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Limonium spp. are known to have sexual and apomixis (asexual reproduction through seeds) reproductive modes. Here, we present dissection protocol developed for ovules of Limonium spp. using differential interference contrast (DIC) microscopy. This protocol permits better handling of ovules and offers certain advantages over earlier techniques particularly in larger ovules. This method also enables observation of meiosis and embryo sac development in intact ovules, and the readily detection of events distinguishing sexual and apomictic development.

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Cytohistological Analyses of Mega-sporogenesis and Gametogenesis in Ovules of Limonium spp.
补血草属胚珠中大孢子发生和配子形成的细胞组织分析

植物科学 > 植物细胞生物学 > 细胞成像
作者: Ana S. Róis
Ana S. RóisAffiliation 1: Linking Landscape, Environment, Agriculture and Food (LEAF), Instituto Superior de Agronomia (ISA), Universidade de Lisboa, Tapada da Ajuda, Lisboa, Portugal
Affiliation 2: School of Psychology and Life Sciences, Universidade Lusófona de Humanidades e Tecnologias (ULHT), Campo Grande, Lisboa, Portugal
Bio-protocol author page: a3647
 and Ana D. Caperta
Ana D. CapertaAffiliation: Linking Landscape, Environment, Agriculture and Food (LEAF), Instituto Superior de Agronomia (ISA), Universidade de Lisboa, Tapada da Ajuda, Lisboa, Portugal
For correspondence: anadelaunay@isa.ulisboa.pt
Bio-protocol author page: a3648
Vol 6, Iss 21, 11/5/2016, 1059 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1983

[Abstract] Limonium spp. are known to have sexual and apomixis (asexual reproduction through seeds) reproductive modes. Here, we present dissection protocol developed for ovules of Limonium spp. using differential interference contrast (DIC) microscopy. This protocol permits better handling of ovules and offers certain advantages over earlier techniques particularly in larger ovules. This method also enables observation of meiosis and embryo sac development in intact ovules, and the readily detection of events distinguishing sexual and apomictic development.

[Background] To describe the events that occur during ovule development it is necessary to cytologically examine ovules. This study can involve microscopic observation of paraffin- or resin-embedded, sectioned material, or cleared organs. The first cytological investigations into ovule and embryo sac development in sexual and apomictic Limonium species were published in the pioneer works of D’Amato (1940; 1949). In these works, flowers were fixed using the Karpechenko’s method, embedded in paraffin, sectioned and stained with Heidenhain’s iron haematoxylin, which stains chromatin and chromosomes in the cell nuclei. Flower buds sectioning using these methods can result in preparations with poor quality, due to partial disruption structural integrity of individual cells. A more facile alternative is clearing formalin:acetic acid:ethyl alcohol fixed organs and staining with pure Mayer’s hemalum (Wallis, 1957; Stelly et al., 1984). This technique requires much less time and labor, particularly for species which usually only form a small ovule within the ovary, which is the case of Limonium spp. However, in both small and large ovules chloral hydrate worked better than methyl salicylate as a clearing solution, because in this latter fluid ovules become quite fragile and difficult to handle during experiments. Our approach with an enzymatic digestion of ovules helps to reveal the central mass of tissue, the nucellus and the two integuments it covers, particularly in large ovules. Examples of meiotic and ameiotic ovules and embryo sacs cleared in chloral hydrate were observed under differential interference contrast optics.

[Abstract]

Materials and Reagents

  1. Glass microscope slides (Belden, Hirschmann, catalog number: 8210101 )
  2. Diagnostic microscope multiwell slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 101432648 EPOXY )
  3. Glass coverslips (24 x 50 mm) (Belden, Hirschmann, catalog number: 8000119 )
  4. Teasing needle (BioQuip Products, catalog number: 4751 )
  5. Micropipette tips (2-20 μl, 20-200 μl, 100-1,000 μl) (Eppendorf)
  6. Six to eight months old Limonium plants in the flowering period
  7. Formaldehyde (36% aqueous solution) (VWR, catalog number: 20909.29 0)
  8. Glacial acetic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10304980 )
  9. Distilled H2O
  10. Ethanol (absolute 99.5%) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP2818500 )
  11. Hematoxylin solution, Mayer’s (Sigma-Aldrich, catalog number: MHS16 )
  12. Chloral hydrate (Sigma-Aldrich, catalog number: 15307 )
  13. Cellulase (Sigma-Aldrich, catalog number: C1184 )
  14. Cellulase ‘Onozuka R-10’ (Serva, catalog number: 16419 )
  15. Pectinase (Sigma-Aldrich, catalog number: 6287 )
  16. Citric acid-1-hydrate (Sigma-Aldrich , catalog number: 33114 )
  17. Sodium citrate dehydrate (AppliChem, catalog number: 131655 )
  18. FAA solution (see Recipes)
  19. Dehydration solutions (see Recipes)
  20. Hydration solutions(see Recipes)
  21. 0.1% chloral hydrate (see Recipes)
  22. Enzymatic mixture (see Recipes)
  23. 1x EB (Enzyme buffer) (see Recipes)

Equipment

  1. Forceps with extra fine tips (Rubis forceps [BioQuip Products, catalog number: 4524 ])
  2. Glass watch glasses (O.D. 25 mm and 40 mm) (Sigma-Aldrich)
  3. Stereo microscope (Leica, model: WILD M3Z LEITZ )
  4. Fluorescence microscope (Carl Zeiss, model: Axioskop 2 )
  5. Differential interference contrast (DIC) optics
  6. Micropipettes (2-20 μl, 20-200 μl, 100-1,000 μl) (Eppendorf)
  7. AxioCam 289 MRc5 digital camera (Carl Zeiss)

Software

  1. Adobe Photoshop 5.0

Procedure

  1. Collect flower buds at different developmental stages ranging from 2 to 6 mm in length (Figure 1A, left – young stage; right – mature stage) using forceps. Flower buds are fixed in 1 ml FAA solution in a watch glass for 24 h at 4 °C. The fixed material may be stored in 1 ml 70% ethanol at -20 °C until use for 3 months. To this end, after fixation the flower buds should be dehydrated in a series of water/ethanol (v/v) solutions, 30%, 50%, 70%, shaking for 30 min each before storage.
    Note: As the flowering season occurs during a short period of the year, storage can be useful to save flower buds for further use.
  2. Dissect pistils from flower buds with forceps onto a microscope slide using a stereomicroscope. Make a sharp longitudinal incision in the ovary (Figure 1B, incision arrowed) with a needle to help subsequent ovule fixation in 1 ml FAA solution for 14-18 h, at 4 °C.
    Note: Alternatively, big ovaries (> 5 mm) can be dissected directly in a drop (5 μl) of FAA, and then proceed to step 3.


    Figure 1. Dissection procedure and obtainment of digested ovules of Limonium spp. A. Flower buds at different developmental stages ranging from 2 to 6 mm (Figure 1A: left – young stage; right – mature stage). B. Pistil showing a longitudinal incision in the ovary (arrowed). C. Stained ovule immediately after dissection. D. Digested ovule mounted in a drop of chloral hydrate, in well of a multiwell slide.


    Figure 2. Examples of meiotic and ameiotic ovules and embryo sacs cleared in chloral hydrate and observed under differential interference contrast (DIC) optics. Figures A-E show sexual diploid (2n = 2x = 16) Limonium ovalifolium plants: accession numbers 2009I4SR (Sra. Remédios, Peniche), and 2009I1PL (Praia da Luz, Lagos), 2009I1PL (Praia da Luz, Lagos). Figures F-J were taken from apomictic tetraploid (2n = 4x = 35, 36) Limonium multiflorum plants: accession numbers 2009I4VF (Vale dos Frades, Lourinhã), 2009I9CR (Cabo Raso, Cascais), 2009I4VF (Vale dos Frades, Lourinhã). A. Megaspore mother cell at interphase with one nucleolus (arrowed). B. Coenomegaspore with four unreduced nuclei, each nucleus with two nucleoli (arrowed) (4-nucleated stage; tetrasporic development). C. Coenomegasporocyte at the beginning of gametogenesis with a cruciate nuclear arrangement. D. Megaspores in a bipolar 1 + 3 arrangement (arrowed) with the embryo sac showing extended vacuolization. E. Detail of an embryo sac of Adoxa-type with the egg cell and inferior polar nuclei arrowed. F. Megasporocyte within ovule showing one nucleolus (arrowed). G. Restitution nuclei after first meiotic division (meiotic diplospory). Nucleoli with distinct dimensions within the same nucleus are arrowed. H. Coenomegasporocyte at the beginning of gametogenesis. I. Detail of the egg cell apparatus (encircled by dashed lines) in a mature embryo sac; antipodal cells towards the chalazal pole are arrowed. J. Parthenogenetic embryo formation within a mature sac. Bars = 10 μm.
    Note: Each image is oriented so that the micropyle is towards the top of the page and the chalaza is towards the bottom. Figures abbreviations: e – embryo; ii – integuments; n – nucleus; nl – nucleolus; nc – nucellus; rn – restitution nucleus; Syn – synergid.

  3. Afterwards, hydrate pistils in watch glasses in a series of progressive 1 ml water/ethanol solutions, 50%, 75% and 100%, shaking for 30 min each.
  4. Stain pistils with 1 ml pure Hemalum Mayer’s solution for 24-48 h in a watch glass, and then place them in 1 ml 0.1% chloral hydrate in a new watch glass during 1-2 h, at room temperature.
    Note: If the ovaries are small (< 3 mm) 24 h in the staining solution are required; for bigger ones 48 h may be needed.
  5. Thereafter, dissect ovules from ovaries (Figure 1C) onto a glass slide under a stereomicroscope. Then, place them in a watch glass for digestion with 500 μl of an enzyme mixture 2% (w/v) cellulase, 2% (w/v) cellulase ‘Onozuka R-10’, and 2% (v/v) pectinase in 1x EB, for 10 (young stages) to 20 min (mature stages) at 37 °C, inside a box on a paper humidified with distilled water.
  6. Remove the enzyme mixture by a micropipette and wash ovules in a watch glass, in 1 ml 1x EB solution for 3-5 min, at room temperature.
  7. Finally, mount ovules in multiwell slides in a drop (5 μl) of chloral hydrate, under a coverslip (Figure 1D).
  8. When the procedure is completed, cytohistological observations are made using a fluorescence microscope with DIC optics using 20x and 100x objectives (Figure 2).
  9. Images are collected with a digital camera and if necessary further processed using Adobe Photoshop 5.0.

Data analysis

Female gametophytes of sexual and/or apomictic origin were determined in accessions from the ISA collections, and cytohistological observations were made under 20x and 100x magnification. For each species the development pattern ovules during megasporogenesis and gametogenesis were examined, and the number and frequency of tetrasporic versus diplosporous embryo sacs were evaluated (c. 700). In sexual species tetrasporic embryo sacs of Gagea-ova, Adoxa- and Drusa-types were found while apomictic species only produced diplosporous meiotically-unreduced eggs of the Rudbeckia-type. Details on numerical analysis of these ovules are published in Róis et al. (2016).

Recipes

  1. FAA solution
    Formaldehyde-acetic acid-ethyl alcohol in proportions of 1:1:18 (38.5% formaldehyde, 5% acetic acid, 70% ethanol)
    For 100 ml mix:
    5 ml of 38.5% formaldehyde
    5 ml of glacial acetic acid
    90 ml of 70% ethanol
    Note: Use always a fresh solution. Do not store it.
  2. Dehydration solutions
    30% EtOH-70% H2O
    50% EtOH-50% H2O
    70% EtOH-30% H2O
    Note: Make fresh solutions every three months.
  3. Hydration solutions
    50% EtOH-50% H2O
    25% EtOH-75% H2O
    100% H2O
    Note: Make fresh solutions every three months.
  4. 0.1% chloral hydrate
    For 50 ml mix:
    50 mg of chloral hydrate
    50 ml of distilled water
    Notes:
    a. Make a fresh solution every three months.
    b. Chloral hydrate is toxic if swallowed. Can be irritating to eyes and skin.
    c. Store in a cool, dry, well ventilated place, in securely closed original container.
  5. Enzymatic mixture
    For 20 ml:
    606 µl pectinase
    0.12 g celulase ‘Onozuka R10’
    1.10 g celulase Sigma C-1184
    Add 20 ml 1x PBS
    Note: Store 500 µl aliquots at -20 °C.
  6. 1x EB (enzyme buffer)
    For 100 ml mix:
    40 ml of 1 M citric acid-1-hydrate
    60 ml of 1 M sodium citrate dehydrate
    pH 4.8
    Note: 1x EB buffer should be kept at 4 °C for 1-2 weeks. For longer periods of time, buffer should be stored at -20 °C.

Acknowledgments

We thank Generosa Teixeira (Faculdade de Farmácia, University of Lisbon) for useful discussions with her on methods. FUNDAÇÃO PARA A CIÊNCIA E TECNOLOGIA provided financial support for this work under project AGR/04129 (LEAF).

References

  1. D’Amato, F. (1940). Contributo all’embriologia delle Plumbaginaceae. Nuovo Giornale Botanico Italiano 47(2): 349-382.
  2. D’Amato, F. (1949). Triploidia e apomissia in Statice oleaefolia Scop. var. confusa Godr. Caryologia 2(1): 71-84.
  3. Róis, A. S., Sadio, F., Paulo, O. S., Teixeira, G., Paes, A. P., Espirito-Santo, D., Sharbel, T. F. and Caperta, A. D. (2016). Phylogeography and modes of reproduction in diploid and tetraploid halophytes of Limonium species (Plumbaginaceae): evidence for a pattern of geographical parthenogenesis. Ann Bot 117(1): 37-50.
  4. Stelly, D. M., Peloquin, S. J., Palmer, R. G. and Crane, C. F. (1984). Mayer’s hemalum-methyl salicylate: a stain-clearing technique for observations within whole ovules. Stain Technol 59(3): 155-161. 
  5. Wallis T.E. 1957. Analytical microscopy. London: J & A Churchill.

材料和试剂

  1. 玻璃显微镜载玻片(Belden,Hirschmann,目录号:8210101)
  2. 诊断显微镜多孔载玻片(Thermo Fisher Scientific,Thermo Scientific ,目录号:101432648 EPOXY)
  3. 玻璃盖玻片(24×50mm)(Belden,Hirschmann,目录号:8000119)
  4. 试用针(BioQuip Products,目录号:4751)
  5. 微量吸头(2-20μl,20-200μl,100-1,000μl)(Eppendorf)
  6. 六至八个月的 Limonium 植物在开花期
  7. 甲醛(36%水溶液)(VWR,目录号:20909.290)
  8. 冰乙酸(Thermo Fisher Scientific,Fisher Scientific,目录号:10304980)
  9. 蒸馏的H 2 O 2 /
  10. 乙醇(绝对99.5%)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP2818500)
  11. 苏木精溶液,Mayer's(Sigma-Aldrich,目录号:MHS16)
  12. 水合氯醛(Sigma-Aldrich,目录号:15307)
  13. 纤维素酶(Sigma-Aldrich,目录号:C1184)
  14. 纤维素酶"Onozuka R-10"(Serva,目录号:16419)
  15. 果胶酶(Sigma-Aldrich,目录号:6287)
  16. 柠檬酸-1-水合物(Sigma-Aldrich,目录号:33114)
  17. 柠檬酸钠脱水物(AppliChem,目录号:131655)
  18. FAA解决方案(参见配方)
  19. 脱水解决方案(参见配方)
  20. 水合解决方案(参见配方)
  21. 0.1%水合氯醛(见配方)
  22. 酶混合物(见配方)
  23. 1x EB(酶缓冲液)(参见配方)

设备

  1. 具有超细尖端的镊子(Rubis镊子[BioQuip Products,目录号:4524])
  2. 玻璃手表眼镜(O.D.25mm和40mm)(Sigma-Aldrich)
  3. 立体显微镜(Leica,型号:WILD M3Z LEITZ)
  4. 荧光显微镜(Carl Zeiss,型号:Axioskop 2)
  5. 差分干涉对比(DIC)光学器件
  6. 微量移液管(2-20μl,20-200μl,100-1,000μl)(Eppendorf)
  7. AxioCam 289 MRc5数码相机(Carl Zeiss)

软件

  1. Adobe Photoshop 5.0

程序

  1. 收集花芽在不同的发育阶段长度从2至6毫米(图1A,左 - 年轻阶段;右 - 成熟阶段)使用镊子。将花芽在4℃下在表面玻璃中的1ml FAA溶液中固定24小时。固定材料可以在-20℃下储存在1ml 70%乙醇中,直到使用3个月。为此,在固定之后,花蕾应当在储存之前在一系列水/乙醇(v/v)溶液(30%,50%,70%)中脱水30分钟,每次振荡30分钟。 注意:由于开花季节发生在一年的短时间内,储存可以有助于节省花芽供进一步使用。
  2. 使用立体显微镜将来自花蕾的雌蕊用镊子解剖到显微镜载玻片上。使用针在卵巢(图1B,切口箭头)用尖锐的纵向切口,帮助后续胚珠固定在1毫升FAA溶液中14-18小时,在4℃。
    注意:或者,可以在FAA的一滴(5μl)中直接分离大卵巢(> 5mm),然后进行步骤3.


    图1.不同发育阶段花蕾在2至6mm范围内的花芽(图1A:左 - 年轻阶段;右 - 成熟阶段)。 B.雌蕊在卵巢中显示纵向切口(箭头)。 C.解剖后立即染色的胚珠。 D.消化的胚珠装在一滴水合氯醛中,在多孔玻片的孔中

    图2.在水合氯醛中清除并在微分干涉对比(DIC)光学下观察到的减数分裂和精子胚珠和胚囊的实例。图AE显示有性二倍体(2×n = 2 x = 16)绿叶植物:登录号2009I4SR(Sra.Remédios,Peniche)和2009I1PL(Praia da Luz,Lagos),2009I1PL(Praia da Luz,拉各斯)。图FJ取自单性生殖四倍体(2×n = 4×x = 35,36)多花青霉植物:登录号2009I4VF(Vale dos Frades ,Lourinh?),2009I9CR(Cabo Raso,Cascais),2009I4VF(Vale dos Frades,Lourinh?)。 A. Megaspore母细胞与一个核仁(箭头)相间。 B.具有四个未还原核的Coenomegaspore,每个核具有两个核仁(箭头)(4-成核阶段;四孢子发育)。 C. Coenomegasporocyte在gametogenesis的开始与十字形核安排。 D.双极1 + 3排列(箭头所示)的巨噬细胞与胚囊显示延长的空泡化。 E.具有卵细胞和下极性核的Adoxa 型胚胚囊的细节箭头。 F.胚珠中的巨细胞显示一个核仁(箭头)。 G.在第一次减数分裂(减数分裂期)后的恢复核。在相同细胞核内具有不同尺寸的核仁具有箭头。 H.coenomegasporocyte在gametogenesis的开始。 I.成熟胚囊中的卵细胞装置(由虚线包围)的细节;反足细胞朝向chalazal极箭头。 J.成熟囊内的单性胚发育。条=10μm。
    注意:每个图像的方向是使墨球朝向页面的顶部,chalaza朝向底部。图缩写:e-胚; ii - 珠被; n核;核仁;复原核; Syn - 协同。

  3. 然后,在一系列渐进的1ml水/乙醇溶液(50%,75%和100%)中的手表眼镜中水合雌蕊,每次摇动30分钟。
  4. 将1ml纯的Hemalum Mayer's溶液在表面玻璃中染色雌蕊24-48小时,然后在室温下1-2小时内将它们放在1ml 0.1%水合氯醛中,放在新的表面玻璃中。
    注意:如果卵巢小(<3mm),则需要染色溶液中24小时;对于较大的48小时可能需要。
  5. 此后,从卵巢解剖胚珠(图1C)到立体显微镜下的载玻片上。然后,将其置于表玻璃中,用500μl2%(w/v)纤维素酶,2%(w/v)纤维素酶"Onozuka R-10"和2%(v/v)果胶酶的酶混合物在1×EB中,在37℃,在用蒸馏水湿润的纸上的盒子内,10(幼龄期)至20分钟(成熟期)。
  6. 用微量移液管除去酶混合物,在室温下,在1ml 1x EB溶液中在表玻璃杯中洗涤胚珠3-5分钟。
  7. 最后,挂在胚珠在多孔载玻片上的一滴(5微升)的水合氯醛,在盖玻片下(图1D)。
  8. 当程序完成时,使用具有DIC光学的荧光显微镜使用20x和100x物镜进行细胞组织学观察(图2)。
  9. 使用数码相机收集图像,如有必要,使用Adobe Photoshop 5.0进行处理。

数据分析

在来自ISA集合的种质中确定性和/或无融合生殖起源的女性配子体,并且在20x和100x放大下进行细胞组织学观察。对于每个物种,检查在胚胎发生和配子形成期间的发育模式胚珠,并且评价四孢子与双孢子胚囊的数量和频率(c.700)。在有性生殖物种的四孢子胚囊中,发现了无鞭毛虫物种仅产生双孢子的减数分裂未被减数的卵的 gagea-ova ,和 drusa 的 rudbeckia 类型。这些胚珠的数值分析的细节公开在Róis等人中。 (2016年)。

食谱

  1. FAA解决方案
    甲醛 - 乙酸 - 乙醇(比例为1:1:18)(38.5%甲醛,5%乙酸,70%乙醇)
    对于100 ml混合:
    5ml 38.5%甲醛
    5ml冰乙酸 90毫升70%的乙醇 注意:始终使用新的解决方案。不要存储它。
  2. 脱水溶液
    30%EtOH-70%H 2 O v/v 50%EtOH-50%H 2 O v/v 70%EtOH-30%H 2 O 2 / 注意:每三个月清洁一次。
  3. 水合溶液
    50%EtOH-50%H 2 O v/v 25%EtOH-75%H 2 O v/v 100%H 2 O·
    注意:每三个月清洁一次。
  4. 0.1%水合氯化
    对于50ml混合物:
    50毫克水合氯醛
    50ml蒸馏水
    注意:
    a。每三个月清新一次。
    b。水合氯醛如果吞咽有毒。可刺激眼睛和皮肤。
    c。存放在阴凉,干燥,通风良好的地方,安全封闭的原装容器中。
  5. 酶混合物
    对于20ml:
    606μl果胶酶
    0.12g纤维素酶"Onozuka R10"
    1.10g纤维素酶Sigma C-1184
    加入20 ml 1x PBS
    注意:将500μl等分试样储存在-20°C。
  6. 1×EB(酶缓冲液)
    对于100 ml混合:
    40ml 1M柠檬酸-1-水合物 60ml的1M脱水柠檬酸钠
    pH 4.8
    注意:1x EB缓冲液应在4℃保存1-2周。较长时间,缓冲液应储存在-20°C。

致谢

我们感谢Generosa Teixeira(里斯本大学Faculdade deFarmácia)与她就方法进行有益的讨论。 FUNDA??OPARA A CIENCIA E TECNOLOGIA为项目AGR/04129(LEAF)下的这项工作提供了资金支持。

参考文献

  1. D'Amato,F。(1940)。  47 47(2):349-382。
  2. D'Amato,F。(1949)。  三叶草在 Scop中的脱落。 var。 confusa Godr。 Caryologia 2(1):71-84。
  3. Róis,AS,Sadio,F.,Paulo,OS,Teixeira,G.,Paes,AP,Espirito-Santo,D.,Sharbel,TF和Caperta,AD(2016)。< a class ="ke-insertfile "href ="http://www.ncbi.nlm.nih.gov/pubmed/26424783"target ="_ blank">珍稀物种(Plumbaginaceae)的二倍体和四倍体盐生植物的系统形成和繁殖模式):地理孤立生殖模式的证据。 117(1):37-50。
  4. Stelly,DM,Peloquin,SJ,Palmer,RG和Crane,CF(1984)。  Mayer's hemalum-methyl salicylate:a stain-clearing technique for observation within whole ovules。 Stain Technol 59(3):155-161。 />
  5. 瓦利斯T.E.分析显微镜。 London:J&丘吉尔。
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How to cite this protocol: Róis, A. S. and Caperta, A. D. (2016). Cytohistological Analyses of Mega-sporogenesis and Gametogenesis in Ovules of Limonium spp.. Bio-protocol 6(21): e1983. DOI: 10.21769/BioProtoc.1983; Full Text



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