木本植物非均质化组织石蜡切片制作方法

doi:10.11983/CBB18051

植物学报 ›› 2018, Vol. 53 ›› Issue (5): 653-660.DOI: 10.11983/CBB18051

木本植物非均质化组织石蜡切片制作方法

周乃富¹, 张俊佩^1,^*(), 刘昊², 查巍巍¹, 裴东¹

¹中国林业科学研究院林业研究所, 林木遗传育种国家重点实验室, 国家林业局林木培育重点实验室, 北京 100091
²天津市林业果树研究所, 天津 300112

收稿日期:2018-03-02 出版日期:2018-09-01 发布日期:2018-11-29
通讯作者: 张俊佩
作者简介:
† 共同第一作者。
基金资助:
燕山特色坚果新品种耐旱丰产技术研究集成与科技示范(No.Z161100000916011)和中国核桃优异种质遗传信息评价(No.RIF- 2013-01)

New Protocols for Paraffin Sections of Heterogeneous Tissues of Woody Plants

Zhou Naifu¹, Zhang Junpei^1,^*(), Liu Hao², Zha Weiwei¹, Pei Dong¹

¹Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry Chinese Academy of Forestry, Beijing 100091, China
²Tianjin Research Institute of Forestry and Pomology, Tianjin 300112, China

Received:2018-03-02 Online:2018-09-01 Published:2018-11-29
Contact: Zhang Junpei
About author:
† These authors contributed equally to this paper

摘要/Abstract

摘要： 在常规石蜡切片技术的基础上, 针对木本植物茎段木质化程度高、硬度大以及各部分组织硬度不均匀等特点, 选取核桃(Juglans regia)茎段以及芽接愈合区域组织为实验材料, 对固定、软化和脱水等关键步骤进行改进, 获得结构完整且染色清晰的茎段组织和嫁接愈合区域组织切片, 可清晰地观察到各部分组织的形态特征和愈合过程中的发育特征, 且缩短了制片周期。采用改良后的实验流程成功获得了苹果(Malus pumila)、桃(Amygdalus persica)、杏(Armeniaca vulgaris)、李(Prunus salicina)和杨(Populus tomentosa)的茎段横截面切片。该方法为从解剖学上研究林木茎段生长机制和形态发育变化提供技术基础, 为非均质化植物材料的石蜡切片制作提供参考。

关键词: 非均质化组织, 改良方案, 核桃, 石蜡切片

Abstract: In view of the characteristics of highly lignified, large hardness and heterogeneous of various parts of woody plants, the stem segments and healing areas of bud grafting of Juglans regia were used as materials with the conventional method of paraffin sectioning. The aspects of fixation, softening, dehydration and other critical steps were modified to obtain cross-section slices with complete organizational structure and clear staining. The morphological and developmental characteristics of the healing period could be observed clearly and shortened the experimental period. The paraffin sections of stem segments of apple, peach, apricot, plum and poplar were obtained by the improved experimental process. The study provides a technical basis for studying the growth mechanism and morphological development of stem segments of woody plants and provides a reference for making paraffin sections of heterogeneous plant materials.

Key words: heterogeneous plant tissue, improved protocol, Juglans regia, paraffin section

周乃富, 张俊佩, 刘昊, 查巍巍, 裴东. 木本植物非均质化组织石蜡切片制作方法. 植物学报, 2018, 53(5): 653-660.

Zhou Naifu, Zhang Junpei, Liu Hao, Zha Weiwei, Pei Dong. New Protocols for Paraffin Sections of Heterogeneous Tissues of Woody Plants. Chinese Bulletin of Botany, 2018, 53(5): 653-660.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18051

https://www.chinbullbotany.com/CN/Y2018/V53/I5/653

图/表 6

图1 核桃茎段完整横截面切片(A) 采用传统的FAA固定, 软化方法1处理; (B) 采用改良后的固定液, 软化方法1处理; (C) 采用传统的FAA固定, 软化方法2处理; (D) 采用改良后的固定液, 软化方法2处理。Bars=1 mm

Figure 1 The intact cross-section slice of walnut stem segment tissue(A) Traditional FAA fixative, according to softening method 1 treatment; (B) Improve fixative, according to softening method 1 treatment; (C) Traditional FAA fixative, according to softening method 2 treatment; (D) Improve fixative, according to softening method 2 treatment. Bars=1 mm

表1 不同固定液和软化方法对核桃切片效果的影响

Table 1 Effect of different types of fixation and softening on paraffin section of Juglans regia

	Combination 1	Combination 2	Combination 3	Combination 4
	FAA fixative solution Softening method 1	Improved fixative solution Softening method 1	FAA fixative solution Softening method 2	Improved fixative solution Softening method 2
Paraffin section	Partial cavity, divided and ripples	Continuous and ripples	Continuous and tidy	Continuous and tidy
Cortex	Brokenness and incomplete	Rupture and incomplete	Partial rupture and complete	Complete
Pith	Partial cavity and removed	Partly dehiscent, not detached	Complete	Complete
Transverse section	Fragmentary and incomplete with unclear outline	Shrinkage deformation, unclear outline	Partly complete with clear outline	Complete and smooth with clear outline

表2 两种不同制片方法关键步骤对比

Table 2 Comparison of key steps of two different slicing methods

Key steps of slicing	Traditional slicing method (method 1)		Improved slicing method (method 2)
Key steps of slicing	Reagent	Time (h)	Reagent	Time (h)
Fixation	FAA fixative	48	Improved fixative	48
Softening	70% ethanol-glycerol	288	Hydrogen peroxide-glacial acetic acid, 70% ethanol-glycerol	48
Dehydration	Different concentrations of ethanol solution	8.5	Different concentrations of tert butanol solution	6.5
Transparency	Ethanol, xylene	3	-	-

图2 不同处理方法得到的核桃茎段切片(A), (B) 采用传统的FAA固定, 软化方法1处理; (C), (D) 采用改良的固定液, 软化方法2处理。(B), (D)分别是(A), (C)的局部放大。(A), (C) Bars=200 μm; (B), (D) Bars=100 μm。CZ: 形成层带; Ph: 韧皮部; Xy: 木质部; Pi: 髓; PC: 薄壁细胞

Figure 2 The stem segment tissue slice of walnut with different treatment methods(A), (B) Traditional FAA fixative, according to softening method 1 treatment; (C), (D) Improve fixative, according to softening method 2 treatment. (B) and (D) are local amplification of (A) and (C). (A), (C) Bars=200 μm; (B), (D) Bars=100 μm. CZ: Cambium zone; Ph: Phloem; Xy: Xylem; Pi: Pith; PC: Parenchyma cell

图3 改进石蜡切片法的核桃方块芽接嫁接部位发育图(A)-(C) 嫁接后6天; (D)-(F) 嫁接后12天。(B), (C)和(E), (F)分别是(A)和(D)的局部放大。(A), (D) Bars=1 mm; (B), (C), (E), (F) Bars=500 μm。CF: 愈伤组织细胞形成; SC: 接穗; ST: 砧木; NL: 坏死细胞层; DVB: 发育中的维管组织

Figure 3 The sliced section of walnut bud healing area using the improved paraffin section method(A)-(C) 6 days after grafted; (D)-(F) 12 days after grafted. (B), (C) and (E), (F) are local amplification of (A) and (D). (A), (D) Bars=1 mm; (B), (C), (E), (F) Bars=500 μm. CF: Callus formation; SC: Scion; ST: Stock; NL: Necrotic layer; DVB: Developing vascular bundles

图4 改进石蜡切片法获得的核桃、苹果、桃、杏、李和杨的茎段切片(A)-(F) 分别为核桃、苹果、桃、杏、李和杨的茎段切片; (G)和(H)、(I)和(J)、(K)和(L)、(M)和(N)、(O)和(P)、(Q)和(R)分别为(A)-(F)的局部放大。(A), (D), (E) Bars=1 mm; (G), (M), (O) Bars=500 μm; (H)-(L), (N), (P)-(R) Bars=200 μm

Figure 4 The sliced sections of walnut, apple, peach, apricot, plum and poplar were obtained by improved paraffin section method(A)-(F) Paraffin sections of stems of walnut, apple, peach, apricot, plum and poplar; (G) and (H), (I) and (J), (K) and (L), (M) and (N), (O) and (P), (Q) and (R) are local amplification of (A)-(F). (A), (D), (E) Bars=1 mm; (G), (M), (O) Bars=500 μm; (H)-(L), (N), (P)-(R) Bars= 200 μm

参考文献 24

1	邓传远, 郑俊鸣, 张万超, 郭素枝, 薛秋华, 叶露莹, 孙建文 (2015). 红海榄木材结构的生态解剖. 植物生态学报 39, 604-615.
2	丁平海, 郗荣庭, 李金空 (1989). 核桃愈伤组织形成条件及形成部位的解剖观察. 河北农业大学学报 12(1), 27-33.
3	冯金玲 (2011). 油茶芽苗砧嫁接体愈合机理研究. 博士论文. 福州: 福建农林大学. pp. 22-28.
4	贺冰, 李志岗, 郝晓娟, 贺运春 (2014). 植物材料快速石蜡制片方法. 植物学报 49, 203-208.
5	侯春春, 徐水 (2009). 浅析影响石蜡切片质量的关键因素. 中国农学通报 25(23), 94-98.
6	黄坚钦, 章滨森, 陆建伟, 付敢伟 (2001). 山核桃嫁接愈合过程的解剖学观察. 浙江林学院学报 18(2), 111-114.
7	李和平 (2009). 植物显微技术(第2版). 北京: 科学出版社. pp. 9-34.
8	李正理 (1987). 植物制片技术(第2版). 北京: 科学出版社. pp. 138-148.
9	林月惠, 李寒冰, 贺新强 (2001). 高度木质化材料的冰冻切片技术. 植物学通报 18, 118-120.
10	刘昊, 宋晓波, 周乃富, 马庆国, 裴东 (2017). 吲哚丁酸对核桃嫩枝扦插生根及内源激素变化的影响. 浙江农林大学学报 34, 1038-1043.
11	刘杏娥, 汪佑宏, 江泽慧, 费本华, 任海青, 田根林, 徐鑫 (2010). 黄藤材发育过程中维管束的变化. 西北林学院学报 25(2), 152-155.
12	鲁丹, 张瑞, 彭方仁, 任莺, 华宏 (2013). 红桤木扦插繁殖技术及生根过程的解剖学观察. 西南林业大学学报 33(2), 24-28.
13	牛田, 王厚新, 李承秀, 张林, 于永畅, 孙芳, 王长宪 (2012). 华北五角枫嫩枝扦插生根形态解剖学分析. 山东林业科技 42(5), 19-21.
14	权金娥, 朱海兰, 张春霞, 张胜, 赵忠 (2014). 四倍体刺槐茎段组织石蜡切片的制作方法. 西北林学院学报 29(3), 140-144.
15	王欣, 隋方功, 初庆刚 (2015). 成熟玉米秆组织结构切片方法的比较研究. 西北植物学报 35, 1052-1056.
16	吴华, 陈娉婷, 袁玲, 徐永荣, 陈龙清 (2011). 蕨类植物石蜡切片制作技术探讨. 湖北农业科学 50, 3767-3769, 3774.
17	吴清韩, 赵庆芳, 朱慧, 马瑞君 (2012). 植物高度木质化叶片表皮细胞制备方法. 植物学报 47, 422-426.
18	闫绍鹏 (2011). 欧美山杨杂种扦插生根的理化与分子机理研究. 博士论文. 哈尔滨: 东北林业大学. pp. 23-26.
19	杨虎彪, 李晓霞, 罗丽娟 (2009). 植物石蜡制片中透明和脱蜡技术的改良. 植物学报 44, 230-235.
20	杨捷频 (2006). 常规石蜡切片方法的改良. 生物学杂志 23, 45-46.
21	曾月星 (1991). 硬重木材的软化方法. 木材工业 5(2), 45-46, 32.
22	Coates PJ (1991). Paraffin section molecular biology: review of current techniques.J Histotechnol 14, 263-269.
23	Day RC (2010). Laser microdissection of paraffin-embedded plant tissues for transcript profiling. In: Hennig L, Köhler C, eds. Plant Developmental Biology: Methods and Protocols. Totowa: Humana Press. pp. 321-346.
24	Takahashi H, Kamakura H, Sato Y, Shiono K, Abiko T, Tsutsumi N, Nagamura Y, Nishizawa NK, Nakazono M (2010). A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection.J Plant Res 123, 807-813.

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木本植物非均质化组织石蜡切片制作方法

New Protocols for Paraffin Sections of Heterogeneous Tissues of Woody Plants

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