A Simple , Cost-Effective Method for Leaf Area Estimation

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.

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.

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 cm 2 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 (cm 2 ) = 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 cm 2 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 cm 2 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 to y = 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).

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 Table 1 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 thirtythree, the R 2 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, (R 2 = 0.996 to 0.998, Figure 2).
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.

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.

Figure 1 :
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 :
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).

Table 1 :
Regression equations and coefficients of determination (R 2 ) between leaf area measured by leaf area meter (y, cm 2 ) and that estimated by millimeter graph paper method (x, cm 2 ) for thirty-three plant species, (n = 10 for each species).Observations were made on detached leaves.
All R 2 values are significant at P < 0.0001.

Table 2 :
Regression equations and coefficients of determination (R 2 ) between leaf area measured by leaf area meter (y, cm 2 ) and that estimated by millimeter graph paper method (x, cm 2 ) for five plant species, (n = 10 for each species).Observations were made on attached leaves.
All R 2 values are significant at P < 0.0001.