On plates

On plates kdorfman Mon, 01/16/2012 - 15:45

Planting on Plates

Make MS plates

  • To get weakling mutants started before potting in soil
  • Or to plant seeds on specified media
  • Or to see roots

Seed Preparation

Seeds must be sterilized before they are put on sterile plates.

After sterilization,

  • Make a line of seeds in agarose above the midline of the plate, either
    • either on the agar directly
    • on flattened cellophane
  • Tape plates shut with Micropore surgical tape
  • Stack plates flat
  • Refrigerate the plates for 2 days in the dark
  • Move plates to growth chamber
  • Stand plates on edge under the lights. Tape several together.

0.1% agarose for plating seeds

  • 100 mL sterile ddH2O
  • 0.1 g agarose . autoclave to melt; shake bottle after autoclaving

Sterilize seeds with EtOH & Tritonx

Sterilize seeds with EtOH & Tritonx kdorfman Mon, 02/14/2022 - 18:31

  • sterilize seeds in a solution of

    • 0.5 mL 70% EtOH,
    • 0.05% TritonX-100,
    • ~3 min mild agitation.

  • Let seeds settle, remove liquid

  • Replace with sterile water, 3X

  • Replace water with sterile 0.1% agar, 3X

  • Plate in a line, either

    • directly on agar, one seed per tiny drop or
    • on flattened cellophane (wet it if necessary)

  • Wrap plates with micropore tape

  • stack plates flat

  • cover in foil, refrigerate, 48 hours

Sterilize seeds with EtOH then dry

Sterilize seeds with EtOH then dry kdorfman Mon, 02/14/2022 - 18:39

  • Put appropriate number of seeds in a sterile microfuge tube

  • Sterilize filter paper with EtOH, let dry in sterile hood (can set it on top of an open sterile petri plate cover

  • Add 95% EtOH to seeds for 5 min, dump seeds onto filter paper

  • let dry, tap onto agar of an MS plate

Sterilize seeds with bleach

Sterilize seeds with bleach kdorfman Mon, 02/14/2022 - 18:37
  • Add ~1mL of bleach solution to each tube
  • Cap and shake
  • Leave tube on its side for 20-30 min, shaking periodically
  • Put upright after last shake (so seeds settle)
  • In laminar flow hood
    • Use a pipetman that has been sprayed with ethanol and air-dried in the hood
    • Remove as much bleach solution as possible from the tube
    • Add ~1 mL sterile ddH2O, mix and let seeds settle again
    • Do at least 4 washes, to dilute out the bleach
    • Add ~1mL sterile 0.1% agarose (so seeds will be suspended in the liquid)

Bleach solution for sterilizing seeds

  • 7 mL sterile ddH2O (must be freshly made!)
  • 3 mL Chlorox
  • 1 uL Triton X-100 (detergent to help get bleach into seed crevices)

0.1% agarose for plating seeds

  • 100 mL sterile ddH2O
  • 0.1 g agarose . autoclave to melt; shake bottle after autoclaving