Through repeated failure I am finally coming to terms with what I have to do to make this system work. Slowly, slowly. I need to separate the things that are desirable from those that are essential. Last time, I reported how seedlings that are three days old instead of two have roots that grow faster and reach a constant rate after a few hours. These are desirable traits. But an older root tends to grow in a crooked line, making it difficult to keep it flat on the agar. I plan to use this system for experiments in which I introduce an inhibitor into the agar and therefore reliable contact with the agar is an essential trait.

For the next experiment with the three-day-olds, I germinated kernels face down, that is with the embryo contacting agar (I explained what I was interested in trying this in the last post). The roots were long and bent, making it impossible to lay them flat on the agar. I aborted the experiment. This is clearly a problem with using a doofus because it holds the kernels but not the roots on the agar.

My first thought was to give up on fast and stable growth and use shorter but straighter, two-day old roots. I set one up, paying careful attention to how well these short roots lay on the agar. Among the germinated seedlings, two had roots that were all but geometrically straight, so much so that the seedling stayed on the agar without any help (Figure 1). Well actually one of them fell down before the first picture (arrow in Figure 1) but the other one stayed aloft throughout. Still, roots on all the other seedlings had gentle bends, big enough to make them ill-fitting on the agar. Frustrated, I plugged the roots into the agar. That held them in place and kept the root in contact with agar. And the average growth rate of the roots in agar was about the same as the one on the surface of the agar, and nicely similar to the group of experiments I have done with two-day-old seedlings.

Well, plugging the roots into the agar could suffice. But it is a little … violent. Before settling on that, I want to go back to placing a wet paper towel over the roots. This has the undoubted advantage of enforcing contact between root and agar. The problem I had with this before was smothering the roots by extending the towel over the tips. I tried to use a thinner strip but with the relatively short two-day-old roots, this was a bit fussy. I realized that I could get thinner and possibly more gentle strips by replacing the paper towel with a Kimwipe (essentially ‘laboratory grade’ Kleenex). These are thin, easily folded and manipulated when wet.

For the upcoming experiments, I will first use three-day-old seedlings and look carefully when I lay the wet Kimwipe strip over them to determine whether this really does, as advertised, nail the connection between root and agar. If so, then the cake is both had and eaten: that is I get older better growing roots that rest on the agar. But if the bending of the three-day-old roots overcomes the Kimwipe pressure, then I’ll repeat the approach with two-day-old roots. And finally if they too balk at a Kleenex on their back, then I’ll plug ‘em in the agar.