Determination of Fe2+ in Rice Leaves (Oryza sativa L.) by Using the Chelator BPDS Alone or Combined with the Chelator EDTA

Abstract

Iron toxicity is a problem in many areas of wetland rice. Since Fe²⁺ is considered to be the toxic form of iron, the objective of this research was to determine the Fe²⁺ concentration in rice leaves using the chelator bathophenanthroline disulfonate (BPDS), disodium salt alone or combined with the chelator ethylenediaminetetraacetate (EDTA), disodium salt, where BPDS should solely chelate the Fe²⁺ and EDTA chelate only Fe³⁺. Thus, the combination of these chelators should stabilize the Fe oxidation states. It was also tested whether the chelators BPDS and EDTA could stabilize the oxidation states of Fe during the extraction of rice leaves. Extractions of rice leaves were carried out using an 1 mM BPDS or BPDS‐EDTA extractant solution. To test the stabilization of the Fe oxidation states by the combination of BPDS with EDTA, the extraction solution for one part of the samples contained 0.07 mM Fe³⁺. An extraction without plant material as control was also taken into consideration. The results indicated that the chelators were able to stabilize the oxidation states of Fe in the control (extraction without plant material). However, in the presence of plant material, Fe³⁺ was partly reduced to Fe²⁺, i.e., the chelators could not stabilize the oxidation states of Fe. Accordingly, we concluded that the BPDS‐EDTA method may function for the Fe²⁺ determination in water and soil, but it is apparently not suited for rice leaves.     

Tolerance of rice cultivars to iron toxicity

Iron toxicity is an important growth‐limiting factor for flooded rice production in various parts of the world, including Brazil. Data related to the reaction of rice cultivars to iron concentrations are limited, especially for large numbers of cultivars. Forty rice cultivars were grown in a greenhouse in nutrient solutions containing 0.09, 0.89, and 1.78 mM Fe (5, 50 and 100 ppm Fe). The effects of excess iron were measured on plant height, root length, and root and shoot dry weight. Root and shoot dry weight were found to be more sensitive to excess iron concentration. Based on dry matter yield, reduction of shoots at higher Fe concentrations compared to the optimum or control treatment, rice cultivars were classified as tolerant, moderately tolerant, moderately susceptible or susceptible.

The effect of Fe concentrations on concentrations and contents of other nutrient was also investigated. Higher concentration of Fe in the nutrient solution exerted an inhibiting effect on the concentrations and contents of almost all macro and micronutrients.     

Multiple applications of FeDTPA or DTPA on micro‐nutrient extraction and potential availability from peat‐based medium

Abstract

The chelate, DTPA, has heen shown to be an effective extraction agent for the micronutrients from peat‐based soilless media. Multiple applications of 2 mM DTPA extracted 95 ppm iron (Fe) after one week and then declined to 16 ppm Fe while 0.2 mM DTPA extracted Fe which increased to 16 ppm after three weeks. Leachate Fe concentrations from 0.02 mM DTPA and 0.02 mM FeDTPA were similar, increasing from 1 to 2 ppm. Leachate Fe concentrations from 0.2 mM FeDTPA increased from 7 to 14 ppm, however, 0.2 mM DTPA decreased from 9 to 3 ppm. The 0 DTPA treatment had a constant 0.1 ppm Fe in the leachates. Manganese (Mn) was rapidly extracted from the medium. Leachate concentration of both copper (Cu) and zinc (Zn) increased slightly above applied concentrations. There were no differences in dry weight or total micronutrient content among geranium (Pelargonium x hortorum ’Aurora') grown with either DTPA or FeDTPA treatments 0.2 mM and below.     

New Citrate-Bicarbonate-Ethylenediaminetetraacetate (CBE) Method for Chemical Extraction of Hydrous Iron Oxides from Plant Root Surfaces

In the present study, the sodium citrate, sodium bicarbonate, and ethylenediaminetetraacetate (CBE) method was evaluated for iron (Fe) extraction from plant root surfaces and compared with the dithionite-citrate-bicarbonate (DCB) method. Iron plaque on root surfaces was induced by growing rice seedlings in soil with 1.8 mM Fe²⁺. Iron plaque was extracted following CBE and DCB methods. The effects of pH, temperature, and incubation time of these methods on Fe extraction from root surfaces were also examined. Iron extraction of CBE and DCB methods did not differ significantly (P < 0.05) at pH between 6 and 8, whereas Fe extraction decreased substantially for further increase of the pH of CBE and DCB solution. In some instances, there were significant differences between CBE and DCB methods in extracellular Fe extraction for temperature and incubation time. The average Fe extraction of CBE and DCB methods were 94% and 81%, respectively, indicating that CBE method would be a better choice for Fe extraction from plant roots. The recommended optimal conditions for CBE method are pH 8, volume of the solution 30 mL, incubation time 30 min, and solution temperature 22 ± 2 °C.     

Interactive effects of salinity and iron deficiency on different rice genotypes

Salinity adversely affects plant growth, photosynthesis, and availability of nutrients including iron. Rice (Oryza sativa L.) is susceptible to soil salinity and highly prone to iron (Fe) deficiency due to lower release of Fe‐chelating compounds under saline conditions. In order to investigate the effects of salinity and low iron supply on growth, photosynthesis, and ionic composition of five rice genotypes (KS‐282, Basmati Pak, Shaheen Basmati, KSK‐434 and 99417), a solution culture experiment was conducted with four treatments (control, 50 mM NaCl, Fe‐deficient, and 50 mM NaCl + Fe‐deficient). Salinity and Fe deficiency reduced shoot and root growth, photosynthetic and transpiration rates, chlorophyll concentration, and stomatal conductance. The reduction in all these parameters was more in the interactive treatment of salinity and low Fe supply. Moreover, a significant increase in shoot and root Na⁺ with corresponding decrease in K⁺ and Fe concentrations was also observed in the combined salinity and Fe‐deficiency treatment. Among the tested genotypes, Basmati Pak was the most sensitive genotype both under salt stress and Fe deficiency. The genotype KS‐282 performed better than other genotypes under salinity stress alone, whereas Shaheen Basmati was the best genotype under Fe deficiency in terms of all the studied parameters.     

Elemental composition of the rice plant as affected by iron toxicity under field conditions

Abstract

Iron (Fe) toxicity is a major nutrient disorder affecting the production of wetland rice in the humid zone of West Africa. Little attention has been given to determining the macro‐ and micronutrient composition of rice plants grown on wetland soils where Fe toxicity is present although results from such study could provide useful information about the involvement of other nutrients in the occurrence of Fe toxicity. A field experiment was conducted in the 1997 dry season (January‐May) at an Fe toxic site in Korhogo, Ivory Coast, to determine the elemental composition of Fe tolerant (CK 4) and susceptible (Bouake 189) lowland rice varieties without and with application of nitrogen (N), phosphorus (P), potassium (K), and zinc (Zn). For both Fe‐tolerant and susceptible varieties, there were no differences in elemental composition of the whole plant rice tops, sampled at 30 and 60 days after transplanting rice seedlings, except for Fe. All the other nutrient element concentrations were adequate. Both Fe‐tolerant and susceptible cultivars had a high Fe content, well above the critical limit (300 mg Fe kg^‐1 plant dry wt). These results along with our observations on the elemental composition of rice plant samples collected from several wetland swamp soils with Fe toxicity in West Africa suggest that “real”; iron toxicity is a single nutrient (Fe) toxicity and not a multiple nutrient deficiency stress.     

Impact of Feedstock,Parboiling Condition,and Nutrient Concentration on Simultaneous Fortification of Two U.S. Long‐Grain Rice Cultivars with Iron and Zinc

This work investigated the effect of parboiling on simultaneous fortification of rice with iron (Fe) and zinc (Zn) using rough rice and brown rice as feedstocks. Three fortificant concentrations (0, 100, and 200 mg/L for both Fe and Zn) were tested, and two long‐grain rice cultivars (CLXL745 and RoyJ) were used as test samples. Cultivar had little impact on the retention of Fe and Zn; steaming combined with soaking significantly increased the migration of Fe and Zn into the endosperm compared with soaking only. The Fe and Zn contents of the resultant parboiled head rice were related to the initial concentrations in the soaking water and were 7.2–17.6 and 21.8–31.9 mg/kg, respectively, when rough rice was used as a feedstock, and they significantly increased to 32.4–84.9 mg/kg for Fe and 45.8–78.4 mg/kg for Zn when brown rice was used as a feedstock. Mineral retention after simulated washing was 87.5–95.1% for Fe and 81.1–84.3% for Zn. Dilute‐HCl extractability as an indicator of mineral bioavailability was 66.2–72.4% for Fe and 83.4–92.0% for Zn. The results indicate that brown rice is a better feedstock than rough rice for mineral fortification via parboiling.     

Classification of rice genotypes based on their mechanisms of adaptation to iron toxicity

Iron (Fe) toxicity is a nutritional disorder that affects lowland rice (Oryza sativa L.). The occurrence of excessive amounts of reduced Fe(II) in the soil solution, its uptake by the rice roots, and its transpiration‐driven transport result in elevated Fe(II) concentrations in leaf cells that catalyze the formation of reactive oxygen species. The oxidative stress causes rusty brown spots on leaves (bronzing) and the reduction of biomass and yield. While the use of resistant genotypes is the most promising approach to address the problem, the stress appears to differentially affect rice plants as a function of plant age, climatic conditions, stress intensity and duration, and the prevailing adaptation mechanism. We comparatively assessed 21 contrasting 6‐week‐old rice genotypes regarding their response (symptom score, biomass, Fe concentrations and uptake) to a 6 d iron pulse of 1500 mg L^–1 Fe(II). Eight selected genotypes were further compared at different stress intensities (0, 500, 1000, and 1500 mg L^–1 Fe(II)) and at different developmental stages (4‐, 6‐, and 8‐week‐old plants). Based on Fe‐induced biomass reduction and leaf‐bronzing score, the tested spectrum was grouped in resistant and sensitive genotypes. Linking bronzing scores to leaf iron concentrations allowed further differentiation into includer and excluder types. Iron precipitation on roots and organ‐specific iron partitioning permitted to classify the adaptation strategies into root exclusion, stem and leaf sheath retention, and leaf blade tissue tolerance. The effectiveness of these strategies differed with stress intensity and developmental stage. The reported findings improve the understanding of Fe‐stress response and provide a basis for future genotype selection or breeding for enhancing Fe‐toxicity resistance in rice.     

Do water regimes affect iron‐plaque formation and microbial communities in the rhizosphere of paddy rice?

Pot experiments were conducted to investigate the effect of soil water regimes on the formation of iron (Fe) plaque on the root surface of rice seedlings (Oryza sativa L.) and on the microbial functional diversity in a paddy soil. The rice seedlings were subjected to three moisture regimes (submergence, 100%, and 60% water‐holding capacity [WHC]), and were grown for 5 and 11 weeks. Aerobic lithotrophic Fe(II)‐oxidizing (FeOB) and acetate‐utilizing Fe(III)‐reducing bacteria (FeRB) in the rhizosphere and non‐rhizosphere soil were determined at 5 weeks using the most probable number (MPN) method. The carbon substrate use patterns of the microbial communities in the rhizosphere and non‐rhizosphere soil samples were determined at 11 weeks using Biolog‐GN2 plates. The amount of Fe plaque (per unit dry root weight) was much higher under submerged conditions than at lower soil moisture contents and decreased with plant age. There was a positive correlation between the amount of Fe plaque and phosphorus accumulated in the Fe plaque at both sampling times (r = 0.98 and 0.92, respectively, n = 12). Numbers of FeOB and FeRB in the submerged soil were lower than in aerobic soil, but by two orders of magnitude higher in the rhizosphere than in the bulk soil. On the other hand, the functional diversity of the rhizosphere microbial communities was much higher than that of the non‐rhizosphere soil, irrespective of soil water regimes. We conclude that soil flooding results in a decreased number and diversity of Fe‐oxidizing/reducing bacteria, while increasing the Fe‐plaque formation.     

Fe Chelates for Remediation of Fe Chlorosis in Strategy I Plants

Abstract

Iron chlorosis is a mineral disorder due to low Fe in the soil solution and the impaired plant uptake mechanism. These effects increased with high pH and bicarbonate buffer. The solution to Fe chlorosis should be made by either improving the Fe uptake mechanism or increasing the amount of Fe in the soil solution. Among Fe fertilizers, only the most stable chelates (EDDHA and analogous) are able to maintain Fe in the soil solution and transport it to the plant root. In commercial products with the same chelating agent, the efficacy depends on the purity and the presence of subproducts with complexing activity, that can be determined by appropriate analytical methods such as HPLC. In commercial products declaring 6% as Fe‐EDDHA, purity varied from 0.5% to 3.5% before 1999, but in 2002 products ranging 3–5.4% chelated Fe are common in the Spanish market. Fe‐o,p‐EDDHA, as a synthesis by‐product with unknown efficacy, is present in all Fe‐EDDHA formulations. Commercial Fe‐EDDHMA products also contain methyl positional isomers. Fe‐EDDHSA synthesis produces condensation products with similar chelating capacity to the Fe‐EDDHSA monomer that can account for more than 50% of the chelated iron in the commercial products. Chelates with different molecules should be compared for their efficacy considering firstly their ability to maintain Fe in solution and secondly their capacity to release iron to the roots. Accepting the turnover hypothesis, their efficacy is also dependent thirdly on the ability of the chelating agent to form the chelate using native iron from the soil. The 1st and 3rd points are related to the chemical stability of the chelate, while plants make better use of iron from the less stable chelates. Plant response is the ultimate evaluation method to compare commercial products with the same chelating agent or different chelates.     

Genotypic differences in concentrations of iron,manganese, copper,and zinc in polished rice grains

Identification of genotypic differences in micronutrient concentrations of staple food crops is essential if plant breeding strategies are to improve human mineral nutrition. The concentrations of zinc (Zn), iron (Fe), copper (Cu), and manganese (Mn) in polished grains of 285 rice (Oryza sativa L.) genotypes and the relationship between concentrations of the four micronutrient elements and concentrations of protein and lysine were examined. Significant differences (P<.01) were found in the concentrations of Zn, Fe, Cu, and Mn in polished rice with a fairly normal distribution among rice genotypes. On average, Cu and Zn concentrations of Indica rice were about 2‐fold higher than Japonica rice, while Fe concentrations of Japonica rice were slightly higher than Indica rice. Among Indica rice genotypes, red rice contained higher Zn than white rice. Protein and lysine concentrations differed considerably among the genotypes, but no close relationship between the micronutrients and protein or lysine concentrations was observed among genotypes. Sixteen genotypes with significantly higher grain Zn, Fe, Cu, and Mn concentrations were identified.     

硒(Ⅳ)预处理下根表铁膜对水稻幼苗吸收和转运汞的影响

采用水培试验的方法研究硒(Se,Ⅳ)预处理下,根表铁膜对水稻幼苗吸收和转运汞(Hg)的影响。将水稻幼苗置于Se0和Se0.5(mg L-1)培养液中培养2周,再用4种不同浓度的Fe2+溶液(0、25、50和100 mg L-1即Fe0、Fe25、Fe50、Fe100)诱导水稻根表形成不同数量的铁膜,随后置于0.3 mg L-1的Hg Cl2培养液中继续培养72 h。结果表明,根表铁膜对水稻幼苗生长无显著影响,但硒可以增加其生物量。碳酸氢钠―柠檬酸三钠―连二亚硫酸钠(DCB)提取液(即根表铁膜)中含铁比例(57.3%~96.2%)显著高于水稻幼苗地上部(1.1%~17.5%)和根部(2.7%~25.9%),水稻幼苗的大部分铁被积累至DCB提取液中。随着根表铁膜数量的增加,根和地上部汞含量均显著降低。在Fe50和Fe100处理中,硒的加入显著减少了地上部和根部的汞含量,也显著降低了汞的分配系数,Se(Ⅳ)预处理能明显提高铁膜固持汞的量。综上所述,Se(Ⅳ)预处理和根表铁膜均能阻碍水稻幼苗对汞的吸收和向地上部的转运,减轻水稻汞胁迫,从而起到保护水稻避免汞毒害的作用。本研究对于提高汞污染区稻米质量和保证粮食安全具有一定的现实意义。     

Nitric acid‐ and o‐phenanthroline‐extractable iron for diagnosis of iron chlorosis in citrus lemon trees

Abstract

Plant analysis for total iron (Fe) is frequency used for diagnosis of Fe‐deficiency chlorosis. However, chlorotic plants frequency contained similar or higher amount of total Fe than the healthy green plants. The objectives of this study were to (i) determine if Fe chlorosis in citrus lemon can be diagnosed by total or active Fe and can be related to the degree of chlorosis, and (ii) determine the optimum extraction time and ratio of extracting solution to plant sample for extracting the active Fe. Leaf samples of different degrees of Fe chlorosis were sampled from different citrus lemon trees from three different sites. Total Fe was extracted with nitric acid (HNO₃) and active Fe with o‐phenanthroline from lemon leaves. An extraction time of 20 and 45 hours and the ratios of the extractor to the sample of 5:l, 10:1, and 20:1 were investigated. The results indicated that an extraction time of 20 hours is enough for extracting the active Fe from citrus lemon leaves by o‐phenanthroline. The amount extracted by all ratios (5:1, 10:1, and 20:1) were detectable and at the same time similarly and consistency showed the differences in degrees of chlorosis in all plant samples. Total Fe content was always higher in moderately and severely chlorotic leaves compared to the green leaves and was not related to the degree of chlorosis. Therefore, total Fe cannot be used as a criteria to differentiate between the Fe‐deficient and non‐deficient plants. On the other hand, active Fe tended to decrease with the increase in the degree of chlorosis. The ratio of active to total Fe was calculated and was found to be closely correlated with the degree of chlorosis. This clearly illustrates the failure of plant analysis for total Fe and the effectiveness of active Fe and/or the ratio of active to total Fe for diagnosing Fe chlorosis.     

Effects of chromium stress on the subcellular distribution and chemical form of Ca,Mg, Fe,and Zn in two rice genotypes

A hydroponic experiment was carried out to study effects of chromium (Cr) stress on the subcellular distribution and chemical form of Ca, Mg, Fe, and Zn in two rice genotypes differing in Cr accumulation. The results showed that Ca, Mg, Fe, and Zn ions were mainly located in cell walls and vacuoles in roots. However, large amounts of metal ions were transferred from the vacuole to the nucleus and to other functional organelles in shoots. Chromium concentrations in the nutrient solution of 50 μM and above significantly decreased Ca concentrations in the chloroplast/trophoplast, the nucleus, and in mitochondria. It further increased Mg concentrations in the nucleus and in mitochondria, as well as Zn and Fe concentrations in the chloroplast/trophoplast. These Cr‐induced changes in ion concentrations were associated with a significant reduction in plant biomass. It is suggested that Cr stress interferes with the functions of mineral nutrients in rice plants, thus causing a serious inhibition of plant growth. The chemical forms of the four nutrients were determined by successive extraction. Except for Ca, which was mainly chelated with insoluble phosphate and oxalic acid, Mg, Zn, and Fe were extractable by 80% ethanol, d‐H₂O, and 1μM NaCl. The results indicated that these low–molecular weight compounds, such as organic acids and amino acids, may play an important role in deposition and translocation of Mg, Zn, and Fe in the xylem system of rice plants.     

Rapid Gas Chromatographic Technique for Quantifying 2‐Acetyl‐1‐Pyrroline and Hexanal in Rice (Oryza sativa,L.)

The aroma of rice plays a role in its consumer acceptability. The popcorn‐like smell of aromatic rice stemming primarily from its 2‐ acetyl‐1‐pyrroline (2‐AP) content is considered desirable by many consumers. Conversely, hexanal has been correlated with off odors in rice that develop from lipid oxidation. A rapid method for 2‐AP and hexanal quantification suitable for use in breeding programs, large‐scale research efforts, and quality assurance programs is needed. While developing such a method, sample preparation (degree of milling, particle size), solvent extraction time and temperature, and gas chromatographic parameters were studied. Particle size had no influence on 2‐AP or hexanal recovered. One extraction solubilized ≈80% of the 2‐AP and 56% of the hexanal present in milled rice. The optimum extraction method was assessed to require 0.3 g of ground brown or milled rice in methylene chloride held at 85°C for 2.5 hr. The complete gas chromatographic run requires ≈25 min, and 50 samples can be analyzed per day. The optimized method's linear response (R² = 0.99) and reproducibility was demonstrated. The stability of 2‐AP and hexanal in frozen milled rice and in refrigerated methylene chloride extracts was excellent for at least six months. Milled and unmilled commercial and breeders' aromatic rice samples contained 10–1,104 ng/g of 2‐AP and 148–2,541 ng/g of hexanal. Genotype had the greatest effect on the 2‐AP and hexanal content of two lines grown over four years and in four states.     

In vivo staining of reduced iron by 2,2′ bipyridine in rice exposed to iron toxicity

A protocol for a novel method to visualize Fe(II) in rice tissues is proposed. The method is based on the selective formation of a purple‐red color complex of 2,2′ bipyridine and Fe(II). Rice genotypes were exposed to 18 mM Fe(II) in nutrient solution for 2 d. Root systems of intact plants were subsequently placed in 2,2′ bipyridine solutions. The formation of the [Fe(bipy)<$>_3^{2+}<$>] color complex was visualized using bifocal microscopy. The method may improve the selection of genotypes during breeding for Fe‐toxicity resistance of rice.     

Intensification of arsenic toxicity to paddy rice by hydrogen sulfide and ferrous iron

Abstract

The phytotoxicity of arsenic to paddy rice was examined by the pot culture method using Utsunomiya grey lowland soil which had received nutrient salts including ammonium sulfate with or without additional rice straw powder as a reducing agent.

By treatment with 50 ppm of arsenic and straw, plant growth was retarded from the beginning of culture, and about 6 weeks later, at the middle of July, small reddish black spots emerged near the tips of expanded green leaves. The spots then increased and spread over the whole leaves resulting in bronzing and final dieback in about the mid-August. On treatment with higher concentrations of arsenic and straw, the plants were more severely injured and died through bronzing at earlier stages. All such dead plants were found to have accumulated abnormally high iron in their leaf tissues. On treatment with lower concentrations of arsenic and straw or in the case of higher arsenic without straw, plant growth and grain yield were reduced with the occurrence of partial bronzing or oranging of leaves and the iron content of the plants was somewhat increased.

These results indicate that arsenic may induce ferrous iron toxicity which intensifies the toxicity of arsenic to paddy rice.     

Assessment of the soil phosphorus–mobilization potential by microbial reduction using the Fe(III)‐reduction test

The mobility of soil P is greatly influenced by the redox potential (Eh), which depends on the reducing activity of soil microorganisms. Standard extraction methods for the determination of the mobile soil P disregard the P mobilization caused by the influence of microorganisms on Eh, while P test methods that include soil microbial activities are lacking. Thus, the Fe(III)‐reduction test was investigated for its suitability to determine the P fraction that is mobilized in soil under reducing conditions (P_Red). In this test, the soil‐microbial reducing activity is measured from the microbial Fe(III) reduction combining a bioassay with 7 d incubation and a chemical extraction using 1M KCl. After the incubation, Eh in 26 different soil samples ranged from –282 to –123 mV. The concentration of P_Red in the soil samples ranged from concentrations below the limit of detection to 84.9 mg kg^–1 and was on average of all soil samples by a factor of 2.4 to 18 smaller than the P fractions determined by standard soil P–extraction methods. As standard agronomic and environmental P extractants, respectively, water (P_H2O), dithionite citrate bicarbonate (P_Dith), ammonium oxalate (P_Ox), ammonium lactate (P_AL), double lactate (P_DL), and sodium bicarbonate (P_Olsen) were selected. The P_Red fraction was not correlated with P_AL, P_DL, P_olsen, and the degree of P saturation, but with P_H2O (r = 0.43*), P_Dith (r = 0.60***), and P_Ox (r = 0.61***). Furthermore, P_Red depended on the concentration of amorphous Fe oxides (Fe_Ox, r = 0.53**) and was closely correlated with the concentration of microbially reduced Fe (Fe_Red, r = 0.94***). This indicated the influence of the Fe(III)‐reducing activity of soil microorganisms on P mobilization. In subsoils, low in Fe(III)‐reducing activity, no P was released by the Fe(III)‐reduction test, which was in contrast to the results from the other chemical extraction methods. Additional alterations of the microbial activity by inhibiting and activating amendments, respectively, clearly affected the microbial Fe(III)‐reducing activity and the associated release of P_Red. Thus, P_Red, determined by the Fe(III)‐reduction test, might be termed as the fraction that is potentially released from soil by microbial reduction.     

Optimizing Seed Sample Size for Zinc and Iron Analysis of Wild and Cultivated Lentil

Zinc and iron deficiencies affect billions of people globally and have direct impact on human health. Zinc and iron concentrations in plant materials are commonly analyzed by flame atomic absorption spectrometry (F-AAS). This research describes the optimal analysis method for precise zinc (Zn) and iron (Fe) analysis of seeds from the seven species of genus Lens. Estimation of Zn and Fe concentration in whole lentil seeds by F-AAS is destructive, using nitric acid digestion of seeds prior to instrument analysis. The procedure proposed for the determination of Zn and Fe concentration uses the minimum amount of lentil seed of all lentil species with suitable standard reference materials. The relative standard deviations of the method were about 5% for both Zn and Fe. The minimum sample amount 0.3 g of wild and 0.5 g of cultivated lentil seed samples was identified for accurate and precise estimation of Zn and Fe concentration.