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Rice Meiotic Chromosome Spread Preparation of Pollen Mother Cells
从水稻花粉母细胞中分离与制备处于减数分裂期细胞染色体   

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Abstract

In this protocol, we describe a simple and efficient method for meiotic chromosome spread preparation in rice pollen mother cells. Meiotic chromosome preparation by spreading itself is an important technique for plant cytogenetics (Higgins et al., 2004; Chelysheva et al., 2012; Wang et al., 2009); furthermore, it is a crucial step for applying other cytogenetic methods including Fluorescence in situ hybridization (FISH) and immunostaining.

Materials and Reagents

  1. Young panicles containing meiocytes of rice (Oryza sativa ssp japonica cv. Zhonghua 11)
  2. 70%, 90% and 100% ethanol (Analytical Reagents)
  3. 60% (v/v) acetic acid (Analytical Reagents)
  4. Carmine (Sigma-Aldrich, catalog number: C1022-25G )
  5. Liquid nitrogen
  6. Vectashield Mounting Medium with DAPI (Vector Laboratories, catalog number: H-1200 )
  7. Carnoy’s fixative (see Recipes)
  8. Aceto-carmine (see Recipes)

Equipment

  1. Stereo microscope
  2. Fluorescence microscope (Zeiss, model: AX10 )
  3. Microscopic slides and cover slips
  4. Dissection needles and fine forceps
  5. Heating block
  6. CCD camera (Hamamatsu Photonics K.K., model: ORCA-R2 C10600 )

Procedure

  1. Fix young panicles by immersing whole young panicles (from 4 cm to 15 cm in length) at proper developmental stages into about 100 ml freshly prepared Carnoy’s solution at room temperature for 2-4 h and store the fixative materials in Carnoy's solution at 4 °C.
  2. Dissect about 15 florets under stereo microscope and remove all parts except anthers on a microscopic slide.
  3. Put the anthers into 20 μl Aceto-carmine stain on a slide, and keep cutting the anthers with a small scalpel until no obvious large debris were observed.
  4. Add 30 μl 60% (v/v) acetic acid to chopped anther on the slide and put the slide on a heating block at 50 °C for 2 min.
  5. Cover the slide with a piece of glass cover slip and put the slide and cover slip under a piece of filter paper, then press the cover slip down tightly with thumb for 10 sec to squash the meiocytes. Keep the slide at -20 °C for at least 30 min (keep the glass slides from cracking when it was placed into liquid nitrogen next step).
  6. Put the squashed slide into liquid nitrogen for 5 min and remove the cover slip with a scalpel.
  7. Dehydrate the slides for 2 min in 40 ml of 70% ethanol, 90% ethanol and 100% ethanol, sequentially.
  8. Add 15 μl VECTASHIELD Mounting Medium with DAPI on the air-dried slide, and seal the slide with a glass cover slip.
  9. Observe the chromosome spreads under fluorescence microscope and capture the images with CCD camera (Figure 1).

Representative data



Figure 1. Male meiotic chromosome spread by DAPI staining at pachytene (A) and diakinesis (B) in wild type. Bar= 5 μm

Recipes

  1. Carnoy’s fixative
    Ethanol: Glacial acetic acid 3: 1 (v/v)
    Freshly prepared
  2. Aceto-carmine
    0.5 g carmine was dissolved in 100 ml boiling 45% acetic acid

Acknowledgments

This protocol was adapted from the previously published paper Li et al. (2013), and was supported by the 863 Project Grant (2012AA10A303), the National Natural Science Foundation of China (31171441 and J1103510), the National Key Basic Research Developments Program (2013CB126900), the Fundamental Research Funds for the Central Universities, and the Program for New Century Excellent Talents in University.

References

  1. Chelysheva, L., Vezon, D., Chambon, A., Gendrot, G., Pereira, L., Lemhemdi, A., Vrielynck, N., Le Guin, S., Novatchkova, M. and Grelon, M. (2012). The Arabidopsis HEI10 is a new ZMM protein related to Zip3. PLoS Genetics 8(7): e1002799.
  2. Higgins, J. D., Armstrong, S. J., Franklin, F. C. and Jones, G. H. (2004). The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis. Genes Dev 18(20): 2557-2570.
  3. Li, X., Chang, Y., Xin, X., Zhu, C., Li, X., Higgins, J. D. and Wu, C. (2013). Replication protein A2c coupled with replication protein A1c regulates crossover formation during meiosis in rice. Plant Cell 25(10): 3885-3899.
  4. Wang, K., Tang, D., Wang, M., Lu, J., Yu, H., Liu, J., Qian, B., Gong, Z., Wang, X. and Chen, J. (2009). MER3 is required for normal meiotic crossover formation, but not for presynaptic alignment in rice. J Cell Sci 122(12): 2055-2063.

简介

在这个协议,我们描述一种简单和有效的方法,在水稻花粉母细胞减数分裂染色体传播准备。 通过铺展本身的减数分裂染色体制备是植物细胞遗传学的重要技术(Higgins等人,2004; Chelysheva等人,2012; Wang等人,/em>,2009); 此外,它是应用其他细胞遗传学方法包括荧光原位杂交(FISH)和免疫染色的关键步骤。

材料和试剂

  1. 含有水稻(Oryza sativa ssp japonica cv。Zhonghua 11)的细胞的幼穗
  2. 70%,90%和100%乙醇(分析试剂)
  3. 60%(v/v)乙酸(分析试剂)
  4. 胭脂红(Sigma-Aldrich,目录号:C1022-25G)
  5. 液氮
  6. 使用DAPI(Vector Laboratories,目录号:H-1200)的Vectashield封固剂
  7. Carnoy的固定剂(见配方)
  8. Aceto-carmine(见食谱)

设备

  1. 立体显微镜
  2. 荧光显微镜(Zeiss,型号:AX10)
  3. 显微镜载玻片和盖玻片
  4. 解剖针和细钳
  5. 加热块
  6. CCD照相机(Hamamatsu Photonics K.K.,型号:ORCA-R2 C10600)

程序

  1. 通过将整个幼小穗(从4​​厘米到15厘米长)以适当的发育阶段在室温下浸入约100毫升新鲜制备的Carnoy's溶液中2-4小时来固定幼穗,并将固定材料储存在4℃的Carnoy's溶液中。
  2. 在立体显微镜下解剖约15朵小花,除去显微镜载玻片上除花药外的所有部分
  3. 将花药放入载玻片上的20μlAceto胭脂红染色,并用小手术刀切割花药,直到没有观察到明显的大碎片。
  4. 加入30μl60%(v/v)乙酸在玻片上切碎花药,将载玻片放在50℃的加热块上2分钟。
  5. 用一块玻璃盖片盖住载玻片,将载玻片和盖片放在一张滤纸下,然后用拇指用拇指压下盖片10秒钟,压扁髓细胞。保持载玻片在-20°C至少30分钟(保持玻璃载片,当它被置于液氮下一步骤开裂)。
  6. 将压扁的幻灯片放入液氮中5分钟,并用手术刀去除盖玻片
  7. 在40ml的70%乙醇,90%乙醇和100%乙醇中依次将载玻片脱水2分钟。
  8. 在空气干燥的载玻片上加入15μl含DAPI的VECTASHIELD固定介质,并用玻璃盖片密封载玻片。
  9. 在荧光显微镜下观察染色体扩散,并用CCD照相机捕获图像(图1)。

代表数据



图1.通过DAPI在雄激素(A)和野生型的diakinesis(B)染色扩散的雄性减数分裂染色体。 Bar = 5μm

食谱

  1. Carnoy的固定剂
    乙醇:冰醋酸3:1(v/v)
    新鲜准备的
  2. 丙酮胭脂红
    将0.5g胭脂红溶于100ml沸腾的45%乙酸中

致谢

该协议改编自以前发表的论文Li (2013),并由863项目拨款(2012AA10A303),中国国家自然科学基金(31171441和J1103510), 国家重点基础研究发展计划(2013CB126900),中央大学基础研究基金,大学新世纪优秀人才计划。

参考文献

  1. Chelysheva,L.,Vezon,D.,Chambon,A.,Gendrot,G.,Pereira,L.,Lemhemdi,A.,Vrielynck,N.,Le Guin,S.,Novatchkova,M.and Grelon, (2012)。 拟南芥 em> HEI10是与Zip3相关的新的ZMM蛋白。

    8(7):e1002799。
  2. Higgins,J.D.,Armstrong,S.J.,Franklin,F.C.and Jones,G.H。(2004)。 拟南芥 MutS同源物AtMSH4在重组的早期阶段发挥作用:证据对于拟南芥中的两类重组。基因Dev 18(20):2557-2570。
  3. Li,X.,Chang,Y.,Xin,X.,Zhu,C.,Li,X.,Higgins,J.D.and Wu, 复制蛋白A2c与复制蛋白A1c偶联调节水稻减数分裂期间的杂交形成。 em> Plant Cell 25(10):3885-3899。
  4. Wang,K.,Tang,D.,Wang,M.,Lu,J.,Yu,H.,Liu,J.,Qian,B.,Gong,Z.,Wang,X.and Chen, 2009)。 正常减数分裂需要MER3 交叉形成,但不适用于水稻的突触前对齐。 122(12):2055-2063。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, X. and Wu, C. (2014). Rice Meiotic Chromosome Spread Preparation of Pollen Mother Cells. Bio-protocol 4(14): e1189. DOI: 10.21769/BioProtoc.1189.
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