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Volume 36 Issue 4
Aug.  2023
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Establishment of Tissue Culture System of the Ancient Trees of Platycladus orientalis

  • Corresponding author: ZHAO Xiu-lian, Zhaoxl@caf.ac.cn
  • Received Date: 2022-11-11
    Accepted Date: 2023-01-18
  • Objective This study aims to establish a tissue culture system for ancient Platycladus orientalis for overcoming the difficulties such as non-thoroughness of disinfection and sterilization, sever browning, and weak meristematic ability. Method In this study, the new shoots of about 3 000-year-old P. orientalis were used as explants, and aseptic explants were obtained through disinfection and sterilization. The tissue culture system for ancient P. orientalis was established by selecting the appropriate culture medium and hormone. Result Disinfecting the explants from ancient P. orientalis using 0.3% HgCl2 for 10 min showed the best sterilization effect, with a pollution rate of 25.81% and the survival rate of 54.82%. The effect of using 3% sucrose as the sugar source for the tissue culture medium of ancient P. orientalis was better than that of 3% glucose. The results of antioxidant and adsorbent tests demonstrated that the browning rate of explants was the lowest under the addition of 3.00 g·L−1 active carbon to the explant initiation medium. The optimal primary culture medium for tissue culture of ancient P. orientalis was 1/2MS + NAA 0.2 mg·L−1 + 6-BA 0.1 mg·L−1, with the induction rate of adventitious buds of 32.05%, rapid bud growth, as well as tender green and robust buds. The optimal proliferation medium was 1/2MS + 6-BA 0.05 mg·L−1 + NAA 0.2 mg·L−1 + KT 0.1 mg·L−1, with the proliferation coefficient reaching 1.93, and rapid growth of green and robust buds. The optimal rooting culture medium was WPM + NAA 0.5 mg·L−1 + IBA 0.5 mg·L−1, which achieved a rooting rate of 9.12%, with an average number of roots per plant of 2.67, and an average root length of 1.23 cm. A total of 24 rooting seedlings were obtained. Two months later, red roots appeared in 2 plants, and the tender buds continued to grow. The tissue-cultured rooting seedlings of ancient P. orientalis were successfully obtained. Conclusion The selection of culture medium and the ratio of auxin to cytokinin are important factors for the proliferation and adventitious root formation of ancient P. orientalis. This study provides a reference for tissue culture of other Cupressaceae trees.
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Establishment of Tissue Culture System of the Ancient Trees of Platycladus orientalis

    Corresponding author: ZHAO Xiu-lian, Zhaoxl@caf.ac.cn
  • 1. State Key Laboratory of Tree Genetics and Breeding, Key Laboratory Tree Breeding and Cultivation of the National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
  • 2. Beijing Green Space Maintenance and Management Affairs Center, Beijing 102211, China
  • 3. Handan Forestry Engineering Project Center, Handan 057650, Hebei, China

Abstract:  Objective This study aims to establish a tissue culture system for ancient Platycladus orientalis for overcoming the difficulties such as non-thoroughness of disinfection and sterilization, sever browning, and weak meristematic ability. Method In this study, the new shoots of about 3 000-year-old P. orientalis were used as explants, and aseptic explants were obtained through disinfection and sterilization. The tissue culture system for ancient P. orientalis was established by selecting the appropriate culture medium and hormone. Result Disinfecting the explants from ancient P. orientalis using 0.3% HgCl2 for 10 min showed the best sterilization effect, with a pollution rate of 25.81% and the survival rate of 54.82%. The effect of using 3% sucrose as the sugar source for the tissue culture medium of ancient P. orientalis was better than that of 3% glucose. The results of antioxidant and adsorbent tests demonstrated that the browning rate of explants was the lowest under the addition of 3.00 g·L−1 active carbon to the explant initiation medium. The optimal primary culture medium for tissue culture of ancient P. orientalis was 1/2MS + NAA 0.2 mg·L−1 + 6-BA 0.1 mg·L−1, with the induction rate of adventitious buds of 32.05%, rapid bud growth, as well as tender green and robust buds. The optimal proliferation medium was 1/2MS + 6-BA 0.05 mg·L−1 + NAA 0.2 mg·L−1 + KT 0.1 mg·L−1, with the proliferation coefficient reaching 1.93, and rapid growth of green and robust buds. The optimal rooting culture medium was WPM + NAA 0.5 mg·L−1 + IBA 0.5 mg·L−1, which achieved a rooting rate of 9.12%, with an average number of roots per plant of 2.67, and an average root length of 1.23 cm. A total of 24 rooting seedlings were obtained. Two months later, red roots appeared in 2 plants, and the tender buds continued to grow. The tissue-cultured rooting seedlings of ancient P. orientalis were successfully obtained. Conclusion The selection of culture medium and the ratio of auxin to cytokinin are important factors for the proliferation and adventitious root formation of ancient P. orientalis. This study provides a reference for tissue culture of other Cupressaceae trees.

  • 侧柏(Platycladus orientalis L.)具有抗逆和长寿等优良特性[1]。陕西省黄帝陵树龄约3 000年的侧柏古树(黄帝手植柏)为中华十大手植古树之首,具有重要的文化价值[2-4],其繁殖材料极其珍贵又极度匮乏,因此,能利用较少的繁殖材料的组织培养技术对于保存侧柏古树的种质资源至关重要[5-6]。目前,国内已经建立了一些古树,如杜鹃(Rhododendron simsii Planch.)、楸树(Catalpa bungei C. A. Mey.)、西府海棠(Malus micromalus Makino)等的组织培养体系[7-9];但大部分的古树,如柿树(Diospyros kaki Thunb.)、侧柏等的组织培养繁殖困难,存在愈伤组织褐化、生根难、组织培养体系不完整等问题[10]。国外在建立古树组织培养体系方面也有一定的研究,Ewald等利用140年生欧洲落叶松(Larix decidua Mill.)嫁接复幼后的新梢为外植体进行组织培养获得了生根苗,但直接利用古树外植体进行组织培养并没有获得生根苗[11]。因此,对古树的外植体组织培养体系的研究尤为重要。

    目前,国内已经有了一些柏科树木组织培养的相关研究[12-17],如臭柏(Sabina vulgaris Ant.)、龙柏(Sabina chinensis L.)等;但组织培养过程中,外植体真菌、细菌聚集,灭菌不彻底,褐化严重等问题仍然存在,对后期外植体的正常生长造成阻碍[15]。NaClO、双氧水、75%乙醇、HgCl2等是常用的消毒和灭菌剂,如用75%酒精和HgCl2配合使用对叉子圆柏(Juniperus sabina L.)的外植体灭菌效果较好[15-16],适合的灭菌剂和灭菌时间是组织培养需要解决的首要问题。在防止褐化方面,大果柏木( Cupressus macrocarpa Hartw.) 培养基中加入聚乙烯基吡咯烷酮[18]、黄扁柏(Chamaecyparis nootkatensis Spach.)培养基中加入抗坏血酸能有效防止褐化[19];但是酸刺柏(Juniperus oxycedrus L.)的培养基中加入活性炭后生根率降低[20],因此,对柏科树木组织培养抗褐化配方还需要进一步研究。

    柏科古树随着年龄的增长,细胞逐渐衰老,枝条次生代谢物质多、营养物质(可溶性糖、可溶性蛋白)和生长相关激素等含量降低,从而导致枝条分裂能力降低[5-6]。如酸刺柏以糖原为4%蔗糖的培养基上生根率较高[21]。激素种类如:IAA、IBA和KT等显著影响柏科树木外植体的分化和生长[15],NAA和BA的配合使用能诱导更多的腓尼基桧(Juniperus phoenicea L.)丛生芽[22],ZT能较好的诱导日本香柏(Thuja occidentalis L.)茎的丛生芽[23]。因此,筛选合适的糖源和激素在组织培养过程中很关键。

    许多植物在组织培养中能获得增殖的幼苗却很难获得生根苗,生根培养也是柏科植物组织培养的瓶颈[8],侧柏古树筛选的生根培养基主要有MS和WPM为基本培养基[5]。绒柏(Chamaecyparis pisifera cv. Squarros)和山达脂柏(Tetraclinis articulata (Vahl.) Masters)的生根培养基分别为SH、MS[13, 24]。前人对侧柏古树的组织培养过程中只获得新芽的增殖配方,却没有获得生根苗。本研究以侧柏古树新梢为外植体,通过改变外植体消毒处理方式、抗褐化剂、不同碳源和植物生长调节剂的种类及浓度配比,探索培养黄帝手植柏无菌苗的适宜条件,旨在为保存侧柏古树的优良种质资源提供参考依据。

    • 外植体选择生长季采自陕西黄帝陵的树龄约3 000年侧柏古树(黄帝手植柏)生长健壮、无病虫害的新梢,用装有冰块的保温箱当日带回实验室进行组织培养。每个处理设3个重复,每个重复20个外植体。

    • 首先将采回的新梢用5% NaClO表面消毒5 min,用自来水冲洗5遍;再用洗涤灵浸泡5 min,用自来水流水冲洗2 h,接着用无菌水将嫩枝在超净工作台里反复冲洗5遍;用75%的酒精浸泡30 s,再用无菌水冲洗5遍;最后使用0.3% HgCl2消毒,消毒时间分别为6、8、10、12 min,再用无菌水冲洗5遍。吸干水分,剪掉下端与消毒剂接触的部分后用于下一步实验。30 d后对污染率(Pollution rate,rp)、死亡率(Mortality rate,rm)和成活率(Survival rate,rs)进行统计。

      式中:np为污染个数,nm为死亡个数,ns 为成活个数,N为接种个数。

    • 在侧柏古树外植体初代培养基中分别添加3%葡萄糖和3%蔗糖,比较不同糖源对侧柏古树外植体褐化的影响。

    • 侧柏古树外植体的培养基中分别添加质量浓度为:0、1.00、3.00、5.00 g·L−1的活性炭、抗坏血酸和聚乙烯吡咯烷酮,对比它们抑制褐化的效果,并在接种14 d后统计侧柏古树外植体的褐化率(Browning rate,rb)。

      式中:nb 为褐化个数,N为接种个数。

    • 侧柏古树选择1/2MS为初代培养基,分别添加6-BA(浓度分别为:0.05、0.10、0.50 mg·L−1)、NAA(浓度分别为:0.05、0.10、0.20 mg·L−1)和3.00 mg·L−1的活性碳。侧柏古树新梢剪成2~3 cm的小段进行接种,增殖培养45 d后统计不定芽诱导率(Adventitious bud inductivity rate,rabi)、平均生长量及生长情况等。

      式中:nabi为不定芽个数,N为接种个数。

    • 增殖培养的目的是在短时间内得到大量适合生根的嫩芽,将初代培养基上培养的组织培养苗的萌芽切成小段进行增殖培养。1/2MS增殖培养基中添加6-BA(浓度分别为:0.02、0.05、0.10 mg·L−1)、KT(浓度分别为:0.05、0.10、0.20 mg·L−1)、NAA(浓度分别为:0.05、0.10、0.20 mg·L−1),培养45 d后分别统计增殖系数、平均生长量及生长情况等。

    • 把增殖培养的组织培养苗分别放入1/2MS、WPM、DKW基本培养基中,培养30 d后统计生根率及生根情况。选用激素NAA(浓度分别为:0、0.20、0.50、1.00 mg·L−1)和IBA(浓度分别为:0.50、1.00、1.50 mg·L−1)。培养40 d后统计生根率(Rooting rate,rr)及生根情况。

      式中:nr 为生根个数,N为接种个数。

    • 生根苗是否能适应外界环境是移植的关键。移植前3 d将生根苗瓶盖打开。移植当天用镊子将小苗取出,并洗掉根上附着的培养基,移植入基质(草炭土:蛭石:珍珠岩=1:1:1)后浇水,将生根苗置于培养箱内培养,每天观察小苗的生长状况,计算炼苗死亡率(Mortality rate of training seedlings,rmts)。

      式中:nmts 为死亡的组织培养苗数,Nts为炼苗的组织培养苗数。

    2.   结果与分析
    • 表1表明:侧柏古树组织培养过程消毒时间过长或过短对组织培养都不利,0.3%HgCl2消毒6 min处理的组织培养苗污染率最高(图1A);当处理消毒12 min时,组织培养苗污染率虽然最低,但是成活率也最低(31.22%);消毒8、10 min的组织培养苗成活率高于其他处理,成活率分别为54.82%和54.98%。因此,消毒处理10 min较适合侧柏古树组织培养。

      消毒时间
      Time of
      sterilization/min
      污染率
      rc/%
      死亡率
      rd/%
      成活率
      rs/%
      640.00 ± 10.31 a16.72 ± 2.68 c43.34 ± 5.94 ab
      825.81 ± 5.89 b19.45 ± 3.58 b54.82 ± 8.71 a
      1024.55 ± 5.26 b20.64 ± 4.19 b54.98 ± 10.87 a
      1224.21 ± 4.62 b44.88 ± 7.52 a31.22 ± 4.92 b
      注:同列不同小写字母代表处理间差异显著(P<0.05);数值为平均值 ± 标准差;下同。
        Notes: Data were presented as means ± SD. Different lowercase letters in the same column represent significant difference at 0.05 level. The same as below.

      Table 1.  Effect of different sterilizing time on sterilizing effect of ancient Platycladus orientalis

      Figure 1.  Tissue culture process of ancient P. orientalis

    • 糖源为外植体生长提供所必需的能量。表2表明:培养基的糖源选择3%葡萄糖和3%蔗糖的侧柏古树外植体的褐化率分别为78.07%和64.85%(P <0.05),结果说明不同糖源的培养基对侧柏古树外植体组织培养褐化有显著影响。

      糖源
      Sugar source
      基本培养基
      Basal medium
      NAA/
      (mg·L−1)
      IBA/
      (mg·L−1)
      褐化率
      rb/%
      3%葡萄糖1/2MS0.050.0578.07 a
      3%蔗糖1/2MS0.050.0564.85 b

      Table 2.  Sugar source screening of ancient P. orientalis

    • 侧柏中含有大量的单宁和多酚物质,使外植体在组织培养过程中容易褐化。表3表明:在培养基中添加3.00 g·L−1的活性炭能显著抑制侧柏古树外植体褐化(P <0.05),褐化率仅为13.45%(图1B);在培养基中添加浓度为5.00 g·L−1活性炭处理的新芽发生率最高,新生芽个数约为5.44。因此,适合的吸附剂能防止侧柏古树外植体组培褐化。

      处理
      Treatments
      质量浓度
      Mass concentration/(g·L−1)
      新生芽数量
      Number of new buds/个
      褐化率
      rb/%
      对照 CK0.001.72 ± 0.64 f25.72 ± 2.43 a
      活性炭(AC)
      Activated carbon
      1.003.45 ± 0.55 cd15.72 ± 3.54 d
      3.004.72 ± 0.78 b13.45 ± 3.45 de
      5.005.44 ± 1.19 a15.64 ± 4.55 d
      抗坏血酸(VC)
      Ascorbic acid
      1.003.64 ± 0.52 cd22.64 ± 6.16 b
      3.003.81 ± 0.61 c18.88 ± 4.17 c
      5.003.45 ± 0.55 cd15.72 ± 3.54 d
      聚乙烯吡咯烷酮(PVP)
      Polyvinyl pyrrolidone
      1.002.45 ± 0.57 e21.45 ± 5.47 b
      3.003.54 ± 0.64 cd16.64 ± 3.41d
      5.003.98 ± 0.52 c18.88 ± 4.75 c

      Table 3.  Effects of anti-browning agents with different concentration on browning of explants from ancient P. orientalis

    • 初代培养用于诱导侧柏古树外植体萌发新芽。表4表明:处理5的初代培养基是:1/2MS + NAA 0.2 mg·L−1 + 6-BA 0.1 mg·L−1 + 活性碳3.00 g·L−1,不定芽诱导率为32.05%,平均生长量为20.66 mm,芽生长较快、粗壮、且绿色,增殖效果显著好于其他处理(P <0.05);处理1、7、8和9的芽诱导效果较差,平均生长量显著低于处理4、5、6(P <0.05)。处理2、3、6的芽诱导效果也差,芽生长较慢。这可能是6-BA的浓度高时,造成愈伤组织生长较多消耗了较多的营养,从而导致外植体生长较弱或者死亡。

      试验号
      Test number
      NAA/
      (mg·L−1)
      6-BA/
      (mg·L−1)
      不定芽诱导率
      rabi/%
      平均生长量
      Average of growth/mm
      外植体生长情况
      The growth state of the explants
      1 0.10 0.05 13.54 ± 0.42 cd 9.37 ± 2.51 ef 芽生长慢,叶绿色,苗矮小
      Buds grew slowly, leaves are green and seedlings are short
      2 0.10 0.10 16.32 ± 0.25 c 11.55 ± 2.64 cd 芽生长较慢,较细弱,叶绿色
      Buds grew slowly, weakly, and leaves are green
      3 0.10 0.20 17.85 ± 0.12 c 10.64 ± 2.09 de 芽生长较慢,绿色,苗矮小
      Buds grew fast, green, and the seedlings are short
      4 0.20 0.05 25.67 ± 0.28 b 13.35 ± 3.18 b 芽生长快,深绿色
      Buds grew fast, dark green
      5 0.20 0.10 32.05 ± 0.41 a 20.66 ± 4.68 a 芽生长较快,粗壮,绿色
      Buds grew fast, strong and green
      6 0.20 0.20 15.32 ± 0.23 c 12.11 ± 2.71 bc 芽生长快,绿色,苗矮小
      Buds grew fast, green, seedlings are short
      7 0.50 0.05 12.54 ± 0.27 cd 10.96 ± 1.88 de 芽生长慢,较细弱,苗矮小
      Buds grew slowly, weakly and seedlings are short
      8 0.50 0.10 9.87 ± 0.13 e 8.72 ± 1.54 f 芽生长慢,较细弱,苗变黄
      Buds grew slowly, weakly and seedlings are short
      9 0.50 0.20 11.45 ± 0.06 cd 7.58 ± 2.31 g 芽生长慢,苗变黄
      Buds grew slowly, seedlings turn yellow

      Table 4.  Selection of primary culture medium for ancient P. orientalis

    • 侧柏古树愈伤组织形成是增殖培养的目的,同时培养适合生根的嫩芽。表5表明:处理5中含有较高浓度的NAA,愈伤组织生长势好,外植体的平均生长量达到18.35 mm,新长出的芽为绿色且生长速度较快(图1C);而高浓度的6-BA的增殖情况较差,形成淡黄绿色松软状愈伤组织。因此,侧柏古树的最佳增殖培养基是:1/2 MS + 6-BA 0.05 mg·L−1 + NAA 0.2 mg·L−1 + KT 0.1 mg·L−1

      试验号
      Test number
      6-BA/
      (mg·L−1)
      NAA/
      (mg·L−1)
      KT/
      (mg·L−1)
      增殖系数
      Proliferation coefficient
      平均生长量
      Average of growth/mm
      外植体生长情况
      The growth state of the explants
      1 0.02 0.05 0.10 1.45 ± 0.32 c 8.92 ± 1.98 cd 愈伤组织质地较差、疏松
      The quality of embryogenic callus was poor, loose
      2 0.02 0.10 0.05 1.19 ± 0.25 cd 10.15 ± 3.12 b 愈伤组织呈浅绿色紧密状
      Compact callus was lightyellow-green
      3 0.02 0.20 0.20 0.81 ± 0.15 d 7.68 ± 1.39 de 愈伤组织质地较差、疏松
      The quality of embryogenic callus was poor, loose
      4 0.05 0.10 0.20 1.52 ± 0.35 b 11.97 ± 2.11 b 愈伤组织诱导率高,生长好
      The inductive rates of callus tissue was highest, and
      the growth was well
      5 0.05 0.20 0.10 1.93 ± 0.42 a 18.35 ± 4.48 a 愈伤组织诱导频率高,愈伤组织生长势好
      Most efficiency for callus initiation, and the growth was well
      6 0.05 0.05 0.05 1.35 ± 0.29 cd 9.36 ± 2.76 c 愈伤组织质地较差、疏松
      The quality of embryogenic callus was poor, loose
      7 0.10 0.20 0.05 1.69 ± 0.37 b 6.47 ± 2.03 ef 形成淡黄绿色松软状愈伤组织
      Spongy callus was lightyellow-green
      8 0.10 0.05 0.20 0.61 ± 0.12 d 7.76 ± 2.38 de 愈伤组织质地较差、疏松
      The quality of embryogenic callus was poor, loose
      9 0.10 0.10 0.10 0.32 ± 0.07 e 6.14 ± 1.25 ef 愈伤组织质地较差、疏松
      The quality of embryogenic callus was poor, loose

      Table 5.  Selection of the proliferation medium for ancient P. orientalis

    • 侧柏古树组织培养生根是整个组织培养的难点,是一个复杂的生长发育过程。表6表明:1/2MS的生根率仅为2.97%,而DKW生根培养基则无不定根形成,只有WPM + IBA 0.2 mg·L−1 + NAA 0.2 mg·L−1中有少量不定根形成,生根率为8.51%,平均根长1.06 cm(图1D)。根系是从愈伤组织处形成不定根,可能是由于侧柏古树外植体衰老,外植体中含有大量次生代谢物抑制生根。

      基本培养基
      Minimal medium
      IBA/
      (mg·L−1)
      NAA/
      (mg·L−1)
      生根率
      rr/%
      平均根长
      Average root length/cm
      生根状况
      Rooting status
      1/2MS IBA 0.2 mg·L−1 + NAA 0.2 mg·L−1 2.97 ± 0.46 b 0.43 ± 0.12 b 愈伤组织多,愈伤组织分化根,白根粗且短
      More callus, the root differentiation of callus,
      thick and short roots
      WPM 8.51 ± 2.58 a 1.06 ± 0.28 a 愈伤组织少,愈伤组织分化根,根细长且壮
      Less callus, the root differentiation of callus,
      fine and strong roots
      DKW 0.00 c 0.00 c 未生根 No root

      Table 6.  Selection of the basic medium for rooting of ancient P. orientalis

    • 在筛选出的侧柏古树外植体生根基本培养基WPM中添加NAA和IBA以促进生根。表7图1E、F)表明:处理4生根过程中愈伤组织较少时容易形成不定根,生根率达9.12%,每株平均生根数为2.67,平均根长为1.23 cm,生根情况显著好于其他处理(P<0.05);处理3的生根率仅为3.26%,每株平均根数1.84,平均根长0.81 cm,处理5和处理6的生根率则分别为3.85%和4.56%,其他处理均没有生根。通过组培总共获得24株生根苗。

      试验号
      Test number
      NAA/
      (mg·L−1)
      IBA/
      (mg·L−1)
      生根率
      rr/%
      平均根数
      Average root number/
      (条·株−1)
      平均根长
      Average root length/cm
      生根状况
      Rooting status
      1 0.20 0.50 0.00 c 0.00 c 0.00 c 愈伤组织少,未生根
      Less callus, no root
      2 0.20 1.00 0.00 c 0.00 c 0.00 c 愈伤组织少,未生根
      Less callus, no root
      3 0.20 1.50 3.26 ± 0.79 b 1.84 ± 0.34 b 0.81 ± 0.21 b 愈伤组织少,愈伤组织分化根,根细长
      Less callus, the root differentiation of callus, fine roots
      4 0.50 0.50 9.12 ± 3.82 a 2.67 ± 0.58 a 1.23 ± 0.35 a 愈伤组织少,愈伤组织分化根,根细长且壮
      Less callus, the root differentiation of callus, fine and
      strong roots
      5 0.50 1.00 3.85 ± 0.94 b 1.32 ± 0.34 b 0.58 ± 0.19 b 愈伤组织多,愈伤组织分化根,白根粗且短
      Less callus, the root differentiation of callus, thick and
      short roots
      6 0.50 1.50 4.56 ± 0.84 b 1.56 ± 0.51 b 0.64 ± 0.18 b 愈伤组织多,愈伤组织分化根,白根粗且短
      More callus, the root differentiation of callus, thick and
      short roots
      7 1.00 0.50 0.00 c 0.00 c 0.00 c 愈伤组织多,未生根
      More callus, no root
      8 1.00 1.00 0.00 c 0.00 c 0.00 c 愈伤组织多,未生根
      More callus, no root
      9 1.00 1.50 0.00 c 0.00 c 0.00 c 愈伤组织多,未生根
      More callus, no root

      Table 7.  Selection of the rooting culture for the ancient P. orientalis

    • 炼苗和移植是组织培养最关键的部分。表8表明:20 d后部分植株的嫩叶长出,30 d的时候死亡率58.38%,并且老叶发黄(图1G-H)。到了50 d时85.22%的植株死亡,60 d时只有2株组织培养生根苗成活。这是因为大多数不定根是从愈伤组织处形成,后期随着愈伤组织的脱落,不定根也随之脱落。整个过程说明侧柏古树组织培养生根苗的根系较弱,生根培养基需要改良,炼苗及移植的环境条件也需要继续优化。

      种植时间
      Planting time/d
      炼苗死亡率
      rmts/%
      生长情况     
      The status of growth     
      10 6.24 ± 1.51 d 叶绿色
      Leaf color green
      20 16.75 ± 2.81 c 部分长出嫩芽
      A small part sprouts
      30 58.38 ± 12.34 b 长出的嫩芽发黄,老叶发黄
      The tender buds turn yellow, the old leaves turn yellow
      40 64.32 ± 13.58 b 嫩芽发黄,老叶干枯
      The tender buds turn yellow and the old leaves wither
      50 85.22 ± 20.31 a 嫩芽和老叶干枯
      Buds and old leaves withered
      60 95.84 ± 22.18 a 只有2株有红根出现,嫩芽继续生长
      Only two plants have red roots and buds continue to grow

      Table 8.  The status of transplanting of tissue culture seedlings of the ancient P. orientalis

    3.   讨论
    • 侧柏古树鳞形叶隐藏有大量的细菌和真菌造成灭菌困难[2-3]。本文先后用NaClO、75%的乙醇对侧柏古树消毒,再用0.3%HgCl2消毒10 min后效果较好。与徐龙光用0.1%HgCl2消毒侧柏古树外植体12 min的结果相似[2]。另外,西府海棠古树、木瓜(Carica papaya L.)古树叶片含有大量的多酚类、醌类物质从而产生褐化现象[9-10]。本研究中,在初代培养基中添加3.00 g·L−1活性炭能较好的防止侧柏古树外植体褐化,与弗吉尼亚栎(Quercus virginiana Mill.)防止外植体褐化的结果一致[25];而天女木兰(Magnolia sieboldii K. Koch)、银杏(Ginkgo biloba L.)则是添加抗坏血酸时防止褐化效果较好[26-27]。本研究糖源筛选发现,用 3%蔗糖的效果较好,与酸刺柏适用的糖源是4%蔗糖的结果相似[21],适合的糖源为组织培养顺利进行奠定基础。

      年龄效应引起的树木再生能力弱是造成侧柏古树生根难的主要原因。适宜的生长调节剂促进不定芽萌发、愈伤组织的增殖、不定根形成。6-BA具有诱导不定根与不定芽的能力[28-29],成年龙柏组织培养的6-BA 浓度在0.1~2.0 mg·L−1范围内分化率较高[14],杉木的最佳初代培养基的6-BA浓度是0.8 mg·L−1[28]。本文筛选的6-BA浓度为0.1 mg·L−1能较好的促进侧柏古树外植体萌发新芽,其效果显著优于之前的柏树组织培养研究。本研究发现,高浓度的6-BA使侧柏古树外植体形成较多的愈伤组织而消耗过多外植体的营养,适合的生长调节剂浓度是组织培养的关键。

      针叶古树含有大量的次生代谢物从而抑制细胞分裂和不定根形成[30-31]。本文将WPM为基本培养基配合NAA和IBA的浓度都为0.5 mg·L−1能使侧柏古树生根率达到9.12%,而在侧柏种胚快繁中用NAA和IBA能诱导侧柏不定根形成[32],NAA的浓度仅为 0.01 mg·L−1。龙柏基本培养基中添加浓度1.0 mg·L−1的IBA生根较好[14],叉子圆柏生根培养基中IBA的浓度为0.5 mg·L−1的生根率较高[16]。表明适合的激素配比刺激形成层分裂、促进不定根形成。

    4.   结论
    • 本文通过对侧柏古树组织培养各个阶段的筛选试验,建立了一套组织培养体系。以侧柏古树的新梢为外植体,使用0.3%HgCl2消毒10 min、培养基中添加3.00 g·L−1活性炭初步解决了外植体褐化的问题,使用筛选后的糖源、初代、增殖、生根培养基获得了侧柏古树的组织培养苗,最终建立完整的侧柏古树组织培养体系,为保存侧柏古树的优良种质资源提供了基础。

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