Measuring water potential would give you quantitative information about the stress level imposed.

Gravitiy method is easy and worked well in my experimnet, something like the below can help.

http://www.sciencedirect.com/science/article/pii/S0098847212001724

Good luck!

Define what you mean by "gradient". I dont think you mean continuous drought treatment. You mean you want steady-state drought? In that case you need to water the two groups either with different frequencies or with different amounts of water and monitor the soil moisture until you reach a steady range of soil moisture for each group and be able to keep it for some time until you make sure you have two water regimes.

Measuring stomatal conductance using a gas exchange system, in conjunction with measurement of stomatal density, would be an effective way of assessing the effects of water stress.

Dear Thilina

1- water stress imposition:

When you look at the literature you will see lots of work where researchers imposed water stress by water withholding. However, the true definition of water stress in a medium stems from physical properties of the medium. I encourage you to read about "field capacity" and "permanent wilting point". By these parameters you can make sure how much water you have in the medium and what fraction of it is accessible to plant. You would need numerous optimization experiments to establish plant growth stage, plant density in pot experiment, and even various types of soil as potting medium. You may want to consider using osmoticum reagents and keep track of water potential of the media to quantify amount of stress imposed. Once you have established a working condition of drought experiment vs. normal or optimal growth condition you are good to go to the easy part of plant characterization.

2- plant characterization:

This part depends on what resources are available to you. You can look at the below-ground tissues and above ground tissues. Root length, root morphology, growth rate, relative water content, water loss rate in excised leaves, photosynthetic potential under drought, stomatal conductance (seemingly best chriteria) etc. etc.

Sincerely

Mohsen

Dear Thilina,

The water stress treatment at long time during nursery culture affects the seedling morphological characteristics. You should sample randomly 10 - 15 seedlings by treatment and measure shoot height (Hs), root collar diameter (RCD). Subsequently, cut the seedlings at the cotyledon insertion point and separate into four fractions [leaves, stem, fine roots (diameter < 2 mm) and tap root (diameter > 2 mm)] and determinate the dry weight of each fraction by means of oven drying at 65 ºC for 48 hours. Finally, three morphological indices can be calculated. Slenderness index (SI) is determined as the relation between shoot height and root collar diameter [SI = Hs / RCD], root/shoot ratio [DWR / DWS] where DWR is root dry weight and DWS is shoot dry weight and Dickson Quality Index (Dickson et al., 1960). The Dickson Quality Index (DQI) incorporate in the analysis of the two quality indices (slenderness index and shoot/root ratio) and is calculated according to the equation [DQI = SMT / (SI + (DWS / DWR))] where SMT is seedling dry weight. Other variables as leaf area, SLA or SLW can be calculated. Specific leaf area (SLA) is calculated as leaf area/leaf dry weight. Specific leaf weight (SLW) is calculated as the ratio between leaf dry weight and leaf area. Several papers show tables with results on seedling morphological characteristics (e.g. Chirino et al. 2008 / Forest Ecology and Management 256:779–785; Luis et al. 2010. Trees 24:1143–1150; Chirino et al. 2011. Plant Soil 344:99–110).

Soil moisture and plant water potential can be used to measure the level of applied water stress. Also several physiological characteristics can be used to monitor the seedling response.

Sincerely

Esteban

Be careful how you use biomass allometric ratios. See the following article: http://www.frontiersin.org/Functional_Plant_Ecology/10.3389/fpls.2012.00259/abstract

As mentioned, it really depends upon available instrumentation. To gauge a short-term stress response you would ideally want some indication of a decrease in mamixal photosynthesis (CO2 uptake or fluorescence). If you don't have the correct instruments, you can visually inspect the leaves for drooping/wilting late in the day and at first light. That may be sufficient to get a general gauge of how dry the soil has to be. These suggestions, like those above, assume that your seedlings have enough leaves to show a response.

Regardless of your plant assessment, you need to take measures of soil water potential both predawn and mid-day.

To my understandings, reproductive growth is the most sensitive stage to environmental stresses. Exposing plants to water deficit at this stage will give you good enough data to analyze. You can make gas exchange measurements along with water potential and osmotic potential, stomata conductance just to mention a few.

The following references are from a series of studies that we created to look at conditions that could address the effects of water stress in western redcedar during typical summer field conditions. This series of trials was designed to look for genetic variation in this species so that the tree improvement program could devise methods for selecting material suitable for performance under drier conditions. These studies broke this issue down into three aspects of plant water stress:

1. Summer time vapor pressure deficit conditions effect on plant performance. Grossnickle, S.C., S. Fan and J.H. Russell. 2005. Variation in gas exchange and water use efficiency patterns among populations of western redcedar. Trees, 19:32-42.

2. Long-term moderate water stress effect on plant performance. Fan, S., Grossnickle, S.C., and Russell, J.H. 2008. Morphological and physiological variation in western redcedar (Thuja plicata Donn. Ex D. Don) populations under contrasting soil water conditions. Trees, 22:671-683.

3. Short-term severe water stress effect on plant performance. Grossnickle, S.C. and J.H. Russell. 2010. Physiological variation among western redcedar (Thuja plicata Donn ex D. Don) populations in response to short-term drought. Ann. of For. Sci. 67:506-517.

Hopefully this information will help you with your program.

Steve

Applicatin of mulch around the plant in the irrigation basin will help

Beside applicatin of mulch, selection of right types of plants which are more drought resistant will help in obtaining good seedling growth and performance.

Maybe you can use PEG (Mr 6000-8000) in different concentrations and use osmometer to measure and calculate water potential of the solution you are using to wet the paper you put the seeds to germinate. Also, you can add PEG to the agar which you may use to grow seedlings. Good luck with your experiments!

Hi thilina

craven and chirino has given an enlightening answers. I believe that you would have got more specific responses had you mentioned the plant species.

Best of luck

Dear Thilina,

first of all, you must to define which parameters you want to measure. If you want controlled environment with different water potential, then you can use PEG, as Ivana advised you, but if you want to simulate field conditions, then soil as a growth medium was preferred. If you choose soil, then you must to have basic information of its characteristics (physico-chemical composition), because the different soils have different holding capacity for water, i.e. heavier, clayey soils have better capacity to hold water for longer time, but they are hard for seed germination and protrusion, while the lighter, sandy soils are poor in water holding capacity, but they are lax and easy for growth of root and shoot). Then you must to determine water field capacity of chosen soil and then, according to determined value you could maintain water stress given as a percentage of water field capacity, only by simple weighting.

You can measure variations in germination percentage, as well as in seedling growth and factors of dessication tolerance, if you are testing potentially tolerant and/or sensitive genotypes.

Did you mean "a gradient of moisture contents"? The "water stress" is an extremely complex phenomenon to be fully quantified. This involves changes in the transcriptome, proteome, metabolome, ... How to create the gradient of something that can't be fully measured? If you insist, you need to choose a parameter as comprehensive as possible, which is the germination test. You can even choose a single factor, such as the concentration of a specific free radical; scavenger enzyme activity; quantitative gene expression (scavenger, dehidrin, heat shock protein, etc). But you have to recognize the limitations of this measure to strip conclusions.

Gamal M. Fahmy

You can simulate the water stress conditions by irrigating the germinated young seedlings with different rainfall treatments. In the desert conditions, the low rainfall ranges from 100 to 150 mm during the whole growth period or life cycle; and this would represent the drought stress to which the seedlings would be subjected. The non-stressed conditions could be created by irrigating the seedlings with 400 to 500 mm during the growth season. The volume of water added in each condition depends on the surface area of the pot or soil in which you planted your test species. As you know if you irrigate with 100 mm rainfall, each 1 square mm of the soil would receive 100 mm (=10 cm height) of rainfall during the growth period. You would convert this to the actual area of your soil used in planting

Hi Fernando, your question is answered by Ivana & Vesna; i just want to add that PEG is best to assess water stress response for seedlings, however, the concentration can greatly vary for species. Wheat can be screened at 20-25% weight/volume of PEG 6000 but not PEG 8000. Similarly for tomato its 10 times less that of wheat. See if ISTA has listed your plant species for germination test, use it for your control; and as per literature use a gradient of PEG concentration for optimization of dose. Once optimized, use that across all material for comparison. e.g. in case of wheat 7 day is final germination, so after recording germination % measure of root length and shoot length can be a vigor indicator or stress level potential when compared relative to control. fresh weight, dry weights and their moisture contents can all be used to establish an index for assessment of drought stress. Material thus screened for water stress sensitive and tolerant may be selectively grown in pots, for adult stage data verification. water stress may be applied at flowering beginning by stopping irrigation 2 to 3 times till partial wilting, flag leaf different parameter can be used to determine tolerance to stress. This will relate seedling data with adult plant data for validation. Best of luck.

I may add, you have to identify what seeds you will test,

In many physiological experiments mannitol has been used to exert water stress. In our studies 0.4 M mannitol was good for rice.

Dear Thilina,

I guess that if you are interested in morphological changes, you plan a mid- to long-term water stress. You can emulate water stress by increasing osmotic water potential in the soil (e.g. with PEG), but this is somehow closer to a salinity stress than to a water stress. To plan the water stress you must know something about the requirements of your plants. One typical approach is to fill some of the pots with a fraction of the daily water use, while the "control" ones can be filled to field capacity. This will create an incremental drought stress, so if you want to keep the seedlings alive you should stabilize the amount of watering in the "drought" plants after they reach a given water stress level (e.g. reduction of stomatal conductance up to 1/2, 1/3 or 1/4 of the control pots). An alternative for long-term studies, and indeed the one closest to real world, is to increase the time spacing between watering. In any case, I recommend to measure gas exchange parameters, particularly stomatal conductance, since they react very fast to water stress. I cannot be more specific without knowing the requirements of your species, but you can see a couple of examples of the mentioned approaches in the following papers:

Progressive drought:

Ferrio JP, Pou A, Florez-Sarasa I, Gessler A, Kodama N, Flexas J, Ribas-Carbo M (2012) The Péclet effect on leaf water enrichment correlates with leaf hydraulic conductance and mesophyll conductance for CO2. Plant, Cell and Environment 35, 611-625.

Long-term drought:

Ruehr NK, Offermann CA, Gessler A, Winkler JB, Ferrio JP, Buchmann N, Barnard RL (2009) Drought effects on allocation of recent carbon: from beech leaves to soil CO2 efflux. New Phytologist 184, 950-961.

An useful review on drought stress experiments:

Hamlyn G. Jones (2007) Monitoring plant and soil water status: established and novel methods revisited and their relevance to studies of drought tolerance

J. Exp. Bot. 58(2): 119-130 doi:10.1093/jxb/erl118

Dear Thilina,

Your question consists of 3 parts: How can you create a water stress gradient (1) for two kinds of seedlings during shot time (2) and evaluate the morphological changes (3)?

The first part: it is possible to use two ways - a) wet and drying filter paper, b) pot with vertical and horizontal gradients (simultaneously) of artificial substrate watering.

The second: the drought duration equals to 7-10 days.

The third: the measurement of root length (in filter paper variant) and leaf length (in the artificial substrate variant).

No PEG, no soil.

Dear Thilina,

Root growth, leaf rolling, chl content are few sensitive parameters used.However, it depends whether you are looking for root or shoot parametres.

PEG induced osmotic stress or soil moisture induced 'soil water stress' are common methods.

Best wishes.

PEG or manitol in different conc.can be used to create water stress gradients

Dear Tilina,

your question is quite broad; at least it includes many aspects of performance- levels(molecular, cell, issure, tisure,orga and whole indivaduls) and aspects (morphological, anatomical, physiological and biochemical).Thus answer is decided by your scientific question and hypothesis. Currently Tare are large amount of works done in this subject. I suggest you first define and restrict your questions.

The following article describes a program where we looked at the effects of long-term drought throughout a growing season. What we found out is that one needs to address a number of criteria.

1. Create edaphic conditions you can control throughout a very extensive timeframe.

2. Create a soil system where your test plants can go through normal seasonal growth patterns.

3. Manipulate the atmospheric conditions to maintain a desired range of typical growing season conditions; with the removal of any precipitation.

4. Define a desired range of soil water conditions that creates enough stress to cause a plant response without creating a water stress condition that completely shuts your plants down.

5. Define a series of measurements parameters that describe the plant response across the entire growing season.

The system we created allowed us to relate our information back to real-world conditions. This was important because this work was conducted for a tree improvement program that was using the data to make operational decisions on how to deploy western redcedar populations across their natural range.

Fan, S., Grossnickle, S.C., and Russell, J.H. 2008. Morphological and physiological variation in western redcedar (Thuja plicata Donn. Ex D. Don) populations under contrasting soil water conditions. Trees, 22:671-683

Hi Fernando, seedling performance under stress conditions can be measured in terms of imbibition seeds,germination velocity index (GVI).vigor index (VI) and mobilization of solutes from cotyledon to seedling,as seedling performance is very much influenced on mobilization of solutes.

Dear Fernando,

Good question. I have tried as suggeested by Ferrio, creating water stress by allowing plant to dry for sucessive periods of time, while maintaining them under controlled environment (assuming water depletion arte is constant). In field condition, we try to monitor soil water (in situ) and apply irrigation at specified allowed water depletion (like IW:CPE ratio), starting from field capacity.

Dear Mr. Thilina R.

Attached an old paper I came across it, you may possible find it useful please!

Seed desiccation assessment methods of: (1) (in vitro) germination, (2) conductivity test and tetrazolium staining may also help!

For creating water stress,PEG or Manitol in different gradients can be usred.Seedling performence can be measured in terms of %germination,germination velocity index (GVI),vigor index (VI) and mobilization efficiency under morphological parameters.For biochemical parameters,sugar and protein contents can be taken for both cotyledon and seedling along with a-amylase and prteinase activity in cotyledon.All these parameters be corelated for seedling performence

In my view, seedlings' performances can be monitored based on the different aspects and divided under several clusters such as physiological, biochemical or etc. What sort of research you would like to conduct? I suggest you to run some agro/ morphological investigations as well as crop growth rate (CGR), relative growth rate (RGR) and net assimilation rate (NAR) to have an entire view of your plants' behaviors.

First at all, I think some are confounding germination with seedling performance. Take into account that you can not use PEG on soil, I did a humidity release curve, I measured water potential in Megapascals in a known soil humidity percentage.

I am assuming you will have treatments that will grow at different rates. Also, drought treatments themselves may change the way the plants get water (change in biomass allocation to roots) or control the use of water (for example, by changing osmostic potential and so stomatal conductance). So the different treatments will use water at differnent rates and so will dry down at different rates. Then watering at specific intervals of time will subject the differnt treatments to different levels of water stress. You might try measuring pre-dawn water potential and rewatering each seedling (or treatment) when it reaches your desired threshold.

The measurement techniques were Germination percentage, Vigour index, Biomass, Isozyme activity (SOD, CAT, POX), Root length & Shoot Length

The best way to measure seedling performance is simply to measure different pant growth parameters such as plant height, number of leaves, number of branches, fresh biomass and shoot to root ratio under different water stress conditions. Moreover, it also depends on the crop plant under test, because the plant physiological parameters are different for different crop cultivars which also depend on the water stress treatments coupled with irrigation water quality especially the total water salinity and the application of NPK fertilizer if applied.

Good sugestion by parthasarathi.Biochemical measurment of another antioxidants as well as Malondealehyde and proline and alfa amylase are my suggestions.

Excellence is Doing an ordinary thing in an extraordinary way

"Some thing is better than nothing"

If we add a para daily after one month it will result in a productive item (paper , review, thesis , chapter, etc.etc.) So start from today

Dear All

I am pleased to inform u , after successful publication of two books i have started compiling another one or two books depending upon response from authors and dedicated scientists like you) in the aspect of crop improvement

The themes are

1. plant breeding in relation to crop improvement( biotic, abiotic stress, quality improvement, etc)

2. Plant bioticjnology aspects of crop improvement( biotic, abiotic stress, quality improvement,) both Marker assisted selection AND TRANSGENIC APPROACH/ CISGENIC APPROACH/ other in vitro techniques etc.)

3. Molecular breeding/QTLS biotic, abiotic stress, quality improvement

4 Plant genetic resources , conservation and its application in crop improvement

5. Crop specific breeding approaches like rice , wheat , maize or other horticultural crops

6. Hybrid technology and status of hybrid breeding in various crop plants

7. Molecular farming

8. Plant plastid/chloroplast engineering

9.Genome sequencing of major crop plants

10. other aspects related to crop improvement

Interested scientists , students are requested to send in their titles and abstracts from the above themes before 30 September 2013 for finalization. Selection of papers will be on first come first serve basis.

Interested scientists who want to be co editors can also send in their interest

For any further query kindly contact

[email protected]

shabirhwani@[email protected]

Kindest regards

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Thanks & Regards

Shabir Hussain Wani Ph.D, F.S.P.R

Assistant Professor

AICRP NSP (Crops), Division of Plant Breeding and Genetics

SKUAST-K, Shalimar, Srinagar, Jammu and Kashmir, India 190025

Mobile : +91 9419035566/ +91 9797001791

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Thanks & Regards

Shabir Hussain Wani Ph.D, F.S.P.R

Assistant Professor

AICRP NSP (Crops), Division of Plant Breeding and Genetics

SKUAST-K, Shalimar, Srinagar, Jammu and Kashmir, India 190025

Mobile : +91 9419035566/ +91 9797001791

http://www.skuastkashmir.a

There are so many parameters to study, but depending on your objectives select the parameters.You can consider morphological, physiological & biochemical .I think ,the parameters suggested by Dr.Parthasarathi will serve your purpose.

First of all Ur query needs two important aspects for an expert to answer.

.1 U plan to do in which crop ?and to what duration U need to gather information?

AS U know it it is soil,plant atmospheric continuum which needs to be addressed for creating a water/drought stress. U may use mannitol which is more desirable in hydroponics as it does not enter through the cell membranes.PEG on the other hand have different MW and the potentials change based on grade. Also, it enters thru plasma membrane and thus have an influence on cell metabolic activities hence, not desirable. In pots or soils and inward or outward situations maintenance of field capacity is needed. The parameters could be Relative water content of seedlings as such and also to some extent proline, Malonaldehyde, sugars or polyols will do initially to establish the stress effects. If you have more sophisticated systems water potential and stomatal conductances are the best even in the seedlings. All these can be done in a systematic way fashion of having four to ten sets with replications one set for destructive sampling, one set for measurements such as stomatal conductances, third a control. Remaining sets you may start with field capacity mositure and stop irrigation take observations on every 24 H alternate days which gradually decreases water content in the soil which can be taken up for soil moisture % on the basis of weight.

Cereals, pulses and small oil seed crops best suited for this type of experimentation. Tree crops U need altogether a different design.

Have a nice experimentation

You can measure the stomatal opening through the porometry. When plants are in water stress, they do not open the stomata.

I want to share our experience with chickpea. Creating a water stress gradient using black soil (vertisol) requires pooling soil that is uniform and homogenous. Later, we need to estimate the the field capacity and the initial soil moisture of the soil lot. You can develop any % level of soil water by applying that much water per kg of soil. We found 19 to 22% range useful for assessing seedling growth variation in Vertisol. This range would vary from soil to soil.

Using PEG in solutions of known stress level is another approach, which is irrelevant in this context.

I do not think I am adding much Info since all the above researcher have provided you with excellent info. However, I have recently run a similar experiment. I did not have at the time the right tools available. Nonetheless, I applied a gradient based on watering frequency. All plants were watered up to field capacity but the gradient focused on watering frequency (three times a week, twice a week etc). Evaluation took place both on morphological (leaf area etc) and physiological (dry weights etch) seedling measurments.

Best wishes

Try the (topographical) tetrazolium, condictivity and/or gemination tests! The germination test can be done in vitro and/or in vivo!

The big issue is if your two seedling sets vary greatly in leaf area. if so then they will dry the soil out at different rates and to different degrees which will confound your results. perhaps you should water with a specific amount of water, maybe even to field capacity, and then monitor leaf water potential and then rewater the seedling or seedling lots when a trageted stress level is reached.

I meant pre-dawn water potential when the water potential is mostly in equilibrium with the soil.

Dear Thilina,

Issues related to the soil dessication have been already explained in the other answers. You will have to face these problems and choose your protocol. I would use the gravimetric method for returning water to pots.

However, since you are working with seedlings, you must be sure that you are really applying water deficiency (WD) to your plants. Furthermore, you should worry about which part of the root system you are applying WD to.

I have worked with seedlings. I tested soil fertility and phenotypic plasticity. Please check:

Habermann & Bressan (2011) Functional Plant Biology, v.38, p.209-218.

In this paper we measured morphological traits, which I think would be similar to what you aim to get. The most important morphological trait was root length. In your case I guess this will fit as well because WD may influence ABA, which influence stomatal conductance (gs), ethylene and roots may respond to the disturbance you're going to provoke.

Think about it and if you have any other question, just let me know.

Best regards,

Gustavo Habermann

Associate Professor of Plant Ecophysiology

São Paulo State University (UNESP)

Rio Claro

Brazil

May I suggest reading the paper in David W Lawlor 2013 Genetic engineering to improve plant performance under drought: physiological evaluation of achievements, limitations, and possibilities. Journal Experimental Botany (details on Research gate) which addresses these points - and dispell some myths about how to control soil water content/potential.

Authors: David W Lawlor

Article Genetic engineering to improve plant performance under droug...

I have just performed such an experiment before to examine the effects of water stress on nitrogen fixation capability of plant. There are different methods to induce water stress on your plants. But regarding my experiences, you can first examine the normal amount of water consumption by your target plants, and try then to make water gradients in your examination. For instance, if the normal water requirement for your target plant is 500 ml, you can have water stress treatments such as 500 ml per week (as control treatment) and 400, 300, 200 and 100 ml per week as water stress treatments.

Just a quick advice. Whatever method you choose, monitor both plant and soil and if you work with pots, water seedlings from below (using underpots or saucers). This will avoid dry soil and dead roots at the bottom.

Follow the literature from the 1970s to early 1990s, it is a huge body. Methods used are from analysis of leaf gas exchange (stomata, photosynthetic capacity), of morphology (height growth, leaf extension, leaf area, subsequent leaf numbers, internode extension etc.)

Drs. Jerry and Carol Baskin at the University of Kentucky (USA) would be able to give you an authoritative reply for this question.

If you have enough leaf canopy monitoring of leaf water potential may be an appropriate choice as it correlates with the available water content in the soil. In experiments on cotton crop we usually maintain LWP at -2.4+/-0.2 MPa for water deficit and at -1.6+/-0.2 MPa for normal irrigation.

I recommend Maldonado et al. (2002) [Maldonado, C., E. Pujado y F.A. Squeo. 2002. Effect of water availability during the growth of Lycopersicon chilense on the capacity of their seeds to germinate at different temperatures and concentrations of manitol and NaCl. . Revista Chilena de Historia Natural 75: 651-660]

Article Effect of water availability during the growth of Lycopersic...

Polyethylene glycol can be tried. Just one can measure % of seed germination! shoot and root tolerance indices! besides basic fresh and dry biomass. Simply petri plate experiment will for this or filter paper method or vermiculite .

From this very simple method you can easily assess the different levels of stress tolerance in plants