Arabidopsis Pollen Germination Medium

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    Introduction

    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 the 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 information about different dilutions that can be made from the stock.

    pH

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

    Composition

    ReagentM.WWorking concentrationStock solutionAdd for 10 ml stockAdd from stock for 1X of 50ml
    Sucrose 342.310%- - 5 g
    H2BO361.80.01%1%0.1 g 0.5 ml
    CaCl2(anhydrous)110.985mM 1M 1.1 g 250 µl
    KCl 74.55mM 1M 0.745 g 250 µl
    MgSO4 .7H2O246.471mM1M2.465 g50 µl
    Noble Agar - 1.50%- - 0.75 g

    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 a 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 a 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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