Development of a digital image analysis method for real-time estimation of chlorophyll content in micropropagated potato plants

The present work describes a digital image analysis method based on leaf color analysis to estimate chlorophyll content of leaves of micropropagated potato plantlets. For estimation of chlorophyll content, a simple leaf digital analysis procedure using a simple digital still camera was applied in parallel to a SPAD chlorophyll content meter. RGB features were extracted from the image and correlated with the SPAD values. None of the mean brightness parameters (RGB) were correlated with the actual chlorophyll content following simple correlation studies. However, a correlation between the chromaticity co-ordinates ‘r’, ‘b’ and chlorophyll content was observed, while co-ordinate ‘g’ was not significantly correlated with chlorophyll content. Linear regression and artificial neural networks (ANN) were applied for correlating the mean brightness (RGB) and mean brightness ratio (rgb) features to chlorophyll content of plantlet leaves determined through a SPAD meter. The chlorophyll content as determined by the SPAD meter was significantly correlated (RMSE = 3.97 and 3.59, respectively, for linear and ANN models) to the rgb values of leaf image analysis. Both the models indicate successful prediction of chlorophyll content of leaves of micropropagated plants with high correlation. The developed RGB-based digital image analysis has the advantage over conventional subjective methods for being objective, fast, non-invasive, and inexpensive. The system could be utilized for real-time estimation of chlorophyll content and subsequent analysis of photosynthetic and hyperhydric status of the micropropagated plants for better ex vitro survival.     

Threshold values of canopy reflectance indices and chlorophyll meter readings for optimal nitrogen nutrition of tomato

Sustainable and optimal economic N management requires correct and timely on‐farm assessment of crop N status to detect N deficiency or excess. Optical sensors are promising tools to assess crop N status throughout a crop or at critical times. The ability of optical sensor measurements of canopy reflectance (Crop Circle ACS 470) and leaf chlorophyll (SPAD 502 chlorophyll meter) to assess crop N status was evaluated weekly throughout an indeterminate tomato crop. Strong linear relationships with the optical sensor measurements were obtained, throughout most of the crop, for both (a) crop N content for ranges of 1.5–4.5%, and (b) the nitrogen nutrition index (NNI) for ranges of 0.4–1.3. The relationships of the optical sensor measurements to crop NNI were generally equal to or slightly better than with crop N content. Indices based on reflectance in the red, the normalised difference vegetation index (NDVI) and the red vegetation index (RVI), were the best predictors of crop N status in terms of goodness of fit, earliness and maintenance of relationships throughout the crop. SPAD chlorophyll readings and reflectance indices based on reflectance in the green, the normalised difference vegetation index on greenness (GNDVI) and the green vegetation index (GVI), were good indicators of crop N status for most of the crop, but with lower goodness of fit in the latter part of the crop. The linear relationships between sensor indices and readings with NNI or crop N content, each week, demonstrated the potential for using proximal canopy reflectance indices such as NDVI and RVI, and chlorophyll meter for monitoring crop N status of indeterminate tomato crops. Threshold values for optimal crop N nutrition for canopy reflectance indices and for chlorophyll meter readings were derived for each day of measurement from the relationships between optical sensor measurements and NNI by solving for NNI = 1. The threshold values obtained for each index and type of measurement varied during the crop cycle. The approach developed for determining threshold values from NNI can facilitate on‐farm use of optical sensors for monitoring crop N status, by enabling assessment of whether crop N status is excessive, deficient or adequate.     

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.     

基于Sentinel-2A影像干旱区棉花叶片SPAD数字制图

植被叶片叶绿素是农业遥感反演的重要参数,叶绿素含量的变化与植被生长环境的胁迫程度、生理变化密切相关,故将植被叶绿素进行实时、动态监测对农业生产极为重要。然而,传统经验模型及叶绿素精准测量存在困难。基于高分辨率的Sentinel-2A数据,在机器学习框架下,利用光谱信息、最适光谱指数和基于PROSAIL辐射传输模型的生物协变量构建3种建模方案(方案1:光谱信息和最适光谱指数联合,方案2:光谱信息和物理模型生物协变量联合,方案3:光谱信息、最适光谱指数和物理模型生物协变量联合)。最终基于优选出的建模方案进行棉花叶片叶绿素相对含量的空间数字制图。结果表明:(1)红边波段参与的最适光谱指数比值植被指数(RVI)与棉花叶片SPAD值相关性最高r=0.767,P^**=0.195;(2)将构建的17个变量进行重要性分析可知,构建的最适光谱指数比值植被指数(RVI)与物理模型生物协变量LAI-Cab对估算模型的精度贡献率较大;(3)建模方案构建植被指数时红边波段被确定为最优波段,在增加精度方面起到决定性作用;通过模型评价标准来分析3种方案可知,预测精度大小顺序为模型方案3>...     

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.     

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.     

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.     

平稳小波变换在冬小麦SPAD高光谱监测中的应用

在2010与2011年度冬小麦生长季通过大田小区试验,利用ASD便携式野外光谱仪和SPAD 502叶绿素计实测冬小麦冠层的高光谱反射率与SPAD值.分析不同SPAD值下的冬小麦冠层光谱特征,建立了基于归一化植被指数（NDVI）与比值植被指数（RVI）、小波能量系数的不同生育期冬小麦SPAD估算模型.结果表明： 随着SPAD值的增大,“绿峰”与“红谷”特征愈加明显.在冬小麦返青期、拔节期、抽穗期、灌浆期NDVI估算SPAD的效果较好,估算模型的R²分别为0.7957、0.8096、0.7557、0.5033.小波能量系数回归模型可以提高冬小麦SPAD的估算精度,在返青期、拔节期、抽穗期、灌浆期以高频、低频小波能量系数为自变量的冬小麦SPAD估算模型的R²分别达到0.9168、0.9154、0.8802、0.9087.     

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.     

Simple and robust methods for remote sensing of canopy chlorophyll content: a comparative analysis of hyperspectral data for different types of vegetation

Canopy chlorophyll content (CCC) is an essential ecophysiological variable for photosynthetic functioning. Remote sensing of CCC is vital for a wide range of ecological and agricultural applications. The objectives of this study were to explore simple and robust algorithms for spectral assessment of CCC. Hyperspectral datasets for six vegetation types (rice, wheat, corn, soybean, sugar beet and natural grass) acquired in four locations (Japan, France, Italy and USA) were analysed. To explore the best predictive model, spectral index approaches using the entire wavebands and multivariable regression approaches were employed. The comprehensive analysis elucidated the accuracy, linearity, sensitivity and applicability of various spectral models. Multivariable regression models using many wavebands proved inferior in applicability to different datasets. A simple model using the ratio spectral index (RSI; R815, R704) with the reflectance at 815 and 704 nm showed the highest accuracy and applicability. Simulation analysis using a physically based reflectance model suggested the biophysical soundness of the results. The model would work as a robust algorithm for canopy‐chlorophyll‐metre and/or remote sensing of CCC in ecosystem and regional scales. The predictive‐ability maps using hyperspectral data allow not only evaluation of the relative significance of wavebands in various sensors but also selection of the optimal wavelengths and effective bandwidths.     

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     

基于叶绿素荧光参数的小麦叶片叶绿素相对含量估算方法

叶绿素含量是植物学和农业相关研究领域常用的生理指标.叶绿素含量和叶片光合功能密切相关,但是现有的叶绿素含量的测定方法无法实现叶绿素含量和光合功能的同步测定和关联分析.为解决该问题,本研究通过测定35个小麦品种旗叶的SPAD值和叶绿素荧光诱导动力学曲线,分别使用不同时间的快速叶绿素荧光动力学曲线的荧光值,以及33个常用荧...     

Genetic analysis of iron chlorosis tolerance in Prunus rootstocks

The high economic losses caused by the occurrence of iron chlorosis in Prunus orchards in the Mediterranean area justifies the implementation of breeding programs to generate high-performance rootstocks for different edaphoclimatic area conditions. For that reason, the genetic control of iron chlorosis tolerance was studied in an F₁ population derived from a three-way interspecific cross between a Myrobalan plum (P 2175) and an almond?×?peach hybrid (Felinem). Several phenotypic measurements were assessed to guarantee an accurate data set for genetic analysis. SPAD (Soil and Plant Analyzer Development) values, chlorophyll concentration, and visual diagnostic symptoms were highly correlated with leaf chlorosis in trees. SPAD value was the most reliable measure, since it was an objective, unbiased, and non-destructive method. Two significant quantitative trait loci (QTLs) involved in SPAD and chlorophyll concentration were identified for Felinem in linkage groups 4 and 6. Both QTLs were detected in four of the six consecutive years of the experiment. For P 2175, two of the three putative QTLs identified, pspad4.1 and chl4.1, were placed in linkage group 4. These QTLs were related to the SPAD values and chlorophyll concentration, respectively, and co-localized with QTLs detected in the Felinem map affecting the same traits. Candidate gene PFIT, related to iron metabolism, was localized within the confidence interval of the QTL in linkage group 4. This research suggests an association of this chromosome region with tolerance to iron chlorosis in Prunus, and it provides a first approach to localize candidate genes involved in tolerance to this abiotic stress.     

A new optical leaf-clip meter for simultaneous non-destructive assessment of leaf chlorophyll and epidermal flavonoids

We have characterized a new commercial chlorophyll (Chl) and flavonoid (Flav) meter called Dualex 4 Scientific (Dx4). We compared this device to two other Chl meters, the SPAD-502 and the CCM-200. In addition, Dx4 was compared to the leaf-clip Dualex 3 that measures only epidermal Flav. Dx4 is factory-calibrated to provide a linear response to increasing leaf Chl content in units of μg cm(-2) , as opposed to both SPAD-502 and CCM-200 that have a non-linear response to leaf Chl content. Our comparative calibration by Chl extraction confirmed these responses. It seems that the linear response of Dx4 derives from the use of 710 nm as the sampling wavelength for transmittance. The major advantage of Dx4 is its simultaneous assessment of Chl and Flav on the same leaf spot. This allows the generation of the nitrogen balance index (NBI) used for crop surveys and nitrogen nutrition management. The Dx4 leaf clip, that incorporates a GPS receiver, can be useful for non-destructive estimation of leaf Chl and Flav contents for ecophysiological research and ground truthing of remote sensing of vegetation. In this work, we also propose a consensus equation for the transformation of SPAD units into leaf Chl content, for general use.     

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.     

Online forecasting chlorophyll a concentrations by an auto-regressive integrated moving average model: Feasibilities and potentials

Algal blooms are commonly observed in freshwater and coastal areas, causing significant damage to drinking water and aquaculture production. Predictive models are effective for algal bloom forecasting and management. In this paper, an auto-regressive integrated moving average (ARIMA) model was developed to predict daily chlorophyll a (Chl a) concentrations, using data from Taihu Lake in China. For comparison, a multivariate linear regression (MVLR) model was also established to predict daily Chl a concentrations using the same data. Results showed that the ARIMA model generally performed better than the MVLR model with respect to the absolute error of peak value, root mean square error and index of agreement. Because the ARIMA model needs only one input variable, it shows greater applicability as an algal bloom early warning system using online sensors of Chl a.     

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.     

Trends in Ocean Colour and Chlorophyll Concentration from 1889 to 2000, Worldwide

Marine primary productivity is an important agent in the global cycling of carbon dioxide, a major ‘greenhouse gas’, and variations in the concentration of the ocean''s phytoplankton biomass can therefore explain trends in the global carbon budget. Since the launch of satellite-mounted sensors globe-wide monitoring of chlorophyll, a phytoplankton biomass proxy, became feasible. Just as satellites, the Forel-Ule (FU) scale record (a hardly explored database of ocean colour) has covered all seas and oceans – but already since 1889. We provide evidence that changes of ocean surface chlorophyll can be reconstructed with confidence from this record. The EcoLight radiative transfer numerical model indicates that the FU index is closely related to chlorophyll concentrations in open ocean regions. The most complete FU record is that of the North Atlantic in terms of coverage over space and in time; this dataset has been used to test the validity of colour changes that can be translated to chlorophyll. The FU and FU-derived chlorophyll data were analysed for monotonously increasing or decreasing trends with the non-parametric Mann-Kendall test, a method to establish the presence of a consistent trend. Our analysis has not revealed a globe-wide trend of increase or decrease in chlorophyll concentration during the past century; ocean regions have apparently responded differentially to changes in meteorological, hydrological and biological conditions at the surface, including potential long-term trends related to global warming. Since 1889, chlorophyll concentrations have decreased in the Indian Ocean and in the Pacific; increased in the Atlantic Ocean, the Mediterranean, the Chinese Sea, and in the seas west and north-west of Japan. This suggests that explanations of chlorophyll changes over long periods should focus on hydrographical and biological characteristics typical of single ocean regions, not on those of ‘the’ ocean.     

Differential Gene Expression Reflects Morphological Characteristics and Physiological Processes in Rice Immunity against Blast Pathogen Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae is a serious pathogen that jeopardises the world’s most important food-security crop. Ten common Malaysian rice varieties were examined for their morphological, physiological and genomic responses to this rice blast pathogen. qPCR quantification was used to assess the growth of the pathogen population in resistant and susceptible rice varieties. The chlorophyll content and photosynthesis were also measured to further understand the disruptive effects that M. oryzae has on infected plants of these varieties. Real-time PCR was used to explore the differential expression of eight blast resistance genes among the ten local varieties. Blast disease has destructive effects on the growth of rice, and the findings of our study provide evidence that the Pikh, Pi9, Pi21, and Osw45 genes are involved in defence responses in the leaves of Malaysian rice at 31 h after inoculation with M. oryzae pathotype P7.2. Both the chlorophyll content and photosynthesis were reduced, but the levels of Pikh gene expression remained constant in susceptible varieties, with a developed pathogen population and mild or severe symptoms. The Pi9, Pi21, and Osw45 genes, however, were simultaneously upregulated in infected rice plants. Therefore, the presence of the Pikh, Pi9, Pi21, and Osw45 genes in the germplasm is useful for improving the resistance of rice varieties.     

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.