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植物的生长和形态具备很强的可塑性,在其生长过程中会形成新的组织,以应对季节和环境的变化[1]。次生维管束是多年生木本植物周期性活动的产物,由维管形成层不断分裂分化形成,其活性随季节更替而变化[2-3]。次生维管组织承担了树木体内物质长距离输导的功能,内含2条主要的长距离输导路径:第一条为木质部,将根系吸收的水分和养分输送到叶片,用于维持蒸腾和光合作用;第二条为韧皮部,将呼吸和生长所需的信号分子和光合产物输送到发育组织和贮藏组织[4]。
温带树木的维管组织在春季复苏,冬季休眠[5]。维管组织在不同时期的形态及生理特性不同[6]。白杨(Populus tomenosa Carr.)活跃期的维管形成层细胞壁中含有较高的甲酯化果胶和较低的酸性果胶,休眠期的维管形成层细胞壁具有较高的酸性果胶,形成层活跃期与休眠期的细胞壁结构也存在很大差异[7]。新生管胞从维管形成层产生后经体积增大,细胞壁加厚,最终发育为成熟管胞[8]。受维管形成层形态和季节的影响,不同时期形成的成熟管胞形态特征如直径、长度及细胞壁厚度等存在明显差异。管胞形态的差异直接影响了管胞的输导能力[9],如冷杉(Abies fabri(Mast.)Craib)的早材输导率约为晚材的11倍,超过90%的输导量来自于早材[10]。同时,树木韧皮部与木质部的内含物及组成成分,如韧皮部的内含多糖颗粒数量、木质部纤维素、木质素、脂类提取物和碳水化合物等,其分布与含量在不同组织和活动期也有显著差异[11]。如云杉(Picea asperata Mast.)的心材比边材含有更多的木质素和更少的纤维素,晚材的半乳糖、葡聚糖和甘露聚糖含量明显高于早材,晚材的果胶含量与亲脂性萃取物较早材更少[12]。辐射松(Pinus radiata D. Don)心材内晚材比早材含有更多的提取物,树脂酸含量也更高[13]。杉木(Cunninghamia lanceolata (Lamb.) Hook.)代谢产物在复苏前后不同,丙酮酸和抗坏血酸含量在复苏后增加[14],并且杉木的早材与晚材的果胶多糖、木聚糖、纤维素和木质素含量差异明显[15]。
马尾松(P. massoniana Lamb.)是一种喜光、不耐荫的速生树种[16],广泛分布于我国南方,是南方面积最大、储量最多的针叶树种之一[17]。目前,有关于马尾松维管组织的发育研究鲜有报道,细胞发育观察也多为马尾松产脂结构的发育研究[18-20],而马尾松次生维管束发育、细胞结构和成分变化仍有待研究。次生维管组织是树木的主体,承担了物质的远距离输导与机械支撑功能。研究维管组织有利于深入揭示树木的次生发育过程。本文通过组织化学分析和细胞离析等手段,研究马尾松纵向输导组织在不同组织的形态、结构及成分变化,旨在揭示维管组织发育过程中细胞的变化特征,为其他树种的维管组织细胞发育研究提供借鉴与参考。
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采集地位于贵州省花溪区(106°39′ N,26°26′ E),为20世纪80年代飞播造林形成的马尾松林。该区为亚热带高原季风湿润气候,温润多雨,年均气温15.3 ℃,土壤以石灰土为主,为喀斯特地貌区。在林内选取5颗胸径30 cm以上,健康的马尾松成年树,于2022年6月,用凿子于马尾松茎干1.2 ~ 1.5 m处取马尾松木质部与韧皮部材料,取样大小为2 cm × 2 cm × 1 cm,将其裁成0.5 cm × 2 cm × 1 cm后放入FAA固定保存,固定时间在3 d以上,用于韧皮部与木质部的细胞离析。用生长锥于茎干1.2 ~ 1.5 m处钻取木芯,要求韧皮部、形成层与木质部连接完整,随后放入FAA固定保存,固定时间在3 d以上,用于木质部细胞离析。
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将所采集木芯样本分2组,各5个样本,第一组将修样后长度保留0.5 ~ 1 cm,要求包含完整维管形成层及木质部;第二组修样后保留2021年与2022年完整木质部。将修样后的试验材料经软化,70%、85%、95%、100%乙醇梯度脱水,二甲苯透明,石蜡浸蜡,包埋后于石蜡切片机(Leica RM2235)切片,切片厚度12 μm,经展片,粘片后放入烘箱中40 ℃烘干备用。切片以番红固绿染色,中性树胶封片,显微镜观察,LAS X成像系统拍照。第二组样本中每个样本各选取3列包含完整2021年管胞的细胞列,以Image J统计2021年的马尾松木质部管胞的弦向壁厚度、径向壁厚度、弦向腔径(弦向细胞腔直径)、径向腔径(径向细胞腔直径)、弦向胞径(弦向细胞直径)和径向胞径(径向细胞直径)。同一样本中不同细胞列的细胞层数会存在较小差异,导致不同重复的数据量不同。为便于数据统计,以细胞层数最少的细胞列为标准,将长细胞列多余数据分别在早晚材细胞列的中值周围随机剔除,并求取均值。
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将裁好的韧皮部材料从固定液中取出,切除周皮。切片观察结果显示,不具输导功能韧皮部约与木质部相距400 ~ 500 μm,因此,在距木质部约500 μm的厚度处将韧皮部切成2块,以富兰克林离析法分别对2块材料进行离析,分别得到2个区域的筛胞。根据木质部的年轮,将木质部的早材和晚材分开,将早晚材转换期管胞刮除干净,每个样本分别切取一部分进行混合离析,得到混合样本的早材管胞和晚材管胞。显微镜观察,DS-Fil成像系统拍照,从离析的早材和晚材管胞中随机选取25个管胞以Image J分别统计管胞上具缘纹孔的纹孔口直径与纹孔缘直径。
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2组切片经二甲苯脱蜡,无水乙醇浸泡3 min,95%乙醇浸泡3 min后,放入间苯三酚染液中浸染3 min,将浓盐酸滴于切片上,可见木质素的显色反应,同时依据酚类物质的自发荧光现象,结合荧光显微镜(LEICA DM3000)观察,DS-Fil成像系统拍照。纤维素检测选用钙荧光白(CFW)荧光染色试剂盒(GENMED GMS 16034.1),吸取5 μL GENMED染色液至离心管中,加入95 μL蒸馏水将其稀释20倍后得到制备的染色液。将包含早晚材与维管形成层的切片经二甲苯脱蜡,梯度乙醇逐级复水后直接将染色液于暗环境下滴于切片上,室温下静置5 min后以移液枪将染色液吸出,随后吸取GENMED清理液(Reagent A)滴于切片上,用移液枪反复吸取混匀,5 min后吸出,重复2次。最后直接置于荧光显微镜(LEICA DM 3000)下观察。以DS-Fil成像系统拍照,Image J量化第二组5张切片在早晚材相接处木质素与纤维素的组织化学显色和荧光面积。
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使用excel、SPSS和Origin 2022进行数据处理。依据数据的分布图像,判断管胞层与管胞各系数间为非线性关系,选用非线性模型进行拟合。通过对比origin非线性拟合函数,最后选取了拟合效果较好的Gaussmod函数对不同管胞层的细胞壁厚度、细胞直径与细胞腔径进行拟合。GaussMod函数:
$ y={y}_{0} + \frac{A}{{t}_{0}}{\mathrm{e}}^{\frac{1}{2}{\left(\frac{w}{{t}_{0}}\right)}^{2}-\frac{x-{x}_{c}}{{t}_{0}}}{\int }_{-\infty }^{\frac{x-{x}_{c}}{w}-\frac{w}{{t}_{0}}}\frac{1}{\sqrt{2\Pi }}{{\rm{e}}}^{-\frac{{y}^{2}}{2}}{d}_{y} $
式中:y为管胞的各项测量数据;x为细胞层;y0为模型对x轴的偏移量;w为模型宽度参数;xc为函数峰值对应的细胞层;A为模型面积;t0为辅助参数。
马尾松次生维管组织的变化特征
Variation Characteristics of Secondary Vascular Bundles in Pinus massoniana
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摘要:
目的 旨在揭示马尾松次生维管组织在发育过程中细胞结构、成分和功能的变化规律。 方法 通过组织化学分析和细胞离析等手段,研究马尾松维管组织的细胞形态、结构、木质素和纤维素的变化。 结果 维管形成层分化形成新生韧皮部和木质部时会逐渐富集纤维素;韧皮部发育过程中筛胞发生形变、细胞壁木质化,韧皮部纤维素含量占比下降。木质部管胞由早材向晚材的发育过程中,纹孔直径降低,纹孔数量下降,次生壁木质化程度增加,胞腔面积下降,纤维素含量占比下降,弦向壁及径向壁增厚。 结论 次生维管组织内木质部与韧皮部的细胞结构和成分随发育进程会发生规律性变化,其中,木质部细胞的胞内及胞间输导能力下降,机械强度上升;次生韧皮部丧失输导能力,机械强度上升。 Abstract:Objective This study is to reveal the changes of cellular structure, composition and function of secondary vascular tissue in Pinus massoniana during development. Methods The change of morphology, structure, lignin and cellulose in the vascular tissue of P. massoniana were studied by histochemical analysis and cell segregation. Results The vascular cambium gradually enriched cellulose when it differentiated into new phloem and xylem. During the development of phloem, the sieve cells were deformed and lignified, and the proportion of cellulose in phloem decreased. During the development of xylem tracheids from early wood to late wood, the diameter of striated pores decreased, the number of striated pores decreased, the lignification degree of secondary wall increased, the cell lumen area decreased, the proportion of cellulose content decreased, and the choroidal and radial walls thickened. Conclusion The cellular composition and structure of secondary vascular tissues change regularly with development. The intracellular and intercellular transport capacity of xylem cells decrease, while the mechanical strength increases. The secondary phloem loses its ability to transport and its mechanical strength increases. -
Key words:
- Pinus massoniana
- / secondary vascular bundle
- / structural change
- / histochemical analysis
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