July 19 End game

In which I describe going four for four on roots and some loose ends.

No post last Sunday because I spent the weekend enjoying bits of the Manchester International Festival (a concert of music by Arvo Pärt – ethereal, and a world premier of a new musical, called wonder.land about going down a rabbit hole of the electronic kind –brilliant!) and bits of the city itself. I have been squeezing in tourism (yesterday was Sheffield) and experiments with more or less equal fervor. So much to see and do.

As for the steady root growth project, discussed most recently in the last post, I finished crunching a root, and hey presto! the first principal component pulls out a rhythm, similar to the others. That makes it four for four. If you think that is still random, then go flip a penny four times. I’d be surprised if you get four heads. Of course, it *can* happen, but I doubt my luck would ever run so hot. I am cautiously optimistic that this rhythmicity is real. In fact, I will not-so-cautiously say that it is the significant finding of my sabbatical.

To have found one thing after a year of work sounds like a poor harvest indeed. And it may be so. Nevertheless, people have been studying root growth for ages, and revealing something none of them have observed is arguably nourishing. Naturally, if the next four roots lack the rhythm altogether then, well, it will be a winter of cabbage.

But this brings me to one of the tasks accomplished in the past week, namely imaging more roots. Before last week, I had six replicate image sequences (“movies”), four of which have been analyzed. During the week, I made three more, bringing the total to nine. And this coming week, I plan yet another three, to make 12. This is a number that will assuage the hunger of my statistical friend for N, even allowing for the fact that one or two of the sequences might prove unusable for an extraneous reason. For example, during the fall, I made a sequence where the root changed its overall elongation rate drastically, evidently responding to an unpleasant dust mite. The point is, 12 roots should be plenty.

I was prevented from doing further analysis by, not one, but two friends begging me to read and comment on their grant proposals. I read their stuff and suggested revisions and the week sailed on by. I did manage though to set coat in the lab. I did a “ticking” experiment for yet another friend who wants to see if their mutant has a root growth phenotype. It doesn’t in their lab but I pointed out that the growth rate of wild-type roots in their lab is about a third of that in my lab. A ticking experiment means marking the position of the root tip with a razor blade on the back of the Petri plate once a day (Monday through Friday), and then scanning the plates. Curiously, it looks as though root growth of their mutant slowed down with time, but I need to measure this to be sure (task this coming week). To be sure, this experiment suffered from the kinds of exploding nonsense that I have been fearing from everything I try during these last weeks. Three of the agar plates had frozen prior to plating and wow! are the roots turned off (well—slowed down). Numerous seeds failed to germinate. Still, after measuring, I might be able to tell my friend something, though the main message will be: “Sorry, I have to do this again back at UMass.”

And there is stem growth. Yes! I did not abandon my other project. This project has not yielded anything that could be called a major finding. What it has yielded has been ‘protocol development’. That is, I now have a system ready for exploitation. Major findings here I come. Still, one thing is missing, and an important thing it is. All of this protocol development has been done by means of a simple assay – namely measuring segment length and width at two time points, by hand. But segment length needs to measured continually and by computer algorithm. That expedient will greatly improve the precision in both space and time, not to mention be easier than tedious ol’ point-and-click with a mouse. Alas, writing such an algorithm far exceeds my basic skills in image analysis. I realized I ought to collect some image sequences anyway, so that once I cajoled a suitable image analyst into helping me, there would be images to analyze. I had made four sequences last month and I made another four this past week to increase the sample size and to allow perhaps that something definite would be gained from the segment growth kinetics once Puff the Magic Algorithm emerges from the mist.

With that accomplished, I also really wanted to see whether I could stimulate growth in width by wiping out the microtubules. I tried this a few months ago with an inhibitor called oryzalin but no such stimulation happened (read about it here). For this recent experiment, I made a couple of enhancements. First, I used a different inhibitor, colchicine, because it is in my view less toxic at high concentrations than is oryzalin and less prone to stick to glassware and dissolve in the cuticle. Second, I cut the stems while they were submerged to minimize the chances that air would be sucked into the xylem with excision and thereby restrict diffusive entry of inhibitors. Third, I let the experiment go for 8 hours in hopes of giving the colchicine enough time to diffuse in.

In the dark, with seeing in one eye through the sniper ‘scope, cutting segments while the stem is submerged is difficult (some difficulties described here). Nevertheless, I feel that I have now got it down ok. Although I would have liked to cut them submerged in solution containing the colchicine so that any sucking-in that occurs upon cutting would not only fail to suck in air but would instead succeed in sucking in the inhibitor. But colchicine is toxic stuff and cutting submerged stems involves getting my fingers wet. So I cut in water and then moved the segments to the washer-dish filled with a treatment solution.

Well, this experiment seemed to go just fine. No overturned dishes, no disastrous failure of the apparatus. Phew! But I have not yet measured the segments so I have no idea whether the colchicine worked. Because it is bound to take time to diffuse in and after that to alter the relevant cell wall structures, I reckon that the radial growth might be simulated after 6 hours, and this would be all but invisible by eye. This week – segment measurement binge (with time points every two hours, it equals five of my previous segment growth experiments in terms of the number of measurements needed). Stay tuned.

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