Lemna Bioassay System

Herbicides are common contaminants in ecosystems affected by modern agriculture. Drift and runoff are almost inevitable. Even in well-managed property such as the Ft Ord BLM Lands, herbicide use to combat invasive species can end up affecting non-target species. Vernal pool habitats are of special concern for the BLM Land managers.

Today you will use the Lemna BioAssay system to estimate the concentration of an unknown amount of the herbicide RoundUp™ (glyphosate).

First you will take a second photograph of your Lemna assay from last week (and save it with a different name than last week's image!). Your instructor will post it up on the data website. Make sure you take the photo in the same orientation each week!!! Make sure you know the concentrations of each well in the photo!!!

Then you will analyze the difference between the two images using ImageJ in the computer lab. The difference in growth (leaf area) will be used to see if there was inhibition relative to the control. Do this part as homework. Summarize the data for all five teams in order to arrive at an average relative inhibition with standard deviation for each concentration of salt.

Then you will go back up to the lab to prepare the herbicide assay. It is essentially the same as the salinity assay, except that one well should be used for the unknown sample. This time you will prepare growth media with increasing concentrations of glyphosate and observing the effect on Lemna or Spirodela growth over a seven day period.

Again, take a photograph when you are done setting up, and make sure it gets to the data website.

Important Note: This is an upper-division lab. You and your partner will be required to think about and plan your experiments!

You should receive a copy of the MSDS for [N-(phosphonomethyl)glycine], also known as glyphosate or Round-UpTM. Read it and we will discuss it.

Round-up usually comes as an 18% solution of active ingredient when you buy it over-the-counter. That is the equivalent of 18 grams in 100 mL w/v.

However we have purchased reagent-grade glyphosate from Sigma-Aldrich and prepared a 15 mM stock solution in water for use in this lab.

The molecular weight of the active ingredient [N-(phosphonomethyl)glycine] is 169.1 so a 15 mM solution is only about 0.25 g per 100 mL or 0.25% w/v.

Today you will be given a microtitre-plate, a stock solution with a known quantity of herbicide, and a sample of water that may have been contaminated with herbicide (unknown quantity).  You will determine if the contamination is significant or not.

Now calculate the dilutions you will need to make in order to prepare the following wells. Write the mL of growth media required plus the uL of glyphosate STOCK SOLUTION. Assume there is a final volume of 15 mL in each well.

Record your preparations carefully. Based on your past experience with Lemna and the Jimage software select an appropriate amount of plant material and make certain to get almost the same amount into each well.

Take a large format, high resolution JPEG of your microtitre plate with a 5 mm strip in at least one of the wells. Then set the plate in the incubator. Then make sure your instructor gets your digital photo up on the data website.

1. What is the molecular weight of glyphosate? What is the concentration of the glyphosate stock solution?
2. How will you prepare 10 uM, 20 uM, 40 uM, and 80 uM glyphosate in Lemna Growth Medium?
3. How much solution will be needed for each well?
4. What is your control in this dose-response experiment?
5. How many plants should you use per well?
6. What are the parameters you should record for this experiment?
7. The photograph is probably the most important part of this experiment. What do you need to think about when taking the photo? Were the photos well-focused last week?
8. Be sure to record every step you take in order to prepare a good discussion for your write-up.
9. Think about how to calculate the growth relative to the control. If you want absolute growth you would subtract day0 from day7. If you want relative growth you would divide day7 by day0. Which do you want? How do you compare the treatments to the control?

1. Title as question.
2. Introduction - How does this herbicide harm plants? Where is this herbicide used? How does the concentration that is damaging to plants compare to the concentration of salt that is damaging to plants? Is this herbicide toxic to animals? Why or why not?
3. Methods - details on how you answered the question including a description of the software and how you used it (the functions and settings used).
4. Materials - what chemicals, organisms, and equipment did you use?
5. Data/Results - should include two photos with captions, table of Image J data from all five plates with title and caption, line graph of growth relative to the control with legend, title and caption. Was there a plant growth inhibitor in the unknown sample? If a BLM Land Manager told you it was glyphosate, could you give them the concentration?
6. Discussion - should include prose description of results as well plausible explanations for the results, should also include suggestions for further experimentation.

Due Thursday, March 16th, together with the Salinity report.