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Arabidopsis Pollen Germination Medium (Leonor 2007)

There are a handful of Pollen Germination Media (PGM) formulated for one of the widely used model plant, Arabidopsis. In this article, we have provided composition of one of the robust PGM. This PGM composition is adopted from a publication by Leonor 2007 (see the reference at the end of the article). If you are a beginner, I highly recommend you to read the full article since it shows the effect of temperature, light, pH and other factors on pollen germination and pollen tube growth. We have placed reagents required for preparation in a tabular form. This also contains information on Molarity of each reagent and their quantities to be added for making 10 mL of stock solution. We have also provided the information about different dilutions that can be made from the stock.

For your convenience, we have given the molecular weights of each reagent and for its most used hydrated forms too.  It is up to the user to determine whether hydrated forms make any difference for the experiments or not.  

pH

The pH of the medium should be adjusted to 7.5 using dilute HCl and KOH.

Composition

pH of the medium is one of the critical factor that determines germination efficiency. For this medium, pH should be 7.5. Achieve this pH using HCl and KOH.
pH of the medium is one of the critical factor that determines germination efficiency. For this medium, pH should be 7.5. Achieve this pH using HCl and KOH.
Reagent M.W Working concentration Stock solution Add for 10 ml stock Add from stock for 1X of 50ml
Sucrose 342.3 10% - - 5 g
H2BO3 61.8 0.01% 1% 0.1 g 0.5 ml
CaCl2(anhydrous) 110.98 5mM 1M 1.1 g 250 µl
CaCl2 .2H2O 147.01 5mM 1M 1.4 g 250 µl
KCl 74.5 5mM 1M 0.745 g 250 µl
MgSO4 120.36 1mM 1M 1.2 g 50 µl
MgSO4 .7H2O 246.47 1mM 1M 2.465 g 50 µl
Noble Agar - 1.50% - - 0.75 g

Click here to get molarity and dilution calculator for making stocks

Reagents

  • Sucrose
  • Boric acid
  • Calcium chloride
  • Potassium chloride
  • Magnesium sulphate
  • Noble agar
  • Distilled water
  • HCl
  • KOH

Instruments and other requirements

  • Weighing balance. paper boats.
  • Graduated cylinder and a glass beaker.
  • pH meter. Magnetic stirrer and magnetic bead/pellet.
  • pipette.
  • Tissue paper.

Procedure

Use ddH2O only for making PGM

You can choose to make 1X (working concentration) or make stocks and use them to make 1X whenever you want. Making stock solutions eliminate the possibility of errors that may happen while measuring very low quantities of reagents. Making working concentration from stocks also helps you to add some other reagents without further diluting working concentration.  

  1. Determine whether you want to start with stock solutions or directly making working concentration.
  2. Weigh the reagents accordingly (Refer to the table above).
  3. Dissolve salts by using magnetic stirrer or vortex mixer.
  4. Adjust pH to 7.5 using HCl and KOH (pH is one of the critical factors)
  5. Make the volume as planned.
  6. Sterilization of media is not required for immediate use. However, doing filter sterilization might increase its shelf life by preventing microbial growth.
  7. Aliquot to smaller volumes if needed (frequent opening and closing in open air might result in microbial growth).
  8. Noble agar is preferable over agar as solidifying agent.

Storage

Store PGM (Pollen Germination Medium) stock solutions at 4˚C for 1-2 weeks.

Reference

Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana.

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