Jan 25th Data

In which my first, actual, accomplished, stem growth experiment is lovingly described

Not quite a whole week in the lab, because on Friday CPIB had their ‘away day’ where group leaders and other mainstays met for the day to discus management and other meta-issues. Having plenty of gray in my beard (along with bits of yesterday’s dinner), I was invited. But four days in the lab is not so bad, especially reviewing, editing, writing and other bumf were all on holiday.

I made progress crunching root velocity profiles, but as this is in medeas res, I’ll hold off describing those issues. The exciting thing was accomplishing the first stem growth experiment in the dark. I have been building up to this since my arrival in August, finishing up with no broken glass last Thursday afternoon was a good moment.

The experiment succeeded, in the sense that there are data. Here they are:

—————Delta L           Delta W

—————-%/h                 %/h

Control           1.0                   1.0

0.3 µM IAA   1.6                   0.5

1 µM IAA       3.7                    0.6

3 µM IAA       3.3                   0.3

The treatments are different concentrations of auxin (indole-3-acetic acid, IAA), with the controls just getting solvent (DMSO). The first column shows the growth rate in length, and the second shows the growth rate in width. The material under observation was segments of the maize mesocotyl and the time over which the growth rate was measured was about 5 hours.

A key point is that, as advertised, IAA increased the rate of growth in length. Well by golly auxin is the growth hormone and had it not done so, I could not call this a success however well the sniper-scope sniped. The next point is that auxin did not stimulate growth in width, and if anything tended to inhibit it. Insofar as almost, but not quite, nobody has measured growth in width in response to auxin, this marks the beginnings of news.

Another way in which the experiment succeeded was that it went more or less as planned. I didn’t have to abort it, or cheat by turning on a light. I was able to use the sniper scope to cut ~2 mm segments in the dark. I collected about 30 of them in a dish of buffer. Then I filled washer-dish #1 with treatment solution, transferred 6 segments, put them on the rack (Inquisitor that I am) before the camera, left the room but without opening the screen of the laptop, snapped the image, then put the washer-dish on the shaker, and repeated. I returned a little more than 5 hours later and took the final pictures, in this case using flashlight and computer screen because any growth inhibition from the light would not matter. The growth rates above come from the difference between initial and final measurements.

If you want to get technical, and of course !  you want to get technical because otherwise you would not be reading this (but by all means skip to the next paragraph), the growth rates shown above are calculated as:

rate = 100*(1/t)*ln(F/I)

Yikes! They say every equation loses a dozen readers, but stay with me! This equation is not so bad and if you are at all interested in understanding plant growth, really important. Let’s take the terms in order. The initial ‘times 100’ just converts the rate to a percentage (you can see that the units of the data are in percent per hour, which for my money is easier to grasp than just ‘per hour’. Next, the 1/t divides by the time interval for growth, that five and something hours (I know the exact time from the image capture software, which happily records wee details like that). F is the final length (or width) and I is the initial length (or width). Then, finally, one takes the natural logarithm (that is what ‘ln’ stands for) of the ratio of those lengths (or widths). Why take the natural log? Ahhh, well, let’s say the initial length were 100 and the final length were 110. Intuitively, we might think of this as a “ten percent” increase. That is about right. But doing that endows the 100 units of the initial material with the entire 10 units of increase. But that is not quite what happened. First, the 100 units grew a little and there was 101 units. Then all 101 units grew a little more and made 102 units. Then those 102 units grew and made 103 units. Now, going from 10o to 101 is 1% but 101 going to 102 is less than 1%, and 102 going to 103 misses 1% by a even more. And so when we get all the way to 110 units, we have had less than a 10% increase, as far as the material is concerned. We could have broken this down even further by looking at an increase of 0.1 unit, and so on. Well that is what taking the natural log does – it finds the percentage increase that when applied continuously produces the measured increase. In this case, ln (110/100) ~ 0.095 (that is ~9.5%, a little less than the 10% increase calculated from the ratio itself.

The experiment had its successes, yet it had its failures too. The biggest one is the growth rate: 3% per hour is sluggish, almost slothful. The intact mesocotyl is almost certainly doing more than twice that, maybe thrice. My job now is to pick up the pace.

Why might have things been slow? There are a few reasons that might be contributing. Here are a few of them.

Maybe 3 µM IAA is not high enough? I can test 10 µM although typically that much is not needed. But I am not abrading so perhaps therein lies a difference?

Maybe the plants were too old? Eventually the mesocotyl growth slows down and at that point, no amount of added auxin is going to perk it up. I have let plants grow a day more and the mesocotyls are larger but what I don’t know is how the growth is distributed over the mesocotyl. I can try using one day younger plants, though in this case the mesocotyls might be only a cm or so in length.

Maybe, I cut too far down from the node? I suspect this is likely to be important. The way I did this was to position the stem over the parallel razor blades (in a block facing up) and carefully push down to cut a 2 mm segment. I started a little below the node. This was easy and so then I cut about 6 segments per stem. I also allowed about 1 mm or perhaps a bit less between each. That puts me around 2 cm away from the node. It is possible that in maize the growth is confined to the region closer to the node. In the dishes with the two high IAA levels, there were a couple of segments that grew a lot more than the rest, which is consistent with this idea. I can test this by using segments closer to the node.

There were other issues too. The solution in the washer-dishes evaporated rather a lot over the 5 h. This is easy to fix with some covers. Another problem is that the amount of solvent (DMSO) in the treatment solutions was rather high. The controls got that but since their growth is so small it might not matter for them but could yet matter for the high growth under auxin. I wound up with high DMSO so that I could pipette accurately, but I could also make extra treatment solution. Instead, I could make dilute aqueous, stocks, but I worry about freezing and thawing them. I know that frozen DMSO is easy on chemicals, but maybe not frozen water? So I am going to burn a little auxin and make larger volumes than I need for the sake of minimizing the DMSO in the treatments and pipetting accurately. It goes against my grain to burn it but I’d wager with 99.9% confidence that my IAA stocks will be tossed out after I leave. Anyway, IAA is not expensive.

Optics:  the images were kind of daggy. The segments lump together sideways, obscuring their widths, or end-to-end, obscuring their length. Also, they were not as sharp as I’d like. I am going to try to improve the optics by putting them into the washer-dish without any solution and taking their picture “dry”. Then adding the treatment solution. The wireless keyboard came in the day after the experiment above and it works great, meaning that I can hit the ‘acquire image’ key inside the experiment room, and so I won’t spend a lot of time with dry segments in the dish. At the end, I can pipette out the treatment solution and image again. It is possible that the residual liquid from this approach will mess things up, but just have to try and see. I might need to adjust the focus of the lens.  Stay tuned!

 

 

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