Happy new year, faithful readers. Well stability has been much in the noosphere of late, but that is no reason to spurn the concept. Indeed, the three experiments I have run since the last entry have all been done to find conditions where the roots grow at a stable rate, alas unsuccessfully. I keep latching on to one thing or another, and being chagrinned when it doesn’t pan out, a disappointment barbed enough to have kept me from writing each week. That along with general slothfulness and of course, the holidays. Anyway, here is what happened.

The first of those three experiments, on Dec 12, was the last chance to salvage the method of supporting the seedlings on a vertical plate by means of a paper towel strip over the roots. The first few times I did this (see posts from October, e.g., this one) gave great results but then in November the results tanked. The roots appeared to be unable to grow between the towel and the agar. For the Dec 12 experiment, I used a narrow strip of towel, enough to support the seedlings but narrow enough that root tips would be uncovered. Here are a pair of images showing the seedlings at the start and end of the experiment.

Evidently, the uncovered roots grew better than the covered ones. But the situation is hardly optimal. Cutting narrow strips of paper towel is fiddly.

For the next experiment, on Dec. 20^th, I decided to replace the towel strip over the roots with a doofus over the kernels. These experiments have two phases. First, I set up kernels for germination; and second, after a few days, I transplant seedlings to a new plate for imaging and then growth measurement. For the germination phase, in the initial experiments, I used ‘jelly rolls’ (description at the end of that post) because they are fast and convenient; but trials showed that it is more reliable to germinate seedlings on plates, perhaps because roots have trouble breathing (i.e., low oxygen) deep inside the roll. For the germination phase, the kernels need to be held in place with the plate positioned vertically and I discovered this could be readily done with a doofus, namely a roll of wet paper towel placed over only the kernels. The experiment on the 20^th extended the use of the doofus to growth imaging.

As always, there is a problem. When I inspect seedlings germinated with a doofus some of the roots grew in air, above the agar. I suppose in the closed petri dish, there is enough water vapor in the air that the roots don’t ‘need’ to contact the agar. But still having the roots spurn the moist agar seemed odd. I discovered I could avoid roots in the air by germinating kernels on spongy paper (the same stuff that I used for jelly rolls) in a petri plate. I cover the spongy paper with a piece of paper towel to prevent roots from burrowing into the spongy material and thence disappearing.

For Dec 20^th, where I would use a doofus to hold up the kernels for the growth phase, I used the same spongy paper for the growth phase too, wanting to minimize any roots growing up off the surface and into the air. Still, using spongy paper for the imaging phase turned out to be … problematic. To image the roots, I had to put the top of the plate toward the camera and condensation formed, obscuring the root tips. Having one or two roots lifting off the surface did not seem a strong enough reason to suffer the poorer image quality from imaging with the spongy paper plates.

As seen on the graph, for both of these substrates, spongy paper (squares) or agar (open circles, ‘free’), roots grew slowly for about 2 hours and then grew faster hour by hour for the rest of the experiment. They never reached a stable rate. Also the top rate, around 1.5 mm/h was slower than the fastest rates I was getting back in October (2 to 2.5 mm/h). Hmm. Why?

For those experiments, the kernels were germinated in a jelly roll, which is kept in the dark prior to the experiment. When, instead, kernels are germinated on a plate, on either agar or spongy paper, they see red light throughout. In fact, I thought a constant light environment was a good reason to avoid jelly rolls. But, I wondered, maybe the darkness predisposed seedlings to faster root growth?

For the Jan. 3 experiment, I germinated kernels in a plate on spongy paper, but in darkness. The seedlings saw red light for a few minutes to set up the experiment but not thereafter. This appeared to increase the rate of root growth overall (figure 2, triangles), although that might just be day to day variation. However, the dark environment still saw the roots growing faster hour by hour. The similarity of the Jan 3 time course to the others is vaguely comforting insofar as it suggests that the roots are more or less oblivious to the history of light exposure (at least for dim red light vs darkness). But growth rate was still unstable.

I might be tempted to say, well, that is the way it crumbles rootwise had it not been for earlier results. I showed this graph before but am repeating here for impact (Fig. 3). On Oct 30^th, and to a lesser extent on Oct 25^th, the roots grew fast and steadily (at least for a goodly period). What is so special about about Oct 30^th?

There is a clue! Colonel Mustard with the wrench in the dining room. I looked at all the growth movies and on the 25^th and 30^th October, the shoots are bigger than in any of the other movies. By far. I need to check my notes (which are in the lab, I am writing at home) but I remember that for some experiments, I germinated kernels over four days instead of three. That, logically, would produce bigger shoots. If this is the case for those two “good” experiments, then perhaps germinating for one more day is the silver bullet needed to kill the instability? It is not unreasonable that it takes a day or two of growth for a root to reach a steady rate. These roots grow for many days. That is what is next – a four day experiment.