• 中国中文核心期刊
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Volume 33 Issue 2
Apr.  2020
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Study on the Factors Affecting Adventitious Shoots Proliferation and in vitro Regeneration of Quercus virginiana

  • Corresponding author: WANG Shu-feng, wangshufeng6609@163.com
  • Received Date: 2019-05-08
    Accepted Date: 2019-12-16
  • Objective To study the factors that affect the adventitious buds development and rooting process, so as to develop an efficient in vitro propagation methodological system in live oak (Quercus virginiana). Method Using stem segments as explants, the effects of basal medium, browning control, the origin of explant, and different hormone combinations on cluster buds proliferation and rooting were studied. Result The results showed that the addition of ascorbic acid, sodium thiosulfate, and powdered activated carbon could effectively reduce the browning range of explants compared with the control (p<0.05), among which 3.00 g·L−1 and 5.00 g·L−1 powdered activated carbon were proved to be the most effective. The growth medium screening test showed that more and longer shoots were achieved on the low salt medium with a quarter of Murashige and Skoog (MS) medium or woody plant medium (WPM). And the browning was also alleviated with the low salt medium in this study. The highest proliferation rate (6.6 buds per explant) was achieved on a quarter of MS medium supplemented with 1.20 mg·L−1 6-benzylaminopurine (BA) after 40 days. In vitro regenerated shoots were rooted on a quarter of MS medium supplemented with 0.50 mg·L−1 indole-3-butyric acid (IBA) and 0.50 mg·L−1naphthalene acetic acid (NAA) with the highest rooting rate 53.33% and more lignified roots. The plantlet was transferred to sterile sand after 3-5 days acclimation in greenhouse and 57.78% survival rate was observed finally. Conclusion Based on the repeated experiments during the past three years, it is confirmed that the basal medium composition and the proportion of hormone combination will be the major factors determining the proliferation of adventitious shoots and in vitro regeneration of Q. virginiana. The basal mediums that contain lower concentration of inorganic salt are proved to be favorable not only for the adventitious shoots proliferation, but also for alleviating the browning of explants. It is concluded that the explants from the field are much easier to become brown than that from sterile seedlings.
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Study on the Factors Affecting Adventitious Shoots Proliferation and in vitro Regeneration of Quercus virginiana

    Corresponding author: WANG Shu-feng, wangshufeng6609@163.com
  • Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Key Laboratory of Tree Breeding of Zhejiang Province, Hangzhou 311400, Zhejiang, China

Abstract:  Objective To study the factors that affect the adventitious buds development and rooting process, so as to develop an efficient in vitro propagation methodological system in live oak (Quercus virginiana). Method Using stem segments as explants, the effects of basal medium, browning control, the origin of explant, and different hormone combinations on cluster buds proliferation and rooting were studied. Result The results showed that the addition of ascorbic acid, sodium thiosulfate, and powdered activated carbon could effectively reduce the browning range of explants compared with the control (p<0.05), among which 3.00 g·L−1 and 5.00 g·L−1 powdered activated carbon were proved to be the most effective. The growth medium screening test showed that more and longer shoots were achieved on the low salt medium with a quarter of Murashige and Skoog (MS) medium or woody plant medium (WPM). And the browning was also alleviated with the low salt medium in this study. The highest proliferation rate (6.6 buds per explant) was achieved on a quarter of MS medium supplemented with 1.20 mg·L−1 6-benzylaminopurine (BA) after 40 days. In vitro regenerated shoots were rooted on a quarter of MS medium supplemented with 0.50 mg·L−1 indole-3-butyric acid (IBA) and 0.50 mg·L−1naphthalene acetic acid (NAA) with the highest rooting rate 53.33% and more lignified roots. The plantlet was transferred to sterile sand after 3-5 days acclimation in greenhouse and 57.78% survival rate was observed finally. Conclusion Based on the repeated experiments during the past three years, it is confirmed that the basal medium composition and the proportion of hormone combination will be the major factors determining the proliferation of adventitious shoots and in vitro regeneration of Q. virginiana. The basal mediums that contain lower concentration of inorganic salt are proved to be favorable not only for the adventitious shoots proliferation, but also for alleviating the browning of explants. It is concluded that the explants from the field are much easier to become brown than that from sterile seedlings.

  • 弗吉尼亚栎(Quercus virginiana Mill)原产美国,为壳斗科(Fagaceae)栎属(Quercus L.)常绿高大乔木,分布于美国东南沿海。弗吉尼亚栎是沿海森林植被群落中的优势树种,其根系深而发达,具有很强的抗风能力,能抵御飓风和暴雨的袭击,耐短期水涝;还耐干旱、高浓度盐雾和土壤盐分,病虫害较少,是难得的沿海防护林优良树种[1-2]。又因其四季常青,树冠开阔,也可作为良好的行道遮阴树种和园林绿化树种。

    弗吉尼亚栎于2001年首次引入我国,在浙江富阳、上虞、慈溪、海宁和上海、江苏等地海涂试种,都表现出良好的适应性[3-4]。目前,国内弗吉尼亚栎苗木大多依靠种子繁殖,由于种子变异大[5],实生苗在生长速度、株形、叶形等方面具有很大差异,导致苗木质量参差不齐,严重制约生产推广。同时,弗吉尼亚栎种子没有休眠期,只要条件合适即可萌发,不耐储存,且存在象甲及动物取食的影响,种子产量极不稳定。无性繁殖对控制弗吉尼亚栎苗木质量及苗木快繁具有重要意义。研究报道,弗吉尼亚栎通过扦插繁殖,可达70%~80%的生根率[6-7],但依然存在生根率不稳定、难以规模化生产等问题。因此,有必要进一步探索、完善弗吉尼亚栎无性繁殖技术,尤其组织培养研究。

    栎类树种组织培养研究始于20世纪80年代末,随着组织培养技术的发展,通过体细胞胚发生和芽再生的方法已实现多种栎树植株再生,培养成功的栎树已近30个种[8],包括欧洲夏栎(Q. robur L.)[9-10]、欧洲栓皮栎(Q. suber L.)[11-12]、冬青栎(Q. ilex L.)[13]、北美红栎(Q. rubra L.)[14]、麻栎(Q. acutissima Carruth.)[15]以及高山栎(Q. semecarpifolia Sm.)[8]等。近年来,国内在大叶栎(Castanopsis fissa (Champ. ex Benth.) Rehd. et Wils.) [16]、辽东栎(Q. liaotungensis Koidz.)[17]、蒙古栎(Q. mongolica Fisch. ex Ledeb)[18]等栎树组织培养上也取得一些突破。弗吉尼亚栎引种以来,国内针对育苗技术、繁殖技术以及对生态因子的响应等方面展开了大量研究[19-22]。培育弗吉尼亚栎无性系苗是当前该树种生产过程中的一大难题,虽然已有试验以扦插繁殖进行无性系选育[6-7],但至今没有应用到生产实践中。本研究以弗吉尼亚栎茎段为外植体,通过不定芽增殖途径,探索弗吉尼亚栎组织培养过程中外植体防褐化措施、不同种类激素及激素配比对不定芽增殖和生根的影响,以期建立弗吉尼亚栎试管苗再生体系,为弗吉尼亚栎无性系选育提供技术支撑。

    • 弗吉尼亚栎种子或带芽茎段。种子来源于浙江上虞弗吉尼亚栎母树林,该母树林位于绍兴市上虞区沥海镇,营建于2004年,造林种源来自美国路易斯安那州。带芽茎段取自中国林科院亚热带林业研究所实验大棚2~4年生地栽苗,取材一般在每年3—5月份,剪取未木质化新萌枝条备用。

    • 种子或茎段经自来水冲洗24 h后,再以75%的酒精消毒30 s(若是大田采取的外植体,加入1~2滴吐温−80),在无菌操作台以升汞浸泡3~10 min(其中幼嫩叶片或顶芽需3 min,种子需8 min),然后以无菌水冲洗3~5次,置于无菌玻璃瓶备用。

    • 选取无虫眼、颗粒饱满的弗吉尼亚栎种子,在自来水中浸泡24 h,弃去漂浮的种子,然后以饱和漂白粉上清液浸泡30 min进行表面消毒,消毒后的一部分种子剥去种皮,于超净工作台内,按1.2.1程序进行消毒,消毒后的种子接种于琼脂培养基,置于(25±2)℃培养室内进行暗培养。

    • 采用3种防褐化试剂[23-24]:抗坏血酸、硫代硫酸钠和活性炭,质量浓度分别为:抗坏血酸2.50、5.00 g·L−1,硫代硫酸钠1.00、2.00 g·L−1,活性炭1.00、3.00、5.00 g·L−1,分别添加于增殖培养基中,对照培养基则不添加任何防褐化试剂。

    • 2012—2017年间,以MS、1/4MS和WPM为基本培养基,添加不同质量浓度的6-苄氨基嘌呤(6-BA)、萘乙酸(NAA)和吲哚乙酸(IAA)进行不定芽增殖试验,6-BA质量浓度为1.00、1.20、1.50 mg·L−1,NAA为0.00、0.10、0.20 mg·L−1,IAA为0.00、0.30、0.50 mg·L−1,设置细胞分裂素和生长激素不同浓度配比,以只添加细胞分裂素(6-BA)的培养基作为对照,激素浓度设置参考文献[9-18]有关浓度配比及项目组前期的大量试验。在无菌条件下,将灭菌的嫩枝剪成1 cm左右带芽茎段,每种处理接种50个茎段,3次重复,分别在接种15、30 d后记录茎段生长、增殖情况和不定芽健康状态等,进行综合评价。培养条件为:温度25±2℃,光照3 000 lx,光暗周期14 h/10 h,培养室相对湿度70%左右。

    • 以1/4MS为基本培养基,添加不同质量浓度的吲哚丁酸(IBA)和NAA进行生根试验,IBA质量浓度为0.50、0.80、1.00 mg·L−1,NAA为0.50、1.00、1.50 mg·L−1,将壮苗培养中生长一致的苗接种于不同培养基,每个处理接种15株,3次重复,接种20 d后统计生根情况及根系状态。

    • 试管苗生根后,先将生根的培养瓶瓶盖打开,置于驯化室中进行驯化,3~5 d后取出小苗,洗净根部,移植于装有灭菌河沙的无纺布容器袋,置于大棚内培养,期间严格控制水分,保持沙子表面湿润。30 d后统计幼苗成活率。

    2.   结果与分析
    • 表1可以看出:3.00、5.00 g·L−1活性炭处理对防止弗吉尼亚栎外植体褐化效果最好,但5.00 g·L−1的活性炭中芽生长的启动时间延长,因此,在培养基中添加3.00 g·L−1的活性炭可有效防止褐化。另外,发现多次反复接种也可以有效防止褐化。外植体第一次接种后,培养3 d,转接到相同培养基,如此转接3次,褐化基本不会发生。

      处理
      Treatments
      质量浓度Mass
      concentration/
      (g·L−1)
      芽启动时间
      Initial time
      for buds/d
      目测褐化发生
      半径Browning
      diameter/cm
      对照CK0.0014.25±0.96 d1.83±0.24 a
      抗坏血酸
      Ascorbic acid
      2.5016.75±0.96 bc1.15±0.24 b
      5.0016.00±0.82 bcd0.58±0.10 c
      硫代硫酸钠
      Sodium thiosulfate
      1.0017.50±1.29 b1.08±0.10 b
      2.0016.00±0.82 bcd0.58±0.10 c
      活性炭
      Activated carbon
      1.0015.25±0.50 cd0.65±0.13 c
      3.0016.25±0.50 bcd0.20±0.14 d
      5.0020.00±0.82 a0.13±0.10 d
        注:表中数据为平均值±标准差,不同小写字母表示不同处理间差异显著(p<0.05)。下同。
        Note: 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.  Effects of anti-browning agents with different concentration on browning of explants and the initial time for buds

    • 试验发现:在添加不同质量浓度激素的MS、1/4MS和WPM培养基中,弗吉尼亚栎茎段均能生长,但在MS培养基中,外植体褐化较严重,在低盐的1/4MS和WPM培养基中,褐化较轻(表2)。

      基本培养基
      Medium
      平均芽长
      Average bud
      length/cm
      平均芽数
      Average bud number /
      (个·外植体−1
      褐化状况
      Browning
      MS2.79±0.16 c3.10±0.99 b+++
      1/4MS3.49±0.27 b4.30±0.82 a+
      WPM4.17±0.31 a4.20±0.79 a+

      Table 2.  Growth difference of stem segments of Q. virginiana on different primary media

      表3可以看出:在供试的33种培养基中,5号、20号、21号培养基对弗吉尼亚栎丛芽发生具有促进作用,特别是5号配方,茎段在该培养基中丛芽发生最多,增殖倍数达6.60,且丛芽生长状态良好。21号培养基虽然增殖倍数也较高,但增殖的芽纤弱,且部分叶片没有展开。

      编号
      Number
      基本培养基
      Basal medium
      质量浓度 Mass concentration/(mg·L−1)增殖倍数
      Proliferation rate
      生长状况
      Growth description
      6-BANAAIAA
      1MS1.00000.16±0.05 k茎段维持绿色,基部褐变Green stem, but basal browning
      21.20000.64±0.05 k茎段维持绿色,基部褐变,部分腋芽增殖
      Green stem, but basal browning, partially proliferation of axillary buds
      31.50000.34±0.07 k茎段维持绿色,基部褐变,部分腋芽增殖
      Green stem, but basal browning, partially proliferation of axillary buds
      41/4MS1.00000.30±0.10 k茎段绿色,无明显变化Green stem, no obvious change
      51.20006.60±1.39 a茎段绿色,丛芽较多,芽健壮Green stem, more multiple shoots, strong shoots
      61.50000.22±0.11 k茎段绿色,无明显变化,部分腋芽增殖
      Green stem, partially proliferation of axillary buds
      71.000.1000.28±0.07 k茎段绿色,腋芽伸长Green stem, elongation in axillary buds
      81.200.1003.75±0.46 cd基部膨大,有丛芽发生,芽健壮
      Basal expanding, multiple shoots occur, strong shoots
      91.500.1000.76±0.28 jk茎段绿色,腋芽生长不明显Green stem, no obvious growth of axillary buds
      101.000.2002.27±1.00 fgh基部膨大,部分有丛芽发生,芽健壮Basal expanding, multiple shoots occur, strong shoots
      111.200.2001.70±0.55 hij基部膨大,部分有丛芽发生Basal expanding, multiple shoots occur partially
      121.500.2004.18±0.90 bc基部膨大,有丛芽发生Basal expanding, multiple shoots occur
      131.0000.302.60±0.54 efgh茎段维持绿色,丛芽少Green stem, less multiple shoots
      141.2000.301.74±1.08 hij茎段维持绿色,丛芽少Green stem, less multiple shoots
      151.5000.303.34±0.58 cde茎段有生长,有丛芽发生Growth in stem, multiple shoots occur
      161.0000.501.96±0.33 ghi有丛芽发生,但叶片卷曲Multiple shoots occur, curl leaves
      171.2000.503.16±0.70 def丛芽较多,叶片纤细More shoots, long and thin leaves
      181.5000.502.91±0.34 defg丛芽较多,叶片纤细More shoots, long and thin leaves
      19WPM1.00003.42±1.78 cde基部膨大,丛芽较少Basal expanding, less multiple shoots
      201.20005.03±0.18 b丛芽较多,叶片展开More shoots, expanding leaves
      211.50006.10±0.77 a丛芽增殖多,但芽纤细More shoots proliferation, long and thin shoots
      221.000.1000.42±0.10 k丛芽较少,基部有愈伤Less shoots, basal callus
      231.200.1002.95±0.28 defg丛芽较少,基部有愈伤组织发生Less multiple shoots, basal callus
      241.500.1002.23±0.19 fgh丛芽发生,较少Less multiple shoots
      251.000.2001.82±0.34 hi部分有丛芽,但基部愈伤组织较多Multiple shoots occur, more basal callus
      261.200.2001.69±0.34 hij部分有丛芽,但基部愈伤组织较多Multiple shoots occur, more basal callus
      271.500.2004.17±0.17 bc丛芽较多,但叶未展开More multiple shoots, non-expanding leaves
      281.0000.302.85±0.44 defg有丛芽,芽细长Multiple shoots occur, long and thin shoots
      291.2000.300.36±0.14 k丛芽少,芽细长Multiple shoots occur, long and thin shoots
      301.5000.301.06±0.13 ijk丛芽少,芽细长,叶片未展开
      Multiple shoots occur, long and thin shoots, nonexpandingleaves
      311.0000.504.15±0.36 bc丛芽较多,叶片纤细More shoots, long and thin leaves
      321.2000.503.61±0.54 cd丛芽较多,芽健壮More shoots, strong shoots
      331.5000.502.53±0.61 efgh丛芽较多,芽较健壮,但基部有愈伤More shoots, strong shoots, but basal callus occur

      Table 3.  Effects of different types and proportion of hormones combination on the development and growth of cluster buds of Q. virginana stem segments

    • 表4可以看出:生根培养20 d 时,单独添加IBA或NAA均导致弗吉尼亚栎丛芽基部愈伤化,虽然部分丛芽也有根的发生,但生根率极低,最高也仅3.33%。在本试验中,生根率最高的激素配比是0.50 mg·L−1IBA+0.50 mg·L−1NAA,该培养基中丛芽的生根率可达53.33%,且每株芽的平均根数达到6条,同时在该培养基中发生的根系状态也与其它不同,该培养基中的根较粗壮,且木质化程度较高。另外,发现在IBA质量浓度不变情况下,提高NAA质量浓度不利于弗吉尼亚栎的生根。

      编号
      Number
      基本培养基
      Basal
      medium
      质量浓度Mass
      concentration/
      (mg·L−1)
      生根率
      Rooting rate/%
      平均根数
      Average root
      number/(条
      ·株 −1)
      平均根长
      Average root
      length/cm
      生长状况
      Growth description
      IBANAA
      11/4 MS0.500.67 d0.67 g0.57 cde基部愈伤化,偶有根发生Basal callus, occasional roots
      20.801.33 d1.33 ef0.37 de基部愈伤化,偶有根发生Basal callus, occasional roots
      31.001.33 d0.67 g0.37基部愈伤化,偶有根发生Basal callus, occasional roots
      40.503.33 d1.00 fg0.57 cd基部愈伤化,偶有根发生Basal callus, occasional roots
      51.000.00 d0.00 g0.00 e基部愈伤化,无根发生Basal callus, no root
      61.501.33 d1.00 fg0.10 e基部愈伤化,偶有根发生Basal callus, occasional roots
      70.500.5053.33 a6.00 a2.53 a基部愈伤不明显,根系健壮,木质化程度高,有根毛
      No obvious callus in the base, strong roots, high lignification,
      root hair occurring
      80.501.0038.00 b4.00 bc1.67 ab基部有愈伤,根细长Basal callus and fine roots
      90.501.5040.67 ab2.33 def1.40 bcd基部愈伤少,根细长,轻微木质化,根毛少
      Less callus, less lignification, fine roots, less root hair
      101.000.5028.67 bc4.33 b1.63 ab根部愈伤组织少,根细长Less callus, fine roots
      111.001.008.67 d3.67 bcd0.70 bcd基部有愈伤,根粗短,无根毛
      Basal callus, short and thick roots, no root hair
      121.001.5022.00 c2.67 cde1.43 bc基部愈伤组织少,根细长Less callus, fine roots
        注:表中数据为2016年5月、7月和10月共3次试验生根情况的平均值。
        Note: Data were presented as means for three repeated experiments conducted in May, July, and October, 2016.

      Table 4.  Effects of different proportions of hormone combination on root development of cluster buds in Q. virginiana

    • 对形成木质化根的弗吉尼亚栎试管苗进行炼苗移植。首先将培养容器盖打开,放在培养室中进行3~5 d炼苗,然后将试管苗取出,以灭菌河沙为介质进行移植,放于大棚中培养。2016—2018年,共计移苗61株,平均成活率57.78%,其中,2017年5月移植的试管苗成活率达83.33%以上。通过对试管苗移植的观察,其成活率与叶片的数量和大小有关,叶片较多的试管苗更易成活,推测可能是由于叶片数量多,使光合产物增多,从而提高了试管苗的活力。

      弗吉尼亚栎试管苗移植的成功说明通过不定芽增殖途径获得试管苗的技术体系是可行的,因此,基于组织培养中外植体防褐化措施、不定芽增殖和丛芽生根等过程影响因子的选择和优化,建立弗吉尼亚栎试管苗再生体系,具体流程见图1

      Figure 1.  Different steps of tissue culture of Q. virginiana

    3.   讨论
    • 栎类树种是离体培养较难的树种之一,影响其组织培养最重要原因之一是外植体的褐化[25],褐化主要与植物组织内的酚类物质有关,木本植物由于本身含丰富木质素、单宁、色素等次生代谢物,因此较草本植物更容易褐化[26]。研究表明,外植体的褐化与基因型、外植体取材部位、取材季节以及外植体本身的年龄等有关,一般而言,年龄越大,木质化程度越高的外植体越容易褐化,幼龄组织褐化相对较轻[23]。然而,位于形态学最上端的顶端分生组织,由于细胞活跃,各种酶活性较高,作为外植体进行组织培养时,也很容易褐化。刘光金等 [27]对3年生大叶栎不同部位茎段进行多酚含量测定发现,半木质化枝条茎段和叶子的多酚类物质含量高于新生萌芽条的茎段与叶子,而同一枝条形态学上端多酚类物质含量高于形态学下端。于艳等[24]研究发现,采用麻栎枝条的幼嫩茎段和半木质化的茎段作为外植体,褐化率较低,萌发率较高;而采用带生长点的茎尖作为外植体,褐化率最高。本研究也发现,从成年弗吉尼亚栎植株上取材的外植体在培养过程中褐化较严重,而无菌苗取材的外植体基本未发现褐化现象。吕秀立等[28]在研究舒玛栎(Q. shumardii Buckl.)组织培养中也发现,从自然条件下生长的枝条取材,无论是顶芽还是腋芽茎段,外植体褐化非常严重,均难以正常的生长。

      组织培养中的防褐化措施,主要从培养基的选择、pH值、硬度、外植体的选择和处理以及吸附剂、抗氧化剂和激素的添加等方面抑制[23],其中,向培养基中添加抗氧化剂和活性炭是最常用的措施,且也是最有效的措施。刘光金等[27]发现,培养基中添加活性炭、抗坏血酸可以有效减轻大叶栎外植体的褐化。于艳等[24]发现,对麻栎茎段褐化抑制效果最好的是1.0 mg·L−1PVP。在本研究中,针对野外取材的外植体,采用活性炭和添加抗氧化剂的方法对褐化进行控制试验,发现活性炭和抗氧化剂对弗吉尼亚栎外植体的褐化均有一定的抑制作用,但总的来说,活性炭的效果要优于抗氧化剂。作者发现,弗吉尼亚栎外植体切口处氧化速度非常快,因此,在接种过程需要尽快将外植体接种到培养基中,而活性炭对已经产生的酚类物质具有很好的吸附作用,推测这可能是活性炭对弗吉尼亚栎外植体更有效的原因。抗氧化剂虽然能够抑制外植体进一步的氧化,但对已经产生的酚类物质无法去除,因此,导致接种前产生的酚类物质在培养基中扩散。

      6-BA在促进栎树不定芽增殖方面的效果已在众多组织培养中得以证明,但不同栎树不定芽增殖阶段对6-BA的浓度反应均有不同。在欧洲夏栎组织培养研究中,Chalupa [9,29]发现,0.88~3.50 μmol·L−1 6-BA在诱导不定芽增殖方面的效果要优于6-糠氨基嘌呤(KT)。Puddephat等[10]认为,0.44 μmol·L−1 6-BA(约0.10 mg·L−1)对夏栎腋芽的诱导、伸长以及数量增殖均有良好的促进效果,而较高浓度6-BA(4.40 μmol·L−1)则会导致玻璃化、基部愈伤化以及叶片的变形扭曲,进而影响芽的伸长生长。在北美红栎组织培养过程中,Vengadesan等[14]发现,4.40 μmol·L−1(约1.00 mg·L−1)6-BA可以促进不定芽的增殖。蒙古栎在含有0.20 mg·L−1 6-BA的培养基中不定芽诱导效果最佳[18],而麻栎不定芽的诱导则需要1.00 mg·L−1 6-BA[30]。本研究发现,1.20 mg·L−1 6-BA对弗吉尼亚栎不定芽的诱导效果最佳,增值倍数可达6.6。因此,作者推测在栎类树种不定芽诱导过程中,6-BA浓度在0.44~4.40 μmol·L−1范围进行试验设计,可以有效缩短试验探索周期。在不定芽诱导和增殖方面,除6-BA单独使用以外,6-BA常与GA、TDZ以及NAA组合使用,也能获得良好的增殖系数[14]。考虑到成本问题,作者在设计弗吉尼亚栎不定芽增殖试验中使用6-BA和NAA组合,但发现NAA在0.50~1.50 mg·L−1条件下,与不同浓度6-BA搭配,增值倍数最高也仅4.18,均不及单独使用6-BA的效果,推测可能是NAA浓度过高,抑制了不定芽的增殖[31]。由于本试验并未考虑6-BA与GA和TDZ的组合,因此,为提高弗吉尼亚栎不定芽增殖系数,今后的研究中应加以补充,以寻找最佳的激素组合。

      试管苗的生根诱导是组织培养是否成功的关键步骤,在栎树生根诱导过程中,激素配比及其施用方法对根系的发生至关重要,其次,培养基组分、光暗周期等均可影响试管苗的生根。研究发现,在栎树试管苗生根诱导过程中,IBA的效果优于NAA[11],但也有采用IBA与NAA组合使用获得良好的生根效果,Chalupa采用IBA(0.30 mg·L−1)和NAA(0.10 mg·L−1)用于夏栎(Q. robur L.)的诱导生根[9]。研究发现,IBA的浓度和施用方法均可影响根系的诱导,大多数研究是在含有激素的培养基中进行连续培养,IBA浓度最低为2.50 μmol·L−1,可用于舒玛栎的生根培养[32];Manzanera等[11]采用5.00 mg·L−1IBA连续培养7~14 d,获得栓皮栎(Q. suber L.)的试管苗生根。当采用高浓度IBA时,一般采用短期浸泡方式诱导生根,Purohit等采用100.00 μmol·L−1 IBA 对栎树不定芽基部进行24 h浸泡,Q. glauca Thunb、Q. leucotrichophora L.和Q. floribunda Lindl. 试管内生根[33-35]。本研究在IBA 0.50 mg·L−1和NAA 0.50 mg·L−1条件下,获得良好的生根率(53.33%),其次为IBA 0.50 mg·L−1和NAA 1.50 mg·L−1,生根率40.67%,但NAA比例提高时,基部愈伤化明显,根系不发达;而在IBA:NAA为1时,获得的根系粗壮,木质化程度高,移苗成活率也高。除此以外,研究也表明,低盐基本培养基有利于栎树试管苗的生根[29,36],不定芽诱导过程中采用WPM和1/4MS两种低盐培养基,发现1/4MS对不定芽的诱导效果要优于WPM,因此,在生根诱导过程中采用1/4MS培养基,也获得良好的生根诱导效果。

    4.   结论
    • 研究表明,野外取材的弗吉尼亚栎外植体易褐化,但可通过添加3.00 g·L−1或5.00 g·L−1的活性碳解决;无论是室内无菌苗来源的外植体,还是野外取材,均可通过不定芽增殖途径获得大量的丛芽,进而获得生根的试管苗。由于弗吉尼亚栎喜砂质土壤,因此,试管苗的移植开始宜采用灭菌河沙,在河沙中成活以后,可移植至含有营养基质的土壤。

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