Coleoptile contributions to plant growth theory

What part did coleoptiles play in your studies of wood?

They came into the study of the differentiation of, say, the tracheids in a conifer, when cell division takes place in the cambium and the primary wall is formed. This is very thin, and is present during the time of growth or dimensional changes of the differentiating cell, before the secondary wall.

A favourite experimental object for studying the changes in the microfibril arrangement in the primary wall during dimensional change was oat coleoptiles. Imagine a germinating wheat seed of any kind. The first leaf as it grows up is enclosed by a slender sheath made up entirely of a single layer thin-walled parenchyma cells, with no differentiation. This outer sheath is the coleoptile, and the reason it is such a beautiful object for physiological study is that when it is first formed, up to about one centimetre long, it elongates entirely by cell division but after that time – and it can extend through about five centimetres long before the leaf breaks through – the elongation is brought about purely by the extension of the cells already formed. So when it is, say, two or three centimetres long, you can cut a bit out and pull the leaf out of the middle, and then you have got a little cylinder of cells. If you put those in a dish, they will extend, according to what you feed them, or the temperature. The coleoptile was ideal, since this is only a primary wall present, to study the arrangement of the microfibrils.

By this time we had an electron microscope so we could see the microfibril orientation, and we had all the ancillary equipment. So we started to grow the coleoptiles to different lengths, using chemical treatment to separate the individual cells one from another, and then looking at what their structure is. (You can look at this in a polarising light microscope too.)

I think you were about to collapse a hypothesis that had been made.

Well, people were going pretty mad with electron microscopy at this time and you could get all sorts of interpretations. This was prior to the development of good ultramicrotomes to allow you to section things, so you often had to break up the cell walls by very violent methods – by ultrasound or by putting them in a Mixmaster or something of that kind.

There had always been an argument about whether, when the extension period of growth in plants occurred, the growth of differentiating cells was generally uniform or tip. The view that the cells grew at their tips was based mainly on electron microscopy, and it had been advanced by two very eminent people – Frey-Wyssling and Kurt Muhlethaler, who were based at the ETH, in Zurich.

We were able to show, however, in a very simple set of observations, that if you isolate these cells you find that they are interconnected, one with the other, by little pores in the wall by which the cytoplasm communicates with the cell next to it. First of all we observed that these pit-fields, which are easily recognised in the optical microscope, did not increase in number during elongation or in distribution as you would expect if there was tip growth. So, since they did not increase in number, as far as we could see, was there a new cell wall forming? Was it different at the tip than at the middle? No, it wasn't. And then, to clinch it, we fed the coleoptile segments with radioactive glucose, C-14 glucose, and observed that the distribution of the newly formed cellulose was the same all over the cell.

Under-the-counter research

Is it true that you were actually growing your coleoptiles under benches?

Yes. There was a sort of red-tape situation in which the Department of Forestry in Canberra was in charge of growing trees and the CSIRO Division of Forest Products just looked at the wood. So we had to keep a very low profile on growing anything. These coleoptiles posed no problem, you could grow them in a dish under the bench. But we had to relate this to wood.

Fortunately, at about that time the CSIRO Executive of the day wanted to assess how their labs were going, so James Bonner – from the California Institute of Technology – visited Plant Industry in Canberra and us in Melbourne. I told him about the compression wood story and that we needed glasshouses to be able to grow the plants under different conditions, and also about the coleoptile stuff. He quite liked all this, and after he had left the lab, the chief of the division came round to me and said, ‘Well, I've talked to Bonner, and you can have your glasshouse for your coleoptiles.' Of course, we had them already, but it meant we could grow little trees, and we put a glasshouse on the roof of the lab then. That gave us a nice experimental set-up.

So in the very early '60s you could get tissue absolutely as you wanted it, and get right to the bench with it.

Yes, that's right. By the way, things have changed now and there is no problem about growing timber for research. We now have a Division of Forestry and Forest Products, all integrated in every way.

Focus questions

• Although Wardrop studied wood, he used oat coleoptiles as a model system. Why was this system useful for studying plant cell wall formation?
• What were the two lines of evidence that Wardrop used to support his idea that during plant cell extension growth was uniform over the cell and was not just occurring at the cell tip?