THE POLLEN TUBE GROWTH TEST (PTGT)
The pollen grain is used by plants to carry male sperm from one flower to another. Once transported to another flower (via bees or wind) the pollen lands on the style and grows a pollen tube, which grows down through the stigma to the ovary at the base of the flower. The sperm then travels down this pollen tube, where it can fertilise the eggs inside the ovary. The diagram below shows a few pollen grains, and the tubes they produce. (fig1)

We use the growth of the pollen tube to tell us about the effect our formulations can have on living cells. The addition of a chemical formulation can slow the growth of the tube; the more formulation is added, the slower it grows. The typical effect of a chemical on a living cell can be described using this chart (pdf), which shows the decrease in pollen growth (100% = no growth) and has four crucial points:

• The bottom of the curve. The chemical has no effect on the living cell below this point
• The Hill slope. The chemical effects the growth of the cell in a linear fashion. Usually the more toxic the chemical, the steeper the hill slope will be
• The EC50. This is the level of chemical (the concentration) at which 50% of the observable effect has occurred. (usually 50% growth)
• The Top of the curve. Adding more chemical above this point will have no more effect on the living cell.

Very well, you ask, but how is this relevant to people? The pollen tube growth test has been compared against the most common other types of toxicity testing, and it shows the best correlation against the Draize rabbit eye test. The graph (pdf) shows a comparison between the Draize and the Pollen tube Growth test. The Draize test involves inserting drops of the chemical into the eyes of rabbits, then looking for signs of irritation, including redness and weeping. When compared with this, the Pollen Tube Growth test does not use animals, is much quicker to provide results, and is less likely to be influenced by individual bias.

So how is the test conducted? Well first, we need tobacco pollen. Tobacco plants are commonly used in universities for research, where the pollen tube test was designed. The tobacco plants are grown at our site in New Plymouth, and the flowers are harvested (fig 2). Back in the lab, the pollen is then collected and can be stored at -20°C for up to 2 years. To run the test, we first make up a dilution series of our formulation. Starting with a 1% solution (or 10,000 parts per million) we dilute by a factor of 10 each time until we get down to 1 ppm.

At the same time, we make up a suspension of the pollen in a special growth media. Then we add about 0.1ml of pollen suspension, and 0.1 ml of one of the chemical samples to a small bottle. This is incubated at 25°C for 18 hours overnight. The following day, the pollen tubes rinsed out into centrifuge tubes (fig 3). This is repeated to make sure we have all the pollen tubes. Then a dye called alcian blue is added, which adheres to the pollen tubes and turns them blue. The next process is washing, in which we suck out most of the blue water, leaving behind the pollen tubes and some of the water. Then we add of fresh clear water and centrifuge to concentrate the pollen tubes at the bottom.

At this time, we add citric acid. This makes the water more acidic, and the acidic conditions change the cellulose so that the blue dye no longer sticks to it. We then end up with colourless pollen tubes, and blue water. But the amount of blue in the
water depends on how many pollen tubes there were, which in turn depended on how much chemical they received. We can measure the amount of blue color there is using a spectrophotometer, which measures how much light is received after passing through a sample of the blue water.

When we plot the numbers from the spectrophotometer against the concentration, we end up with the kind of graph demonstated. Approximately 5 points are plotted, and a computer program finds the model with the best fit. We then use this model to determine the EC50. (you can see how on the graph). The EC50 is the number we use to compare formulations against one another.

To finish off with, we will show you some of the results we can obtain with the pollen tube growth test. These three graphs (pdf) show three formulations of the same active chemical. The first is of an emulsifiable concentrate, which is very toxic to the pollen tubes; having an EC50 of only 1 ppm! It is also known that this formulation is very irritating to skin. The second graph shows a competitors product, and it has a much higher EC50 of about 100 ppm. This product is much less irritating, and is easily used with appropriate safety gear. Our product is the last one shown, and it has an EC50 of 600 ppm. This is much higher than the competitors product, so we can conclude that it should be even safer to use than theirs.