12
1 绪论12-26
    1.1 植物组织培养概述12-16
        1.1.1 植物组织培养发展简史12-13
        1.1.2 植物组织培养的应用及发展方向13-16
    1.2 笋用竹的研究进展16-20
        1.2.1 笋用竹的形态特征16-17
        1.2.2 笋用竹的生态习性17-18
        1.2.3 笋用竹的类型18-19
        1.2.4 笋用竹的世界分布概况19-20
        1.2.5 中国笋用竹品种资源及分布概况20
    1.3 笋用竹的繁殖方式20-21
        1.3.1 种子繁殖20
        1.3.2 分蔸移栽20
        1.3.3 截秆20-21
        1.3.4 扦插21
        1.3.5 移鞭21
        1.3.6 空中诱根21
        1.3.7 组织培养21
    1.4 竹子组织培养研究进展21-26
        1.4.1 国内外研究现状21-23
        1.4.2 国内外竹子组织培养技术研究23-26
2 研究的目的意义、技术路线、研究内容和方法26-28
    2.1 研究的目的和意义26
    2.2 技术路线26
    2.3 主要研究内容26-27
    2.4 研究方法27-28
3 材料与方法28-36
    3.1 实验材料28
        3.1.1 实验植物材料28
        3.1.2 实验植物材料预处理28
        3.1.3 基本培养基28
        3.1.4 培养条件28
    3.2 实验方法28-36
        3.2.1 外植体的消毒和选择28-29
        3.2.2 初代培养29-32
        3.2.3 继代培养32-34
        3.2.4 几种防褐化措施对外植体褐变的影响34-36
4 结果与分析36-62
    4.1 外植体的消毒和选择36-39
        4.1.1 不同消毒方式对材料消毒效果的影响36-38
        4.1.2 不同采样时间对笋用竹组织培养的影响38-39
    4.2 初代培养39-52
        4.2.1 茎段节芽萌芽的诱导39-43
        4.2.2 以地下茎和嫩枝条的嫩茎段诱导愈伤组织43-52
    4.3 继代培养52-59
        4.3.1 不定芽的增殖培养52-56
        4.3.2 愈伤组织的增殖培养56-59
    4.4 几种防褐化措施对外植体褐变的影响59-62
        4.4.1 不同预处理对组织培养褐变的影响59-60
        4.4.2 不同种类不同浓度抗氧化剂对组织培养褐变的影响60
        4.4.3 不同浓度AgNO_3对组织培养褐变的影响60-61
        4.4.4 不同光照对组织培养褐变的影响61
        4.4.5 不同温度对组织培养褐变的影响61-62
5 结论与讨论62-66
    5.1 外植体的消毒与选择62
    5.2 初代培养62
        5.2.1 以节芽诱导萌芽62
        5.2.2 以地下茎和嫩枝条的嫩茎段诱导愈伤组织62
    5.3 继代培养62-63
        5.3.1 不定芽的增殖62-63
        5.3.2 愈伤组织的增殖63
    5.4 防褐变措施63-64
        5.4.1 预处理对组织培养褐变的影响63
        5.4.2 不同种类不同浓度抗氧化剂对组织培养褐变的影响63
        5.4.3 不同浓度AgNO_3对组织培养褐变的影响63
        5.4.4 培养条件对褐变的影响63-64
    5.5 讨论64-66
        5.5.1 外植体的消毒与选择64
        5.5.2 初代培养64
        5.5.3 继代培养64-65
        5.5.4 褐变问题65-66
6 创新点66-67
参考文献67-74
附录A 培养基配方74-76
附录B 攻读学位期间的主要学术成果76-77
致谢77
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