14/9 A diversity Problem
In which I rant about the individualistic tendancies of the maize seedlings and introduce readers to the word, mesocotyl
Here it is, another week has gone by and it is an exaggeration to say that accomplishments have been modest. People speak of “the least publishable unit” for the smallest, thinnest, slightest bit of data that can be dressed up into a paper; what is the least thing that can be called an accomplishment?
Well, I don’t want to argue or go all philosophical – here is one thing that went down.
For the stem growth project, I need a good source of material. Plant material that is. I decided to use maize (aka corn, or if you will Zea mays). Maize seed (“kernels”) are easy to get, they germinate rapidly, and the seedling stem grows vigorously and is relatively large. As an aside, the seedling stem has a special word, all its own, the mesocotyl. It is privileged with its own coinage because this internode of the stem is built by the embryo whereas all other stem internodes are built by the shoot meristem of the plant. Getting back to the story, the large size of the maize mesocotyl (more than a millimeter in diameter) is salient because I will be working with them in the total darkness. Visibility will be limiting, even with my infrared gadget. Vigorous growth is good too, but of course it is not a given how they will respond when cut and incubated in a dish. But maize seems as good a place to start as any, and several weeks ago, I got ~1ooo kernels.
Being a person who likes a simple approach, not to say the path of least resistance, to sprout kernels, I got a small plastic box, put some wet paper in the bottom, added the kernels—embryo side up, and put the whole thing in complete darkness for 5 days. Upon opening the box, the good news was that most of them germinated. But the bad news was that they germinated whenever they felt like it, so to speak, with the result that a few of the seedlings were super tall, and others had barely lifted off the kernel. This is a problem because for reproducible experiments, it helps to use similar material and it looked like for ten seedlings, only two of them were similar enough to each other to use. Not a good yield.
So, how to improve the uniformity? I remembered back to my Ph.D., for which I used heaps of maize. In those days, I would soak the seed in running tap water for 4 hours before putting in on wet paper for germination. I did this because that is the way it was done in the lab. I think that was a waste of water. Some seeds are endowed by the momma plant with a germination inhibitor and, for such species, that compound could be leached out by the running water; but maize isn’t one like that. Anyway, in later years, I dispensed with the soaking altogether, and just put kernels on wet paper, and off they went.
I was disappointed by the non-uniform germination here. It might be that the seed batch I have is elderly or perhaps genotypically heterozygous? But it also might be the moisture regime. I tried soaking kernels in still water for a few hours, and this made no difference. But what about the “paper” in the box? What I had here was hard blue paper, more or less the consistency of a cereal box box, but a little thicker. CIPB have stacks of it around for another project. It holds water, but it does not get particularly soft, so it doesn’t cuddle the kernel. In my own lab, I have special “germination paper” which is a hundred (OK, I never counted, probably not quite that many) sheets of Kleenex-like tissue crimped together at the sides. When wet, it makes a soggy mass into which the seeds sink. They stay nice and moist.
Amazingly, I found a sheet of this super Kleenex, abandoned on top of an instrument. Possibly a bit dirty from the lab air but available. So I tried that and the blue card stuff, side by side. The seedlings on the blue card were all slower than those on the Kleenex but all of them were too diverse. Grrr. OK, now, it is time for jellyrolls. I got into jellyrolls for work on roots, because it makes they grow rather straight. For all I know, I invented the jellyroll but it isn’t anything I patented. But besides straight roots, the jelly keeps the kernels moist. In the jellyroll method, I cut a strip of the paper a foot long by a few inches wide, and wet it well. I lay the kernels down along one of the long edges, so the root pole of the embryo points toward the rest of the strip. Happily with maize kernels, it is easy to see where the embryo is and which way it points. Then I roll it up, and stand the roll up in the box with the shoot pole of the seeds facing up. Sorry—no picture, and searching the internet for jellyroll did not help!
I made three kinds of jellyroll this past Wednesday: one with super-Kleenex, one with the hard blue cardboard, and one with the standard-issue paper towels, which happen also to be blue (the technical term for them is “blue roll”). I put them all in the box and into the dark. On Monday, I will see whether the diversity of these rowdy kernels has been tamed by the jellyroll. And whether one or another of the substrates does any better. But if not, then I need to find a source of different maize. Maybe fresher seed? or perhaps seed of an inbred line, which should (in theory) be less diverse. Heck—that is the definition of an inbred line, genetically exactly the same, though of course environmental conditions at the time of seed set on the ear can influence the subsequent germination behavior.
The other accomplishments were all more or less financial. I figured out which camera and lens to order. This was a pain thanks to various protectionist legal policies which make it impossible to order something like that from the USA at the USA price. I need the camera and lens for the project but figuring which one and above all how to get it the most economical way, while necessary, is not the stuff of interesting blog posts! So I’ll just say, see you next week.