Rain is the blog’s friend: plenty of yard work I ought to be doing but not in this downpour. I shall leave the rake alone. This week, I did the experiment foreshadowed last time, designed to test that remaining loophole. I think it worked. I have to waffle because closing the loophole did something unexpected. Yes indeed, science is one endless game of whack-a-mole.

The loophole is this. These segment-growth experiments are modeled on (a polite way to say copied from) classic work where seedling stem segments are used to study responses to hormones. My innovation is to use digital-imaging rather than position transducers to measure growth; but otherwise the same protocol. Seeds are germinated and when the seedlings have a vigorously growing stem (e.g., a mesocotyl in the case of maize, around 4 days after sowing), segments (5 to 10 mm long) are cut and used for experiments. For example, the growth hormone auxin can be added and the growth response of the segments measured.

Now, in all these protocols, before adding the auxin, the segments are allowed to cool their heels in water (or some equivalently inert solution) for an hour before the auxin is added. When I was doing this in Nottingham working literally in total darkness, I ditched the rest period because it would been a hassle, a hassle for which there didn’t seem to be a good reason. In the literature, when explained at all, the rest period is said to allow the endogenous auxin to drain away by continued auxin transport. But since I planned to be adding plenty of auxin myself, I didn’t see the point.

But maybe that was just an ad hoc rationalization for a step that was empirically useful? Now that I am working in red light, adding a rest period is easy enough. That was what the past week’s experiment was. Cut segments, and before putting them into the various auxin treatments, put them in water for an hour (the rest period). Image them right after the auxin was added and four hours later; from the images, measure the change in their length.

Auxin made them get bigger as the growth hormone is supposed to do. In the experiments I have done previously, some segments increased their length by 40% in that four-hour window but others stayed the same length, and yet others grew in between these extremes. This is the variability that I have been moaning about. In last week’s results no segment grew that fast but given that red light is known to slow down the growth of the mesocotyl, this seems acceptable. More importantly the variation between segments, though certainly not zero, seemed to be moderate.

I have to say “seemed” because of something else that happened. In the before and after picture, I could not reliably identify the individual segments. It is more surprising that I could do this in previous experiments because, after all, if you have seen one 3 mm hunk of seedling stem you have seen them all. But in the previous experiments where the segments were placed in their washer dishes immediately after cutting, they seem to stick together and their relative orientations are similar in both initial and final pictures. This added to small peculiarities of shape or texture let me be pretty sure who was who. However, I think because I floated the segments and then arbitrarily put them into their treatment washer-dishes, they owed no allegiance to any of their neighbors and their idiosyncrasies were not enough to distinguish them reliably.

Still, the segments at the first time point are reasonable similar in length to each other (they are cut on a jig with razor blades fixed in place). Therefore, the variation in the final length gives some evidence into the variation in the growth response. And the variation seemed to be less than it had been. I could not evaluate this quantitatively because of the overall lower growth rate as well as not having data for individual segments. But good enough to make me think that the rest period is helpful.

I want to try this with coleoptiles. This is another seedling stem-like organ (in botanical terms, a coleoptile is a leaf, but it looks and feels and behaves like a stem; sort of like how a tomato looks and feels like a vegetable though botanically of course is a fruit). The thing about coleoptiles is that their growth is accelerated by red light, so they might be able to make more growth hay with the auxin compared to the mesocotyl. The seeds are sown. We shall see.