University of Missouri and USDA-ARS

The solid-state chlorophyll meter: a novel instrument for rapidly and accurately determining the chlorophyll concentrations in seedling leaves
--Brent Krugh, Lisa Bickham and Donald Miles

The amount of chlorophyll per unit leaf area in maize is a good indicator of the overall condition of the plant. Healthy plants, those capable of maximum growth, generally can be expected to have larger amounts of chlorophyll than unhealthy plants. Therefore, determination of the chlorophyll content of a leaf can be used to detect and study plant mutations, stress, and nutritional state. The standard method for determining the amount of chlorophyll in a leaf sample is to homogenize the leaf tissue in 80% acetone, measure the absorbance at 663 and 645 nm, and then calculate the chlorophyll concentration using the specific absorption coefficients for chlorophyll a and b (MacKinney, J. Biol. Chem. 140:315-322, 1941; Arnon, Plant Physiol. 24:1-15, 1949). Although this method works well, it has two drawbacks. First, this method is time consuming, especially when there are numerous specimens to analyze. Secondly, the leaf specimen for which the chlorophyll amount has been determined is destroyed, thus making further study of that specimen impossible.

The Minolta Chlorophyll Meter SPAD-502 (Spectrum Technologies, 12010 S. Aero Dr., Plainfield, IL, 60544, 1-800-248-8873) (Minolta Corporation, 101 Williams Drive, New Jersey 07446, USA) is a lightweight handheld meter which allows one to quickly read the chlorophyll concentration of a leaf with no damage (Fig. 1). The SPAD-502 was initially developed to aid rice growers in determining when their crops were in need of nitrogen supplementation (Turner and Jund, Agron. J. 83:926-928, 1991). They found a direct correlation between available nitrogen and leaf chlorophyll during the pre-panicle initiation and panicle differentiation growth stages. The meter utilizes two LEDs which emit light onto the upper surface of a leaf; a red LED with a peak wavelength of 650 nm and an infrared LED with a peak wavelength of 940 nm. The light enters the leaf where a portion of the light is absorbed by chlorophyll and the remainder is transmitted through the leaf where it contacts a silicon photodiode detector and is converted into an electrical signal. The amount of light reaching the photodiode detector is inversely proportional to the amount of chlorophyll in the light path. After the signal is processed the absorbance is displayed in arbitrary units from 0 to 199. The procedure takes only seconds to perform and the meter is equipped to store 30 readings, average the data, and make data deletions when necessary. To assure accuracy and consistency the meter is calibrated prior to each use with a standard calibration filter which is supplied with the meter.

Since the SPAD-502 meter gives the data only in arbitrary units, it is more useful and meaningful if the data were correlated to actual amounts of chlorophyll per unit area of leaf tissue. In order to accomplish this, leaf disks were excised from 8-15 day old B73 maize (Zea mays L.) seedlings. The leaf disks were used to obtain SPAD values and for the calculation of total chlorophyll. A section of leaf was selected and a circular disk 0.87 cm in diameter was cut from the section. SPAD values were obtained from five locations on the leaf disk and averaged. The disk was homogenized in 80% acetone to extract chlorophyll and then, after a brief centrifugation to remove leaf material, the absorbance was measured at 663 nm and 645 nm. Using these absorbance values, the chlorophyll concentration was calculated with the formula described by Arnon (1949). The SPAD values were plotted against the calculated chlorophyll concentrations (adjusted to a leaf disk area of 1.0 cm2) and when a line was fit to the data points, a linear relationship with a 0.96 correlation resulted (Fig. 2).

In addition, we also used the SPAD-502 to address the common practice of classifying maize mutants as "yellow", "yellow-green", "green", or similar phenotype. The usual method of making these classifications is to simply look at the plant and decide what general color it appears. This method seems somewhat ambiguous and assigns no real parameters to these classifications. To address this situation, we used 10 day old seedlings of the maize mutant Oy-700, which segregates into three phenotypes designated "yellow", "yellow green" (oil yellow), and "green" (Hopkins et al., Z. Pflanzenphysiol. 99: 417-426, 1980). One hundred seeds were planted and on the tenth day, each was visually assigned to one of the three color classifications: two were yellow, 24 yellow-green, and 70 green. In order to maintain some sense of uniformity in numbers, 25 of the green plants were randomly chosen to be included with the 24 yellow-green and 2 yellow plants in this study. Five SPAD values were obtained from the second leaf of each plant ranging in a random array from mid leaf to the leaf tip. Regardless of where the measurement was attempted, the SPAD-502 was unable to detect any chlorophyll in the yellow plants. The five SPAD values obtained from each of the yellow-green and green plants were averaged and used to generate a plot for each color classification (Figs. 3 and 4). To generate plots for the data, whole numbers throughout the range of averages for each color classification were grouped in consecutive pairs (categories) and plotted against the number of individual averages falling within each category (#/category). For example category 12/13 would represent all SPAD averages from 12.00 to 13.99. The mean SPAD value averages were determined to be 17.35 for yellow-green plants and 40.02 for green plants. In both cases, greater than 95% of the SPAD value averages were within two standard deviations. Our data suggest the possibility of using the Minolta Chlorophyll Meter SPAD-502 to develop a color classification system that would be more precise than the visual method of making these assignments. For instance, due to the inability of the SPAD-502 to measure any chlorophyll in the yellow plants, a possible "yellow" category would be SPAD values very near 0. Furthermore, yellow-green and green categories could be set up encompassing their respective mean of the SPAD value averages (Fig. 5).

It is important to note that these data were obtained from 8-15 day old maize seedlings and due to variations in leaf thickness and morphology, it may not be applicable to other species or developmental stages of plants. However, the data may prove useful when applied to maize research. It will enable researchers to use the Minolta Chlorophyll Meter SPAD-502 on maize seedlings and then refer to the plot (Fig. 2) as a standard curve and obtain "real" chlorophyll concentrations per unit area. These data may allow this instrument to be used more extensively and in a broader range of analyses. For instance, the same method could be used to create standard curves for other species and developmental stages of plants. Aside from its initial intended use (to monitor the levels of nutrients that affect leaf greening), the SPAD-502 could be used to monitor the effects of environmental pollutants on chlorophyll content of plants or to study and identify plants carrying mutations that affect chlorophyll biosynthesis. Furthermore, the instrument could be used to more accurately and reproducibly classify the effects of mutant genes as "green", "yellow-green", "yellow" etc., thus replacing the visual method of making these judgments. Ranges of SPAD values could be assigned for each color classification (Fig. 5) resulting in a clear, concise classification system.

The Minolta Chlorophyll Meter SPAD-502 can be used to rapidly determine chlorophyll concentrations in plant leaves without damage to the leaf. Initially, one was limited to the arbitrary units which the instrument displays. However, the data and graphs presented above show that there is a linear relationship between the SPAD values and the total chlorophyll (calculated by conventional methods) in maize seedling leaves. This relationship makes it possible to use the graph as a standard curve and determine actual amounts of chlorophyll per unit area from SPAD values. The method presented above can be used to construct standard curves for other species and developmental stages of plants which may not correlate directly to our data due to differences in leaf thickness and morphology. Furthermore, we have shown that it may be possible to assign real parameters to the color classifications that are now typically determined visually. Finally, our data indicate the possibility for a wide variety of uses for the Minolta Chlorophyll Meter SPAD-502 including the detection and classification of mutant plant lineages.

Figure 1. The Minolta Chlorophyll meter SPAD-502 being used to determine the chlorophyll concentration of a maize seedling leaf.

Figure 2. Graphic representation of the correlation of SPAD values with chlorophyll concentration per cm2 in maize seedling leaves.

Figure 3. Histogram showing the number of representative "green" plants falling in each category constructed using the averages of the five SPAD readings from each plant.

Figure 4. Histogram showing the number of representative "yellow-green" plants in each category constructed using the averages of the five SPAD values from each plant.

Figure 5. Graphic representation of the correlation of SPAD values with chlorophyll concentration per cm2 in maize seedling leaves. Possible ranges for a color classification scheme are marked on the graph.

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors

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