Darshan Panda: Even if there is a calibration curve available for rice, this calibration curve (obtained under environmental conditions different to yours) must be used with care. If you already have chlorophyll data and SPAD data, your best option may be to use your own calibration curve.

SPAD readings can be affected by factors such as leaf anatomy, and leaf anatomy may be influenced by factors such as drought, salinity or light level. I am sending you a paper showing effects of light level on SPAD readings in bell pepper.

Thanks Juan! I will go through your paper and come back to you.

estimated Darshan Panda

In response to a question posted on ResearchGate last time, I kept the information I will mention at once.

Measuring Chlorophyll (& Anthocyanin) Concentration

What level of accuracy you need? You may try a extremely easy procedure by crushing 50 mg of leaves in 10 mL 80% acetone (aqueous). The homogenate is then filtered through a paper filter (standard nitrocellulose filter), and measure the absorption of the filtrate at 645 and 663 nm. You need the Arnon equation, easily found in internet. See for example http://employees.csbsju.edu/SSAUPE/biol327/Lab/photosyn/chlorophyll_quant.htm.

 Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; [email protected].

Background Information:

Chlorophyll can easily be quantified with a spectrophotometer based on the Beer-Lambert Law and the extinction coefficient for chlorophyll. In a classic paper, Arnon (1949) reported the following equations for quantification of the total chlorophyll, chlorophyll a and chlorophyll b content in an 80% acetone extract:

Total chlorophyll (µg/ml) = 20.2 (A645) + 8.02 (A663)

Chlorophyll a (µg/ml) = 12.7 (A663) - 2.69 (A645)

Chlorophyll b (µg/ml) = 22.9 (A645) - 4.68 (A663)

where A663 is the solution absorbance at 663 nm andA645 is the absorption at 645.

Unfortunately, the Arnon equations are not particularly accurate (Porra, 2002). Other equations have been derived that minimize the problems with the Arnon equations. For example, Lichtenthaler & Welburn (1983) report the following equations to determine chlorophyll a, chlorophyll b and carotenoid content in 80% acetone extracts:

Chlorophyll a (µg/ml) = 12.21 (A663) - 2.81 (A646)

Chlorophyll b (µg/ml) = 20.13 (A646) - 5.03 (A663)

Carotenoids (µg/ml) = (1000A470 - 3.27[chl a] - 104[chl b])/227

And, more recently Porra (2002) reports the following equations in buffered aqueous 80% acetone:

Chlorophyll a (µg/ml) = 12.25 (A663.6) - 2.55 (A646.6)

Chlorophyll b (µg/ml) = 20.31 (A646.6) - 4.91 (A663.6)

Total chl (µg/ml) = 17.76 (A646.6) + 7.34 (A663.6)

If the absorbance is greater than 0.8 then the solutions should be diluted with fresh 80% acetone and remeasured.

Sims and Gamon (2002) used a solution acetone/Tris buffer (80:20 volume; pH = 7.8) to extract tissue and reported equations corrected for the presence of anthocyanins. They report:

anthocyanin (µmol ml-1) = 0.08173 A537 - 0.00697 A647 - 0.002228 A663

Chl a (µmol ml-1) = 0.01373 A663 - 0.000897 A537 - 0.003046 A647

Chl b (µmol ml-1) = 0.02405 A647 - 0.004305 A537 - 0.005507 A663

Carotenoids (µmol ml-1) = (A470 - (17.1 x (Chl a + Chl b) - 9.479 x anthocyanin))/119.26

Since the anthocyanin concentration estimated in the extraction medium (80% acetone) is not reliable, they report equations for determining anthocyanin in methanol/HCL/water (90:1:1, vol:vol:vol):

Anthocyanin absorbance (corrected) = A529 - (0.228 A650) and then insert the corrected anthocyanin absorbance in the Beer-Lambert expression, A = ecl, assuming a molar absorbance coefficient at 529 nm or 30,000 l mol-1 cm-1.

References:

Arnon, D (1949) Plant Physiology 24: 1- 15.

Lichtenthaler, HK and AR Wellburn (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochemical Society Transactions 11: 591 - 592.

Porra, RJ (2002) The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynthesis Research 73: 149 - 156.

Sims, DA and JA Gamon (2002) Relationships between leaf pigment content and spectral reflectance across a wide range of species, leaf structures and developmental stages. Remote Sensing of Environment 81: 337 - 354.

Hello!

Thanks for the information Jose but I already have the protocol to calculate the chlorophyll by the destructive method. SPAD meter is a non destructive method to determine the relative amount of chlorophyll in the leaf. There are some papers which gives us an methodology to determine the concentration of chlorophyll from the SPAD reading ( which lies from 0-50). To do this I need a calibration curve for rice. Please check this link and let me know if you need the full paper to check the calibration curves.

http://link.springer.com/article/10.1007%2Fs11120-010-9606-0

Hi Darshan,

I don't think using others' calibration curve is a good idea even if there is one available. Because you cannot compare your experimental setup, genotype's chlorophyll content and treatment. In one of my experiments with heat stress in wheat, I had made my SPAD vs. chlorophyll content calibration curve for wheat using different representative leaves of varrying greeness of 41 different cultivars (The same genotypes on which the SPAD measurements was done in the experiment).And, used this curve to convert SPAD value to Chlorophyll content. You can find more about the method in Sharma et al. 2015, Physiologia Plantarum:153(2):284-298.

I hope this will help you.

Hello! Thanks Dr.Sharma! Can you l please email me the paper at [email protected]?

Dear Darshan,

Sorry, but I must say your question is strange. You need to calibrate the SPAD measurements in terms of chlorophyll content for the leaf material you are working with, and as others have said, it is not useful to use a published calibration. Growth conditions (e.g. nitrogen supply), greatly affect chlorophyll per unit leaf area. Make well-replicated SPAD readings on a range of leaves from different treatments, of different age etc.  Cut out the area on which the reading was made and measure chlorophyll by one of the methods given by others. The Milburn and Lichtenthaler method is good. Then do a proper statistical correlations and test if the different leaf ages or conditions affect the  correlation. If not, then the SPAD can be used as a general method. If there are differences then you will need a calibration for those.  Of course you can compare your correlations with others but only `for interest'!

Hope this helps

Best wishes

David (Lawlor)  ex Rothamsted UK

Thanks David!