Abstract Whole mount clearing is a routine method in morphological study, which allows observation of plant internal structure without section. Using high refractive index materials as medium, clearing techniques reduce light scattering, acquire enhancive light quantity and increase depth of field and vertical planes in a particular focal plane, to facilitate the samples transparency for observation. Nevertheless, clearing materials may disturb the osmosis and pH of sample medium, which is adverse to cells morphology. So far, the effective clearing techniques have been widely used in several studies with ovule and leaf. However, the current protocol is not reliable enough for root tip clearing, because the thin cell wall is vulnerable under the treatment of clearing solutions, resulting in abnormal root tips and cells plasmolysis. To achieve a stable and optimized clearing method for root tip, we established a standard protocol via evaluation of root tip morphology, plasmolysis and cells clarity in Arabidopsis thaliana. With these improved clearing methods, we developed an optimized clearing observation system (including clearing time, pH and composition) for root tip, which could provide a reliable technique for vulnerable tissues clearing. Keywords：morphogenesis;whole mount clearing;root tip;Arabidopsis thaliana
PDF (9273KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 引用本文 马龙, 李桂林, 李师鹏, 蒋苏. 根尖整体透明技术改良. 植物学报, 2020, 55(5): 596-604 doi:10.11983/CBB20016 Ma Long, Li Guilin, Li Shipeng, Jiang Su. An Improved Protocol for Whole Mount Clearing of Plant Root Tip. Chinese Bulletin of Botany, 2020, 55(5): 596-604 doi:10.11983/CBB20016
Table 1 表1 表1原始pH、拟南芥培养基常用pH、中性pH透明液HCG-1和HCG-2 Table 1Solution HCG-1 and HCG-2 in original pH, the pH used for Arabidopsis thaliana culture and neutral pH
No.
HCG-1
Composition
pH
1
H2O
9 mL
1.2
Chloral hydrate
24 g
Glycerol
3 mL
2
H2O
9 mL
5.8
Chloral hydrate
24 g
Glycerol
3 mL
3
H2O
9 mL
7.2
Chloral hydrate
24 g
Glycerol
3 mL
HCG-2
Composition
pH
4
H2O
9 mL
1.8
Chloral hydrate
12 g
Glycerol
3 mL
5
H2O
9 mL
5.8
Chloral hydrate
12 g
Glycerol
3 mL
6
H2O
9 mL
7.2
Chloral hydrate
12 g
Glycerol
3 mL
HCG: Clearing solution that consists of H2O, chloral hydrate and glycerol. HCG: 由水、水合三氯乙醛和甘油组成的透明液。
分别采用透明液HCG-1 (A)和HCG-2 (B)对拟南芥幼苗透明5、15和25分钟后QC细胞清晰的根尖所占比例。 Figure 3The effects of clearing times on clarity of quiescent center (QC) cells of Arabidopsis thaliana root tips
Proportion of root tips with clear QC cells of Arabidopsis thaliana seedlings cleared by HCG-1 (A) and HCG-2 (B) for 5, 15, 25 min, respectively.
分别采用透明液HCG-1 (A)和HCG-2 (B)对拟南芥幼苗透明5、15和25分钟后根尖伸长区表皮细胞质壁分离比例(* P<0.05, ** P<0.01, *** P<0.001)。 Figure 4The effects of clearing times on plasmolysis of Arabidopsis thaliana root tip cells
Proportion of plasmolytic cells of Arabidopsis thaliana seedlings cleared by HCG-1 (A) and HCG-2 (B) for 5, 15, 25 min, respectively (* P<0.05, ** P<0.01, *** P<0.001).
分别采用透明液HCG-1 (A)和HCG-2 (B)在pH1.2/1.8、pH5.8和pH7.2条件下, 对拟南芥幼苗透明5分钟后, 根尖伸长区表皮细胞质壁分离比例和静止中心(QC)细胞清晰的根尖所占比例。不同大写字母表示差异极显著(P<0.01)。Bars=50 μm Figure 5The effects of pH values on Arabidopsis thaliana root tip clearing
Proportion of plasmolytic cells and proportion of root tips with clear quiescent center (QC) cells of Arabidopsis thaliana seedlings cleared by HCG-1 (A) and HCG-2 (B) for 5 min under pH1.2/1.8, pH5.8, pH7.2, respectively. Different uppercase letters indicate extremely significant differences (P<0.01). Bars=50 μm
分别采用透明液HCG-1和HCG-2在pH7.2条件下, 对拟南芥幼苗透明5分钟后, 根尖伸长区表皮细胞质壁分离比例和QC细胞清晰的根尖所占比例(** P<0.01, *** P<0.001)。Bars=50 μm Figure 6The comparison of solution HCG-1 with HCG-2 on Arabidopsis thaliana root tip clearing
Proportion of plasmolytic cells and proportion of root tips with clear QC cells of Arabidopsis thaliana seedlings cleared by HCG-1 and HCG-2 for 5 min under pH7.2 (** P<0.01, *** P< 0.001). Bars=50 μm
分别采用饱和三氯乙醛溶液、透明液HCG-1、透明液HCG-2和25%甘油溶液对拟南芥幼苗透明5分钟后的根尖形态。Bars= 50 μm Figure 7The comparison of root tips cleared by gradient concentrations of chloral solutions
Morphology of root tips of Arabidopsis thaliana seedlings cleared by saturated chloral, HCG-1, HCG-2 and 25% glycerol solutions for 5 min, respectively. Bars=50 μm
采用透明液HCG-2 (pH7.2)对拟南芥3、9和15日龄幼苗根尖透明5分钟。Bars=50 μm Figure 8Arabidopsis thaliana seedlings in different growth stages were used to identify the optimized protocol
3-day-old, 9-day-old and 15-day-old seedlings of Arabidopsis thaliana were cleared by HCG-2 (pH7.2) for 5 min. Bars=50 μm
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TakatsukaH, UmedaM ( 2014). Hormonal control of cell division and elongation along differentiation trajectories in roots J Exp Bot 65, 2633-2643. DOI:10.1093/jxb/ert485URLPMID:24474807 [本文引用: 1] The continuous development of roots is supported by a sustainable system for cell production and growth at the root tip. In the stem cell niche that consists of a quiescent centre and surrounding stem cells, an undifferentiated state and low mitotic activity are preserved by the action of auxin and abscisic acid. Stem cell daughters divide several times in the proximal meristem, where auxin and gibberellin mainly promote cell proliferation. Cells then elongate with the help of gibberellin, and become finally differentiated as a constituent of a cell file in the elongation/differentiation zone. In the model plant Arabidopsis thaliana, the transition zone is located between the proximal meristem and the elongation/differentiation zone, and plays an important role in switching from mitosis to the endoreplication that causes DNA polyploidization. Recent studies have shown that cytokinins are essentially required for this transition by antagonizing auxin signalling and promoting degradation of mitotic regulators. In each root zone, different phytohormones interact with one another and coordinately control cell proliferation, cell elongation, cell differentiation, and endoreplication. Such hormonal networks maintain the elaborate structure of the root tip under various environmental conditions. In this review, we summarize and discuss key issues related to hormonal regulation of root growth, and describe how phytohormones are associated with the control of cell cycle machinery.
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VillaniTS, KorochAR, SimonJE ( 2013). An improved clearing and mounting solution to replace chloral hydrate in microscopic applications Appl Plant Sci 1, apps. 1300016. DOI:10.3732/apps.1300016URL [本文引用: 5]
Leukamenin E调节拟南芥幼苗生长发育的模式及其机制 2 2017
... 整体透明技术是研究植物内部形态结构的重要手段, 可以在不破坏整体结构的情况下对植物样品进行原位观察, 该技术已广泛应用于植物科学研究的诸多领域.植物整体样品之所以不透明, 是由于其组织中的水分、蛋白质、糖类和脂质等多种不同物质的折光系数不同, 当光线通过这些组分的时候会发生散射, 组织样品越厚, 散射就越强, 透射越少, 从而使光学观察受阻(Kurihara et al., 2015; 王培新等, 2016).整体透明技术的原理是利用某些具有高折射率的试剂处理待测样品, 使透射光通过样品时的散射减少, 光通量增加, 并降低球面色差, 以获得较高分辨率的图像(Haseloff, 2003); 此外, 高折射率的介质能够增加视野深度, 使更多层面上的图像进入聚焦平面, 从而达到组织样品透明的效果(Villani et al., 2013).虽然提高透明剂的浓度可以改善透明效果, 但由于透明剂(如水合三氯乙醛)能够改变透明液的渗透势和pH值, 从而对样品细胞形态的保持产生不利影响.因此, 根据材料性质选择适宜的透明液和透明方法是整体透明技术的关键.虽然目前已有多种透明液应用于植物组织透明研究, 但大多适用于叶片或胚珠等细胞壁较厚或液泡较少的组织材料(Lersten, 1986; 杨弘远, 1986, 1988; Liberato et al., 2005; 郝建华和强胜, 2007; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017).根尖作为一种不断保持分裂生长的组织, 细胞壁较薄, 使用现有的透明方法较难维持细胞形态, 造成透明效果的稳定性差(Richmond et al., 1980; Derbyshire et al., 2007; Li et al., 2010, 2013; Villani et al., 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 不仅影响观察对象的清晰度, 还会造成根尖形态不正常和细胞发生质壁分离.虽然整体透明技术已广泛应用于模式植物根尖发育研究(Bougourd et al., 2000; Li et al., 2010, 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 但如何在不影响根尖形态和细胞渗透势的前提下达到对根尖组织最优的透明观察效果, 仍需深入探索实践.本研究以拟南芥(Arabidopsis thaliana)为实验材料, 建立了根尖透明观察体系的评价标准, 即根尖形态是否正常、伸长区细胞是否发生质壁分离及静止中心细胞(QC细胞)的清晰度; 并利用这3个指标, 对透明时间、透明液成分配比和pH值3个要素进行改良, 优化出最适合拟南芥根尖的整体透明观察方法. ...
... 整体透明技术是研究植物内部形态结构的重要手段, 可以在不破坏整体结构的情况下对植物样品进行原位观察, 该技术已广泛应用于植物科学研究的诸多领域.植物整体样品之所以不透明, 是由于其组织中的水分、蛋白质、糖类和脂质等多种不同物质的折光系数不同, 当光线通过这些组分的时候会发生散射, 组织样品越厚, 散射就越强, 透射越少, 从而使光学观察受阻(Kurihara et al., 2015; 王培新等, 2016).整体透明技术的原理是利用某些具有高折射率的试剂处理待测样品, 使透射光通过样品时的散射减少, 光通量增加, 并降低球面色差, 以获得较高分辨率的图像(Haseloff, 2003); 此外, 高折射率的介质能够增加视野深度, 使更多层面上的图像进入聚焦平面, 从而达到组织样品透明的效果(Villani et al., 2013).虽然提高透明剂的浓度可以改善透明效果, 但由于透明剂(如水合三氯乙醛)能够改变透明液的渗透势和pH值, 从而对样品细胞形态的保持产生不利影响.因此, 根据材料性质选择适宜的透明液和透明方法是整体透明技术的关键.虽然目前已有多种透明液应用于植物组织透明研究, 但大多适用于叶片或胚珠等细胞壁较厚或液泡较少的组织材料(Lersten, 1986; 杨弘远, 1986, 1988; Liberato et al., 2005; 郝建华和强胜, 2007; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017).根尖作为一种不断保持分裂生长的组织, 细胞壁较薄, 使用现有的透明方法较难维持细胞形态, 造成透明效果的稳定性差(Richmond et al., 1980; Derbyshire et al., 2007; Li et al., 2010, 2013; Villani et al., 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 不仅影响观察对象的清晰度, 还会造成根尖形态不正常和细胞发生质壁分离.虽然整体透明技术已广泛应用于模式植物根尖发育研究(Bougourd et al., 2000; Li et al., 2010, 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 但如何在不影响根尖形态和细胞渗透势的前提下达到对根尖组织最优的透明观察效果, 仍需深入探索实践.本研究以拟南芥(Arabidopsis thaliana)为实验材料, 建立了根尖透明观察体系的评价标准, 即根尖形态是否正常、伸长区细胞是否发生质壁分离及静止中心细胞(QC细胞)的清晰度; 并利用这3个指标, 对透明时间、透明液成分配比和pH值3个要素进行改良, 优化出最适合拟南芥根尖的整体透明观察方法. ...
... 三氯乙醛和甘油是透明液中的常用成分.三氯乙醛可以提高溶液的折射率, 其在甘油溶液中的折射率达1.428, 接近于玻璃的折射率1.550 (Haseloff, 2003; Villani et al., 2013).这种较高的折射率一方面有助于增大观察体系的数值孔径, 提高分辨率; 另一方面使样品内不同组分的折光率趋于统一, 降低了光线的散射, 提高了光通量.在透明液中加入甘油能够减缓临时装片中水分的蒸发, 防止透明液结晶化(Villani et al., 2013).虽然各种透明液的主要成分相似, 但是不同植物组织样品的整体透明方法不尽相同.目前, 针对胚珠和叶片的整体透明方法较为成熟, 已有Herr 41/2透明、Hoyer氏溶液透明和水杨酸甲酯透明等多种透明体系(Hoyer, 1882; Herr, 1971, 1993; Crane, 1978; Bruzzese and Hasan, 1983; 杨弘远, 1986, 1988; 于明明等, 2009; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017), 但在这些透明体系中并未提出标准化的透明效果评价方案.之前对于胚珠和叶片透明体系的研究是以能够清晰地观察到细胞轮廓作为唯一的评价标准(李彦坤等, 2011; 赵林姝等, 2014; Ursache et al., 2018).相比于叶片, 根尖细胞在胞外渗透压升高时, 胞内膨压降低幅度较大, 使细胞更容易发生质壁分离, 甚至引起根尖整体形态异常(Itoh et al., 1987; Shabala et al., 2000; Shabala and Lew, 2002; Lang et al., 2014; Kim et al., 2018).透明液恰恰形成了一个高渗的胞外环境, 这在以往的研究中经常被忽视(李彦坤等, 2011; Villani et al., 2013; 赵林姝等, 2014).因此, 本文针对透明后的根尖形态、细胞质壁分离情况和透明清晰度, 首次提出了评价拟南芥根尖透明体系的3个量化指标: 形态正常的根尖所占比例、伸长区表皮细胞质壁分离比例及QC细胞清晰的根尖所占比例.综合这三方面评价结果可以更加严谨、完善地对根尖整体透明方法进行分析和改良, 为脆弱组织材料的整体透明研究提供改进依据. ...
... ; 赵林姝等, 2014; Ursache et al., 2018).相比于叶片, 根尖细胞在胞外渗透压升高时, 胞内膨压降低幅度较大, 使细胞更容易发生质壁分离, 甚至引起根尖整体形态异常(Itoh et al., 1987; Shabala et al., 2000; Shabala and Lew, 2002; Lang et al., 2014; Kim et al., 2018).透明液恰恰形成了一个高渗的胞外环境, 这在以往的研究中经常被忽视(李彦坤等, 2011; Villani et al., 2013; 赵林姝等, 2014).因此, 本文针对透明后的根尖形态、细胞质壁分离情况和透明清晰度, 首次提出了评价拟南芥根尖透明体系的3个量化指标: 形态正常的根尖所占比例、伸长区表皮细胞质壁分离比例及QC细胞清晰的根尖所占比例.综合这三方面评价结果可以更加严谨、完善地对根尖整体透明方法进行分析和改良, 为脆弱组织材料的整体透明研究提供改进依据. ...
Analysis of cell division and elongation underlying the developmental acceleration of root growth in Arabidopsis thaliana 1 1998
... 在植物的发育过程研究中, 经常需要对不同苗龄大小的根尖进行透明处理.理论上, 幼苗期的生长发育形成了拟南芥的初生结构, 在这一时期不同苗龄的根尖形态一样(Beemster and Baskin, 1998; Verbelen et al., 2006; Ivanov and Dubrovsky, 2013; Takatsuka and Umeda, 2014; Pavelescu et al., 2018).为了科学严谨地验证本文所筛选出的优选透明方案是否适合整体透明实验中常用的苗龄材料(如3至15日龄幼苗), 我们利用筛选到的透明液HCG-2 (pH7.2)对拟南芥3、9和15日龄幼苗根尖进行透明.结果(图8)表明, 3、9和15日龄幼苗在根尖形态和透明效果上无明显差异.因此, 对于根尖这一特定组织材料, 苗龄大小不会对透明产生不确定性影响.我们改良所得透明液HCG-2 (pH7.2)、透明5分钟的透明方案适用于不同苗龄拟南芥根尖的整体透明. ...
An aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos 2 2000
... 整体透明技术是研究植物内部形态结构的重要手段, 可以在不破坏整体结构的情况下对植物样品进行原位观察, 该技术已广泛应用于植物科学研究的诸多领域.植物整体样品之所以不透明, 是由于其组织中的水分、蛋白质、糖类和脂质等多种不同物质的折光系数不同, 当光线通过这些组分的时候会发生散射, 组织样品越厚, 散射就越强, 透射越少, 从而使光学观察受阻(Kurihara et al., 2015; 王培新等, 2016).整体透明技术的原理是利用某些具有高折射率的试剂处理待测样品, 使透射光通过样品时的散射减少, 光通量增加, 并降低球面色差, 以获得较高分辨率的图像(Haseloff, 2003); 此外, 高折射率的介质能够增加视野深度, 使更多层面上的图像进入聚焦平面, 从而达到组织样品透明的效果(Villani et al., 2013).虽然提高透明剂的浓度可以改善透明效果, 但由于透明剂(如水合三氯乙醛)能够改变透明液的渗透势和pH值, 从而对样品细胞形态的保持产生不利影响.因此, 根据材料性质选择适宜的透明液和透明方法是整体透明技术的关键.虽然目前已有多种透明液应用于植物组织透明研究, 但大多适用于叶片或胚珠等细胞壁较厚或液泡较少的组织材料(Lersten, 1986; 杨弘远, 1986, 1988; Liberato et al., 2005; 郝建华和强胜, 2007; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017).根尖作为一种不断保持分裂生长的组织, 细胞壁较薄, 使用现有的透明方法较难维持细胞形态, 造成透明效果的稳定性差(Richmond et al., 1980; Derbyshire et al., 2007; Li et al., 2010, 2013; Villani et al., 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 不仅影响观察对象的清晰度, 还会造成根尖形态不正常和细胞发生质壁分离.虽然整体透明技术已广泛应用于模式植物根尖发育研究(Bougourd et al., 2000; Li et al., 2010, 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 但如何在不影响根尖形态和细胞渗透势的前提下达到对根尖组织最优的透明观察效果, 仍需深入探索实践.本研究以拟南芥(Arabidopsis thaliana)为实验材料, 建立了根尖透明观察体系的评价标准, 即根尖形态是否正常、伸长区细胞是否发生质壁分离及静止中心细胞(QC细胞)的清晰度; 并利用这3个指标, 对透明时间、透明液成分配比和pH值3个要素进行改良, 优化出最适合拟南芥根尖的整体透明观察方法. ...
Leaf-clearing and staining techniques for the observation of conidiophores in the Phyllactinioideae (Erysiphaceae) 1 2005
... 整体透明技术是研究植物内部形态结构的重要手段, 可以在不破坏整体结构的情况下对植物样品进行原位观察, 该技术已广泛应用于植物科学研究的诸多领域.植物整体样品之所以不透明, 是由于其组织中的水分、蛋白质、糖类和脂质等多种不同物质的折光系数不同, 当光线通过这些组分的时候会发生散射, 组织样品越厚, 散射就越强, 透射越少, 从而使光学观察受阻(Kurihara et al., 2015; 王培新等, 2016).整体透明技术的原理是利用某些具有高折射率的试剂处理待测样品, 使透射光通过样品时的散射减少, 光通量增加, 并降低球面色差, 以获得较高分辨率的图像(Haseloff, 2003); 此外, 高折射率的介质能够增加视野深度, 使更多层面上的图像进入聚焦平面, 从而达到组织样品透明的效果(Villani et al., 2013).虽然提高透明剂的浓度可以改善透明效果, 但由于透明剂(如水合三氯乙醛)能够改变透明液的渗透势和pH值, 从而对样品细胞形态的保持产生不利影响.因此, 根据材料性质选择适宜的透明液和透明方法是整体透明技术的关键.虽然目前已有多种透明液应用于植物组织透明研究, 但大多适用于叶片或胚珠等细胞壁较厚或液泡较少的组织材料(Lersten, 1986; 杨弘远, 1986, 1988; Liberato et al., 2005; 郝建华和强胜, 2007; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017).根尖作为一种不断保持分裂生长的组织, 细胞壁较薄, 使用现有的透明方法较难维持细胞形态, 造成透明效果的稳定性差(Richmond et al., 1980; Derbyshire et al., 2007; Li et al., 2010, 2013; Villani et al., 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 不仅影响观察对象的清晰度, 还会造成根尖形态不正常和细胞发生质壁分离.虽然整体透明技术已广泛应用于模式植物根尖发育研究(Bougourd et al., 2000; Li et al., 2010, 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 但如何在不影响根尖形态和细胞渗透势的前提下达到对根尖组织最优的透明观察效果, 仍需深入探索实践.本研究以拟南芥(Arabidopsis thaliana)为实验材料, 建立了根尖透明观察体系的评价标准, 即根尖形态是否正常、伸长区细胞是否发生质壁分离及静止中心细胞(QC细胞)的清晰度; 并利用这3个指标, 对透明时间、透明液成分配比和pH值3个要素进行改良, 优化出最适合拟南芥根尖的整体透明观察方法. ...
A Sizer model for cell differentiation in Arabidopsis thaliana root growth 1 2018
... 在植物的发育过程研究中, 经常需要对不同苗龄大小的根尖进行透明处理.理论上, 幼苗期的生长发育形成了拟南芥的初生结构, 在这一时期不同苗龄的根尖形态一样(Beemster and Baskin, 1998; Verbelen et al., 2006; Ivanov and Dubrovsky, 2013; Takatsuka and Umeda, 2014; Pavelescu et al., 2018).为了科学严谨地验证本文所筛选出的优选透明方案是否适合整体透明实验中常用的苗龄材料(如3至15日龄幼苗), 我们利用筛选到的透明液HCG-2 (pH7.2)对拟南芥3、9和15日龄幼苗根尖进行透明.结果(图8)表明, 3、9和15日龄幼苗在根尖形态和透明效果上无明显差异.因此, 对于根尖这一特定组织材料, 苗龄大小不会对透明产生不确定性影响.我们改良所得透明液HCG-2 (pH7.2)、透明5分钟的透明方案适用于不同苗龄拟南芥根尖的整体透明. ...
Cell expansion patterns and directionality of wall mechanical properties in Nitella 1 1980
... 整体透明技术是研究植物内部形态结构的重要手段, 可以在不破坏整体结构的情况下对植物样品进行原位观察, 该技术已广泛应用于植物科学研究的诸多领域.植物整体样品之所以不透明, 是由于其组织中的水分、蛋白质、糖类和脂质等多种不同物质的折光系数不同, 当光线通过这些组分的时候会发生散射, 组织样品越厚, 散射就越强, 透射越少, 从而使光学观察受阻(Kurihara et al., 2015; 王培新等, 2016).整体透明技术的原理是利用某些具有高折射率的试剂处理待测样品, 使透射光通过样品时的散射减少, 光通量增加, 并降低球面色差, 以获得较高分辨率的图像(Haseloff, 2003); 此外, 高折射率的介质能够增加视野深度, 使更多层面上的图像进入聚焦平面, 从而达到组织样品透明的效果(Villani et al., 2013).虽然提高透明剂的浓度可以改善透明效果, 但由于透明剂(如水合三氯乙醛)能够改变透明液的渗透势和pH值, 从而对样品细胞形态的保持产生不利影响.因此, 根据材料性质选择适宜的透明液和透明方法是整体透明技术的关键.虽然目前已有多种透明液应用于植物组织透明研究, 但大多适用于叶片或胚珠等细胞壁较厚或液泡较少的组织材料(Lersten, 1986; 杨弘远, 1986, 1988; Liberato et al., 2005; 郝建华和强胜, 2007; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017).根尖作为一种不断保持分裂生长的组织, 细胞壁较薄, 使用现有的透明方法较难维持细胞形态, 造成透明效果的稳定性差(Richmond et al., 1980; Derbyshire et al., 2007; Li et al., 2010, 2013; Villani et al., 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 不仅影响观察对象的清晰度, 还会造成根尖形态不正常和细胞发生质壁分离.虽然整体透明技术已广泛应用于模式植物根尖发育研究(Bougourd et al., 2000; Li et al., 2010, 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 但如何在不影响根尖形态和细胞渗透势的前提下达到对根尖组织最优的透明观察效果, 仍需深入探索实践.本研究以拟南芥(Arabidopsis thaliana)为实验材料, 建立了根尖透明观察体系的评价标准, 即根尖形态是否正常、伸长区细胞是否发生质壁分离及静止中心细胞(QC细胞)的清晰度; 并利用这3个指标, 对透明时间、透明液成分配比和pH值3个要素进行改良, 优化出最适合拟南芥根尖的整体透明观察方法. ...
Ion-specific mechanisms of osmoregulation in bean mesophyll cells 1 2000
... 三氯乙醛和甘油是透明液中的常用成分.三氯乙醛可以提高溶液的折射率, 其在甘油溶液中的折射率达1.428, 接近于玻璃的折射率1.550 (Haseloff, 2003; Villani et al., 2013).这种较高的折射率一方面有助于增大观察体系的数值孔径, 提高分辨率; 另一方面使样品内不同组分的折光率趋于统一, 降低了光线的散射, 提高了光通量.在透明液中加入甘油能够减缓临时装片中水分的蒸发, 防止透明液结晶化(Villani et al., 2013).虽然各种透明液的主要成分相似, 但是不同植物组织样品的整体透明方法不尽相同.目前, 针对胚珠和叶片的整体透明方法较为成熟, 已有Herr 41/2透明、Hoyer氏溶液透明和水杨酸甲酯透明等多种透明体系(Hoyer, 1882; Herr, 1971, 1993; Crane, 1978; Bruzzese and Hasan, 1983; 杨弘远, 1986, 1988; 于明明等, 2009; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017), 但在这些透明体系中并未提出标准化的透明效果评价方案.之前对于胚珠和叶片透明体系的研究是以能够清晰地观察到细胞轮廓作为唯一的评价标准(李彦坤等, 2011; 赵林姝等, 2014; Ursache et al., 2018).相比于叶片, 根尖细胞在胞外渗透压升高时, 胞内膨压降低幅度较大, 使细胞更容易发生质壁分离, 甚至引起根尖整体形态异常(Itoh et al., 1987; Shabala et al., 2000; Shabala and Lew, 2002; Lang et al., 2014; Kim et al., 2018).透明液恰恰形成了一个高渗的胞外环境, 这在以往的研究中经常被忽视(李彦坤等, 2011; Villani et al., 2013; 赵林姝等, 2014).因此, 本文针对透明后的根尖形态、细胞质壁分离情况和透明清晰度, 首次提出了评价拟南芥根尖透明体系的3个量化指标: 形态正常的根尖所占比例、伸长区表皮细胞质壁分离比例及QC细胞清晰的根尖所占比例.综合这三方面评价结果可以更加严谨、完善地对根尖整体透明方法进行分析和改良, 为脆弱组织材料的整体透明研究提供改进依据. ...
Turgor regulation in osmotically stressed Arabidopsis epidermal root cells. Direct support for the role of inorganic ion uptake as revealed by concurrent flux and cell turgor measurements 1 2002
... 三氯乙醛和甘油是透明液中的常用成分.三氯乙醛可以提高溶液的折射率, 其在甘油溶液中的折射率达1.428, 接近于玻璃的折射率1.550 (Haseloff, 2003; Villani et al., 2013).这种较高的折射率一方面有助于增大观察体系的数值孔径, 提高分辨率; 另一方面使样品内不同组分的折光率趋于统一, 降低了光线的散射, 提高了光通量.在透明液中加入甘油能够减缓临时装片中水分的蒸发, 防止透明液结晶化(Villani et al., 2013).虽然各种透明液的主要成分相似, 但是不同植物组织样品的整体透明方法不尽相同.目前, 针对胚珠和叶片的整体透明方法较为成熟, 已有Herr 41/2透明、Hoyer氏溶液透明和水杨酸甲酯透明等多种透明体系(Hoyer, 1882; Herr, 1971, 1993; Crane, 1978; Bruzzese and Hasan, 1983; 杨弘远, 1986, 1988; 于明明等, 2009; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017), 但在这些透明体系中并未提出标准化的透明效果评价方案.之前对于胚珠和叶片透明体系的研究是以能够清晰地观察到细胞轮廓作为唯一的评价标准(李彦坤等, 2011; 赵林姝等, 2014; Ursache et al., 2018).相比于叶片, 根尖细胞在胞外渗透压升高时, 胞内膨压降低幅度较大, 使细胞更容易发生质壁分离, 甚至引起根尖整体形态异常(Itoh et al., 1987; Shabala et al., 2000; Shabala and Lew, 2002; Lang et al., 2014; Kim et al., 2018).透明液恰恰形成了一个高渗的胞外环境, 这在以往的研究中经常被忽视(李彦坤等, 2011; Villani et al., 2013; 赵林姝等, 2014).因此, 本文针对透明后的根尖形态、细胞质壁分离情况和透明清晰度, 首次提出了评价拟南芥根尖透明体系的3个量化指标: 形态正常的根尖所占比例、伸长区表皮细胞质壁分离比例及QC细胞清晰的根尖所占比例.综合这三方面评价结果可以更加严谨、完善地对根尖整体透明方法进行分析和改良, 为脆弱组织材料的整体透明研究提供改进依据. ...
Hormonal control of cell division and elongation along differentiation trajectories in roots 1 2014
... 在植物的发育过程研究中, 经常需要对不同苗龄大小的根尖进行透明处理.理论上, 幼苗期的生长发育形成了拟南芥的初生结构, 在这一时期不同苗龄的根尖形态一样(Beemster and Baskin, 1998; Verbelen et al., 2006; Ivanov and Dubrovsky, 2013; Takatsuka and Umeda, 2014; Pavelescu et al., 2018).为了科学严谨地验证本文所筛选出的优选透明方案是否适合整体透明实验中常用的苗龄材料(如3至15日龄幼苗), 我们利用筛选到的透明液HCG-2 (pH7.2)对拟南芥3、9和15日龄幼苗根尖进行透明.结果(图8)表明, 3、9和15日龄幼苗在根尖形态和透明效果上无明显差异.因此, 对于根尖这一特定组织材料, 苗龄大小不会对透明产生不确定性影响.我们改良所得透明液HCG-2 (pH7.2)、透明5分钟的透明方案适用于不同苗龄拟南芥根尖的整体透明. ...
A protocol for combining fluorescent proteins with histological stains for diverse cell wall components 1 2018
... 三氯乙醛和甘油是透明液中的常用成分.三氯乙醛可以提高溶液的折射率, 其在甘油溶液中的折射率达1.428, 接近于玻璃的折射率1.550 (Haseloff, 2003; Villani et al., 2013).这种较高的折射率一方面有助于增大观察体系的数值孔径, 提高分辨率; 另一方面使样品内不同组分的折光率趋于统一, 降低了光线的散射, 提高了光通量.在透明液中加入甘油能够减缓临时装片中水分的蒸发, 防止透明液结晶化(Villani et al., 2013).虽然各种透明液的主要成分相似, 但是不同植物组织样品的整体透明方法不尽相同.目前, 针对胚珠和叶片的整体透明方法较为成熟, 已有Herr 41/2透明、Hoyer氏溶液透明和水杨酸甲酯透明等多种透明体系(Hoyer, 1882; Herr, 1971, 1993; Crane, 1978; Bruzzese and Hasan, 1983; 杨弘远, 1986, 1988; 于明明等, 2009; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017), 但在这些透明体系中并未提出标准化的透明效果评价方案.之前对于胚珠和叶片透明体系的研究是以能够清晰地观察到细胞轮廓作为唯一的评价标准(李彦坤等, 2011; 赵林姝等, 2014; Ursache et al., 2018).相比于叶片, 根尖细胞在胞外渗透压升高时, 胞内膨压降低幅度较大, 使细胞更容易发生质壁分离, 甚至引起根尖整体形态异常(Itoh et al., 1987; Shabala et al., 2000; Shabala and Lew, 2002; Lang et al., 2014; Kim et al., 2018).透明液恰恰形成了一个高渗的胞外环境, 这在以往的研究中经常被忽视(李彦坤等, 2011; Villani et al., 2013; 赵林姝等, 2014).因此, 本文针对透明后的根尖形态、细胞质壁分离情况和透明清晰度, 首次提出了评价拟南芥根尖透明体系的3个量化指标: 形态正常的根尖所占比例、伸长区表皮细胞质壁分离比例及QC细胞清晰的根尖所占比例.综合这三方面评价结果可以更加严谨、完善地对根尖整体透明方法进行分析和改良, 为脆弱组织材料的整体透明研究提供改进依据. ...
The root apex of Arabidopsis thaliana consists of four distinct zones of growth activities 1 2006
... 在植物的发育过程研究中, 经常需要对不同苗龄大小的根尖进行透明处理.理论上, 幼苗期的生长发育形成了拟南芥的初生结构, 在这一时期不同苗龄的根尖形态一样(Beemster and Baskin, 1998; Verbelen et al., 2006; Ivanov and Dubrovsky, 2013; Takatsuka and Umeda, 2014; Pavelescu et al., 2018).为了科学严谨地验证本文所筛选出的优选透明方案是否适合整体透明实验中常用的苗龄材料(如3至15日龄幼苗), 我们利用筛选到的透明液HCG-2 (pH7.2)对拟南芥3、9和15日龄幼苗根尖进行透明.结果(图8)表明, 3、9和15日龄幼苗在根尖形态和透明效果上无明显差异.因此, 对于根尖这一特定组织材料, 苗龄大小不会对透明产生不确定性影响.我们改良所得透明液HCG-2 (pH7.2)、透明5分钟的透明方案适用于不同苗龄拟南芥根尖的整体透明. ...
An improved clearing and mounting solution to replace chloral hydrate in microscopic applications 5 2013
... 整体透明技术是研究植物内部形态结构的重要手段, 可以在不破坏整体结构的情况下对植物样品进行原位观察, 该技术已广泛应用于植物科学研究的诸多领域.植物整体样品之所以不透明, 是由于其组织中的水分、蛋白质、糖类和脂质等多种不同物质的折光系数不同, 当光线通过这些组分的时候会发生散射, 组织样品越厚, 散射就越强, 透射越少, 从而使光学观察受阻(Kurihara et al., 2015; 王培新等, 2016).整体透明技术的原理是利用某些具有高折射率的试剂处理待测样品, 使透射光通过样品时的散射减少, 光通量增加, 并降低球面色差, 以获得较高分辨率的图像(Haseloff, 2003); 此外, 高折射率的介质能够增加视野深度, 使更多层面上的图像进入聚焦平面, 从而达到组织样品透明的效果(Villani et al., 2013).虽然提高透明剂的浓度可以改善透明效果, 但由于透明剂(如水合三氯乙醛)能够改变透明液的渗透势和pH值, 从而对样品细胞形态的保持产生不利影响.因此, 根据材料性质选择适宜的透明液和透明方法是整体透明技术的关键.虽然目前已有多种透明液应用于植物组织透明研究, 但大多适用于叶片或胚珠等细胞壁较厚或液泡较少的组织材料(Lersten, 1986; 杨弘远, 1986, 1988; Liberato et al., 2005; 郝建华和强胜, 2007; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017).根尖作为一种不断保持分裂生长的组织, 细胞壁较薄, 使用现有的透明方法较难维持细胞形态, 造成透明效果的稳定性差(Richmond et al., 1980; Derbyshire et al., 2007; Li et al., 2010, 2013; Villani et al., 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 不仅影响观察对象的清晰度, 还会造成根尖形态不正常和细胞发生质壁分离.虽然整体透明技术已广泛应用于模式植物根尖发育研究(Bougourd et al., 2000; Li et al., 2010, 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 但如何在不影响根尖形态和细胞渗透势的前提下达到对根尖组织最优的透明观察效果, 仍需深入探索实践.本研究以拟南芥(Arabidopsis thaliana)为实验材料, 建立了根尖透明观察体系的评价标准, 即根尖形态是否正常、伸长区细胞是否发生质壁分离及静止中心细胞(QC细胞)的清晰度; 并利用这3个指标, 对透明时间、透明液成分配比和pH值3个要素进行改良, 优化出最适合拟南芥根尖的整体透明观察方法. ...
... ; Villani et al., 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 不仅影响观察对象的清晰度, 还会造成根尖形态不正常和细胞发生质壁分离.虽然整体透明技术已广泛应用于模式植物根尖发育研究(Bougourd et al., 2000; Li et al., 2010, 2013; 程蔚玲等, 2017; 李芳芳等, 2018), 但如何在不影响根尖形态和细胞渗透势的前提下达到对根尖组织最优的透明观察效果, 仍需深入探索实践.本研究以拟南芥(Arabidopsis thaliana)为实验材料, 建立了根尖透明观察体系的评价标准, 即根尖形态是否正常、伸长区细胞是否发生质壁分离及静止中心细胞(QC细胞)的清晰度; 并利用这3个指标, 对透明时间、透明液成分配比和pH值3个要素进行改良, 优化出最适合拟南芥根尖的整体透明观察方法. ...
... 三氯乙醛和甘油是透明液中的常用成分.三氯乙醛可以提高溶液的折射率, 其在甘油溶液中的折射率达1.428, 接近于玻璃的折射率1.550 (Haseloff, 2003; Villani et al., 2013).这种较高的折射率一方面有助于增大观察体系的数值孔径, 提高分辨率; 另一方面使样品内不同组分的折光率趋于统一, 降低了光线的散射, 提高了光通量.在透明液中加入甘油能够减缓临时装片中水分的蒸发, 防止透明液结晶化(Villani et al., 2013).虽然各种透明液的主要成分相似, 但是不同植物组织样品的整体透明方法不尽相同.目前, 针对胚珠和叶片的整体透明方法较为成熟, 已有Herr 41/2透明、Hoyer氏溶液透明和水杨酸甲酯透明等多种透明体系(Hoyer, 1882; Herr, 1971, 1993; Crane, 1978; Bruzzese and Hasan, 1983; 杨弘远, 1986, 1988; 于明明等, 2009; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017), 但在这些透明体系中并未提出标准化的透明效果评价方案.之前对于胚珠和叶片透明体系的研究是以能够清晰地观察到细胞轮廓作为唯一的评价标准(李彦坤等, 2011; 赵林姝等, 2014; Ursache et al., 2018).相比于叶片, 根尖细胞在胞外渗透压升高时, 胞内膨压降低幅度较大, 使细胞更容易发生质壁分离, 甚至引起根尖整体形态异常(Itoh et al., 1987; Shabala et al., 2000; Shabala and Lew, 2002; Lang et al., 2014; Kim et al., 2018).透明液恰恰形成了一个高渗的胞外环境, 这在以往的研究中经常被忽视(李彦坤等, 2011; Villani et al., 2013; 赵林姝等, 2014).因此, 本文针对透明后的根尖形态、细胞质壁分离情况和透明清晰度, 首次提出了评价拟南芥根尖透明体系的3个量化指标: 形态正常的根尖所占比例、伸长区表皮细胞质壁分离比例及QC细胞清晰的根尖所占比例.综合这三方面评价结果可以更加严谨、完善地对根尖整体透明方法进行分析和改良, 为脆弱组织材料的整体透明研究提供改进依据. ...
... ).这种较高的折射率一方面有助于增大观察体系的数值孔径, 提高分辨率; 另一方面使样品内不同组分的折光率趋于统一, 降低了光线的散射, 提高了光通量.在透明液中加入甘油能够减缓临时装片中水分的蒸发, 防止透明液结晶化(Villani et al., 2013).虽然各种透明液的主要成分相似, 但是不同植物组织样品的整体透明方法不尽相同.目前, 针对胚珠和叶片的整体透明方法较为成熟, 已有Herr 41/2透明、Hoyer氏溶液透明和水杨酸甲酯透明等多种透明体系(Hoyer, 1882; Herr, 1971, 1993; Crane, 1978; Bruzzese and Hasan, 1983; 杨弘远, 1986, 1988; 于明明等, 2009; 王文婧等, 2011; 赵林姝等, 2014; 任媛媛和朱炎, 2017), 但在这些透明体系中并未提出标准化的透明效果评价方案.之前对于胚珠和叶片透明体系的研究是以能够清晰地观察到细胞轮廓作为唯一的评价标准(李彦坤等, 2011; 赵林姝等, 2014; Ursache et al., 2018).相比于叶片, 根尖细胞在胞外渗透压升高时, 胞内膨压降低幅度较大, 使细胞更容易发生质壁分离, 甚至引起根尖整体形态异常(Itoh et al., 1987; Shabala et al., 2000; Shabala and Lew, 2002; Lang et al., 2014; Kim et al., 2018).透明液恰恰形成了一个高渗的胞外环境, 这在以往的研究中经常被忽视(李彦坤等, 2011; Villani et al., 2013; 赵林姝等, 2014).因此, 本文针对透明后的根尖形态、细胞质壁分离情况和透明清晰度, 首次提出了评价拟南芥根尖透明体系的3个量化指标: 形态正常的根尖所占比例、伸长区表皮细胞质壁分离比例及QC细胞清晰的根尖所占比例.综合这三方面评价结果可以更加严谨、完善地对根尖整体透明方法进行分析和改良, 为脆弱组织材料的整体透明研究提供改进依据. ...
,*齐鲁师范学院, 济南 250200
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