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Depending on the species, embryo sacs can be difficult to observe. Ovule clearing allows precise observation of whole tissues under Differential interference contrast (DIC) microscopy. The use of Methyl Salicylate as a clearing agent has proved to be particularly reliable for Solanaceous species.

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Ovule Clearing Method for Solanaceous Species
茄属胚珠整体透明技术

植物科学 > 植物细胞生物学 > 细胞染色
作者: Audrey Loubert-Hudon
Audrey Loubert-HudonAffiliation: Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, Montréal, Québec, Canada
Bio-protocol author page: a943
 and Daniel P. Matton
Daniel P. MattonAffiliation: Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, Montréal, Québec, Canada
For correspondence: dp.matton@umontreal.ca
Bio-protocol author page: a944
Vol 3, Iss 21, 11/5/2013, 2932 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.951

[Abstract] Depending on the species, embryo sacs can be difficult to observe. Ovule clearing allows precise observation of whole tissues under Differential interference contrast (DIC) microscopy. The use of Methyl Salicylate as a clearing agent has proved to be particularly reliable for Solanaceous species.

Keywords: Solanum(茄), Ovule(胚珠), Reproduction(繁殖), Embryo sac(胚囊), Differential interference contrast (DIC)(微分干涉对比(DIC))

[Abstract]

Materials and Reagents

  1. Flower buds
  2. 37% Formaldehyde
  3. Glacial acetic acid
  4. Methyl Salicylate
  5. Ethanol (≥ 95%)
  6. Fixative solution (FAA) (see Recipes)
  7. Clearing solution (see Recipes)

Equipment

  1. Forceps
  2. Microscope with differential interference contrast (DIC)
  3. Printed coated microscopy slides with wells (to avoid crushing the tissues)
  4. Cover slips

Procedure

  1. Carefully collect ovaries from flower buds or flowers at anthesis with precision forceps. During the process, keep ovaries and flowers on ice.


    Figure 1. Organs and cells types of Solanaceous species’ flower and female gametophyte.  a. Longitudinal sections of a Solanaceous flower. b. Transversal sections of a Solanaceous flower. c. Description of ovule cells types and their visualization in cleared ovules (false colors are used to emphasize cells types).

  2. To facilitate penetration of the fixative and clearing solutions, the pericarp has to be removed. Under a magnifying glass, cut the top of the ovary’s pericarp with precision forceps or scalpel to allow its removal (see Figure 2).


    Figure 2. Steps by steps procedure to remove the pericarp of Solanaceous species
    a. Collect flowers at the desired stage of development and keep them on ice; b. Remove the upper part of the flower carefully by hand to keep only the ovary still attached to the pedicel; c. d. Cut the top of the ovary’s pericarp with precision forceps or scalpel to allow its removal; e. f. With the precision forceps, pinch under the bunch of ovules and put them directly in fixative solution.  

  3. A bunch of ovules can then be carefully taken and fixed in FAA solution overnight. Gentle agitation at 4 °C helps to homogenize the fixation, but is not crucial.
  4. The day after, fixed ovaries are incubated in 95-100% ethanol for at least one hour, still with gentle agitation at room temperature.
  5. The ovaries are then transferred in increasing ratio of Methyl Salicylate/EtOH solutions for 30 minutes each (1:3, 1:1, 3:1) at room temperature. Again, gentle agitation will help, but is not crucial.
  6. The tissues are afterwards left in 100% Methyl Salicylate at room temperature. Make sure the tubes are well closed to avoid Methyl Salicylate evaporation.
  7. The ovule can now be observed under differential interference contrast (DIC) microscopy. To avoid crushing the tissues, printed coated slides with wells are very useful. With precision forceps, carefully separate the ovules on the slide. Methyl Salicylate (100%) is used as mounting solution and slides are sealed with clear nail polish.

Recipes

  1. Fixative solution (FAA)
    1% formaldehyde
    0.5% glacial acetic acid
    50% ethanol
    Note: It can be prepared in advance and stored at room temperature.
  2. Clearing solution (see Procedure step 5, for concentration/ratio)
    Methyl Salicylate
    Ethanol

Acknowledgments

This protocol is adapted from Chevalier et al. (2013) and Estrada-Luna et al. (2004).

References

  1. Chevalier, E., Loubert-Hudon, A. and Matton, D. P. (2013). ScRALF3, a secreted RALF-like peptide involved in cell-cell communication between the sporophyte and the female gametophyte in a solanaceous species. Plant J 73(6): 1019-1033.    
  2. Estrada-Luna, A., Garcia-Aguilar, M. and Vielle-Calzada, J. P. (2004). Female reproductive development and pollen tube growth in diploid genotypes of Solanum cardiophyllum Lindl. Sexual Plant Reproduction 17(3): 117-124.

材料和试剂

  1. 花芽
  2. 37%甲醛
  3. 冰醋酸
  4. 水杨酸甲酯
  5. 乙醇(≥95%)
  6. 固定溶液(FAA)(参见配方)
  7. 清除解决方案(参见配方)

设备

  1. 镊子
  2. 微分干涉对比显微镜(DIC)
  3. 带孔的印刷涂层显微镜载玻片(以避免破碎组织)
  4. 盖玻片

程序

  1. 小心地收集卵巢从花蕾或花在开花的精密镊子。 在这个过程中,保持卵巢和花在冰上

    图1. 茄科物种的花和雌配子体的器官和细胞类型。 一个。 一种Solanaceous 花的纵向切片。 b。 花的横向部分。 C。 描述胚珠细胞类型及其在透明胚珠中的可视化(假色用于强调细胞类型)
  2. 为了促进固定剂和清除溶液的渗透,必须除去果皮。 在放大镜下,用精密镊子或手术刀切开卵巢的顶部,以允许其去除(见 图2)。


    图2.逐步操作步骤,以清除 物种 的种皮
    一个。收集花在所需的发展阶段,并保持在冰上; b。用手小心去除花的上部,保持只有卵巢仍附着在花梗上; C。 d。用精密镊子或手术刀切割卵巢的果皮顶部,以便去除; e。 F。用精密镊子,夹在胚珠下面并将其直接放在固定溶液中。  

  3. 然后可以小心地取出一束胚珠,并在FAA溶液中固定过夜。在4℃下温和搅拌有助于均质化固定,但不是至关重要的。
  4. 第二天,将固定的卵巢在95-100%乙醇中孵育至少1小时,仍然在室温下轻轻搅拌。
  5. 然后将卵巢在室温下以增加比例的水杨酸甲酯/EtOH溶液转移30分钟(1:3,1:1,3:1)。再次,温柔的激动会有所帮助,但并不重要。
  6. 组织随后在室温下留在100%水杨酸甲酯中。确保管道密封良好,以避免水杨酸甲酯蒸发
  7. 现在可以在微分干涉对比(DIC)显微镜下观察胚珠。 为了避免破碎组织,用孔印刷涂布的载玻片是非常有用的。 用精密镊子,仔细分离幻灯片上的胚珠。 水杨酸甲酯(100%)用作安装溶液,载玻片用清澈的指甲油密封。

食谱

  1. 固定溶液(FAA)
    1%甲醛
    0.5%冰醋酸 50%乙醇
    注意:可以提前准备并在室温下储存。
  2. 清除溶液(见过程步骤5,浓度/比率)
    水杨酸甲酯
    乙醇

致谢

该方案改编自Chevalier等人(2013)和Estrada-Luna等人(2004)。

参考文献

  1. Chevalier,E.,Loubert-Hudon,A.和Matton,D.P。(2013)。 ScRALF3,一种分泌的RALF样肽,涉及孢子体与雌性配子体之间的细胞间通讯 in a eman solanaceous species。 Plant J 73(6):1019-1033。    
  2. Estrada-Luna,A.,Garcia-Aguilar,M。和Vielle-Calzada,J.P。(2004)。 二倍体基因型中的雌性生殖发育和花粉管生长 > Solanum cardiophyllum Lindl。 性植物繁殖 17(3):117-124。
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How to cite this protocol: Loubert-Hudon, A. and Matton, D. P. (2013). Ovule Clearing Method for Solanaceous Species. Bio-protocol 3(21): e951. DOI: 10.21769/BioProtoc.951; Full Text



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