拟南芥花器官和角果的全装清和染色

Summary

在本议定书中, 我们描述了适当解剖拟南芥花和角果的技术, 一些基本的清洁技术, 以及对生殖结构的全装观察的染色程序。

Abstract

由于其强大的分子遗传学研究工具,拟南芥是植物生物学中最突出的模型物种之一, 尤其是植物繁殖生物学。然而, 植物形态学, 解剖和超微结构分析传统上涉及耗时的嵌入和切片程序, 明亮的领域, 扫描和电子显微镜。最近在共焦荧光显微术, 先进的3维计算机辅助显微分析和不断细化的分子技术, 用于微加工的全装标本, 已导致增加的需求开发高效、最小的样品处理技术。在本议定书中, 我们描述了正确解剖拟南芥花和角果的技术, 基本的清除技术, 以及对生殖结构进行整体观测的一些染色程序。

Introduction

花卉是被子植物最重要的定义器官之一。开花植物出现了一些90–130百万年前的¹, 和多样化如此之快, 他们迅速的外观被描述为一个 “可恶的神秘” 由查尔斯·达尔文²。植物研究人员在花卉开发中的兴趣是多种多样的。一些研究集中于了解花的进化起源, 或特定的解剖, 结构和功能性质的发展花卉^3,4,5,6.花卉形态和结构的高度变异, 以及依赖于它们的性和无性繁殖模式, 使花卉具有高度复杂的结构。这导致了不同的努力描绘花卉器官的解剖和结构特征, 使用光和电子显微技术, 可以结合基因和分子调查⁷。此外, 作为水果和种子的来源, 花卉对人类和动物的营养至关重要。因此, 花卉和果实发育的特征对应用研究有许多影响, 包括在不断变化的环境下为日益增长的人类人口和生态保护战略提供粮食保障^8,9,10。

在拟南芥中的花发育始于花诱导和营养分生组织向花序 (花) 分生的转化。花分化在花序分枝¹¹的侧面侧向展开。花器官分化从外到花的中心逐渐形成同心轮生, 最终发育成萼片、花瓣、雄蕊和心皮⁷。这些花器官在不同的植物种类履行独特的营养, 保护和功能 (例如, 授粉吸引力) 角色, 与性器官保持男性和女性配子体发育的发展,^分别12^,13. 依次, 配子体发育分别区分了一对雄性 (精子) 和雌性配子 (卵和中央细胞), 它们结合在双重受精形成下一代, 受精卵和初生胚乳, 一个终端组织支持开发胚胎^14,15。果实和种子的发育支持胚胎的生长、成熟和保存, 最终它的扩散。已经进行了广泛的研究, 以描述不同植物种类的花和胚胎发育, 特别是模型物种拟南芥^7,16,17。

早期显微分析花卉开发是基于耗时的样品处理和观察技术, 如石蜡或树脂嵌入和切片, 结合光或电子显微镜。这些传统的显微技术通常与分子遗传学研究相结合, 如突变体的显微分析, RNA 的局部化通过原位杂交, 或蛋白质的免疫检测。最近在广域和共焦荧光显微学, 在先进的3维计算机辅助图像分析, 和不断细化的分子方法, 可用于微加工的全装标本, 已导致需要高效、最小的样品处理技术, 优先于定量分析。近年来, 对全芒动物标本的清洁技术的发展取得了长足的进展。它们通过使用水性尿素或糖基试剂 (例如、刻度、SeeDB、立方)^18、19、20或选择性地去除脂质 (使用洗涤剂 SDS), 使样品透明在稳定水凝胶中嵌入样品;脂质的去除可以通过被动扩散 (例如、修改的清晰度协议²¹、协议-边缘-轮辋²²) 或通过电泳 (原始清晰度协议²³和动作的²⁴) 来实现。在这种快速进步的鼓舞下, 一些衍生技术也正在涌现, 用于植物。

在本方法中, 本文重点介绍了模型拟南芥, 我们描述了正确解剖花芽、花朵和幼角果的程序, 并对各种染色和观察程序的全贴装样品进行了清理, 使用经典或最新的基于 SDS 的结算方法。给出了淀粉、胼胝和染色质染色的例子。虽然这些程序可能需要进一步的改进和适应, 当与其他物种使用, 我们希望他们将为进一步研究这些简单但关键的方法, 这是许多研究项目的出发点。

Protocol

1. 花和角果固定 从植物中收获的花朵和角果在第一朵花的开头同步。注: 在这里使用的实验条件下, 植物开始开花大约21天后, 从 Murashige 和 Skoog (MS) 板块移植到土壤。种子被分层为3–4天在4°c 并且发芽或生长在 MS 板材在22°c/16 h 光和18°c/8 h 黑暗期间8到10天, 在被保留的盆栽下在营养丰富的土壤的幼苗在同样条件下 (图 1)。复制的数量取决于具体的研究目标, 但推荐?…

Representative Results

拟南芥属于 Brassicacea 家族, 具有伞房花序(图 1) 中排列的两性花的花序。每朵花有四萼片, 四个花瓣, 六雄蕊 (四长和两个短), 和一个合心皮雌蕊由两个先天融合的心皮 (图1F-H) 安排在四同心螺纹 25, 26.拟南芥有 tenuinucellate, 胚珠胚珠排列在顶叶胎的两行 (每行?…

Discussion

在整个花发育阶段, 在拟南芥的单个花序内存在许多花芽, 这为旨在表征治疗效果或发育特征的研究提供了独特的机会。同时跨越不同阶段的花卉发展。一个好参考点在不同的各自的植物之间是开头第一朵花的主要花序。植物被对待以这样方式开花是尽可能同步的 (例如, 3–4天分层在4°c 最大化同步种子发芽并且因而更加甚而开花), 并且只有在同一天打开第一朵花的植物是在处理和复?…

Materials

Reagents and Materials
Ethanol	Scharlau	ET00102500
Acetic Acid	Applichem	A3686,2500	100% Molecular biology grade
Glacial Acetic Acid	Sigma-Aldrich	320099	Molecular Biology Grade
Methanol	Scharlau	ME03062500
Formaldehyde Solution	Sigma-Aldrich	F1635
Propionic acid	Sigma-Aldrich	81910-250 ml
Chloral hydrate	Sigma-Aldrich	15307
Glycerol	Roth	3783.1
Gum arabic	Fluka	51198
Lactic acid	Fluka	69773
Phenol	Sigma-Aldrich	77607-250ML	We used liquid phenol (use the density to find the required volume for your solution)
Clove oil	Sigma-Aldrich	C8392-100ML
Xylene	Roth	4436.1
Iodine	Fluka	57665
Potassium iodide	Merck	5043
Malachite Green	Fluka	63160
Fuchsin acid	Fluka	84600
Orange G	Sigma	7252
Sodium Dodecyl Sulfate	Sigma-Aldrich	L3771	Molecular Biology Grade
Sodium hydroxide	Sigma-Aldrich	71690
Sodium di-Hydrogen Phosphate	Applichem	A1047,1000
Sodium phosphate dibasic	Sigma-Aldrich	S9763-1KG
Potassium phosphate	Sigma-Aldrich	04347
EDTA	Applichem	A2937,1000
Calcofluor	Sigma	F6259	Fluorescent brightener 28
Auramine	Chroma	10120
DAPI	Sigma	D9542	toxic
Triton-X-100	Sigma	T8787
Aniline blue	Merck	1275
MS medium	Carolina	19-57030
Nutrient-rich substrate	Einheitserde	ED73
Watch maker's glass			No specific brand
15 ml falcon centrifuge tubes	VWR	62406-200
Dumond Forceps	Actimed	0208-5SPSF-PS
Forceps	DUMONT BIOLOGY	0108-5
Syringe	BD	BD Plastipak 300013	1 ml
Preparation needle	BD	BD Microlance 304000
Microscope slides	Thermo Scientific	10143562CE	cut edges
Coverslips	Thermo Scientific	DV40008
Humid box			A plastic box with damp paper towel and slide supports inside
Name	Company	Catalog Number	Comments
Solutions
Fixatives
Carnoy's (Farmer's) fixative			Absolute ethanol : glacial acetic acid, 3:1 (ml:ml)
Methanol/acetic acid fixative			50 % (v/v) methanol, 10 % (v/v) glacial acetic acid in deionized water
FPA50 fixative			Formalin, propionic acid, 50% ethanol; 5:5:90 (ml:ml:ml)
Clearing solutions
Chloral hydrate/glycerol			Chloral hydrate : glycerol : water, 8:1:2 (g:ml:ml). Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50
Modified Hoyer			Gum arabic 7.5 g, chloral hydrate 100 g, glycerol 5 ml , water 30 ml. Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50
Herr's 4½ clearing fluid			Lactic acid, chloral hydrate, phenol crystals, clove oil, xylene; 2:2:2:2:1, by weight. Can be used for all flower developmental stages (especially for stamen development) and for silique development with DIC microscopy. The best fixative is the formaline based FPA50
SDS/NaOH solution			Mix-dilute the the SDS and the NaOH stock solution to 1% SDS / 0.2 N NaOH (10x dilution). For all stages of flower and silique developmental stages. The best fixative is the methano/acetic acid fixative; the other two fixatives can also be used. Can be combined with calcofluor, auramine, DAPI, and aniline blue staining solution.
SDS stock solution			10 % (w/v) sodium dodecyl sulphate. Dissolve 10 g sodium dodecyl sulphate in 80 ml deionized water and make up to 100 ml with deionized water.
NaOH stock solution			2 N NaOH solution: dissolve 4 g of NaOH in 40 ml of deionized water and make up to 100 ml with deionized water
Combined clearing and staining solutions
Herr's IKI-4½			To a standard 4½ (9 g in total) add: 100 mg iodine, 500 mg potassium iodide. This clearing solution can be used for all flower developmental stages and for silique development, either for increasing contrast or for characterizing starch dynamics. Use FPA50 for structural analysis and Carnoy's fixative for quantitative starch analysis.
Alexander staining			Ethanol 95% 10ml, malachite green (1% in 95% EtOH) 1 ml, fuchsin acid (1% in ddH2O) 5ml, orange G (1% in ddH2O) 0.5ml, phenol 5g, chloral hydrate 5g, glacial acetic acid 2ml, glycerol 25ml . This clearing/staining alone or in combination with Herr's 4½ solution can be used to evaluate pollen abortion in flowers with mature and tricellular pollen grains. It's used on freshly harved non-fixed material.
Staining solutions
Calcofluor solution			Calcofluor 0.007% in water (g:ml). Originally used as an optical brightner. Can be used for staining cellulose, carboxylated polysaccharides and callose in cell walls. Frequently used to stain the intine of the pollen grain. All three fixatives can be used with this solution.
Auramine solution			Auramine 0.01% in water (g:ml). This lipophilic fluorscent dye can be used for staining cuticles, cutin, and exine among others. All three fixatives can be used with this solution
Calcofluor-Auramine mixture			Auramine solution : Calcofluor solution, 3:1. Can be used for a combined staining by both solutions. Other proportions can be assayed maintaining a smaller proportion of calcofluor with respect to auramine.
DAPI solution			DAPI 0.4 ug/ml, 0.1 M sodium phosphate buffer (pH 7), 0.1% Triton-X-100, 1 mM EDTA. This solution can be used for staining chromosome spreads during male and female meiosis, and cell nuclei of any tissue. Frequently used for studying pollen grain development. Carnoy's and methanol/ acetic acid are the best fixatives for this solution. Formaldehyde-based fixatives such as FPA50 may interfere with the staining. Excitation in the UV and maximum emission around 461 nm.
Sodium phosphate buffer (0.1 M)			Proton receptor: 0.2 M Na2HPO4, proton donor: 0.2 M NaH2PO4, ratio proton donor / proton receptor: 1.364 ( for a pH 7)
Aniline blue solution			0.1% (w/v) aniline blue, 108 mM K3PO4 (pH 11), 2% glycerol. This solution can be used for staining callose and cellulose of many stages of development (e.g callose deposition in male and female terads, callose plugs in pollen tubes). Excitation in the UV and maximum emission around 455. It can also be excited at 514 nm with emission in the red for cell content staining.