There is no ideal way to perform drought test in root system. Some people took the whole seedling plants out of soil, washed and kept in water before stress treatment by dehydration. Other people just used hydroponics to growth the plant and then use PEG or manitol to induce osmotic stress. An-in-between approach was to grow plant in the pots then let the root outgrow the soil pots into the water trays, cut this roots for dehydration test. We used all the 3 above-- mentioned methods. The last one I used with soybean, you can't practically do it with Arabidopsis. Hope this helps.

If you use PEG to induce drought stress, make sure you use a high molecular weight PEG, say 6000, as smaller molecular weight PEGs can enter the root and cause damage. Also the roots should not be damaged in any way as this also allows PEG to enter into the root and not act as an osmoticum.

Neil Turner

That depends on what you mean by drought conditions and what do you want to do with the roots. We have worked with field-grown plants (i.e. submitted to natural dought) and unearthed the roots by direct excavation to measure biomass or root length, potted plants with easy to wash substrates like perlite, vermicullite, sand (with the problem that dorught is then very quicky, hence unnatural), or just spend a lot of time washing the roots, which everyone tries to avoid but is at the end unavoidable, as all root properties are affected by the substrate the grow in, so any plat growing in unnatural substrates have unnatural roots. This can be acceptable, I guess, if you are not interested in roots per se, but in drought effects in the shoot (but, then, why bother about washing or not? just forget the roots); but if you are interested in the roots, they should grow in a meaningful substrate, which can be an artificial substrate if you are interested in drought in potted plants, as for gardening purpose, greenhouse growth etc. The problem with hydroponics for root growth is not only the risks with PEG pointed out by Neil, but also the fact that a hydroponic grown root is very different from a root grown in the soil, and not only in its morphology. So my advice would be: forget about not washing the roots, just use the substrate or soil type that reasonably eases root processing and is in agreement with what you want to test in your roots and plants.

Thanks everyone for the answers. Dung LE, I used to use the last methodology you mentioned, but this was criticized by reviewers since as Xavier explains it do not represent a good approach for real conditions of drought.

Neil, we use PEG 8000 to be sure that PEG dose not enter the root, thank for your advice.

Santiago and colleagues,

we use a mini-rhizotron approach for 2-D root growth analysis along with drought stress treatments. We use a vermiculite:peat (1:1 v/v) substrate contained between two glasses with rubber separators (4 mm) to allow root growth and works fine with grapevine clonal plants (5 month old). This resembles an acrylamide gel electrophoresis system, and is cheap and you can measure lots of root architectural traits.

Good luck with your experiments!

I guess it depends on what you want to measure...

I have published a few experiments on drought stress and deficit irrigation treatments and we have washed the roots. It is true that we have not just focused on root physiology but we are always interested in measuring some parameters. So far, we have always (quickly) washed the roots upon terminating the plants; then, dry them with tissue paper before getting some measurements such as root length, and after that dry them in the oven to get the root dry weight. That's fine also when we want to do a root mineral analysis, for example. For other analysis such as abscisic acid, since we just need about 1 g of fresh weight, we just collect it before washing them (we normally use sand as substrate, so it goes off easily when it's a small amount of root material).

I have had experience in terms of effects of salt stress on root characteristics so that I cultivated alfalfa in hydroponics system and I removed plants from sand so easy without damage for roots. I use of WinRHIZO Pro 2007a as a root analyzer so that I measured many parameters. I recommended that WinRHIZO Pro 2007a can be the best instrument for your work.

the mini-rhizotron idea sounds interesting... I'm going to look at that.

We often used the Win Rhizo but we have the basic option... basically we use it for root length... it is true that it's use is limited compared to the Pro version.

If you look at win rhizo for minirizhotrons, check also thie win rhizotron: it has many options to manually select what you measure in each root, root section etc. In my opinion, it is too much detail if you work with high quantities of roots, but as with any other methodology you will have to decide, with the available work time, if you analyse more/bigger samples with less detail (then check win rhizo) or less/smaller samples with more detail, were win rhizotron could be indicated.

Just adding to Xavier and Juan comments, we set up the minirhizotron size to a scan in order to have the system scaled -up for winRhizo software.

Indeed win Rhizo has plenty of features that are beyond common data (i.e. insertion angle) and sometimes is a hard work to obtain a simple picture in a sea of roots.

So we use to focus in a clean portion of the image to acquire data, which might give you a quantified estimation of root growth when challenged to any treatment...

Take a look at this paper where we took container-grown seedlings through either through a severe or moderate drought. This work was attempting to either rapidly or moderately dry down seedlings by controlling the amount, and type, of media around the root systems, thereby not damaging the root systems. With the severe drought approach (i.e. enclosing root plug in a plastic bag and hung in the dark) we achieved water stress within 24h and with the moderate stress (i.e. planting seedlings into pure sand in a defined container size and at 40% field capacity) we achieved water stress in 4 days. In both cases the root systems were not damaged.

Grossnickle, S.C., R.S. Folk, S.R. Abrams, D.I. Dunstan and P.A. Rose. 1996. Performance of interior spruce seedlings treated with abscisic acid analogs. Can. J. For. Res. 26:2061-2070

Using hydroponics (solution culture) as a medium to grow plant is totally different to that grown in soil. This is mainly due to differences in matrix potential, soil resistance to root growth etc between two media, which could have differential effect on root growth, physiology and interaction between root and its growth media.

I know this is pretty basic info but, as far a growth is concerned both the impacts of the drought on the shoots and roots must be considered toghether because the ratio of root:shoot changes with plant developement. So harvests over time and then analyzing the allometric reationhips is a good way to go if destrcutive harvests are possible.

Surely osmotic stress including salinity and drought condition influence on morphological root parameters mostly. As my experiences use of WinRHIZO Pro 2007a can give u a lot of date from morphological parameters.

I would then first verify why one takes roots as subject. I think this makes sense in competition studies as it happens in the field for many species nearby or in dense plantations. Root extent and their hydraulic conductance covary with genotype and conditions. In addition biological competition may modify survival chance under stress. I therefore think you have field data that support the controlled environment study or I would spend some time on field exp. under stress, taking note of system complexity in trems of leaf area and species composition.

the discussions on measuring ''growth, morphology and distribution'' of roots is quite interesting thus far. I too am interested in growth dynamics of roots and keen on using either the Rhizo or this new gadget that can take root x-rays (X-ray micro-Computed Tomography) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615952/