Non-destructive evaluation of chlorophyll content in quinoa and amaranth leaves by simple and multiple regression analysis of RGB image components

Leaf chlorophyll content provides valuable information about physiological status of plants; it is directly linked to photosynthetic potential and primary production. In vitro assessment by wet chemical extraction is the standard method for leaf chlorophyll determination. This measurement is expensive, laborious, and time consuming. Over the years alternative methods, rapid and non-destructive, have been explored. The aim of this work was to evaluate the applicability of a fast and non-invasive field method for estimation of chlorophyll content in quinoa and amaranth leaves based on RGB components analysis of digital images acquired with a standard SLR camera. Digital images of leaves from different genotypes of quinoa and amaranth were acquired directly in the field. Mean values of each RGB component were evaluated via image analysis software and correlated to leaf chlorophyll provided by standard laboratory procedure. Single and multiple regression models using RGB color components as independent variables have been tested and validated. The performance of the proposed method was compared to that of the widely used non-destructive SPAD method. Sensitivity of the best regression models for different genotypes of quinoa and amaranth was also checked. Color data acquisition of the leaves in the field with a digital camera was quick, more effective, and lower cost than SPAD. The proposed RGB models provided better correlation (highest R ²) and prediction (lowest RMSEP) of the true value of foliar chlorophyll content and had a lower amount of noise in the whole range of chlorophyll studied compared with SPAD and other leaf image processing based models when applied to quinoa and amaranth.     

Estimation of the chlorophyll content of micropropagated potato plants using RGB based image analysis

A method has been developed for rapid and non invasive determination of chlorophyll content of leaves of micropropagated potato plants using RGB based image analysis. Among the trichomatic colors, R and G negatively correlated with the chlorophyll content, while a positive correlation was observed with B chromate. Compared to mean brightness value, the use of mean brightness ratio considerably improved the relationship of the tricolors with chlorophyll content. The brightness values and ratios of the primary colors are modeled as linear correlation functions for chlorophyll content. A significant correlation was observed between the model predicted chlorophyll content with the chlorophyll content measured by chlorophyll content meter. Spectral properties such as luminosity and saturation were also found to be negatively correlated with the chlorophyll content. The relationship was improved by combining the mean brightness ratio at B band region with luminosity. The potential of the imaging system in micropropagation has been discussed.     

Intelligent image analysis (IIA) using artificial neural network (ANN) for non-invasive estimation of chlorophyll content in micropropagated plants of potato

An artificial neural network (ANN) was used to analyze photometric features extracted from the digitized images of leaves from in vitro-regenerated potato plants for non-invasive estimation of chlorophyll content. A MATLAB®-based, feed-forward, backpropagation-type network was developed for an input layer (three input elements), with one hidden layer (one node) and one output layer representing the predicted chlorophyll content. A significant influence of training function during optimization of ANN modeling was observed. Among the 11 training functions tested, “trainlm” was found to be the best on the basis of comparative analysis of root-mean-square error (RMSE) at zero epoch. A significant correlation between the model-predicted and Soil-Plant Analysis Development (SPAD) meter-measured relative chlorophyll contents was obtained when the mean brightness ratio (rgb) parameters were used. Compared to a red (R), green (G), and blue (B) color space model, the rgb model exhibited better performance with a significant correlation (R ² = 0.85). Incorporation of photometric features, such as luminosity (L), blue (B)/L, and green (G)/L, with rgb failed to improve the performance of the network. The developed Intelligent image analysis (IIA) system was able to estimate in real time the chlorophyll content of in vitro-regenerated leaves for assessment of plant nutrient status during micropropagation.     

Association of specific leaf weight, an estimate of chlorophyll, and chlorophyll concentration with apparent photosynthesis in soybean

Increasing specific leaf weight (SLW) may improve leaf apparent photosynthesis (AP) in soybean [Glycine max (L.) Merr.] but screening for SLW and AP is laborious. The Objectives of this study were (i) to determine the time course of SLW and chlorophyll concentration in experimental lines selected for differences in SLW and (ii) to evaluate the potential use of the Minolta 502 SPAD meter as a rapid estimator of SLW, AP and chlorophyll concentration in leaves of soybean. In 1991 and 1992, sixteen experimental lines representing extremes in SLW were grown at Urbana, IL, and West Lafayette, IN, with three replications at each location. SPAD values, SLW and AP were measured at the R2 (full flower), R4 (full pod) and R5 (beginning seed) growth stages. In 1992 SLW, SPAD values and chlorophyll concentration were measured weekly. Seasonal patterns of SPAD values, SLW, and chlorophyll concentration were very similar through R5. After R5, SLW continued to increase but SPAD values and chlorophyll concentration declined. SPAD values and SLW were highly correlated at the R2, R4 and R5 stages at both locations and in both years. Environmental conditions during this research were not suitable for maximum AP expression, which is likely why AP and SPAD values were correlated only at the R4 growth stage at Urbana in 1992. SPAD measurements were consistent across diverse environments and effectively separated the high SLW lines from the low SLW lines. Measuring with the Minolta 502 SPAD meter is rapid, simple and non-destructive and could be an alternative method for direct selection for SLW.Abbreviations AP- leaf apparent photosynthesis - CV- coefficient of variation - Rug- leaf rugosity - SLW- specific leaf weight - SPAD-L- SPAD value of the most recently expanded terminal leaflet - SPAD-P- SPAD value of leaflet used to measure AP - SPAD-S- SPAD value of leaflets used to measure SLW - SPAD-U- SPAD value of the terminal leaflet one node above the most recently expanded terminal leaflet The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged     

叶绿素计SPAD-502在林业上应用

叶绿素是植物光合作用的色素,传统方法测定叶绿素一般采用分光光度法.本研究采用便携式叶绿素计SPAD-502测定落叶松人工林下4个主要阔叶树种绿色度（SPAD值）的季节变化,并与分光光度法测定的叶绿素含量进行相关性分析.结果表明,SPAD值与叶绿素含量具有显著的相关性,SPAD值能较好地反映树木叶绿素含量的变化.因此,使用叶绿素计测定树木的叶绿素含量是完全可行的,在一定条件下可代替叶绿素含量的直接测定.由于叶绿素计SPAD-502携带方便、测定简便、迅速,且不损坏叶片,应在林业研究中积极推广使用.     

SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement

Non-destructive assessment of chlorophyll content has recently been widely done by chlorophyll meters based on measurement of leaf transmittance (e.g. the SPAD-502 chlorophyll meter measures the leaf transmittance at 650 and 940 nm). However, the leaf transmittance depends not only on the content of chlorophylls but also on their distribution in leaves. The chlorophyll distribution within leaves is co-determined by chloroplast arrangement in cells that depends on light conditions. When tobacco leaves were exposed to a strong blue light (about 340 μmol of photons m⁻² s⁻¹), a very pronounced increase in the leaf transmittance was observed as chloroplasts migrated from face position (along cell walls perpendicular to the incident light) to side position (along cell walls parallel to the incoming light) and the SPAD reading decreased markedly. This effect was more pronounced in the leaves of young tobacco plants compared with old ones; the difference between SPAD values in face and side position reached even about 35%. It is shown how the chloroplast movement changes a relationship between the SPAD readings and real chlorophyll content. For an elimination of the chloroplast movement effect, it can be recommended to measure the SPAD values in leaves with a defined chloroplasts arrangement.     

Anthocyanin contribution to chlorophyll meter readings and its correction

Leaf chlorophyll content is an important physiological parameter which can serve as an indicator of nutritional status, plant stress or senescence. Signals proportional to the chlorophyll content can be measured non-destructively with instruments detecting leaf transmittance (e.g., SPAD-502) or reflectance (e.g., showing normalized differential vegetation index, NDVI) in red and near infrared spectral regions. The measurements are based on the assumption that only chlorophylls absorb in the examined red regions. However, there is a question whether accumulation of other pigments (e.g., anthocyanins) could in some cases affect the chlorophyll meter readings. To answer this question, we cultivated tomato plants (Solanum lycopersicum L.) for a long time under low light conditions and then exposed them for several weeks (4 h a day) to high sunlight containing the UV-A spectral region. The senescent leaves of these plants evolved a high relative content of anthocyanins and visually revealed a distinct blue color. The SPAD and NDVI data were collected and the spectra of diffusive transmittance and reflectance of the leaves were measured using an integration sphere. The content of anthocyanins and chlorophylls was measured analytically. Our results show that SPAD and NDVI measurement can be significantly affected by the accumulated anthocyanins in the leaves with relatively high anthocyanin content. To describe theoretically this effect of anthocyanins, concepts of a specific absorbance and a leaf spectral polarity were developed. Corrective procedures of the chlorophyll meter readings for the anthocyanin contribution are suggested both for the transmittance and reflectance mode.     

Visible foliar injury caused by ozone alters the relationship between SPAD meter readings and chlorophyll concentrations in cutleaf coneflower

The ability of the SPAD-502 chlorophyll meter to quantify chlorophyll amounts in ozone-affected leaves of cutleaf coneflower (Rudbeckia laciniata var. digitata) was assessed in this study. When relatively uninjured leaves were measured (percent leaf area affected by stipple less than 6%), SPAD meter readings were linearly related to total chlorophyll with an adjusted R ² of 0.84. However, when leaves with foliar injury (characterized as a purple to brownish stipple on the upper leaf surface affecting more than 6% of the leaf area) were added, likelihood ratio tests showed that it was no longer possible to use the same equation to obtain chlorophyll estimations for both classes of leaves. Either an equation with a common slope or a common intercept was necessary. We suspect several factors are involved in altering the calibration of the SPAD meter for measuring chlorophyll amounts in visibly ozone-injured leaves, with the most likely being changes in either light absorption or scattering resulting from tissue necrosis.     

Comparison of Chlorophyll Meter Readings with Leaf Chlorophyll Concentration in Amaranthus vlitus: Correlation with Physiological Processes1

The portable chlorophyll meter (SPAD-502) has been successfully used for a rapid and direct estimation of total chlorophyll content (TCHL) in the leaves of some crops. In this work, SPAD-502 meter readings and TCHL concentration were compared for the leaves of Amaranthus vlitus L., a common weed. SPAD readings were linearly and positively correlated to TCHL concentration in the leaves. A linear correlation was also shown between SPAD-502 readings and some physiological parameters of the leaves, such as photosynthesis, transpiration, and stomatal conductance.     

A novel method for the estimation of soybean chlorophyll content using a smartphone and image analysis

The development of smartphones, specifically their cameras, and imaging technologies has enabled their use as sensors/measurement tools. Here we aimed to evaluate the applicability of a fast and noninvasive method for the estimation of total chlorophyll (Chl), Chl a, Chl b, and carotenoids (Car) content of soybean plants using a smartphone camera. Single leaf disc images were obtained using a smartphone camera. Subsequently, for the same leaf discs, a Chl meter was used to obtain the relative index of Chl and the photosynthetic pigments were then determined using a classic method. The RGB, HSB and CIELab color models were extracted from the smartphone images and correlated to Chl values obtained using a Chl meter and by a standard laboratory protocol. The smartphone camera was sensitive enough to capture successfully a broad range of Chl and Car contents seen in soybean leaves. Although there was a variation between color models, some of the proposed regressions (e.g., the S and b index from HSB and Lab color models and NRI [RGB model]) were very close to the Chl meter values. Based on our findings, smartphones can be used for rapid and accurate estimation of soybean and Car contents in soybean leaves.     

A non-destructive determination of leaf chlorophyll in Vitis vinifera

A portable leaf greenness meter (SPAD-501) has been used to provide a rapid and non-destructive measurement of leaf chlorophyll in Vitis vinifera. Leaf extracted chlorophyll was related linearly to SPAD readings. It is suggested that separate linear equations should be developed for each cultivar so as to maximise the accuracy of estimating leaf chlorophyll content as a function of SPAD readings.     

Cultivar-specific impairment of strawberry growth,photosynthesis, carbohydrate and nitrogen accumulation by ozone

In a 2-year study, fruiting plants of strawberry (Fragaria × ananassa Duch.) cv. ‘Korona’ and ‘Elsanta’ were exposed for 2 months to 78 ppb ozone on average or filtered air without ozone in controlled environment chambers. Plant growth, photosynthesis, carbohydrate accumulation, and macronutrient concentrations were investigated in order to demonstrate cultivar-specific differences in the ozone sensitivity of ‘Korona’ and ‘Elsanta’ on the whole plant level. Moreover, the hypothesis was tested whether properties of the root system in strawberry were involved in ozone tolerance, for example, the roots’ ability to store or make available carbohydrates and their capacity to secure plants’ supply with nitrogen during a stress situation. In strawberry, ozone reduced leaf area by reducing leaf number. Moreover, specific leaf area (SLA) and relative leaf water content were reduced. Net photosynthesis was only slightly impaired, but activity of Rubisco and chlorophyll content in older leaves of cv. ‘Elsanta’ were significantly reduced. The most important, indirect impairment of photosynthesis was the reduction of plants’ total leaf area, which resulted in a decrease in plant biomass. The reduction of root biomass, the root/shoot ratio, and also the distribution of carbohydrates indicated a partitioning priority of the shoot at expense of the root system. Cultivar ‘Elsanta’ was characterized by significantly lower carbohydrate levels in ozone-exposed leaves, whereas levels remained fairly stable in ‘Korona’ leaves. In addition, nitrogen concentrations in leaves and roots decreased significantly in ‘Elsanta’, not in ‘Korona’. The reduced nitrogen concentration in leaves may be related with the more distinct reduction in Rubisco activity and chlorophyll content in older leaves of ‘Elsanta’.     

Use of a SPAD-502 meter to measure leaf chlorophyll concentration in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The SPAD-502 meter is a hand-held device that is widely used for the rapid, accurate and non-destructive measurement of leaf chlorophyll concentrations. It has been employed extensively in both research and agricultural applications, with a range of different plant species. However, its utility has not been fully exploited in relation to the most intensively studied model organism for plant science research, Arabidopsis thaliana. Measurements with the SPAD-502 meter produce relative SPAD meter values that are proportional to the amount of chlorophyll present in the leaf. In order to convert these values into absolute units of chlorophyll concentration, calibration curves must be derived and utilized. Here, we present calibration equations for Arabidopsis that can be used to convert SPAD values into total chlorophyll per unit leaf area (nmol/cm²; R ² = 0.9960) or per unit fresh weight of leaf tissue (nmol/mg; R ² = 0.9809). These relationships were derived using a series of Arabidopsis chloroplast biogenesis mutants that exhibit chlorophyll deficiencies of varying severity, and were verified by the subsequent analysis of senescent or light-stressed leaves. Our results revealed that the converted SPAD values differ from photometric measurements of solvent-extracted chlorophyll by just ~6% on average.     

Chlorophyll meter and leaf colour chart to estimate chlorophyll content, leaf colour, and yield of cassava

A field experiment was conducted with two cassava cultivars and eight levels of nitrogen to examine the relationship between extractable chlorophyll (Chl) content of cassava leaves and both the Chl meter value (SPAD) and leaf colour chart (LCC) score. The SPAD, LCC, and Chl a+b content were influenced by leaf position, growth stage, cultivar (cv.), and N fertilization. The cvs. and N fertilization had significant effect on SPAD, LCC, and Chl a+b content of youngest fully expanded leaf (leaf 1) blade in most cases. An F-test indicated that common equations pooled across cvs., N fertilization, and growth stages could be used to describe the relationships between Chl a+b content and LCC and between SPAD and LCC, but not between SPAD and Chl a+b content. Relationships between tuber yield and SPAD, LCC, and Chl a+b content were significant (p<0.05) and positive at 30 and 60 d after planting. Thus LCC and SPAD can be used to estimate leaf Chl content which is an indicator of leaf N status.     

Using a single-board computer as a low-cost instrument for SPAD value estimation through colour images and chlorophyll-related spectral indices

The leaf chlorophyll content is a major indicator of plant stress. Therefore, it is often used for the evaluation of crop status to adjust agricultural management to ensure high quality yield while concurrently applying water and agrochemicals in a sustainable manner. Since laboratory procedures for their assessment are time-consuming and destructive, nondestructive methods have been developed recently based on known vegetation spectral response characteristics. In addition to various vegetation indices derived from remotely sensed data, hand-held sensors such as SPAD-502 are currently widely used for in-field sampling to gain precise information for decision-making in terms of best-fitting agricultural management. However, the costs of such commercial devices can be limiting for farmers. The low-cost alternatives that have been developed recently exploit widely accessible digital cameras with sensors sensitive to the visible region of the electromagnetic spectrum. Digital numbers extracted from colour images in RGB channels serve as the input for broadband “chlorophyll index” calculations. Major constraints regarding digital cameras are, however, the natural light illuminance and the necessity of data postprocessing. In the framework of this study, a novel technological solution was developed to address these issues. A Raspberry Pi single-board computer together with a Pi Camera and a simple LED incorporated in a 3D print case created a prototype called Rasp2SPAD, which was programmed to acquire and analyse a colour image. The prototype and its setup were further tested on the experimental plant material of the winter rapeseed. A set of 22 chlorophyll-related parameters across various colour representation models were generated, from which an SPAD value was modelled using i) a simple linear model, ii) a generalized linear model, and iii) an artificial neural network. The blue (Cb) and red (Cr) chroma components of the YUV colour space were found to be most suitable for SPAD value modelling. Calibration equations were determined, and the results reached relatively high accuracy (mean absolute deviance 1.85 and R-squared 0.81 for simple linear model) while keeping the costs significantly low compared to the most commonly used commercial sensor. In this way, a simple and cheap methodology was introduced to bring the results of research closer to practice, which should help first spread the precision agriculture concept to a wider audience and second allow them to utilize with it.     

刨花楠和华东润楠叶绿素含量分异特征及与净光合速率的关系

采用SPAD-502叶绿素计、LI-6400型便携式光合分析仪与分光光度法分别对种子源于江西遂川(MPS)和福建建瓯(MPJ)的3年生刨花楠与江西上犹(MLG)的3年生华东润楠苗木叶片的SPAD值、净光合速率(P_n)和叶绿素a+b含量(C_t) 进行了测定.结果表明：3种苗木叶片叶绿素含量存在显著差异,其SPAD值、叶绿素a+b、叶绿素a与叶绿素b含量均为MPS-1.受叶肉组织发育成熟程度影响,当年生新叶的叶绿素含量低于2年生叶片.MPS与MPJ刨花楠同一叶龄叶片不同冠层间的C_t次序为上冠层<中冠层<下冠层,华东润楠则为上冠层<下冠层<中冠层.同一叶片不同观测部位间的SPAD值次序为尖端<中部<基部.叶片的SPAD值与叶绿素a+b含量呈显著正相关,与P_n虽存在正相关,但不显著.     

Simultaneous estimation of chlorophyll a and lipid contents in microalgae by three-color analysis

A method of rapid determination of chlorophyll a and lipid contents of microalgae based on colorimetric analysis of the digital images of the microalgae is proposed. The color variation of microalgae during cultivation is evaluated by the brightness of the three primary colors (red, green, and blue). The brightness values of the three primary colors are modeled as two linear correlation functions (RGB model) for microalgal chlorophyll a and lipid contents, respectively. The chlorophyll a and lipid contents predicted by the proposed model are compared with that determined by the standard methods. The good agreement of the model predictions with experimental results is demonstrated with a squared correlation coefficient (R(2)) of 0.99 for chlorophyll a and lipid. The reliability of the RGB model was verified in real cultivations of the microalgae in a photobioreactor. Growth dynamics, contents of chlorophyll a and lipid corresponded very well with previously reported studies.     

Use of a SPAD meter to estimate chlorophyll content in Eugenia uniflora L. leaves as affected by contrasting light environments and soil flooding

In three separate experiments, the effectiveness of a SPAD-502 portable chlorophyll (Chl) meter was evaluated for estimating Chl content in leaves of Eugenia uniflora seedlings in different light environments and subjected to soil flooding. In the first experiment, plants were grown in partial or full sunlight. In the second experiment plants were grown in full sunlight for six months and then transferred to partial sunlight or kept in full sunlight. In the third experiment plants were grown in a shade house (40% of full sunlight) for six months and then transferred to partial shade (25–30% of full sunlight) or full sunlight. In each experiment, plants in each light environment were either flooded or not flooded. Non-linear regression models were used to relate SPAD values to leaf Chl content using a combination of the data obtained from all three experiments. There were no significant effects of flooding treatments or interactions between light and flooding treatments on any variable analyzed. Light environment significantly affected SPAD values, chlorophyll a (Chl a), chlorophyll b (Chl b), and total chlorophyll [Chl (a+b)] contents in Experiment I (p≤0.01) and Experiment III (p≤0.05). The relationships between SPAD values and Chl contents were very similar among the three experiments and did not appear to be influenced by light or flooding treatments. There were high positive exponential relationships between SPAD values and Chl (a+b), Chl a, and Chl b contents.     

Relationships between early vigour, grain yield, leaf structure and stable isotope composition in field grown barley

Fast growth and early development in barley are used in breeding programmes to improve the water use efficiency and transpiration efficiency of this crop in Mediterranean conditions. Here, we examine the use of several simple traits based on the structure and stable isotope composition of seedling leaves to assess differences in early vigour, phenology and grain yield, and also the interaction with low temperatures in barley. A set of 260 F₈ lines of two-row barley (Hordeum vulgare L.) derived from the cross of Tadmor and WI 2291 were cultivated in two locations in northwest Syria. Total chlorophyll content on an area basis (SPAD) and specific leaf dry weight (SLDW) were measured in recently fully expanded intact leaves of seedlings. Total leaf area and total dry weight per seedling were evaluated in the same seedlings. The stable isotope compositions of carbon and nitrogen (δ¹³C and δ¹⁵N, respectively) were analyzed in the same leaves on a subset of 75 genotypes. Number of days from planting to heading and grain yield were recorded at both sites. The grain yield measured at both locations was positively correlated with the SPAD value of seedlings, but showed no relationship with SLDW. Days to heading was negatively correlated with SPAD values. Regarding early vigour, a negative relationship between the SLDW and the total leaf area of seedlings was observed. However, no relationship between the δ¹³C of seedlings and early vigour was observed, except when only the genotypes most resistant to low temperatures (i.e. showing the highest SPAD values) were considered. This subset of genotypes showed negative relationships between δ¹³C and either total leaf area or total dry weight. In addition, δ¹⁵N was negatively correlated with SPAD only within the high-SPAD genotypes. This suggests that within the genotypes resistant to low temperatures, those with higher chlorophyll content assimilate more nitrogen from nitrate.     

Effect of PEG-6000 imposed drought stress on RNA content,relative water content (RWC), and chlorophyll content in peanut leaves and roots

Drought, one of the environmental stresses, plays crucial role in reduction in plant production on majority of agricultural fields of world, In order to evaluate drought stress on RNA content Relative water content (RWC), and chlorophyll content, Water deficit was induced by Polyethylene glycol (PEG) in peanut (Arachis hypogaea), accession number ICGV 91114. In this current study we evaluate RNA content and Relative water content (RWC) both in leaves and roots and chlorophyll content in leaf. The present study was undertaken with the aim to investigate the effect of water deficit imposed by PEG-6000, 40 old day seedlings were treated with varying concentrations of polyethylene glycol-6000 (PEG-6000; w/v-5%, 10%, 15% & 20%) for 24 h. The results showed that RNA content and Relative water content (RWC) content was significantly reduced in both leaves and roots with increased concentration of PEG, In leaves, a concentration dependent decline in chlorophyll content with increasing concentration of polyethylene glycol-6000 (PEG-6000). Reduction in chlorophyll ‘a’ level was to a greater extent than the chlorophyll ‘b’. Thus, this attributes can be used as screening tool for drought tolerance in peanut.