An Improved Protocol for Whole Mount Clearing of Plant Root Tip

doi:10.11983/CBB20016

Abstract

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.

Key words: morphogenesis, whole mount clearing, root tip, Arabidopsis thaliana

Long Ma, Guilin Li, Shipeng Li, Su Jiang. An Improved Protocol for Whole Mount Clearing of Plant Root Tip[J]. Chinese Bulletin of Botany, 2020, 55(5): 596-604.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB20016

https://www.chinbullbotany.com/EN/Y2020/V55/I5/596

Figures/Tables 9

Table 1 Solution 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

Figure 1 Criteria of Arabidopsis thaliana root tip clearing Root tips morphology (A), elongation zone epidermal cells plasmolysis (B) and quiescent center (QC) cells clarity (C) of cleared Arabidopsis thaliana Col-0 seedlings ((A) Bars=100 μm; (B) Bars=50 μm; (C) Bars=20 μm).

Figure 2 The effects of clearing times on Arabidopsis thaliana root tip morphology Proportion of normal root tips of Arabidopsis thaliana seedlings cleared by HCG-1 (A) and HCG-2 (B) for 5, 15, 25 min, respectively.

Figure 3 The 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.

Figure 4 The 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).

Figure 5 The 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

Figure 6 The 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

Figure 7 The 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

Figure 8 Arabidopsis 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

References 39

[1]	程蔚玲, 丁兰, 李金平, 刘国安, 杨宁 ( 2017). Leukamenin E调节拟南芥幼苗生长发育的模式及其机制. 生态学杂志 36, 676-686.
[2]	郝建华, 强胜 ( 2007). 整体透明技术在植物生物学中的应用实例及其剖析. 植物学通报 24, 490-497.
[3]	李芳芳, 杨娜, 钱猛, 甘立军 ( 2018). 生长素参与三十烷醇诱导的拟南芥侧根发育. 南京农业大学学报 41, 473-480.
[4]	李彦坤, 臧巩固, 赵立宁, 李育君, 唐蜻, 程超华 ( 2011). 整体透明技术观察悬铃叶苎麻胚胎发育的方法研究. 中国麻业科学 33, 142-146.
[5]	任媛媛, 朱炎 ( 2017). INO80参与拟南芥气孔数量调控的分子机制. 复旦学报(自然科学版) 56, 653-661.
[6]	王培新, 张丹, 尚爱加, 侯冰 ( 2016). 组织透明技术. 神经解剖学杂志 32, 124-128.
[7]	王文婧, 刘婷, 郭磊, 刘春明 ( 2011). SLC/AGO1基因控制拟南芥细胞分裂与定向伸长. 植物学报 46, 370-378.
[8]	杨弘远 ( 1986). 用整体染色与透明技术观察胚囊、胚、胚乳和胚状体. 植物学报 28, 575-581.
[9]	杨弘远 ( 1988). 植物胚胎学中的整体透明技术. 植物学通报 5(2), 114-116.
[10]	于明明, 李兴国, 张宪省 ( 2009). APETALA1启动子驱动AtIPT4在转基因拟南芥中表达导致花和花器官发育异常. 植物学报 44, 59-68.
[11]	赵林姝, 刘录祥, 古佳玉, 郭会君, 李军辉, 谢永盾, 赵世荣 ( 2014). 一种小麦叶片气孔保卫细胞观察样品的制备方法. 植物学报 49, 120-126.
[12]	Beemster GTS, Baskin TI ( 1998). Analysis of cell division and elongation underlying the developmental acceleration of root growth in Arabidopsis thaliana. Plant Physiol 116, 1515-1526. DOI URL PMID
[13]	Bougourd S, Marrison J, Haseloff J ( 2000). An aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos. Plant J 24, 543-550. URL PMID
[14]	Bruzzese E, Hasan S ( 1983). A whole leaf clearing and staining technique for host specificity studies of rust fungi. Plant Pathol 32, 335-338. DOI URL
[15]	Crane CF ( 1978). Apomixis and Crossing Incompatibilities in Some Zephyrantheae. Ph.D. thesis. Austin: University of Texas.
[16]	Derbyshire P, Findlay K, McCann MC, Roberts K ( 2007). Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness. J Exp Bot 58, 2079-2089. DOI URL PMID
[17]	Hager A ( 2003). Role of the plasma membrane H⁺-ATPase in auxin-induced elongation growth: historical and new aspects. J Plant Res 116, 483-505. DOI URL PMID
[18]	Haseloff J ( 2003). Old botanical techniques for new microscopes. BioTechniques 34, 1174-1182. DOI URL PMID
[19]	Herr JMJ ( 1971). A new clearing-squash technique for the study of ovule development in angiosperms. Am J Bot 58, 785-790. DOI URL
[20]	Herr JMJ (1993). Clearing techniques for the study of vascular plant tissues in whole structures and thick sections. In: Tested Studies for Laboratory Teaching. Proceedings of the Fifth Workshop/Conference of the Association for Biology Laboratory Education (ABLE). Toronto: ABLE. pp. 63-84.
[21]	Hoyer H ( 1882). Beiträge zur histologischen Technik. 3. Einschlussflüssigkeiten. Biologisches Zentralblatt 2, 23-24.
[22]	Itoh K, Nakamura Y, Kawata H, Yamada T, Ohta E, Sakata M ( 1987). Effect of osmotic stress on turgor pressure in mung bean root cells. Plant Cell Physiol 28, 987-994.
[23]	Ivanov VB, Dubrovsky JG ( 2013). Longitudinal zonation pattern in plant roots: conflicts and solutions. Trends Plant Sci 18, 237-243. DOI URL PMID
[24]	Janicka-Russak M (2011). Plant plasma membrane H⁺- ATPase in adaptation of plants to abiotic stresses. In: Shanker A, ed. Abiotic Stress Response in Plants—Physiological, Biochemical and Genetic Perspectives. Rijeka: Intech Open Press. pp. 197-218.
[25]	Kim YX, Stumpf B, Sung J, Lee SJ ( 2018). The relationship between turgor pressure change and cell hydraulics of midrib parenchyma cells in the leaves of Zea mays. Cells 7, 180. DOI URL
[26]	Kurihara D, Mizuta Y, Sato Y, Higashiyama T ( 2015). ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development 142, 4168-4179. DOI URL PMID
[27]	Lang I, Sassmann S, Schmidt B, Komis G ( 2014). Plasmolysis: loss of turgor and beyond. Plants (Basel) 3, 583-593. DOI URL PMID
[28]	Lersten NR ( 1986). Modified clearing method to show sieve tubes in minor veins of leaves. Stain Technol 61, 231-234. DOI URL PMID
[29]	Li SP, Chen M, Yu DL, Ren SC, Sun SF, Liu LD, Ketelaar T, Emons AMC, Liu CM ( 2013). EXO70A1-mediated vesicle trafficking is critical for tracheary element development in Arabidopsis. Plant Cell 25, 1774-1786. URL PMID
[30]	Li SP, van Os GMA, Ren SC, Yu DL, Ketelaar T, Emons AMC, Liu CM ( 2010). Expression and functional analyses of EXO70 genes in Arabidopsis implicate their roles in regulating cell type-specific exocytosis. Plant Physiol 154, 1819-1830. DOI URL PMID
[31]	Liberato JR, Barreto RW, Shivas RG ( 2005). Leaf-clearing and staining techniques for the observation of conidiophores in the Phyllactinioideae (Erysiphaceae). Australas Plant Pathol 34, 401-404. DOI URL
[32]	Pavelescu I, Vilarrasa-Blasi J, Planas-Riverola A, González-García MP, Caño-Delgado AI, Ibañes M ( 2018). A Sizer model for cell differentiation in Arabidopsis thaliana root growth. Mol Syst Biol 14, e7687. DOI URL PMID
[33]	Richmond PA, Métraux JP, Taiz L ( 1980). Cell expansion patterns and directionality of wall mechanical properties in Nitella. Plant Physiol 65, 211-217. DOI URL PMID
[34]	Shabala S, Babourina O, Newman I ( 2000). Ion-specific mechanisms of osmoregulation in bean mesophyll cells. J Exp Bot 51, 1243-1253. URL PMID
[35]	Shabala SN, Lew RR ( 2002). 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. Plant Physiol 129, 290-299. DOI URL PMID
[36]	Takatsuka H, Umeda M ( 2014). Hormonal control of cell division and elongation along differentiation trajectories in roots. J Exp Bot 65, 2633-2643. DOI URL PMID
[37]	Ursache R, Andersen TG, Marhavý P, Geldner N ( 2018). A protocol for combining fluorescent proteins with histological stains for diverse cell wall components. Plant J 93, 399-412. URL PMID
[38]	Verbelen JP, de Cnodder T, Le J, Vissenberg K, Baluška F ( 2006). The root apex of Arabidopsis thaliana consists of four distinct zones of growth activities. Plant Signal Behav 1, 296-304. DOI URL PMID
[39]	Villani TS, Koroch AR, Simon JE ( 2013). An improved clearing and mounting solution to replace chloral hydrate in microscopic applications. Appl Plant Sci 1, apps. 1300016. DOI URL