Hi Anju,

first of all, it is important to separate the tissue of interest and process the samples in order to stabilize them as rapidly as possible to keep the sugars in the form and quantity they were at the moment of harvest. You can dissect your samples, freeze them rapidly by liquid nitrogen and store them at -80 °C in order to suspend metabolic reactions. You could also inactivate them by the use of heat.

However, oven-drying may turn out in a lowering of total WSC compared to the use of fresh tissues. The decrease has been found higher in monosaccharide than in fructan and glucan. Sucrose concentration of dried samples seems to be similar to fresh ones.

After that, sugars need to be extracted from frozen or dried tissues using water or ethanol. Tissues are first ground and then processed with the appropriate solvent. Grinding methods may change depending on the degree of dryness of your samples. Multiple extractions are usually necessary to recover as many sugar as possible. Extraction with boiling 95% (v/v) ethanol allows extraction of soluble sugars and subsequent hydrolysis and analysis of starch content. For extraction of oligomeric sugars (i.e fructans), ethanol concentration should be reduced to no more than 70% (v/v).

After extraction step, you need to analyze the samples. Several methods exist and the choice of the properest depends on the rigor you want to obtain your results with and on the availability of appropriate laboratory instruments.

Colorimetric assays are relatively simple but tend to be less sensitive than others and do not provide information regarding single sugar concentration.

Enzymatic assays provide specific sugar concentration and are relatively cheap but require multiple readings to evaluate sugar concentration.

Gas chromatographic assays provide information regarding numerous sugars in a single run and are quite sensitive. However, they require sample derivatization prior to analysis and may not be appropriate for quantitative analysis of oligomeric sugars. Absolute quantization of all sugars could be difficult to obtain because sugar derivatization may be incomplete for certain sugars.

HPLC may be the most suitable method. It allows a sensitive detection and quantification of numerous sugars in a single run; most detectors do not require derivatization of sugars prior to injection. By the use of an appropriate extraction solvent, separations may be optimized to evaluate concentration of monomeric to oligomeric sugars. This method was also used for trehalose detection in plants as stress tolerance factor.

You’ll find detailed protocols on several articles.

All the best

Hi Anju,

To measure water soluble carbohydrate you can use either the phenol-sulfuric acid  (Dubois et al.1956) method or by using Anthrone reagent (Yemm, EW. and Willis, AJ. (1954) The estimation of carbohydrates in plant extracts by anthrone The Biochemical Journal 57, 508–514). Look at the references and find out which one will be suitable for your sample. In past while I worked with drought stressed tissue I used the latter one. Regarding the sample numbers you can use a microplate reader where in you can measure a large number of samples at a time. Remember that you are dealing with conc. acids, so you need to standardize  the assay along with your sample before putting  a large batch of samples into a plate reader. However, if you want to know specific carbohydrate, then go for HPLC. Hope this will help. You can find more information to assay carbohydrates by using Phenol-sulfuric acid, please refer my paper Mishra et al. 2009. Hope this will help. If any question, please feel free to contact me.

Good luck 

Total soluble sugar content can be analyzed by phenol-sulphuric acid method of

Dey et al.,(1990) with slight modification by using 50mg fresh sample. Absorbance values were determined at 485nm wavelength. D-Glucose was used to make

standard curve for determination of sample total sugar values. 

Dey, P.M. 1990. Oligosaccharides. In: (2ndEd.), Methods in Plant Biochemistry. Carbohydrates.Academic Press, London. pp. 189-218.

Thank you very much Alessandro Romano for your detailed explanation. One more question: Do you have any idea which part of the stem should be focused for extracting WSC in wheat stem under draught stress?

Thank you very much Sasmita Mishra and Kamran Iqbal Shinwari for your answer. Sasmita, would you please provide me the full reference for your Mishra et al. 2009 paper?

Hi Anju,

due probably to the fact that water soluble carbohydrates contribute to the grain filling both under favorable conditions and, especially, under drought stress, they are mainly stored in the most distal stem segments, the nearest to the ear, thus the peduncle, and the underlying internode.  You could cut your culms at a slightly lower point (i.e. third internode from the top) and evaluate its contribution in terms of sugar concentration. Performing analyses on segments with different length may give you an idea about the appropriate biomass to be used.

First you have to sequentially extract Water Soluble Carbohydrate from wheat stem ground tissue in 80% ethanol and water (see van Herwaarden et al., 1998a for details) followed by determination in the extract using the anthrone method of Yemm and Willis (1954) with fructose as the standard. You can see the attached paper. 

I Think the better will be the use of HPLC depending if you have one of them. Another possibilities is to pay a service in some certificate lab. Phenol sulfuric method or Anthrone metod are another possibilities but for 600 samples is very hard.

Probably enzymes and genes related to fructan turnover may play a role over fructan concentration, and this can be obtained by subtraction of HPLC data for monosaccarides from WCS total value from anthrone or other WSC method that accounts for total sugars. It seems no an easy way at end http://onlinelibrary.wiley.com/doi/10.1111/nph.13030/full