I recently executed an experiment in which I collocated 20 lentil seeds in independent Petri boxes with an agar agar solution, all of it located in a clinostat (a slowly rotating device used to simulate microgravity conditions, where they rotated perpendicularly to the ground (gravity). The objective was to see in what direction did the roots grow under the effects of microgravity, since geotropism didn't have a fixed direction to head to. At the end of an entire cycle, the forces (gravity in 360°) applied to the seeds would turn out to be equally strong, in opposite directions, so it would come down to nearly zero. However, after many executions, without having any functional problem with the clinostat, the seeds simply didn't grow roots while being in the agar agar in the Petri boxes. I tried different agar agar solutions, different concentrations, more & less quantity so that the seeds would have a thin air layer; and I also added copper sulphate to prevent fungi from appearing, also varying its concentration. Afterwards, I tried using an already germinated seed so that the radicle had direct access to the solution and to the air layer. Nevertheless, after several executions, only a few seeds grew their roots and I din't find a concrete explanation for this experiment not being successful.

I expected having the roots in the experimental group to grow in many different directions, not being constant. As in the other hand, I expected the control group to grow roots in the same direction, going downwards, being in the same system (clinostat) without having the rotations going on.

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    $\begingroup$ I am looking at an old study on soil symbiotes that used lentils on agar, their method is described as {Surface-sterilized (1 min in 70% ethanol and 3–5 min in 3% NaOCl) and pregerminated (48 h on 1% water agar)} if that helps. $\endgroup$ – John Mar 10 '17 at 2:36

It sounds like your first issue is with seed germination. I am assuming you are doing a seed sterilization. You may want to try allowing them to imbibe water for about an hour after the sterilization process, and then wrapping them with aluminum foil and placing them in a fridge for a day or two. I've done this with Medicago, which is another legume. However, not sure if it works for your lentils.

The root may also have trouble developing given the micro-nutrients in the media. I am curious what recipe for media you are using, as the amount of agar may also affect the ability for these roots to penetrate the media. I have had trouble germinating legume seeds when there were 100+ seeds in a plate as they inhibit each others growth.

It's hard to give a clear answer, but try optimizing your seed sterilization protocol and media recipe and best of luck!

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