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[Bio101] Arabidopsis Pollen Tube Germination
[Bio101] 拟南芥花粉管萌发实验   

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Abstract

This method uses a PEG-supplemented liquid solution to germinate separated Aradidopsis pollen. It thus eliminated the need for humidity control.

Keywords: Pollen tube(花粉管), Arabidopsis(拟南芥), In vitro(体外), PEG3350(PEG3350), In vitro germination(离体萌发)

Materials and Reagents
  1. Plant growth
    1. Stratify surface-sterilized seeds on 1x Johnson’s medium at 4 ˚C for at least 3 days to synchronize the flowering time. Transfer the plates to growth chamber with sufficient illumination (e.g. 12-16 h light/day, growth chamber on the 2nd or 3rd floor is OK).
    2. 1 week seedlings are transferred to soil pots. I usually grow up to 8 seedlings per standard pot. Mutant and wildtype should be put side by side to avoid position effect in the growth chamber. 
    3. Water the plants twice a week (Tuesday and Friday, for example), and apply 0.5x Johnson’s medium once every other week. The plants may begin to flower 3-4 weeks after transferring. The fresh flowers on the first inflorescence will be good for pollen germination experiments (note: The time to pick flowers is very critical).

  2. Preparation of germination plates
    1. Prepare germination medium plates as following (Fan et al., 2001)

      Stock
      Final Conc.
      V stock/40 ml
      MES-Tris (pH 5.8 adjusted with Tris base)
      200 mM
      5 mM
      1 ml
      KCl
      1 M
      1 mM
      40 μl
      MgSO4
      0.5 M
      0.8 mM
      64 μl
      Boric acid
      100 mM
      1.5 mM
      600 μl
      CaCl2
      0.5 M
      10 mM
      800 μl
      Sucrose

      5% w/v
      2 g
      PEG4000

      15% w/v
      6 g
      Note:
      1. Mix well when add MgSO4 and CaCl2 to avoid precipitation.
      2. 5 mM Ca might be better than 10 mM sometimes.
      3. You can make pollen germination medium (PGM) without Ca, use it to prepare the pollen resuspension. The unwanted germination before time 0 can be avoided this way.

Equipment

  1. Incubator with humidity control or saturated humidity
  2. Nikon microscope
  3. Scion Image (free download from NCBI website)
  4. 8-well chamber (Lab-Tek International, catalog number: 155411 or VWR International, catalog number: 43300-774)

Procedure

  1. Collect 20-50 freshly opened flowers (stage 13-15, in which the long filaments are just level with stigma and petals) in 1.5 ml tube, let dry on RT for 0.5 h (tube cap opened).
    Note: You can remove all open flowers from the plant 16-24 h before the pollen experiment. By this way just simply pick all flowers without spending time on identifying right stage (warning: wounding response may happen). Flowers picked in the morning are better than in afternoon.
  2. Add 1 ml germination medium to submerge the flowers. Vortex at maximal speed for 1 min.
  3. Concentrate the pollens by 500 x g for 5 min, at RT centrifuge (3,000 rpm on mini-centrifuge). Carefully remove the supernatant and floating flower residues, resuspend the pollen pellet in 1 ml germination medium (with Ca if you used –Ca PGM previously) by vortex. 
  4. Use 10 μl suspension for pollen amount estimation and purity check under light microscope (typical yield of 10 μl from 20 flowers is somewhere 2,000-5,000 grains).
  5. Germinate the pollen grains at 25-28 °C for 6 h or over night in the chambers of a chambered coverlip (200 μl pollen suspension/ 8-well chamber); no agitation. 
  6. Take photos of the germinated tubes using 10x lens on Nikon microscope (inverted is better). Since the tube is transparent, phase contrast will give good pictures.
  7. Analyze the tube germination (rate and length) using Scion Image. A normal distribution is expected for each population of pollen tube length, the P value should be less than 0.05 for student’s test.

References

  1. Fan, L. M., Wang, Y. F., Wang, H. and Wu, W. H. (2001). In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts. J Exp Bot 52(361): 1603-1614.
  2. Lalanne, E., Honys, D., Johnson, A., Borner, G. H., Lilley, K. S., Dupree, P., Grossniklaus, U. and Twell, D. (2004). SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required for pollen germination and tube growth in Arabidopsis. Plant Cell 16(1): 229-240.
  3. Mouline, K., Very, A. A., Gaymard, F., Boucherez, J., Pilot, G., Devic, M., Bouchez, D., Thibaud, J. B. and Sentenac, H. (2002). Pollen tube development and competitive ability are impaired by disruption of a Shaker K(+) channel in Arabidopsis. Genes Dev 16(3): 339-350.
  4. Thorsness, M. K., Kandasamy, M. K., Nasrallah, M. E. and Nasrallah, J. B. (1993). Genetic ablation of floral cells in Arabidopsis. Plant Cell 5(3): 253-261.

简介

该方法使用PEG补充的液体溶液来发芽分离的假sis花粉。 因此,消除了对湿度控制的需要。

关键字:花粉管, 拟南芥, 体外, PEG3350, 离体萌发

材料和试剂

  1. 植物生长
    1. 在1J Johnson's培养基上,在4℃下将表面灭菌的种子分层至少3天,以使开花时间同步。将板以足够的照明(例如12-16小时光/天,2或3楼的生长室可以)转移到生长室。
    2. 1周的幼苗转移到土壤盆中。我通常每个标准锅长达8个幼苗。突变体和野生型应并排放置,以避免在生长室中产生位置效应。
    3. 每周两次给植物浇灌植物(例如星期二和星期五),并每隔一周施用0.5x约翰逊的培养基。植物可能在转移后3-4周开始花。第一个花序上的鲜花对花粉萌发实验是很好的(注意:花时间非常重要的是)。

  2. 萌发板的制备
    1. 准备萌发培养基板如下(Fan et al。,2001)

      库存
      Final Conc。
      V库存/ 40 ml
      MES-Tris(用Tris碱调节pH 5.8)
      200 mM
      5 mM
      1 ml
      KCl
      1 M
      1 mM
      40μl
      MgSO 4
      0.5 M
      0.8 mM
      64μl
      硼酸
      100 mM
      1.5 mM
      600μl
      CaCl 2
      0.5 M
      10 mM
      800μl
      蔗糖

      5%w / v
      2 g
      PEG4000

      15%w / v
      6克
      注意:
      1. > 以避免降水。
      2. 有时候,5mM Ca可能优于10mM
      3. 您可以使花粉萌发培养基(PGM)不含Ca,用它来制备花粉再悬浮液。在这种情况下,可以避免在时间0之前不想要的萌发。

设备

  1. 具有湿度控制或饱和湿度的孵化器
  2. 尼康显微镜
  3. Scion Image(从NCBI网站免费下载)
  4. 8孔腔室(Lab-Tek International,目录号:155411或VWR International,目录号:43300-774)

程序

  1. 收集20-50新鲜开花(阶段13-15,其中长丝刚好与柱头和花瓣水平)在1.5毫升管中,在室温下干燥0.5小时(管帽打开)。
    注意:您可以在花粉实验前16-24小时从植物中清除所有开花。通过这种方式,只需选择所有的花朵,而不用花时间识别正确的阶段(警告:可能会发生伤员的反应)。早晨选择的鲜花比下午好。
  2. 加入1 ml萌发培养基淹没花。以最大速度涡旋1分钟。
  3. 在室温离心机(小型离心机上3000rpm)下将花粉浓缩500分钟5分钟。小心地去除上清液和漂浮的花残留物,将花粉沉淀物重新悬浮在1ml发芽培养基中(如果您以前使用-Ca PGM,则使用Ca)。
  4. 在光学显微镜下使用10μl悬浮液进行花粉量估计和纯度检查(20朵花的典型产量为10微克,约为2,000-5,000粒)。
  5. 在室温下,在25-28℃下将花粉粒发芽6小时或过夜(200微升花粉悬浮液/ 8孔室);没有激动。
  6. 使用Nikon显微镜上的10x镜头拍摄发芽管的照片(倒置更好)。由于管是透明的,相位对比将给出良好的图像。
  7. 使用Scion Image分析管萌发(速率和长度)。花粉管长度的每个群体预期正常分布,学生考试的P值应小于0.05。

参考文献

  1. Fan,L.M.,Wang,Y.F.,Wang,H.and Wu,W.H。(2001)。 体外拟南芥花粉萌发和表征内向钾电流< em>拟南芥花粉粒原生质体。 J Exp Bot 52(361):1603-1614。
  2. Lalanne,E.,Honys,D.,Johnson,A.,Borner,G.H.,Lilley,K.S.,Dupree,P.,Grossniklaus,U.and Twell,D。(2004)。 糖基磷脂酰肌醇锚定生物合成途径的两个组分的SETH1和SETH2是花粉萌发和管生长所必需的在拟南芥中。植物细胞 16(1):229-240。
  3. Mouline,K.,Very,A.A.,Gaymard,F.,Boucherez,J.,Pilot,G.,Devic,M.,Bouchez,D.,Thibaud,J.B。和Sentenac,H。(2002)。 花粉管发育和竞争能力受到震动K(+)通道扰乱的影响>拟南芥。 Genes Dev 16(3):339-350。
  4. Thorsness,M.K.,Kandasamy,M.K.Nasrallah,M.E.and Nasrallah,J.B。(1993)。 拟南芥中花细胞的遗传消融 >植物细胞 5(3):253-261。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, X. (2011). Arabidopsis Pollen Tube Germination. Bio-protocol Bio101: e73. DOI: 10.21769/BioProtoc.73;
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