Non-matrix Matched Glass Disk Calibration Standards Improve XRF Micronutrient Analysis of Wheat Grain across Five Laboratories in India

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Within the HarvestPlus program there are many collaborators currently using X-Ray Fluorescence (XRF) spectroscopy to measure Fe and Zn in their target crops. In India, five HarvestPlus wheat collaborators have laboratories that conduct this analysis and their throughput has increased significantly. The benefits of using XRF are its ease of use, minimal sample preparation and high throughput analysis. The lack of commercially available calibration standards has led to a need for alternative calibration arrangements for many of the instruments. Consequently, the majority of instruments have either been installed with an electronic transfer of an original grain calibration set developed by a preferred lab, or a locally supplied calibration. Unfortunately, neither of these methods has been entirely successful. The electronic transfer is unable to account for small variations between the instruments, whereas the use of a locally provided calibration set is heavily reliant on the accuracy of the reference analysis method, which is particularly difficult to achieve when analyzing low levels of micronutrient. Consequently, we have developed a calibration method that uses non-matrix matched glass disks. Here we present the validation of this method and show this calibration approach can improve the reproducibility and accuracy of whole grain wheat analysis on 5 different XRF instruments across the HarvestPlus breeding program

Calcium Biofortification of Crops–Challenges and Projected Benefits

Despite Calcium (Ca) being an essential nutrient for humans, deficiency of Ca is becoming an ensuing public health problem worldwide. Breeding staple crops with higher Ca concentrations is a sustainable long-term strategy for alleviating Ca deficiency, and particular criteria for a successful breeding initiative need to be in place. This paper discusses current challenges and projected benefits of Ca-biofortified crops. The most important features of Ca nutrition in plants are presented along with explicit recommendations for additional exploration of this important issue. In order for Ca-biofortified crops to be successfully developed, tested, and effectively implemented in most vulnerable populations, further research is required

Biofortification of major crop plants with iron and zinc - achievements and future directions

Biofortification is a long-term strategy of delivering more iron (Fe) and zinc (Zn) to those most in need. Plant breeding programs within the CGIAR and NARS have made major advances in Fe- and Zn-dense variety development and there have been successful releases of new biofortified varieties. Recent research effort has led to a substantial improvement in our knowledge of Fe and Zn homeostasis and gene regulation, resulting in the identification of candidate genes for marker assisted selection. International cooperation between the agricultural and nutrition community has been strengthened, with numerous implementation and partnership strategies developed and employed over the years. The evidence on the effectiveness of Fe and Zn biofortified crops is slowly building up and the results are encouraging. Biofortification continues to be scaled out and further work is required to reach the general aim of eradicating the hidden hunger of Fe and Zn deficiency in the world’s population and ensuring nutritional security

High-throughput measurement methodologies for developing nutrient-dense crops

With the development of nutrient-dense crops comes the need for analytical methodologies to enable rapid and accurate analysis of the micronutrients of interest. The analysis of provitamin A carotenoids (pVACs) and the minerals iron (Fe) and zinc (Zn) are the focus of this chapter with the considerations and commonly employed methods discussed. When analyzing samples there are various considerations to minimise analyte degradation (in the case of provitamin A) and reduce possible contamination from external sources (for Fe and Zn). Spectroscopic and chromatographic analyses are the most common analysis approaches utilised when screening for carotenoids. Spectroscopic analyses including near-infrared spectroscopy (NIRS) and iCheck are rapid and require minimal samples preparation and provide fast analysis times. The carotenoids present in the sample is dependent on the crop analyzed and resulting number and concentration of carotenoids present will impact the final decision on suitable analysis techniques. For example, in crops with high concentrations of non-pVACs, chromatographic analysis is necessary in order to accurately quantify the micronutrients. This process is able to accurately identify and quantify individual carotenoids, but requires extensive sample preparation and often long chromatographic separation analysis. When analyzing the minerals Fe and Zn, these same techniques are not suitable, but it is still important to ensure careful sample preparation to deliver accurate analytical results. Degradation of these micronutrients is not a concern, however, possible contamination from soil/ dust/ insects can lead to inaccurate results. Commonly employed analysis such as atomic absorption spectroscopy (AAS) and Inductively Coupled Plasma-Optical Emission Spectrometry ICP-OES or Inductively Coupled Plasma-Mass spectrometry (ICP-MS) require sample digestion prior to analysis and highly pure reagents and gases. These techniques are able to analyze multiple elements and have high accuracy and sensitivity but require specialised facilities and highly trained staff. The use of high-throughput analyses to complement these high-accuracy methods include colorimetric and X-ray flourescence (XRF) technologies. These approaches enable much higher throughput with simple sample preparation and enable screening for micronutrient concentration without the need for specialised facilities.Keywords: Screening, Analysis, Carotenoid, Provitamin A, Iron, Zinc, Micronutrient analysi

Improved techniques for measurement of nanolitre volumes of phloem exudate from aphid stylectomy

Background: When conducting aphid stylectomy, measuring accurate rates of phloem exudation is difficult because the volumes collected are in the nanolitre (nl) range. In a new method, exudate volume was calculated from optical measurement of droplet diameter as it forms on the tip of a severed aphid stylet. Evaporation was shown to decrease the accuracy of the measurement but was countered with the addition of water-saturated mineral oil. Volume measurements by optical estimation of the volume of a sphere suspended in oil was affected by the curvature of the oil surface. In contrast, measuring the exudate volume from optical measurement of droplet-diameter as formed on the tip of a severed aphid stylet, removes any inaccuracies due to oil surface curvature. A modified technique is proposed for measuring exudate volumes without oil by estimating the flow rate from photo-sequences of the collection period; a correction for evaporation is applied later. Results: A change in oil volume of ±1.75% from an optimum volume of 285 μl had a statistically significant effect on droplet measurement, under or over-estimating droplet volume due to optical effects caused by the oil surface. Using microscope image capture and measurement software, a modified method for measuring phloem volume in air was developed, by reducing air exposure during measurement to approximately 5 s for each measurement. Phloem volumes were measured using both techniques with measurements in air being on average 19.9 nl less (SD 18.87, p<0.001) than those made in oil, and there was a strong linear relationship (R2=0.942) between the techniques. This linear relationship enabled the development of a correction equation with no significant difference at the 5% level between corrected volumes and actual volumes measured under oil. Conclusions: This study showed that oil has a significant role in countering evaporation but oil volume must be carefully optimised for optical measurement of droplets to ensure measurement accuracy. A linear correction factor was generated to correct the volumes measured in air for loss due to evaporation and the method provides for a much simpler alternative to previous approaches for measuring exudation rates and volumes from a cut aphid stylet.Lachlan J Palmer, Lyndon T Palmer, Jeremy Pritchard, Robin D Graham and James CR Stangouli

Characterisation of ethylene pathway components in non-climacteric capsicum

Background: Climacteric fruit exhibit high ethylene and respiration levels during ripening but these levels are limited in non-climacteric fruit. Even though capsicum is in the same family as the well-characterised climacteric tomato (Solanaceae), it is non-climacteric and does not ripen normally in response to ethylene or if harvested when mature green. However, ripening progresses normally in capsicum fruit when they are harvested during or after what is called the ‘Breaker stage’. Whether ethylene, and components of the ethylene pathway such as 1-aminocyclopropane 1-carboxylate (ACC) oxidase (ACO), ACC synthase (ACS) and the ethylene receptor (ETR), contribute to non-climacteric ripening in capsicum has not been studied in detail. To elucidate the behaviour of ethylene pathway components in capsicum during ripening, further analysis is therefore needed. The effects of ethylene or inhibitors of ethylene perception, such as 1-methylcyclopropene, on capsicum fruit ripening and the ethylene pathway components may also shed some light on the role of ethylene in non-climacteric ripening. Results: The expression of several isoforms of ACO, ACS and ETR were limited during capsicum ripening except one ACO isoform (CaACO4). ACS activity and ACC content were also low in capsicum despite the increase in ACO activity during the onset of ripening. Ethylene did not stimulate capsicum ripening but 1-methylcyclopropene treatment delayed the ripening of Breaker-harvested fruit. Some of the ACO, ACS and ETR isoforms were also differentially expressed upon treatment with ethylene or 1-methylcyclopropene. Conclusions: ACS activity may be the rate limiting step in the ethylene pathway of capsicum which restricts ACC content. The differential expression of several ethylene pathway components during ripening and upon ethylene or 1-methylclopropene treatment suggests that the ethylene pathway may be regulated differently in non-climacteric capsicum compared to the climacteric tomato. Ethylene independent pathways may also exist in non-climacteric ripening as evidenced by the up-regulation of CaACO4 during ripening onset despite being negatively regulated by ethylene exposure. However, some level of ethylene perception may still be needed to induce ripening especially during the Breaker stage. A model of capsicum ripening is also presented to illustrate the probable role of ethylene in this non-climacteric fruit.Wan M Aizat, Jason A Able, James CR Stangoulis and Amanda J Abl

The influence of food consumption and socio-economic factors on the relationship between zinc and iron intake and status in a healthy population

Objective: To examine Zn and Fe nutritional status of a healthy population by means of anthropometric, dietary and biochemical measurements and to investigate the relationship of usual Zn and Fe dietary intakes to Zn and Fe status. In addition, to examine the impact of food choices and socio-economic factors on Fe and Zn dietary intakes and status with the aim to identify groups at risk of dietary deficiency and suggest factors that may influence the status of these nutrients. Design: Food consumption was assessed by 24 h recall questionnaires. Twenty biochemical parameters were measured, of which Hb, haematocrit, erythrocyte count and plasma concentrations of Fe and Zn were directly related to Fe and Zn nutrition. The prevalence of study participants with inadequate micronutrient intakes was calculated using the Estimated Average Requirement cut-point method. Setting: Serbia, Europe. Subjects: Apparently healthy adults (25-65 years of age). Results: Mean daily Zn and Fe intakes were 9.1mg and 11.6 mg for males and 7.3 mg and 9.4 mg for females, respectively. Five per cent of the study population had inadequate dietary Fe intake and 15-25% had inadequate Zn intake. Lower Hb concentrations were measured in women with lower Zn intakes. No differences in Fe and Zn intakes and status among various socio-economic groups were observed, except for Fe intake between the low-income and affluent groups. Conclusions: Regular follow-ups are needed to ensure that potential deficiencies of Zn and Fe do get recognized and addressed in a timely manner

Genetic dissection of zinc, iron, copper, manganese and phosphorus in wheat (Triticum aestivum L.) grain and rachis at two developmental stages

The development of high-yielding wheat genotypes containing micronutrient-dense grains are the main prio- rities of biofortification programs. At the International Maize and Wheat Improvement Center, breeders have successfully crossed high zinc progenitors including synthetic hexaploid wheat, T. dicoccum, T. spelta and landraces to generate high-zinc varieties. In this study, we report a genome-wide association using a wheat diversity panel to dissect the genetics controlling zinc, iron, copper, manganese and phosphorus concentrations in the grain and rachis during grain development and at physiological maturity. Significant marker-trait associations (MTAs) were identified for each nutrient using multi-locus mixed model methodologies. For mature grain, markers that showed significant pleiotropic effects were found on chromo- somes 1A, 3B and 5B, of which those on chromosome 5B at ∼95.5 cM were consistent over two growing seasons. Co-located MTAs were identified for the nutrient concentrations in developing grain, rachis and mature grain on multiple chromosomes. The identified genomic regions included putative candidate genes involved in metal uptake and transport and storage protein processing. These findings add to our understanding of the genetics of the five important nutrients in wheat grain and provide information on genetic markers for selecting high mi- cronutrient genotypes.Suong T. Cua, Georgia Guilda, Alison Nicolsona, Govindan Velub, Ravi Singhb, James Stangouli

QTL Mapping of Grain Zn and Fe Concentrations in Two Hexaploid Wheat RIL Populations with Ample Transgressive Segregation

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these termsMore than 50% of undernourished children live in Asia and more than 25% live in Africa. Coupled with an inadequate food supply, mineral deficiencies are widespread in these populations; particularly zinc (Zn) and iron (Fe) deficiencies that lead to retarded growth, adverse effects on both the immune system and an individual's cognitive abilities. Biofortification is one solution aimed at reducing the incidence of these deficiencies. To efficiently breed a biofortified wheat variety, it is important to generate knowledge of the genomic regions associated with grain Zn (GZn) and Fe (GFe) concentration. This allows for the introgression of favorable alleles into elite germplasm. In this study we evaluated two bi-parental populations of 188 recombinant inbred lines (RILs) displaying a significant range of transgressive segregation for GZn and GFe during three crop cycles in CIMMYT, Mexico. Parents of the RILs were derived from Triticum spelta L. and synthetic hexaploid wheat crosses. QTL analysis identified a number of significant QTL with a region denominated as QGZn.cimmyt-7B_1P2 on chromosome 7B explaining the largest (32.7%) proportion of phenotypic variance (PVE) for GZn and leading to an average additive effect of −1.3. The QTL with the largest average additive effect for GFe (−0.161) was found on chromosome 4A (QGFe.cimmyt-4A_P2), with 21.14% of the PVE. The region QGZn.cimmyt-7B_1P2 co-localized closest to the region QGZn.cimmyt-7B_1P1 in a consensus map built from the linkage maps of both populations. Pleiotropic or tightly linked QTL were also found on chromosome 3B, however of minor effects and PVE between 4.3 and 10.9%. Further efforts are required to utilize the QTL information in marker assisted backcrossing schemes for wheat biofortification. A strategy to follow is to intercross the transgressive individuals from both populations and then utilize them as sources in biofortification breeding pipelines