The Wembley Weedkiller Challenge

Technical and teaching notes

Determining the concentration of the synthetic plant hormone 2,4-D required as a cost effective germination inhibitor for a weed killer preparation on sports pitches.

Seeds, like other parts of a plant, are adapted to their ecological niche and are affected by environmental changes whether these are chemical or physical.

Seed experiments can be used to look at plant adaptation, effects of commercial herbicides, climate change, conservation, crop selection etc

Apparatus

  • Distilled water
  • Laminated graph paper
  • Agar
  • Fine pointed tweezers
  • Petri dishes
  • Method and results sheet
  • 2,4-D solution (supplied by Philip Harris Ed. Suppliers)
  • Cress Seed (fast germination - Suttons Speedy seeds)
  • Safety glasses
  • Marker pens

Preparation of materials

Preparation of the 2,4-D Agar

  1. Make up 1 litre of 1% Agar ( 10g in a litre of distilled water)
  2. Microwave for approx. 16 min. until boiling, stirring intermittently.

BEWARE: AGAR BECOMES SUPER CRITICALLY HEATED AND CAN SUDDENLY BOIL OVER WITH A GENTLE TAP OR KNOCK

  1. Allow to cool to around about 70°C
  2. Prepare the 2,4-D dilution series by setting up 7 test tubes containing 9 ml distilled water and one containing 10ml.
  3. Pipette (plastic Pasteur pipette) 250 µl 2,4-D solution into the test tube containing 10 ml water.

BEWARE: 2,4-D IS TOXIC. GLOVES ANDEYE PROTECTION ARE RECOMMENDED. STORE AWAY FROM CHILDREN.

  1. Mix well by drawing the solution up and down in a Pasteur pipette.
  2. Remove 1 ml of this solution and transfer to the next test tube containing 9 ml water and mix thoroughly.
  3. Continue alliquoting 1 ml of the dilution into the next test tube of water.

Preparation of the plates

  1. Set out 40 plates in 8 piles of 5 petridishes (This gives 1 plate per 30 students and 2 for the faster students and a complete demonstration set.)
  1. Label each petridish on the LID around the edge with the date, time and the 2,4-D concentration. This will be: control, 2.06 x ………….10-9, 10-8, 10-7, 10-6, 10-5, 104, 10-3µl2,4-D/litre.
  1. Pour approx. 20 ml agar into each dish in the pile labelled control. (To compare plates accurately, 9 ml distilled water should be added to the 100 ml agar and mixed before pouring.)
  1. Using a measuring cylinder measure 100 ml of the agar into a beaker and add

9 ml of 10-9 µl 2,4-D solution. Mix well.

  1. Immediately pour approximately 20 ml of this solution into each Petri dish in the pile marked 10-9 µl 2,4-D
  1. Continue along the 2,4-D dilution series pouring the 9 ml of 2,4-D solution into 100 ml agar, mixing and immediately pouring into the corresponding labelled Petri dishes.
  1. Repeat with the other dilutions of 2,4-D until all the piles have been poured with the corresponding concentration of 2,4-D agar.
  1. Leave to set at room temperature. (About half an hour with lids off to prevent condensation.)

Lids can be replaced and the plates either used immediately or stored in the fridge for one week before use by the students. Allow to reach room temperature before sowing seeds. Plates stored longer than this may start to grow mould and bacterial spores.

Expected Results

Radicle growth of germinating cress seeds after 48 hours, with and without auxin – the naturally occurring plant growth hormone. Without auxin - radicle growth is 5 mm which is normal growth. With auxin - radicle growth is 1 mm indicating inhibition of root growth.

Extension / Coursework

The quick version

Set up as previously and after 20 to 24 hours record the number of seeds with radicle emergence.

Plot this on a graph to determine inhibition level of 2,4-D on seed germination.

  • Try with different rapid commercial seeds eg parsnip, turnip or UK natives eg dandelion, henbane, dock, nettles plantain.
  • Effect of dark and light on germination?
  • Effect of different wavelengths of light? Either infra red v red or different wavelengths of visible light.
  • Effect of exudates of Beech nuts or Pine needles on germination (Allelopathy)?
  • Effect of the allelopathic chemical(extract in luke warm water) on different seeds.
  • Accelerated ageing of seeds adapted to different climates?. Relate to length of storage in a seed bank.
  • Comparative germination rates of ecologically different plants within the same genus and relate germination to the ecological adaptation for those conditions.
  • Effect of pollutants upon germination?
  • Effect of ‘dirty water’ on germinating horticultural seeds?
  • Effect of nutrients eg mineral salts on germination?
  • Effect of carbohydrates on germination?
  • Effect of temperature rise (relate to climate change) on germination?
  • Demonstration of crop selection for fast, even germination rates compared to their wild counterparts.
  • DO YOU GET WHAT IT SAYS ON THE TIN? Comparison of brands of herbicide on the market and their relative strengths.

Further investigation: determination of the auxin strength obtained naturally from willow shoots.

Extract auxin from willow shoots by soaking willow shoots overnight in distilled water. The container should be kept in the dark and the solution of the auxin extract should be in a light proof vessel as auxin readily breaks down in the light.

Use the experimental data from the effect of 2,4-D on cress germination as a standard assay for the affect of differing concentrations of 2,4-D. Determine a concentration of 2,4-D which totally inhibits radicle growth or conversely determine the concentration which allows optimum radicle growth.

Make a serial dilution of the auxin from the highest extracted strength to about 10-6 in 1% agar.

Plate out the cress seeds and determine the concentration of auxin which either totally inhibits germination or has no affect on radicle growth and compare with the 2,4-D assay.

KEEP THESE PLATES IN THE DARK TO PREVENT THE AUXIN BREAKDOWN.

Use this information to determine equivalent strength of auxin to 2,4-D.

The ‘home produced’ auxin can now be used semi quantitatively in other experiments eg. Shoot tip growth, phototropic stimulation etc