- My research question was what environmental signals activate root
branching in plants? You can come up with a laundry list of lots of
different hypotheses that could answer the question of what the key
environmental signal is.

And some of those environmental signals, of course, we'd ruled out
already. So one environmental signal was the agar, or the gelling
agent is what's patterning development and preliminary data already
existed to suggest that that was probably unlikely. But the other
hypotheses that we had, such as water availability being in the key
signal or physical contact of the root with a solid substrate. We
didn't have too much preliminary data to rule out one of those over
the other.

So, with a project like mine, where you have a lot of different
hypotheses that you want to test, it can be very easy to get
overwhelmed a little bit, just because there's so many different tests
that you can imagine, how do you prioritize which one you want to do
first? And for me, it really came down to what was technically
feasible. Because there was some hypotheses that appeared to be
relatively straightforward, in terms of how you would go about testing
them and some that would require fabrication of very complicated
setups in order to devise the perfect experiment to test this
particular hypothesis.

So with that in mind, I took my list of all these different hypotheses
and basically ranked them in terms of how easy it would be to do the
experiment. And I did the easiest ones first and the ones that were
more challenging, I saved for later. You could have one hypothesis
that is water availability is the key signal that's patterning root
development. Once you come up with that hypothesis, what kind of
experimental system exists, where we can actually manipulate how much
water is available around the root? So that was something that I had
to come up with. So the experiment that I ultimately executed was
something that we be dubbed affectionately in the lab, the Agar
Sandwich.

And this idea came to me, pretty surreptitiously, there was a postdoc
in the lab that was just troving literature on root development, and
stumbled upon this paper that had this really interesting setup where
these researchers grew rice roots between two agar gel surfaces. It's
basically like a sandwich, where you have two agar surfaces on either
side and you have the primary root of the plant, growing in the middle
between them. And by altering the composition of these two agar
surfaces, you can impose chemical gradients across the circumference
of the main root. It seemed like a very elegant experimental setup to
test what I was trying to test, which was if you altered the water
environment around the root, how does it affect root patterning? And
the other useful thing about the agar sandwich is that there's an
internal control, for every sample. So there's one variable that's
constant across all the different samples, and that is on one side of
the sandwich, you always have the same composition of the media.

So you can see, in an unperturbed state, what is the baseline
development of the root in all these different conditions. And if you
see that that is consistent, then you can ask, on the other side of
the root, how is it responding to whatever my treatment is? How do you
change the water availability? So plant cells take up water pretty
much by osmosis so by increasing the concentration of solutes, outside
of the cell, that makes it harder for that cell to take up water. So
if you imagine these two agar surfaces. If one of these media has a
higher concentration of solutes, whatever that solute may be, it will
be harder for the root to take up water from that side. So all I had
to do was add some inert solute to one side of this agar sandwich to
change the water availability, across the diameter.

I had basically two sets of agar sandwiches, two conditions. One where
the water availability on the two sides of the root, was the same. So
I was expecting the number of branches to emerge in either direction,
to be equal. And then in another condition, I had a water gradient
between these two sides of the sandwich. So on one side there was high
water availability and on the other side, there was low water
availability and after I put my plants into that setup, I came back a
few days later and what I saw was, the side of the sandwich that had
this low water availability, very strongly suppressed how many root
branches emerged toward that side, compared to what was occurring in
my other condition, where the two water availabilities were the same.