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

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

植物科学 > 植物生理学 > 组织分析
作者: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford , USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
5/20/2011, 10254 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.73

[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)

[Abstract] 该方法是使用PEG补充液体基来复苏拟南芥花粉,从而不需要考虑对湿度的控制。

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.

材料和试剂

 

1.        植物生长

1)       表面消毒的种子在1X Johnson’s培养基上4?C至少春化3天使开花时间同步. 将平板放入有效光照的生长室中(比如12-16光照/白天,生长室在二楼或三楼都没有问题)

2)       1周大小的幼苗移到盆土中生长. 我通常每一标准盆种八颗苗子. 突变体和野生型应该挨着放避免温室位置效应的影响.

3)       一周内浇两次水(比如周二和周五), 每隔一周用0.5X Johnson’s 培养基施肥一次. 幼苗在移苗后3-4周开始开花. 第一层花絮的新鲜的花朵是花粉萌发实验的好材料(注意:取花的时间非常重要).

2.        准备萌发的平板

如下所示准备萌发平板(from Fan et al. 2001):

 

Stock

Final Concentration

V stock/40ml

MES-Tris(pH5.8 adjusted with Tris base)

200mM

5mM

1ml

KCl

1M

1mM

40ul

MgSO4

0.5M

0.8mM

64ul

Boric acid

100mM

1.5mM

600 ul

CaCl2

0.5M

10mM

800 ul

Sucrose

 

5% w/v

2g

PEG4000

 

15%  w/v

6g

 

注意:

a)       当加入MgSO4 and CaCl2时充分混匀避免产生沉淀.

b)      有时5mM Ca 10mM 的效果好.

c)       可以制备没有Ca 的花粉萌发培养基(PGM) ,用它可以准备花粉悬浮.  0时间点前的不想要的萌发可通过这种方法避免.

 

设备

 

1.        带有湿度控制或饱和湿度的培养室.

 

步骤

 

1.        收集20-50个新鲜的开发的花朵 (阶段 13-15,在这个阶段长丝和气孔和花瓣水平)放到1.5ml离心管中,在室温下开盖干燥半小时.

注意:  在花粉实验16-24小时前你可以移除所有开放的花. 这种方法可以简单的选取所有的花避免在辨别正确阶段上花费时间(警告:创伤应答可能产生). 早上取花要比下午好一些.

2.        加入1ml萌发培养基浸没花朵.最大速度的涡旋1分钟.

3.        收集花粉在常温下500g 离心(3,000rpm小型离心机).

4.        小心的去除上清和漂浮的花残余,1 ml萌发培养基(加Ca如果你先前用Ca PGM)通过涡旋重悬花粉球. 10 ul 悬浮液用于花粉数量估计和在光显微镜下检查纯度 (标准情况下从20朵花中取10ul2000-5000个花粉)

5.        在一个分室coverlip 的室中25-28oC 6小时或过夜萌发花粉 (200 ul 花粉悬浮液/ 8- , Lab-Tek 155411 or VWR 43300-774). 不要震荡.

6.        用尼康显微镜10倍镜头拍萌发管(倒置效果更好). 因为管事透明的,相位差将拍摄更好的照片.

7.        分析管萌发(率和持续时间)用Scion Image (可以从NCBI 网站上免费下载). 每一个种群花粉管长度预计是一个标准正态分布, the P value 对于学生实验应该低于0.05.

 

References

 

1.        Fan L.M., Wang Y.F., Wang H., 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): 1603-14. 

2.        Lalanne E., Honys D., Johnson A., Borner G.H., Lilley K.S., Dupree P., Grossniklaus U., 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): 229-40. 

3.        Mouline K., Very A.A., Gaymard F., Boucherez J., Pilot G., Devic M., Bouchez D., Thibaud J.B., Sentenac H. (2002). Pollen tube development and competitive ability are impaired by disruption of a Shaker K(+) channel in Arabidopsis. Genes and Development 16(3): 339-50. 

4.        Thorsness M.K., Kandasamy M.K., Nasrallah M.E., Nasrallah J.B. (1993). Genetic Ablation of Floral Cells in Arabidopsis. Plant Cell 5(3): 253-61. 

 

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How to cite this protocol: Li, X. (2011). Arabidopsis Pollen Tube Germination. Bio-protocol Bio101: e73. DOI: 10.21769/BioProtoc.73; Full Text



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