Hi,

I can think of hydroponic culture (culture in a liquid composition-controlled solution), which would allow full control of the solution (you could change it in one go, or create gradients of solutions with pumps). However you can trace root growth to a limited extent this way.

Alternatively, if you prefer solid support, you could grow it on paper (filter paper, which would retain some chemicals, or something like nylon, which should not retain much) on plates with solid medium. This would allow you to move the paper disks to different conditions, including medium composition, aeration, etc. If you do this, I would suggest finding a support disc with high contrast (i.e. black-ish), so you can photograph the roots and analyse them with a software (i.e. smartroot). That support should be 'plant-friendly' and ideally not have a chemical effect on the plant.

If you take the second approach, you can put your plates horizontally, so your roots would grow horizontally, or vertically (It think about 65C is a good choice, better thank 90C, I would suggest checking some literature), so you could trace root growth in a more 'natural' way.

Cheers,

Ruben

Hi Jolien,

I have used a simple method as alternative to hydroponic cultures, perhaps it helps.

Cheers, Maren

Hello!

I am not sure how early in the development of the plant you want to monitor the root.

In case you are looking at few hours to few days after germination, it is common practice to grow your seedlings in liquid Murashige-Skoog medium (MS) in sterile conditions.

By doing so, one can add any soluble chemical for short term treatment, and then wash the seedlings with MS medium and place back in fresh MS medium free of the chemical compound used as treatment.

At any time of the culture, seedlings can be harvested (for RNA, DNA and/or protein extractions) and/or observed by different microscopy techniques.

Alternatively, if one would like to avoid liquid cultures, it is also common to grow young seedlings on solid MS medium (then containing 0,8% agar) in sterile boxes.

The chemical for short term treatment can be put in the MS medium just before letting it solidify.

In this case, to stop the treatment, one can simply transfer the seedlings (in sterile conditions) from MS medium plates containing the chemical of interest to MS medium plates that do not contain it (maybe with a washing step in liquid sterile MS medium in between).

Note that when growing seedlings on solid medium in vertical sterile plates, all the seedlings should be placed at the same height in the plate (to receive the same amount of light).

Good luck!

Hi Jolien

We have developed a 'microphenotyping' method for chemical biology/chemical genetics applications that might be suitable for your purposes. It is a novel technology that allows the detailed analysis of root (and shoot) development under conditions where chemical treatments can be applied in a 96-well format. The basics of the method are described in Fig. 1 of Forde et al. 2013 Plant Journal 75, 1-10 (link provided).

In brief, Arabidopsis seed is sown on the surface of nutrient agar contained in strips of 0.3 ml microtubes (Framestrips from 4titude.co.uk) whose bottoms have been excised. The Framestrips (each with 8 tubes) are placed in 96-well microtitre plates and the chemical treatments are applied through the bottoms of the tubes - chemicals diffuse very easily through the agar and the roots grow into a concentration gradient of the chemicals. Root growth (and both root morphology and root architecture) can be observed very clearly by lifting up the Framestrips and viewing the clear-walled tubes from the side (see pics in the above paper). Growth rates can also be quantified by marking the walls of the tubes at intervals. We are currently developing an automated robotic version of this microphenotyping platform for large-scale screening studies.  Please contact me if you need further experimental details.

cheers

Brian

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739925/

To Ruben Alvarez-Fernandez: If you grow your plants on filter disks vertically, do the roots still grow through this disk?  Otherwise they cannot be submitted to different (chemical) conditions as they don't contact the supplemented media. Maybe the chemicals are absorbed by the filter, but then it acquires a wash up in between. If they grow trhough it, still the roots will encounter some mechanical stress when they are pulled out of the medium. But offcourse, stress levels will be greatly reduced in comparison with using a forceps.

To Brian Gordon Forde: Your method looks very intresting. I didn't think about the possibility of diffusion of chemicals through the agar. The only constraint this could have on first sight is that it might be difficult to score the germination timing of different plant lines and even plants within one line as you can't  follow up visually this process.  At least during early development it is quite difficult to determine their stage visually. Apart from that, this method looks promising.

Thanx to all for your answers!

Hi Jolien

There are different ways of using our microphenotyping system and if you wanted to observe the timing of germination you could easily do so by observing the same Framestrips from above (the top of each agar-filled tube is like a little Petri dish).  I've attached a couple of photos  taken from above to illustrate - although for your purposes you might want to make sure the seeds are more spread out. If I've understood your requirements this should work.

A further note - please don't use MS medium, or even 0.5 x MS. Yes, a lot of people do, but MS is designed for tissue culture and the concentrations of nutrients are extremely high (e.g. 30 mM N) and completely non-physiological, so are inappropriate for normal plant growth. There are plenty of alternative media such as those designed for hydroponic culture. We use a diluted version of Gamborg's B5.

Good luck!

Brian

Hi Jolien,

As I said, I haven't experimented myself with my own suggestions :) However, I can imagine some pros/cons:

hydroponic pros: fast chemical exchange, no gradients

cons: evaporation if the system is low volume, not sealed and the plants are grown there for long enough. If the seedlings are taking from the hydroponic into a 2D surface for imaging, they may experiment some dehydration and physical stress. Roots will grow in liquid, so good aeration has to be provided to prevent hypoxia.

filter pros: minimal physical stress, the roots grow on the filter (I'm thinking of a micron-pore size) and the filter and not the plant itself is manipulated. If the filter is thin and non-adsorbent for the chemical in use (ask suppliers), chemicals may be exchanged readily and at most a simple immersion in liquid growth medium without chemicals would wash them off (I would do the same if I was working with hydroponic).

cons: roots grow on filter surface, so root hair development and other processes will be affected. Chemical gradients may be formed, although I suppose that diffusion would be good enough.

Any system will have its advantages and its limitations, so appropriate controlling (positives and negatives) is key to obtain convincing results. Therefore, one has to design the experimental system around the biological question that is asked. In other words, I'm pretty curious about your bio-question.

Good luck!

Ruben

Maybe you could have a look at RootArray (Busch et al., 2012). It's a microfluidics based platform normally used for in vivo gene expression imaging, but it might be very suitable for rapid exchange of liquid media, and subsequent semi-automated high throughput imaging.