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Stage V: Auxin and Plant Cloning

A practical introduction to auxin

Auxin movement is important for gravity and light perception and response in plants. Auxin is also an important tool in agriculture being used to both clone and kill plants. Many pioneering studies of plants in orbit have been achieved using plant stem cell cultures. Growing stem cells and cloning plants is easy. Understanding how auxin affects plants at a cellular cell is difficult and requires computers to model and make simulations.

Length of Stage: \~ 7 Days

Supplies:

  • Auxin powder (lots of brands Bonide, MiracleGro "FastRoot to choose from and compare against each other in experiments)
  • Plant what you want to clone (Tomatoes, chili’s, lavender, rose, whatever you want or pre-cut kitchen Rosemary from the supermarket can work)

Simple protocols for Auxin experiments and plant cloning

  • There are many protocols for taking cuttings online so first compare, contrast and discuss as a group.
  • The most important thing is to compare a few different variations (such as different light environments or different age) and record what happens in your Science Journal.
  • What variables?
  • Age of the plant
  • Species
  • Size of the canopy left intact
  • Method of auxin application
    • Powder
    • Liquid solution
    • Brand
    • Concentration

Advanced plant cell culture cloning protocol

This is a real scientific protocol, so there are a lot of variations to this protocol on the internet and in various labs it has been adapted for different purposes. The amount of hormones varies depending on the type of plant you are using. Write down your steps in your Science Journal so your methods are reproducible.

Making a media of 1 L

  • 1Liter of water,
  • 4.4g MS salts,
  • 30g sucrose,
  • 1ml MS vitamins,
  • 300 ul 2,4-D (Auxin)

Mix ingredients with 1L with 1L H20 then adjust the pH to 5.72

Alter pH by adding drops of potassium hydroxide

Final concentration = 1.0% agar (10g in 1L )

Example: 5g of agar in a 500mL bottle.

Autoclave for 30 min at 123C (or cook in a pressure cooker for \~35min)

Check your agar is dissolved (if not cooked for longer, might be a batch effect).

After autoclaving, allow to cool to \~45C

Add 100 ul kinetin, 667 ul 1M NaOH (to prepare 1M NaOH: Add 1.99g NaOH in 50 mL H2O) and 2,4-D (1mg/mL) (2,4-Dichlorophenoxy acetic acid = Auxin!).

Preparing Auxin stocks

  • Dissolve 400mg 2,4-D in 400mL 70% methanol (may also use ethanol)
  • Must be stored at -20C

Cloning/cuttings protocol

  • Pour a moderate amount of agar in selected plates (use rockwool, perlite or soil).
  • Cut small shoot of plant
  • Dip wound in Auxin powder
  • Gently press stem into the media making sure the stem is embedded (Do not crush seeds, use minimal pressure)
  • Cover plates with lids and seal with micropore medical tape.
  • Store plants in your growth environment (Blue light help some species establish roots better).
  • Don’t let them dry out.
  • After they’ve made roots move the new plants into a soil pots or hydroponics and enjoy.