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Measuring Auxin Transport Capacity in Seedling Roots of Medicago truncatula

测量蒺藜苜蓿幼苗根部的生长素运输能力

Jason Liang Pin Ng Jason Liang Pin Ng
UM Ulrike Mathesius

发布: 2016年06月20日第6卷第12期 DOI: 10.21769/BioProtoc.1842 浏览次数: 10240

评审: Marisa RosaScott A M McAdamMoritz Bomer

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Abstract

Measurement of auxin transport capacity provides quantitative data on the physiological machinery involved in auxin transport within plants. This technique is easy to perform and gives quick results. Radiolabelled auxin (indole-3-acetic-acid) is fed into the roots of Medicago truncatula via an agar block. The resulting radioactivity from radiolabelled auxin uptake in the roots is measured with a liquid scintillation counter. Here, we describe the measurement of auxin transport capacity around the nodulation susceptible zone in young seedling roots of M. truncatula in response to rhizobia inoculation. Similar assays could be adapted in other plant species and to answer other biological questions.

Keywords: Indole-3-acetic acid (吲哚乙酸) search Nodulation (结瘤) search Acropetal auxin transport (向顶生长素的极性运输) search Basipetal auxin transport (使用生长素极性运输) search

Materials and Reagents

  1. Petri dish (150 x 15 mm) (BD, catalog number: 351058 )
    Note: Currently, it is “Corning, catalog number: 351058”.
  2. Petri dish (35 x 10 mm) (BD, catalog number: 351008 )
    Note: Currently, it is “Corning, catalog number: 351008”.
  3. SandBlaster fine 320 grit sandpaper (3M, catalog number: 70-0710-1785-2 )
  4. 50 ml Falcon tube (Greiner Bio One International GmbH, catalog number: 227261 )
  5. Waste bottle
  6. Aluminium foil (Confoil)
  7. 1.5 ml Eppendorf tube
  8. Kimwipes
  9. 6 ml scintillation vials (Pico Prias vial) (PerkinElmer, catalog number: 6000192 )
  10. Parafilm strips
  11. Medicago truncatula seeds (cultivar Jemalong A17) (SARDI)
  12. Sinorhizobium meliloti (rhizobia) culture
  13. Sodium hypochlorite solution (Bleach) (Pelikan)
  14. Sterilised distilled water
  15. Ethanol (EMD Millipore Corporation, EMSURE®)
  16. Agarose (AMRESCO, catalog number: 0710 )
  17. Emulsifier-safe, 2 x 5 L (scintillation fluid) (PerkinElmer, catalog number: 6013389 )
  18. CaCl2.2H2O (Sigma-Aldrich, catalog number: C3306 )
  19. Magnesium sulfate heptahydrate (MgSO4.7H2O) (Sigma-Aldrich, catalog number: M1880 )
  20. KH2SO4 (Sigma-Aldrich, catalog number: P0772 )
  21. Na2HPO4.2H2O (Sigma-Aldrich, catalog number: 71643 )
  22. Fe citrate (Sigma-Aldrich, Fluka®, catalog number: 44941 )
    Note: It is also named “Iron(III) citrate tribasic monohydrate” on Sigma-Aldrich website.
  23. H3BO3 (Sigma-Aldrich, catalog number: B6768 )
  24. MnSO4.4H2O (Merckmillipore, catalog number: 102786 )
  25. ZnSO4.7H2O (Sigma-Aldrich, catalog number: Z4750 )
  26. CuSO4.5H2O (Sigma-Aldrich, catalog number: C7631 )
  27. H2MoO4 (Sigma-Aldrich, catalog number: 232084 )
  28. Na2MoO4.2H2O (Sigma-Aldrich, catalog number: 331058 )
  29. Agar (TEKNOVA, catalog number: A7777 )
  30. Tritium-labelled indole-3-acetic acid (3H-IAA) blocks (pH 4.8) (American Radiolabelled Chemicals, catalog number: ART 0340 )
  31. Fahraeus media (see Recipes)
  32. 3H-IAA blocks (see Recipes)

Equipment

  1. Plant growth chamber, set at 25 °C, 16/8 h day/night cycle with 150 μmol m−2 s−1 light intensity (Thermo Fisher Scientific)
  2. Scintillation counter (PerkinElmer, model: Tri-Carb 2800TR )
  3. Scintillation racks (PerkinElmer)
  4. Pipettes (Eppendorf AG)
  5. Scissors (ECKERSLEY’S)
  6. Fine forceps (Dumont and Fils) (D'OUTILS DUMONT S.A.)
  7. Rotating shaker (SeouLin Bioscience, model: MyLabTM Intelli-Mixer )
  8. Platform shaker (Eppendorf AG, New BrunswickTM, model: INNOVA 2100 )
  9. Microwave

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Ng, J. L. P. and Mathesius, U. (2016). Measuring Auxin Transport Capacity in Seedling Roots of Medicago truncatula. Bio-protocol 6(12): e1842. DOI: 10.21769/BioProtoc.1842.

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分类

植物科学 > 植物生物化学 > 植物激素

植物科学 > 植物生理学 > 组织分析

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