LEAF AREA IN GRASS CEREALS IN RELATION TO LEAF PARAMETERS; CASE STUDY IN WHEAT

. The study determined values of the correction factor (CF) for four wheat cultivars, in order to determine with greater accuracy the leaf surface by the non-destructive method, based on leaf parameters (length, width). The wheat cultivars Ciprian, Dacic, Glosa and Padureni were considered in the study. The plant samples came from cultures made within SCDA Lovrin, Romania. For each wheat cultivar, 100 leaf samples were taken (flag leaf). The dimensions of the leaves (length – L, width – w) were obtained by measurement (±0.5 mm). By scanning, the scanned leaf surface (SLA) was obtained. The values of the correction factor were obtained by calculation (CF=0.79 for Ciprian cultivar; CF=0.80 for Dacic cultivar, CF=0.78 for Glosa cultivar; CF=0.79 for the Padureni cultivar). The leaf area was calculated with high precision for each wheat cultivar, as a relationship between L, w and CF (RMSEP=1.32042 for Ciprian cultivar; RMSEP=2.41317 for Dacic cultivar; RMSEP=2.21665 for Glosa cultivar; RMSEP=1.30141 for Padureni cultivar). The fit between MLA (measured leaf area) and SLA (scanned leaf area) was described by linear equations, with the highest precision in the case of the Padureni wheat cultivat (R 2 = 0.933), a cultivar in whose case the RMSEP value also confirmed the higher precision for calculating the MLA. The correlation analysis on the series of data related to each wheat cultivar studied, highlighted various levels of correlations, in conditions of statistical safety. The correlation analysis on the entire data series (400 values) highlighted very strong negative correlations between the calculated ratios (r=-0.977 between L/w and w/L) and very strong positive correlations at the leaf area level (r=0.920 between MLA and SLA). High variability was recorded in the case of MLA for the Ciprian cultivar (CV MLA =19.61861) and for the Padureni cultivar (CV MLA =27.07106), in the case of SLA for the Dacic cultivar (CV SLA =22.72838), and Glosa cultivar (CV SLA =18.52030).


INTRODUCTION
Determining the leaf area for plants in general, and for crop plants in particular, is important in relation to different influencing factors and study perspectives (Fender et al., 2011;Weraduwage et al., 2015;Wang et al., 2019).
The non-destructive methods for determining the leaf area are of high interest, as a result of the multiple advantages they present (Campillo et al., 2010;Suárez et al., 2022;Huaccha-Castillo et al., 2023).
Within the non-destructive methods, the determination of the leaf area based on the leaf size parameters has the advantage of low costs, accessibility, work speed and sufficiently high precision, the implementation of some applications on mobile devices posibilities etc. (Shi et al., 2018;Liu et al., 2019;He et al., 2020).
As a result of the advantages, leaf area determination based on leaf sizes has been used in different plant species (Schrader et al., 2021;Koyama and Smith, 2022).
As a result of the typology of plant leaves, for precision leaf area, coefficients or correction factors in the calculation relationship, or other calculation artifices were most frequently used to compensate the geometric shape that frames the leaf (resulting from L x w) and the really occupied by the leaf (Sala et al., 2017;Shi et al., 2020;Yu et al., 2020;Zhang, 2020;Schrader et al., 2021).
Within cultivated plant species, due to the large number of genotypes within the same species, although the shape of the leaves fits into a general pattern, there are variations between genotypes, which makes it necessary to find out the specific values for the correction factors, in relation to the genotype, or with certain groups of genotypes, based on the typology of the leaves.
Within grass cereals, wheat occupies a very important place in culture, as a result of the importance of grain production in human nutrition, animal feed, pastry, industrialization, etc. (Shewry and Hey, 2015).
The variability of wheat leaves was studied in relation to genetic factors, morphological and physiological aspects, vegetation conditions, nutritional status, stress factors, etc. (Constantinescu et al., 2018;Elshafei et al., 2019;Moraga et al., 2022).
For grass cereals, studies were communicated regarding the determination of the leaf area based on L and w, through different methods and techniques, among which some based on the size of the leaves (Chanda and Singh, 2002;Ahmad et al., 2015).
This study had as its main objective to determine the correction coefficient for four wheat cultivars, useful for calculating the leaf area by non-destructive methods, based on the leaves dimensions(length, width), in order to evaluate the reliability of determining the leaf area, as well as some interrelationships between determined leaf parameters.

MATERIAL AND METHODS
The study was carried out under the specific conditions of SCDA Lovrin, Romania.The biological material was represented by four wheat cultivars: Ciprian, Dacic, Glosa and Padureni, cultivated under similar field conditions.
In relation to the purpose of the study, leaf samples were taken, the flag leaf, 100 leaves for each cultivar.The leaves were scanned, and resulted the scanned leaf area (SLA), in high precision conditions, considered as a reference for subsequent calculations.
Each leaf was measured to obtain the values for leaf length (L) and width (w); the measurement precision was ±0.5 mm.
Wheat leaves have a linear shape, figure 1, and to determine the leaf area based on the leaves dimensions (L, w) a correction factor (CF) is required, relation (1).
(1) From the analysis, data series were obtained for the measured leaf area (MLA) based on leaf parameters in relation to different values of the correction factor (CF).In order to find out the optimal value of CF for each wheat cultivar studied, the series of MLA values were analyzed compared to SLA, and different error values resulted.The distribution of data series (MLA, SLA) was analyzed.
In order to validate the optimal values for CF, the minimum errors (ME) were calculated, as well as appropriate statistical parameters, such as RMSEP, equation (2), p, R 2 . (2) The degree of matching between MLA and SLA was also evaluated, as well as the level of correlation between leaf parameters and leaf surface.For the characterization of the leaf surface, the L/w and w/L ratios were calculated for each wheat cultivar considered in the study (for all leaf samples).
The calculation module in EXCEL and dedicated software (Hammer et al., 2001; JASP, 2022) were used for data processing and analysis.

RESULTS AND DISCUSSIONS
The primary data regarding the leaf sizes (L, w) were obtained by measurement (±0.5 mm precision), and based on them the ratios between the leaf sizes (L/w and w/L) were calculated for each wheat cultivar (series of 100 leaves for each cultivar).By scanning, the scanned leaf area (SLA) was obtained for each wheat cultivar.To determine the measured leaf area (MLA) based on the L and w parameters, it was necessary to find out the correction factor (CF) for each wheat cultivar.Based on the model proposed by Sala et al. (2015) the value of the correction factor (CF) was determined for each wheat cultivar, considered in this study.The general values of the basic statistical parameters of the complete series of data (400 data for the four cultivars) are presented in table 1.
The values obtained for the correction factor (CF), leaf area (SLA, MLA), mean errors (ME) and the RMSEP safety parameter, corresponding to each wheat cultivar studied, are presented in table 2. The determined SLA and MLA values presented normal distributions (normal type histograms), according to the diagrams in figure 2. The error values (ME) resulting as the difference between SLA and MLA (corresponding to the values of the correction factor CF), for each analyzed leaf, are presented on the wheat cultivars studied, in figure 3.
According to ANOVA test, F>Fcrit, p=0 (Alpha=0.001).In order to evaluate the level of precision of the calculated MLA values, the matching degree between the measured leaf area (MLA), calculated based on the leaf parameters (L, w) and the correction factor (CF, optimal values for each cultivar) and scanned leaf area (SLA) was analyzed, for the entire series of 100 leaves determined for each cultivar.
In the case of the Ciprian wheat cultivar, the fit between MLA and SLA was described by the linear equation ( 3), under conditions of R 2 =0.848, p<0.001,F=546.6.In the case of the Dacic wheat cultivar, the fit between MLA and SLA was described by equation ( 4), under conditions of R 2 =0.776, p<0.001,F=339.03.In the case of the Glosa cultivar, the fit between MLA and SLA was described by equation ( 5), under conditions of R 2 =0.673, p<0.001,F=201.3.In the case of the Padureni wheat cultivar, the fit between MLA and SLA was described by equation ( 6), under conditions of R 2 =0.933, p<0.001,F=1086.1.Under the conditions of the entire data series analysis (the four wheat cultivars, 400 leaves), the fit relationship between MLA and SLA was described by equation (7), under conditions of R 2 =0.847, p<0.001,F=2197.6.The graphic representation of the distribution of MLA in relation to SLA and of the fitting line, are presented in figure 4 for each wheat cultivar, and in figure 5 in the synthesis form.
(3) (4) (  From the analysis of the MLA values distribution in relation to SLA, figure 4, and of the associated statistical parameters, a better degree of fit was found in the case of the Padureni cultivar, compared to the other wheat cultivars studied. The correlation analysis highlighted for each wheat cultivar a variable number of correlations, positive or negative, of different intensity levels, between the basic leaf parameters, the calculated ratios and the determined leaf area, table 3.  The correlation analysis on the entire data series, including all four cultivars (400 values for each considered parameter), led to the diagram in figure 6.
In the complex diagram resulting from the analysis, the values of the correlation coefficient (r), the distribution of values for each studied parameter, and the interdependence between the studied parameters, with the statistical safety threshold (confidence and prediction interval, 95%) are presented.The analysis was made regarding the variability of the basic leaf parameter values (L, w), of the determined leaf area values (SLA, MLA) for each wheat cultivar.The analysis was done based on the coefficient of variation (CV), and the data are presented in table 4.
Based on the comparative analysis between the cultivars studied, the Padureni variety recorded the highest values of the coefficient of variation, for the length of the leaves (CVL=16.21858),for the width of the leaves (CVw=14.01973),for the scanned leaf area (CVSLA=25.42976)and for the measured leaf area (CVMLA=27.07106).Analyzing the situation of the level of variability in each cultivar, high variability was presented by the measured leaf area of the Ciprian cultivar (CVMLA=19.61861),the scanned leaf area of the Dacic cultivar (CVSLA=22.72838),the scanned leaf area of the Glosa cultivar (CVSLA=18.52030)and the the leaf mass measured in the Padureni cultivar (CVMLA=27.07106).In the study conditions, the Glosa cultivar presented lower values for the length of the leaves (L=11.00-22.50±0.21cm) and the Padureni cultivar presented higher values (L=13.50-28.00±0.35cm).For the leaf width parameter, lower values were recorded for the Ciprian cultivar (w=0.90 -1.50±0.01cm), and higher values were recorded for the Pădureni cultivar (w=1.00 -1.90±0.02cm).
For each of the cultivar studied, the value of the correction coefficient was determined, and based on them it was possible to determine with high precision the leaf area based on leaf parameters (MLA).The best level of match between MLA (obtained by calculation) and SLA was recorded in the Pădureni cultivar (R 2 =0.933, p<0.001,F=1086.1), a fact that may indicate a more harmonious relationship between the leaf parameters.
Values of the correction factor in relation to the complexity of the leaf lamina, the purpose of the study, the technology used, were also determined in the case of other plant species, in comparative studies on eucalyptus and pinus species (Lopes et al., 2016), on poblano pepper (Mendoza-Pérez et al., 2017), in energetic poplar clones (Cândea-Crăciun et al., 2018), in apple banana (Nowembabazi et al., 2021), but also in other species.Studies were also carried out that analyzed on a general scale leaf surface indices, methods and applications, aspects that confirm the importance of the safety of information related to the geometry of plant leaves (Fang et al., 2019).
Chanda and Singh (2002) used a correction factor with a value of 0.75 to calculate the leaf area in wheat, based on the leaf parameters (length, width) and obtained values of the leaf area, under conditions of statistical safety and acceptable errors, in depending on the varieties under study.
Relationships of interdependence and proportionality of the leaf area and leaf parameters (length, width, leaf size ratios) have been reported in different species of Poaceae in order to describe allometric relationships at leaf level, and in the leaves evolution (Shi et al., 2019).
Models for estimating the leaf area, based on leaf parameters, have been reported for different plant species (Schrader et al., 2021;Zheng et al., 2022;Huaccha-Castillo et al., 2023), in conditions of statistical safety in relation with the species, type of leaves, parameters considered in the obtained models.
The values of the correction factor obtained, specific for each wheat cultivar, are useful for determining with high precision the leaf surface of the respective cultivars, by non-destructive methods, in different studies.
Also, the method for determining the correction factor values, used in this study, can be adapted and used for other grass cereal species, but also for other crop plants, in order to determine the specific values for the correction factor in relation to different cultivated genotypes, which will facilitate the determination of the leaf area with much higher accuracy.

CONCLUSIONS
The approach method facilitated the determination of the correction factor values for each wheat cultivar studied, CF=0.79 for the Ciprian cultivar, CF=0.80 for the Dacic cultivar, CF=0.78 for the Glosa cultivar and CF=0.79 for the Padureni cultivar.
Based on the leaf parameters (L, w) and the specific correction factor, it was possible to calculate the leaf area (MLA) with high precision; RMSEP=1.32042 for the Cyprian cultivar; RMSEP=2.41317 for the Dacic cultivar; RMSEP=2.21665 for the Glosa cultivar; RMSEP=1.30141 for the Padureni cultivar.

Figure 2 .Figure 3 .
Normal type distribution of SLA and MLA values in the wheat cultivars studied Ciprian Dacic Glosa Padureni Graphical distribution of errors (ME) in relation to the correction factor for wheat cultivars studied

Figure 5 .
Figure 5. Distribution of MLA values in relation to SLA, and of the fit line, general analysis for the wheat cultivars studied; color codes: green -Ciprian cultivar; red -Dacic cultivar; blue -Glosa cultivar; purple -Padureni cultivar recorded in all four studied cultivars, a moderate correlation in the Ciprian cultivars, and weaker correlations in intensity were recorded in each of the studied cultivars.

Figure 6 .
Figure 6.Correlation diagram between foliar parameters in the wheat cultivars studied

Table 1 .
Statistical parameter values for the complete data series

Table 2 .
The values of SLA, MLA and safety parameters, associated with the correction factor (CF) for each Distribution of MLA values in relation to SLA, and of the fit lines for the wheat cultivars studied

Table 3 .
The table of correlations between foliar parameters in the wheat cultivars studied

Table 4 .
The coefficient of variation values for parameters studied in wheat, four cultivars