Soran University ـFaculty of Education
Department of General Science
Third stage
Plant
Group: A
Chlorophyll assays
Preparation :- Supervised :-
1.Chra Taher Mr. Muhammad
2022-2023
Abstract:-
Chlorophyll content decreases in plants under stress conditions, therefore it is used commonly as an indicator
of plant health. Arabidopsis thaliana offers a convenient and fast way to test physiological phenotypes of
mutations and treatments. However, chlorophyll measurements with conventional solvent extraction are not
applicable to Arabidopsis leaves due to their small size, especially when grown on culture dishes
Introduction:
The chlorophyll content of leaves is an indirect indicator of the health and nutritional status of the plant . Traditional
methods to calculate the chlorophyll content include a destructive chemical extraction and a non-destructive
measurement of chlorophyll fluorescence. The former method, while direct, is tedious and unsuitable for continuous
monitoring individual plants because of its destructive manner. The latter method needs expensive instruments of
which none are presently suitable for small leaves such as the commonly used Arabidopsis cotyledons. It is important
to develop a non-destructive method to estimate chlorophyll content for Arabidopsis because it is a genetic model
plant, however traditional chlorophyll extraction is not useful due to the small size of the Arabidopsis leaves grown
on agar plates. Recently, digital photographic imaging showed great promise for quantitating plant phenotypes .
Indirect methods are available but none are yet suitable for Arabidopsis. Sass et al. developed a protocol to convert
the RGB values of a color image into a hue saturation value (HSV), and showed that the hue value was correlated to
the chlorophyll content estimated by a destructive method.
Materials and tools needed:
1.Test tubes
2.plant leaves
3.Pipettes
4.80% acetone
5.Distilled water
6.Spectrophotometer
7.Glass cuvettes
8.Mortar and pestle
Procedure :_
Our experiment on chlorophyll How to determine the amount of chlorophyll in a plant? Chlorophyll is found in plants
whose main function is to absorb sunlight and convert the energy into chemical energy. Chlorophyll has other
functions, including the green color of leaves We have the main type of chlorophyll a.b.c..d Here we are talking about
chlorophyll a.b. so chlorophyll b. a because it is found in plants and c.d is more common in other organisms. This
process is called extraction. For chlorophyll, acetone is used because acetone can extract 100% of the substances in
the plant. The acetone should not be 100% 0 acetone and 20% Water First, remove half a gram of leaves, then cut
half a gram of leaves into pieces in a mortar, then grind slightly in the mortar or beat until more pieces are obtained
After 24 hours, start determining the amounts with a spectrophotometer with a screen. Before adding the chlorophyll
to the test tube, add water to calculate the water content Then we zero the apparatus, then we add the chlorophyll,
then we determine the ratios of both chlorophylls (A, b).
Conclusion :
This method is a simple, fast, inexpensive, and nondestructive estimation of chlorophyll content of Arabidopsis
seedlings. This method lead to the discovery that G proteins are important in sensing the C/N balance to control
chlorophyll content in Arabidopsis.
Results:_
We provide a nondestructive method for chlorophyll measurement whereby the red, green and blue (RGB) values of
a color leaf image is used to estimate the chlorophyll content from Arabidopsis leaves. The method accommodates
different profiles of digital cameras by incorporating the ColorChecker chart to make the digital negative profiles, to
adjust the white balance, and to calibrate the exposure rate differences caused by the environment so that this method
is applicable in any environment. We chose an exponential function model to estimate chlorophyll content from the
RGB values, and fitted the model parameters with physical measurements of chlorophyll contents. As proof of utility,
this method was used to estimate chlorophyll content of G protein mutants grown on different sugar to nitrogen ratios.
Calcultio:_
Chl A=[12.7 × 2.553 – 2.69 × 2.354] ×5/1000×0.5
Chlorophyll a= 0.0652271 mg/L
Chlorophyll a=0.1304542 mg/g
Chl B=[ 22.9 × 2.354 – 4.68 × 2.553] ×5/1000×0.5
Chlorophyll b=0.1048964 mg/L
Chlorophyll b=0.2097928 mg/g
ChlA+B= 0.1304542 + 0.2097928
Chl a+b= 0.340247 mg/g
