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Preparation of Mitotic and Meiotic Metaphase Chromosomes from Young Leaves and Flower Buds of Coccinia grandis
用红瓜幼叶和花蕾制备有丝分裂和减数分裂中期染色体   

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参见作者原研究论文

本实验方案简略版
BMC Plant Biology
Dec 2014

Abstract

Somatic chromosomes are usually studied from the root tip cells of the plants for cytological investigations. In dioecious plant species like Coccinia grandis, it is very difficult to get meristematic root tip cells from the mature plants of the respective sex forms. In this report, young leaves of the respective sexual phenotypes were used as tissue samples for mitotic chromosome analysis. For meiotic preparation, flower buds of appropriate size were selected for chromosomal studies. Following protocols could be effectively used for routine chromosome preparations in other plant species as well.

Keywords: Sex chromosomes (性染色体), Mitosis (分裂), Meiosis (减数), Dioecious (dioecious), Coccinia grandis (coccinia巨)

Materials and Reagents

  1. Blotting papers
  2. Glass slides and cover-slips
  3. Specimen tube
  4. 1st, 2nd and 3rd leaf from the shoot apical region for mitotic study
    Note: Young leaves can be harvested from seedlings as well as from mature plants.
  5. Flower buds of 7 and 8 stages for meiotic study
    Note: Assigned according to the length of flower buds.
  6. Saturated solution of para-dichlorobenzene (Sigma-Aldrich, catalog number: D56829 )
    Note: It is also named “1,4-Dichlorobenzene” on Sigma-Aldrich website.
  7. Glacial acetic acid (SRL, catalog number: 85801 )
  8. Hydrochloric Acid (HCl)
  9. Orcein (Sigma-Aldrich, catalog number: O7380 )
  10. Carmine (Sigma-Aldrich, catalog number: C1022 )
  11. Absolute ethanol (99%) (Merck Millipore Corporation, catalog number: 100983 )
  12. 2% w/v aceto-orcein stain (see Recipes)
  13. 1% w/v aceto-carmine stain (see Recipes)

Equipment

  1. Compound microscope (ZEISS, model: Primo-star )
  2. Refrigerator (household refrigerator having a freezer compartment with sub-zero temperature)

Procedure

  1. Mitotic metaphase chromosome study (Video 1)

    Video 1. The MS-preparation of mitotic and meiotic metaphase chromosomes

    1. Selected young leaves (Figure 1A) from healthy plants grown in experimental garden were collected at 9-10 AM for pre-treatment.
    2. Only apical region of the young leaves was cut (Figure 1B) and kept in a specimen tube containing cold saturated solution of para-dichlorobenzene for 5 min at 0 °C (in freezer compartment of normal household refrigerator) followed by 6 h pre-treatment at 10-12 °C (in the freeze compartment of normal household refrigerator).
    3. Pre-treated leaf tips were then thoroughly washed with distilled water and fixed in acetic acid: ethanol mixture (1:3) for overnight at room temperature.
    4. Leaf tips were transferred in 45% acetic acid for 15 min and then washed with distilled water, followed by hydrolysis with 5 N HCl for 20 min in refrigerator.
    5. Leaf tips were again thoroughly washed with distilled water and then immersed in 45% acetic acid for 5 min followed by staining with 2% aceto-orcein for overnight.
    6. Leaf tips were then squashed and mounted in 45% acetic acid (by putting a coverslip on leaf tip and applying gradual pressure with thumb in order to spread out the cells) and observed under a microscope.
    7. Photomicrographs were taken with Zeiss primo-star Microscope (any compound microscope can be used having 40x or 100x objective with oil immersion) and suitably enlarged (Figure1 C-E).


      Figure 1. A-B. Tissue samples for mitotic chromosome study in Coccinia grandis; A. 1st, 2nd and 3rd leaves were selected for pre-treatment; B. Only apical regions of young leaves were selected and the basal regions were discarded; C. Mitotic metaphase male plant; D. Female plant; E. Gynomonoecious plant. Scale bar=5 µm

  2. Meiotic metaphase chromosome study
    1. Flower buds of seventh and eighth developmental stages (Figure 2A-B) were collected directly from the field at 11-11:30 AM during April-May.
    2. Sepals and petals were removed from the selected flower buds and the anthers were kept in 45% acetic acid for 5 min.
    3. A part of the fused anthers was smeared in a drop of 2% aceto-carmine stain.
    4. After 10 min, the prepared slide was slightly warmed by passing over a flame.
    5. After 30 min, the stained cells were washed by adding drops of 45% acetic acid on one side of the cover glass and by drawing the access fluid from the opposite side of the cover glass with the help of a strip of blotting paper; this step is crucial for clearing the cytoplasmic background of the stained pollen mother cells.
    6. Finally, the prepared slide is wrapped in a fold of blotting paper to remove access of 45% acetic acid leaving only a thin film of fluid in between slide and cover-slip.
    7. Photomicrographs were taken with Leica Microscope and suitably enlarged (Figure 2C-D).


      Figure 2. A. Stages of male flower buds of Coccinia grandis for meiotic preparation; B. Gynomonoecious hermaphrodite flower buds; C. Meiotic metaphase chromosome of male (Arrow indicates end to end pairing of X and Y chromosomes.); D. Gynomonoecious plant. Scale bar=1 cm for A and B, 5 µm for C and D,

Notes

Material should be collected on a bright sunny day to ensure proper mitotic stage development. Cloudy or rainy day should be avoided for material collection.

Recipes

  1. 2% w/v aceto-orcein stain
    Add orcein powder to boiling 45% glacial acetic acid, cool rapidly, and then filter into a dark glass bottle using any laboratory grade filter paper.
  2. 1% w/v aceto-carmine stain
    Add carmine powder to boiling 45% glacial acetic acid, cool rapidly, and then filter into a dark glass bottle using any laboratory grade filter paper.

Acknowledgments

Financial support from Department of Biotechnology (DBT), Govt. of India is thankfully acknowledged. Core support from IISER Pune is also acknowledged.

References

  1. Bhowmick, B. K., Jha, T. B. and Jha, S. (2012). Chromosome analysis in the dioecious cucurbit Coccinia grandis (L.) Voigt. Chromosome Science 15: 9-15.
  2. Ghadge, A. G., Karmakar, K., Devani, R. S., Banerjee, J., Mohanasundaram, B., Sinha, R. K., Sinha, S. and Banerjee, A. K. (2014). Flower development, pollen fertility and sex expression analyses of three sexual phenotypes of Coccinia grandis. BMC Plant Biol 14: 325.
  3. Kumar, L. S. and Vishveshwaraiah, S. (1952). Sex mechanism in Coccinia indica wight and arn. Nature 170(4321): 330-331.
  4. Roy, R. P. and Roy, P. M. (1971). Mechanism of sex determination in Cocciniaindica. J Indian Bot Soc 50A: 391-400.
  5. Sousa, A., Fuchs, J. and Renner, S. S. (2013). Molecular cytogenetics (FISH, GISH) of Coccinia grandis: a ca. 3 myr-old species of cucurbitaceae with the largest Y/autosome divergence in flowering plants. Cytogenet Genome Res 139(2): 107-118.

简介

通常从植物的根尖细胞研究体细胞染色体用于细胞学研究。 在雌雄异种植物如Coccinia grandis中,很难从相应性别形式的成熟植物中获得分生组织根尖细胞。 在本报告中,各个性表型的幼叶用作有丝分裂染色体分析的组织样品。 对于减数分裂制备,选择适当大小的花芽用于染色体研究。 以下方案也可以有效地用于其他植物物种中的常规染色体制备。

关键字:性染色体, 分裂, 减数, dioecious, coccinia巨

材料和试剂

  1. 印刷纸
  2. 玻璃载玻片和盖玻片
  3. 试管
  4. 1 st ,2 nd 和3 rd 叶进行有丝分裂研究
    注意:年轻的叶子可以从幼苗和成熟的植物收获。
  5. 减数分裂研究的7和8阶段的花芽
    注意:根据花蕾的长度分配。
  6. 饱和的对二氯苯溶液(Sigma-Aldrich,目录号:D56829)
    注意:在Sigma-Aldrich网站上也将其命名为"1,4-二氯苯"。
  7. 冰醋酸(SRL,目录号:85801)
  8. 盐酸(HCl)
  9. Orcein(Sigma-Aldrich,目录号:O7380)
  10. 胭脂红(Sigma-Aldrich,目录号:C1022)
  11. 无水乙醇(99%)(Merck Millipore Corporation,目录号:100983)
  12. 2%w/v乙酰乳酸凝胶(见配方)
  13. 1%w/v胭脂红染色剂(见配方)

设备

  1. 复合显微镜(ZEISS,型号:Primo-star)
  2. 冰箱(具有零度以下的冷冻室的家用冰箱)

程序

  1. 有丝分裂中期染色体研究(视频1)

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    1. 在9-10AM收集来自生长在实验花园中的健康植物的选择的幼叶(图1A)用于预处理
    2. 只有年轻叶的顶端区域被切割(图1B)并保持 在含有冷饱和溶液的样品管中 对 - 二氯苯在0℃下5分钟(在正常的冷冻室中) ?家用冰箱),然后在10-12℃下预处理6小时(in 正常家用冰箱的冷冻室)。
    3. 然后用蒸馏水彻底洗涤预处理的叶尖 并在乙酸:乙醇混合物(1:3)中在室温下固定过夜 温度
    4. 将叶尖转移到45%乙酸中15分钟 ?然后用蒸馏水洗涤,随后用5水解 ?N HCl在冰箱中20分钟
    5. 叶提示了 用蒸馏水彻底洗涤,然后浸入45%乙酸中 酸处理5分钟,然后用2%乙酰辅酶素染色过夜。
    6. 然后将叶尖压扁并安装在45%乙酸中( 将盖玻片放在叶尖上并用拇指施加渐进压力 ?以展开细胞)并在显微镜下观察
    7. 显微照片用Zeiss primo-star显微镜(任何 可以使用具有40x或100x物镜与油的复合显微镜 浸没)和适当放大(图1C-E)



      图1。 A-B。 在Coccinia grandis中有丝分裂染色体研究的组织样品; A. 1 st , ?选择2个和3个叶片用于预处理; B.只有顶端 选择幼叶的区域,并且基底区域 抛弃C.有丝分裂中期雄性植物; D.雌性植物; E. 雌雄同株植物。比例尺=5μm

  2. 减数分裂中期染色体研究
    1. 第七和第八发育阶段的花芽(图2A-B) 在4月至5月的11-11:30 AM直接从田间收集
    2. 从选定的花芽除去萼片和花瓣,将花药保持在45%乙酸中5分钟。
    3. 将一部分融合的花药涂抹在一滴2%的胭脂红染色剂中。
    4. 10分钟后,将制备的载玻片通过火焰稍微温热
    5. 30分钟后,将染色的细胞通过滴加45% 乙酸在盖玻片的一侧上并通过拉动通路 借助于条带从盖玻璃的相对侧流体 ?的吸墨纸;这一步是清除细胞质的关键 被弄脏的花粉母细胞的背景。
    6. 最后, 将制备的载玻片包裹在吸墨纸的折叠中以除去通道 的45%乙酸,在载玻片之间仅留下流体的薄膜 和盖滑。
    7. 显微照片用Leica显微镜拍摄并适当放大(图2C-D)。




      图2. A。减数分裂的 Coccinia grandis 的雄花蕾阶段 ?制备; B.雌雄同株雌花。减数分裂 中期染色体(箭头指示X的端对端配对 和Y染色体。 D.雌雄同株植物。比例尺= 1厘米的A和 B,对于C和D为5μm,

笔记

材料应在明亮晴朗的日子收集,以确保适当的有丝分裂期发展。阴天或阴雨天应避免材料收集。

食谱

  1. 2%w/v乙酰乳酸凝胶染色 将奶粉加入沸腾的45%冰醋酸中,迅速冷却,然后使用任何实验室级滤纸过滤到深色玻璃瓶中。
  2. 1%w/v丙酮红染料
    将胭脂红粉末加入沸腾的45%冰醋酸中,快速冷却,然后使用任何实验室级滤纸过滤到深色玻璃瓶中。

致谢

生物技术部(DBT),政府的财政支持。的承认。还承认来自IISER Pune的核心支持。

参考文献

  1. Bhowmick,B.K.,Jha,T.B.and Jha,S。(2012)。 异种葫芦中的染色体分析 Coccinia grandis (L.)Voigt。 染色体科学 15:9-15
  2. Ghadge,A.G.,Karmakar,K.,Devani,R.S。,Banerjee,J.,Mohanasundaram,B.,Sinha,R.K.,Sinha,S.and Banerjee,A.K。 Coccinia grandis的三种性表型的花发育,花粉生育力和性表达分析 。 BMC Plant Biol 14:325。
  3. Kumar,L.S。和Vishveshwaraiah,S。(1952)。 Coccinia indica中的性机制 wight and arn。 < em> Nature 170(4321):330-331。
  4. Roy,R.P.and Roy,P.M。(1971)。 Cocciniaindica的性别决定机制。 J Indian Bot Soc 50A:391-400。
  5. Sousa,A.,Fuchs,J.and Renner,S.S。(2013)。 Coccinia grandis的分子细胞遗传学(FISH,GISH):在开花植物中具有最大Y /常染色体分歧的3个月龄的葫芦科植物。 Cytogenet Genome Res 139(2):107-118。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sinha, S., Karmakar, K., Devani, R. S., Banerjee, J., Sinha, R. K. and Banerjee, A. K. (2016). Preparation of Mitotic and Meiotic Metaphase Chromosomes from Young Leaves and Flower Buds of Coccinia grandis. Bio-protocol 6(7): e1771. DOI: 10.21769/BioProtoc.1771.
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