# A Simple, Cost-Effective Method for Leaf Area Estimation

Authors: S. K. Pandey; Hema Singh
Journal: Journal of Botany
 (2011)

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## Abstract

Easy, accurate,
inexpensive, and nondestructive methods to
determine individual leaf area of plants are a
useful tool in physiological and agronomic
studies. This paper introduces a
cost-effective alternative (called here
millimeter graph paper method) for standard
electronic leaf area meter, using a millimeter
graph paper. Investigations were carried out
during August–October, 2009-2010, on 33
species, in the Botanical garden of the Banaras
Hindu University at Varanasi, India. Estimates
of leaf area were obtained by the equation, leaf
area (cm2) = x/y, where x is the
weight (g) of the area covered by the leaf
outline on a millimeter graph paper, and y is
the weight of one cm2 of the same
graph paper. These estimates were then compared
with destructive measurements obtained through a
leaf area meter; the two sets of estimates were
significantly and linearly related with each
other, and hence the millimeter graph paper
method can be used for estimating leaf area in
lieu of leaf area meter. The important
characteristics of this cost-efficient technique
are its easiness and suitability for precise,
non-destructive estimates. This model can
estimate accurately the leaf area of plants in
many experiments without the use of any
expensive instruments.


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## Body

## 1. Introduction

Leaf area is an important variable for most ecophysiological studies in terrestrial ecosystems concerning light interception, evapotranspiration, photosynthetic efficiency, fertilizers, and irrigation response and plant growth (Blanco and Folegatti [1]). The easy, economic, and precise estimate of leaf surface area has been a concern to plant scientists for a long time. Plant physiologists require leaf area measurements for studying primary production in plants (Sestak et al. [2]; Tieszen [3]; Bleasdale [4]). Ecologists use leaf area relations for elucidating competition among different plant species (Harper [5]). Leaf area estimate is valuable in studies of plant nutrition, plant competition, plant-soil-water relations, plant protection measures, respiration, light reflectance, and heat transfer in plants (Mohsenin [6]), and thus it is an important parameter in understanding photosynthesis, light interception, water and nutrient use, and crop growth and yield potential (Smart [7]; Williams [8]). Leaf area estimation is often costly, time-consuming, and destructive (Marshall [9]). Sestak et al. [2] provided an extensive description of the most common methodology available till date that includes counting squares on millimeter graph paper, hand-planimetry, the gravimetric method, dot counting, photoelectric planimetry, air-flow, linear measurements of leaves, leaf weighing, detached leaf counting, and the rating method. Well-known electronic meters can only be used if the plants have sparse and nonfragile leaves (Tieszen [3]; Bleadsdale [4]). A variety of computerized image analysis equipments and software are also available (Brodny et al. [10]). They measure quickly, accurately and nondestructively using a portable scanning planimeter (Daughtry [11]); however, the method is suitable only for small plants with few leaves (Nyakwende et al. [12]) and is expensive (Bignami and Rossini [13]). Several combinations of measurements and models relating length and width to area have been developed for several fruit trees, such as grape (Montero et al. [14]; Williams and Martinson [15]), avocado (Uzun and Celik [16]), pistachio (Ranjbar and Damme [17]), Cherry (H. Demirsoy and L. Demirsoy [18]), peach (Demirsoy et al. [19]), and Chestnut (Serdar and Demirsoy [20]). Some studies also use petiole length (Manivel and Weaver [21]) and leaf weight (Sepulveda and Kliewer [22]; Montero et al. [14]) for area measurement. The most common approach is to develop ratios and regression estimators by using easily measured leaf parameters such as length and width (Kvet and Marshall [23]). Lu et al. [24] proposed that the simple and linear relationships between leaf area and leaf dimensions (length, width) could be useful for nondestructive estimation of leaf area. Estimating leaf area from equations using leaf dimensions is an inexpensive, rapid, and nondestructive alternative for accurately assessing leaf area. Nondestructive models for leaf area determination have been established for many species such as maize (Stewart and Dwyer [25]), bean (Bhatt and Chanda [26]), taro (Lu et al. [24]), white clover (Gamper [27]), sugar beet (Tsialtas and Maslaris [28, 29]), sunflower (Kvet and Marshall [23], Rouphael et al. [30]), radish (Salerno et al. [31]), zucchini (Rouphael et al. [32]), strawberry (Demirsoy et al. [33]), grapevines (Manivel and Weaver [21]; Montero et al. [14], Williams and Martinson [15]), kiwi (Mendoza-de Gyves et al. [34]), chestnut (Serdar and Demirsoy [20]), hazelnut  (Cristofori et al. [35]), eggplant (Rivera et al. [36]), faba bean (Peksen [37]), stevia (Ramesh et al. [38]), persimmon (Cristofori et al. [39]), medlar (Mendoza-de Gyves et al. [40]), small fruits (Fallovo et al. [41]), euphorbia (Fascella et al. [42]), saffron (Kumar [43]), ginger (Kandiannan et al. [44]), roses (Rouphael et al. [45]), and watermelon (Rouphael et al. [46]).However, leaves may have complex shapes making leaf area determination using ratios of leaf parameters difficult, time consuming, and subject to larger errors. Therefore, the aim of this study was to develop an equation for leaf area estimate which is insensitive to changes in leaf shape, and is cost-effective. In this paper, a millimeter graph paper method is described, and its reliability is tested using an electronic leaf area meter.


## 2. Materials and Methods

Thirty-two (twelve- to thirty-five-year old) tree species and one (six-year old) shrub species growing at the Botanical garden of the Banaras Hindu University, Varanasi (25°18′ N and 80°01′ E, at 126 m above sea level, mean annual rainfall 1100 mm), were selected for the study. Leaves were sampled from different levels of the canopy, ten each from the thirty-three species, during the full-foliage period (August–October) in 2009-2010. Each leaf was spread over millimeter graph paper, and the outline of leaf was drawn. The leaf area of each leaf was measured using a leaf area meter (SYSTRONICS, Leaf Area Meter-211) having a sensor and read-out unit. Using the paper knife, the area of the millimeter graph paper covered by the outline was cut and weighed on an electronic balance. One cm2 of the same millimeter graph paper was also cut and weighed. Leaves of some species were sampled more than once.The following equation was used to calculate the leaf area nondestructively:Leaf area (cm2) = x/y, where x is the weight of the graph paper covered by the leaf outline (g) and y is the weight (g), of the cm2 area of the graph paper. In addition, areas of ten leaves each from five species were measured using the leaf area meter while still attached to the plants. Outline of these attached leaf samples were also drawn on the millimeter graph paper. The area of the graph paper covered by the outline was cut and weighed. A one cm2 of the millimeter graph paper was also cut and weighed. There were six hundred forty detached leaf samples (of thirty-three plant species), and fifty attached leaf samples (five plant species). Size of the leaves varied from 3.20 to 285.06 cm.The two sets of estimates (leaf area meter and millimeter graph paper) were related according toy=a+bx, where y is the leaf area estimated by leaf area meter and x is the leaf area estimated by millimeter graph paper. The independent variable here was the leaf area estimated by millimeter graph paper, and dependent variable was leaf area estimated by leaf area meter (SYSTRONICS, Leaf Area Meter-211). The regression equations were calculated by using Sigmaplot (ver.11).


## 3. Results and Discussion

Relationships between leaf area of detached leaves estimated by leaf area meter (dependent variable) and that estimated by millimeter graph paper method (independent variable) for thirty-three plant species as given in Table1 show that the two sets of estimates are strongly related with each other for each of the thirty-three species and that the nondestructive estimates by millimeter graph paper method are as good as those obtained destructively by leaf area meter method (Figure 1). For individual species, the coefficient of determination between the two sets of estimates varied between 0.933 and 0.998 and collectively across the thirty-three, the R2 was as high as 0.999. These relationships were also tested on attached leaf samples for five species (Table 2). Relationships were again linear and significant, (R2 = 0.996 to 0.998, Figure 2).Table 1
Regression equations and coefficients of determination (R2) between leaf area measured by leaf area meter (y, cm2) and that estimated by millimeter graph paper method (x, cm2) for thirty-three plant species, (n=10 for each species). Observations were made on detached leaves.
S. no.SpeciesMonth of samplingRegression equationR21Albizia lebbeckSeptemberY=0.742X+0.7410.933Albizia lebbeckSeptemberY=0.785X+0.4780.859Albizia lebbeckOctoberY=1.093X-1.6850.9862Anacardium occidentaleAugustY=0.986X-1.1590.995Anacardium occidentaleSeptemberY=1.000X-2.1470.9913Anthocephalus cadambaSeptemberY=0.986X-1.4950.9974Artocarpus heterophyllusOctoberY=0.984X-0.9470.998Artocarpus heterophyllusOctoberY=0.972X-0.8940.9935Artocarpus lakoochaAugustY=1.085X-2.7530.9776Bombax ceibaAugustY=0.955X-0.6490.993Bombax ceibaOctoberY=0.961X-0.7360.9967Buchanania lanzanAugustY=0.975X-0.0120.9988Butea monospermaAugustY=0.836X-0.6320.953Butea monospermaOctoberY=0.985X-0.9520.9989Cassia fistulaAugustY=0.942X-0.3210.992Cassia fistulaSeptemberY=0.988X-1.5100.99710Crescentia cujeteSeptemberY=0.976X-0.4260.998Crescentia cujeteAugustY=0.983X-1.4580.99511Diospyros melanoxylonOctoberY=0.973X-0.9540.99712Eucalyptus globulusSeptemberY=0.948X-0.3300.99113Ficus benghalensisSeptemberY=0.971X-0.7630.99714Ficus caricaAugustY=0.986X-1.1700.99415Ficus religiosaSeptemberY=1.000X-1.8790.997Ficus religiosaOctoberY=0.991X-2.2960.997Ficus religiosaOctoberY=0.995X-1.8630.99816Hibiscus rosa-sinensisOctoberY=0.970X-0.8130.99517Holoptelea integrifoliaAugustY=0.982X-1.3670.998Holoptelea integrifoliaOctoberY=0.962X-0.4780.99818Litchi chinensisAugustY=0.989X-2.0090.995Litchi chinensisAugustY=0.970X-0.8160.996Litchi chinensisOctoberY=0.964X-0.8620.99319Madhuca indicaAugustY=0.991X-1.9250.995Madhuca indicaOctoberY=0.990X-1.9330.99820Mangifera indicaAugustY=0.997X-1.8010.998Mangifera indicaOctoberY=0.983X-1.2410.996Mangifera indicaOctoberY=0.996X-1.1770.99821Melia azadirachtaAugustY=1.007X-1.3380.977Melia azadirachtaSeptemberY=1.023X-1.1220.96922Morus albaAugustY=0.973X-0.7350.996Morus albaSeptemberY=0.978X-0.5340.99823Polyalthia longifoliaAugustY=0.966X-0.6440.998Polyalthia longifoliaAugustY=0.967X-0.8870.998Polyalthia longifoliaSeptemberY=1.014X-2.3660.99324Populus albaAugustY=0.956X-0.7080.99325Psidium guajavaSeptemberY=0.969X-0.6000.994Psidium guajavaSeptemberY=0.959X-0.6340.99726Sapindus emarginatusAugustY=0.98X-1.0970.997Sapindus emarginatusSeptemberY=0.967X-0.7870.989Sapindus emarginatusSeptemberY=0.976X-1.0250.997Sapindus emarginatusSeptemberY=0.997X-1.4740.998Sapindus emarginatusAugustY=0.987X-1.2020.99227Shorea robustaAugustY=0.97X-0.7350.997Shorea robustaOctoberY=0.997X-2.6290.99828Sporadic pinataAugustY=0.959X-0.4750.995Sporadic pinataOctoberY=1.024X-3.0140.99629Sterculia colorataOctoberY=1.011X-3.6510.998Sterculia colorataAugustY=1.002X-2.9120.99830Syzygium jambolanumSeptemberY=0.964X-0.0650.996Syzygium jambolanumSeptemberY=1.002X-1.8870.997Syzygium jambolanumOctoberY=0.976X-0.7090.99831Tectona grandisSeptemberY=0.996X-2.1800.99832Terminalia chebulaOctoberY=0.979X-0.7670.99833Terminalia tomentosaAugustY=0.977X-0.6760.997Terminalia tomentosaOctoberY=0.974X-1.0890.992AllR2 values are significant at P<0.0001.Table 2
Regression equations and coefficients of determination (R2) between leaf area measured by leaf area meter (y, cm2) and that estimated by millimeter graph paper method (x, cm2) for five plant species, (n=10 for each species). Observations were made on attached leaves.
S. no.SpeciesMonth of samplingRegression equationR21Crescentia cujeteAugustY=0.953X-1.2470.9982Hibiscus rosa-sinensisOctoberY=0.984X-2.4360.9963Morus albaSeptemberY=0.979X-1.5800.9984Populus albaAugustY=0.967X-1.5370.9985Psidium guajavaSeptemberY=1.030X-2.4830.997AllR2 values are significant at P<0.0001.Figure 1
Relationship between leaf area of detached leaves measured by leaf area meter and that estimated by millimeter graph paper method across thirty-three plant species (n=640).Figure 2
Relationship between leaf area of attached leaves measured by leaf area meter and that estimated by millimeter graph paper method across five plant species (n=50).Easily measured leaf parameters such as length and width, and their combinations have been used for nondestructive leaf area estimation, though the accuracy of the predictions is dependent on the variation of the leaf shape due to differential genotypes (Cristofori et al. [35], Cristofori et al. [39], Zhang and Liu [47]). The ratio of length to width is highly variable among the species due to complexity in the leaf shapes. On the other hand, the method using leaf outline on millimeter graph paper can be successfully used to estimate leaf area across variety of species. Some important factors which affect the accuracy of the millimeter graph paper method are the lack of proper spread of leaf over millimeter graph paper, absence of accurate drawing of leaf margins, lack of even cutting of the drawn outline, and lack of precision in weighing. The errors originating from the leaves not being perfectly flat, overlying leaflets, and similar factors are common to both the millimeter graph paper and leaf area meter. The millimeter graph paper method is faster and can be applied to attached leaves (nondestructive) and anywhere as in forest or agricultural field.


## 4. Conclusion

The millimeter graph paper method described in this paper was used to estimate individual leaf area of thirty-three woody species. The estimates had significant linear relationships with the estimates obtained by using sophisticated leaf area meter. The millimeter graph paper method can estimate precisely and in large quantities leaf area of plants in many experimental comparisons without the use of costly instruments.



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