WIPPER: an accurate and efficient field phenotyping platform for large-scale applications

More accurate, rapid, and easy phenotyping tools are required to match the recent advances in high-throughput genotyping for accelerating breeding and genetic analysis. The conventional data recording in field notebooks and then inputting data to computers for further analysis is inefficient, time-consuming, laborious, and prone to human error. Here, we report WIPPER (for Wireless Plant Phenotyper), a new phenotyping platform that combines field phenotyping and data recording with the aid of Bluetooth communication, thus saving time and labor not only for field data recoding but also for inputting data to computers. Additionally, it eliminates the risk of human error associated with phenotyping and inputting data. We applied WIPPER to 100 individuals of a rice recombinant inbred line (RIL) for measuring leaf width and relative chlorophyll content (SPAD value), and were able to record an accurate data in a significantly reduced time compared with the conventional method of data collection. We are currently using WIPPER for routine management of rice germplasm including recording and documenting information on phenotypic data, seeds, and DNA for their accelerated utilization in crop breeding.     

High-throughput phenotyping early plant vigour of winter wheat

In contrast to high-throughput genotyping which can manage a large number of plants at relatively low cost, phenotyping of many individual genotypes in field trials is still laborious and expensive. Early plant vigour, as an early selection criterion, is a trait that is visually scored due to a lack of suitable phenotyping methods for an accurate detection of this trait in large field trials. A high-throughput phenotyping technique for scoring early plant vigour would enhance the breeding process. This study was conducted to develop a method for scoring phenotypic differences in early plant vigour of 50 winter wheat (Triticum aestivum L.) cultivars in a 2-years experiment using a vehicle based multispectral active sensor and two commercially available active sensors, GreenSeeker and CropCircle. Pixel analysis of RGB images revealed to be the most feasible and superior method compared to other possible reference methods. A comparison between the two years 2011 and 2012 confirmed that early plant vigour was affected by genotypic differences. A novel spectral plant vigour index (EPVI) was found to accurately reflect the plant vigour at tillering. Different methods were applied to identify optimal combinations of wavelengths to predict early plant vigour, including multivariate modelling and prediction, contour maps for identifying all possible simple ratios and testing of combined indices. The EPVI and the relative amount of green pixels (RAGP) derived from digital images were significantly related with r² = 0.98 to each other in both years. A total of 200 plots, 12 m in length, could be measured within 75 min. The EPVI was shown to be an accurate scoring method for the high-throughput screening of large field trials. The rapidity and accuracy of this novel method may contribute to enhanced selection at early growth stages.     

Development of a plant conveyance system using an AGV and a self-designed plant-handling device: A case study of DIY plant phenotyping

Plant phenotyping technology has been actively developed in recent years, but the introduction of these technologies into the field of agronomic research has not progressed as expected, in part due to the need for flexibility and low cost. “DIY” (Do It Yourself) methodologies are an efficient way to overcome such obstacles. Devices with modular functionality are critical to DIY experimentation, allowing researchers flexibility of design. In this study, we developed a plant conveyance system using a commercial AGV (Automated Guided Vehicle) as a case study of DIY plant phenotyping. The convey module consists of two devices, a running device and a plant-handling device. The running device was developed based on a commercial AGV Kit. The plant-handling device, plant stands, and pot attachments were originally designed and fabricated by us and our associates. Software was also developed for connecting the devices and operating the system. The run route was set with magnetic tape, which can be easily changed or rerouted. Our plant delivery system was developed with low cost and having high flexibility, as a unit that can contribute to others’ DIY’ plant research efforts as well as our own. It is expected that the developed devices will contribute to diverse phenotype observations of plants in the greenhouse as well as to other important functions in plant breeding and agricultural production.     

Assessing genetic variation of maize (Zea mays) root DNA density under contrasting water supply

Optimizing root systems is one crucial point in drought tolerance breeding of plants. Introducing root-related traits to breeding programmes is time-consuming and laborious. Most of the commonly used methods are not suitable to be applied in a larger amount of plants. Here we present a study applying a DNA-based root phenotyping method (root DNA density; RDD) for phenotyping the root system of maize. Twenty-one maize inbred lines were investigated in a rain-out shelter experiment and 19 maize inbred lines in a greenhouse experiment under well-watered and drought conditions. Beside other commonly used root traits, agronomic traits of the plants were recorded and compared to RDD. Within root traits, RDD showed high significant genotypic variation and the highest repeatabilities of up to 72.4%. In contrast to most agronomic traits, repeatabilities increased under drought conditions. Values showed also good correlations between rain-out shelter and greenhouse trial, indicating the potential of this method for obtaining comparable results across different environments.     

Mitigating the impact of selective phenotyping in training populations on the prediction ability by multi-trait pedigree and genomic selection models

Training populations for pedigree and genomic prediction in plant breeding programmes are largely updated with superior genotypes from multi-environment trials, where they are tested with the goal of variety development. Such a selective phenotyping has, however, a negative impact on prediction abilities, especially when only a subset of breeding lines can be tested, as for laborious and costly to phenotype traits. This study focused, thus, on investigating the impact of selective phenotyping in the training population of an applied wheat breeding programme, and assessing the potential to mitigate this impact by pedigree and genomic multi-trait prediction models as well as bi-directionally selected training populations for several baking quality parameters. Combining both pedigree and genomic information in multi-trait prediction models with pre-existing phenotypic information for protein content and sedimentation value compensated for the observed loss in prediction ability, while entering few inferior breeding lines into the training population further mitigated the impact of selective phenotyping and even led to a slight increase of prediction ability in comparison to a randomly chosen training population.     

High-throughput field crop phenotyping: current status and challenges

In contrast to the rapid advances made in plant genotyping, plant phenotyping is considered a bottleneck in plant science. This has promoted high-throughput plant phenotyping (HTP) studies, resulting in an exponential increase in phenotyping-related publications. The development of HTP was originally intended for use as indoor HTP technologies for model plant species under controlled environments. However, this subsequently shifted to HTP for use in crops in fields. Although HTP in fields is much more difficult to conduct due to unstable environmental conditions compared to HTP in controlled environments, recent advances in HTP technology have allowed these difficulties to be overcome, allowing for rapid, efficient, non-destructive, non-invasive, quantitative, repeatable, and objective phenotyping. Recent HTP developments have been accelerated by the advances in data analysis, sensors, and robot technologies, including machine learning, image analysis, three dimensional (3D) reconstruction, image sensors, laser sensors, environmental sensors, and drones, along with high-speed computational resources. This article provides an overview of recent HTP technologies, focusing mainly on canopy-based phenotypes of major crops, such as canopy height, canopy coverage, canopy biomass, and canopy stressed appearance, in addition to crop organ detection and counting in the fields. Current topics in field HTP are also presented, followed by a discussion on the low rates of adoption of HTP in practical breeding programs.     

Root phenotyping: important and minimum information required for root modeling in crop plants

As plants cannot relocate, they require effective root systems for water and nutrient uptake. Root development plasticity enables plants to adapt to different environmental conditions. Research on improvements in crop root systems is limited in comparison with that in shoots as the former are difficult to image. Breeding more effective root systems is proposed as the “second green revolution”. There are several recent publications on root system architecture (RSA), but the methods used to analyze the RSA have not been standardized. Here, we introduce traditional and current root-imaging methods and discuss root structure phenotyping. Some important root structures have not been standardized as roots are easily affected by rhizosphere conditions and exhibit greater plasticity than shoots; moreover, root morphology significantly varies even in the same genotype. For these reasons, it is difficult to define the ideal root systems for breeding. In this review, we introduce several types of software to analyze roots and identify important root parameters by modeling to simplify the root system characterization. These parameters can be extracted from photographs captured in the field. This modeling approach is applicable to various legacy root data stored in old or unpublished formats. Standardization of RSA data could help estimate root ideotypes.     

Drought Tolerance Screening Under Controlled Conditions Predicts Ranking of Water‐Limited Yield of Field‐Grown Soybean Genotypes

Drought is a major limitation to crop yields worldwide. Screening for soybean yield under water deficit is often a bottleneck in breeding programmes. We assessed the validity of a standardized drought tolerance screening method to predict water‐limited field performance of soybean in NW Argentina. First, to determine the phenological period when yield of glasshouse‐grown plants was more sensitive to water deficit, we applied treatments during 21 days in V₇, R₃ or R₅ stages, being the period from R₅ to R₆ the most critical for yield. Afterwards, two glasshouse experiments were carried out to quantify the tolerance of either eight or four genotypes, respectively, by applying a controlled water deficit of constant intensity during the critical period. Finally, yield data obtained in field trials in Argentina across several locations and seasons classified according to rainfall were analysed. Drought Susceptibility Index was calculated for each experiment and for field data, and rankings of tolerance were similar in all cases. This standardized method, which can be automated for high‐throughput phenotyping, could represent a useful tool in breeding programmes for identifying soybean cultivars with improved performance under drought conditions.     

Marker‐assisted backcross breeding for improvement of drought tolerance in bread wheat (Triticum aestivum L. em Thell)

Drought stress is presently a major productivity limiting factor in wheat. This study developed five wheat lines with inbuilt tolerance to drought stress using marker‐assisted backcross breeding (MABB) approach employing three linked quantitative trait loci (QTLs) in an initial population of 516 BC₁F₁ plants. The high‐yielding wheat cultivar ‘HD2733’ grown over last few years extensively in the eastern plains of India is largely sensitive to drought and is used as the recurrent parent. ‘HI1500’ released for water‐limiting conditions and carrying drought‐tolerant QTLs was used as donor parent. MABB lines were advanced using foreground and background selection, coupled with stringent phenotyping. We identified 29 lines that were homozygous for targeted QTLs in different combinations with background recovery range of 89.2%–95.4%. Further evaluation of selected lines for physiological traits and distinctness, uniformity and stability (DUS) characters under rainfed condition identified five potential varieties for national varietal evaluation programme in the zone. The report is first of its kind in implementing known QTLs for the development of drought‐tolerant wheat lines through MABB approach.     

Breeding for insect resistance in oilseed rape: Challenges,current knowledge and perspectives

Dealing with insect pests is one of the biggest challenges facing the cultivation of oilseed rape (Brassica napus, OSR). Insect pests are usually controlled using insecticides, but the increasing occurrence of insecticide‐resistant populations and the socio‐economic context argue against the sole use of these substances. Plant resistance is a classical and proven alternative crop protection strategy that is the basic tool of integrated pest management. However, no insect‐resistant OSR cultivar is currently available on the market. Here, I review some of the constraints that make phenotyping for insect resistance particularly challenging with OSR and give perspectives to develop this resistance. Some studies have proved that breeding OSR for insect resistance could be achieved, and three strategies have been considered: introducing resistance transgenes into the OSR genome, exploiting natural variation in resistance already present in B. napus and introgressing resistance from other brassicaceous species. The white mustard, Sinapis alba, seems a particularly promising source of resistance against most of the OSR insect pests.     

Evaluation of a semi‐controlled test as a selection tool for frost tolerance in durum wheat (Triticum durum)

Most durum wheat (Triticum durum) varieties possess only low winter hardiness due to their frost susceptibility. In North America and Central Europe, durum wheat is therefore typically sown in spring to circumvent the local winter conditions. However, the yield potential of durum in these regions could be much better exploited if durum varieties with increased frost tolerance were available, which could be sown in autumn. A factor limiting breeding for increased frost tolerance is the variation in the occurrence of frost stress across years. The ‘Weihenstephaner Auswinterungsanlage’ is a semi‐controlled test that exposes the plants to all weather conditions. Snow coverage of the plants, serving as frost protection, is prevented by the movable glass lid of the semi‐controlled test. In this study, different scorings for frost tolerance based on this semi‐controlled test were evaluated and compared with frost tolerance data in the field. Our results illustrate the potential of the ‘Weihenstephaner Auswinterungsanlage’ as an indirect selection tool for frost tolerance in durum breeding programmes, especially when regular frost tolerance data from the field are not available.     

Correlations between visual biomass scores and forage yield in space planted red clover (<Emphasis Type="Italic">Trifolium pratense</Emphasis> L.) breeding nurseries

Red clover (Trifolium pratense L.) forage yield remains a prime breeding target for improved variety development. In a world of decreasing forage legume breeding resources, rapidly and cheaply phenotyping plants for the highly quantitative trait of forage yield is vital. Many red clover selection programs are based on space planted nurseries. The objectives of this study were to determine: (1) the accuracy of visual forage yield scores in predicting actual forage yield; (2) the nature of the relationship between visual scores and actual measurements; and (3) The repeatability of visual scores between different evaluators. Twenty-seven halfsib families were transplanted at two locations in three replicates of six plant plots. Individual plant fresh weights and visual scores, by two evaluators, for forage yield were taken. On an individual plant basis visual forage yield scores showed an exponential relationship with actual fresh weights. Individual plant visual scores were very accurate with a pseudo-R ² of 0.79 observed for the exponential model. On an entry mean basis using a linear model, visual scores could explain 90% of the variation of actual fresh weights. Agreement among evaluators scoring the same plants was very high with coefficients of determination at 0.84 for individual plants and as high as 0.96 on an entry mean basis. This study suggests that visual scores of plants in space planted red clover breeding nurseries are basically as accurate as measuring actual yields and that plants can be consistently scored the same by different evaluators.     

Maize genotypes with deep root systems tolerate salt stress better than those with shallow root systems during early growth

Maize (Zea mays L.) is susceptible to salinity but shows genotypic variation for salt tolerance. How maize genotypes with contrasting root morphological traits respond to salt stress remains unclear. This study assessed genotypic variation in salinity tolerance of 20 maize genotypes with contrasting root systems exposed to NaCl for 10 days (0, 50 mM or 100 mM NaCl, added in four increments every other day from 14 days after transplanting, DAT) in a semi-hydroponic phenotyping system in a temperature-controlled greenhouse. Considerable variation was observed for each of the 12 measured shoot and root traits among the 20 genotypes under NaCl treatments. Salt stress significantly decreased biomass production by up to 54% in shoots and 37% in roots compared with the non-saline control. The 20 genotypes were classified as salt-tolerant (8 genotypes), moderately tolerant (5) and salt-sensitive (7) genotypes based on the mean shoot dry weight ratio (the ratio of shoot dry weight at 100 mM NaCl and non-saline control) ± one standard error. The more salt-tolerant genotypes (such as Jindan52) had less reductions in growth, and lower shoot Na⁺ contents and higher shoot K⁺/Na⁺ ratios under salt stress. The declared salt tolerance was positively correlated with shoot height, shoot dry weight and primary root depth, and negatively correlated with shoot Na⁺ content at 100 mM NaCl. Primary root depth is critical for identifying salt responsiveness in maize plants and could be suggested as a selection criterion for screening salt tolerance of maize during early growth. The selected salt-tolerant genotypes have potentials for cultivation in saline soils and for developing high-yielding salt-tolerant maize hybrids in future breeding programmes.     

Precision phenotyping of imidazolinone-induced chlorosis in sunflower

Chlorosis level is a useful parameter to assess imidazolinone resistance in sunflower (Helianthus annuus L.). The aim of this study was to quantify chlorosis through two different methods in sunflower plantlets treated with imazapyr. The genotypes used in this study were two inbred lines reported to be different in their resistance to imidazolinones. Chlorosis was evaluated by spectrophotometrical quantification of photosynthetic leaf pigments and by a bioinformatics-based color analysis. A protocol for pigment extraction was presented which improved pigment stability. Chlorophyll amount decreased significantly when both genotypes were treated with 10 μM of imazapyr. Leaf color was characterized using Tomato Analyzer^® color test software. A significant positive correlation between color reduction and chlorophyll concentration was found. It suggests that leaf color measurement could be an accurate method to estimate chlorosis and infer chlorophyll levels in sunflower plants. These results highlight a strong relationship between imidazolinone-induced chlorosis and variations in leaf color and in chlorophyll concentration. Both methods are quantitative, rapid, simple, and reproducible. Thus, they could be useful tools for phenotyping and screening large number of plants when breeding for imidazolinone resistance in this species.     

Genomics-assisted breeding: The next-generation wheat breeding era

Common wheat provides approximately 20% of the total dietary calorie intake of human beings. Recent technological advances in whole-genome sequencing and their application in wheat and its progenitor species provide new opportunities to uncover the genetic variation of wheat traits and to accelerate the traditional breeding (TB) strategies in the context of genomics-assisted breeding (GAB). Integration of TB, marker-assisted selection (MAS) and genomic selection (GS) with high-density SNP markers is expected to accelerate the breeding process and to further enhance genetic gain. With the assistance of the next- or third-generation sequencing technologies and high-throughput phenotyping platforms, GAB can now realistically be considered in the following area: (i) genome sequencing and high-quality assembly to uncover new variations, (ii) whole-genome sequence-based association studies, (iii) gene function (or functional gene) identification and (iv) integration of whole genomic breeding information, utilizing multi-omics data and different breeding strategies. We argue that GAB is becoming the preferred strategy in pursuit of new wheat cultivars with superior traits on high yielding, high nutritional quality, climate-resilience and so on.     

利用卡那霉素间接鉴定法进行大规模的棉花转基因育种技术

:( 1 )将脱脂棉撕成小条沾取 0 .0 5%的卡那霉素溶液 ,粘附于棉花植株倒 2新生叶上。 5d后所有沾有卡那霉素溶液的感虫棉株的叶片均出现明显的黄色斑块 ,而转 Bt基因抗虫棉的叶片仍为正常绿色 ,无任何症状。 ( 2 )转 Bt基因抗虫棉种子接种于加有 1 0 0 0 mg· l- 1卡那霉素的 MS培养基中。四天后 ,抗虫棉子叶均表现为正常绿色 ,而常规品种子叶全为黄色 ,表明卡那霉素间接鉴定法是鉴定转 Bt基因抗虫棉的简便而又准确的方法     

The release of transgenic plants from containment, and the move towards their widespread use in agriculture

Summary The use of transgenic plants in breeding makes it possible to utilise a wide variety of novel genes from unrelated plants, microbes and animals. Because of the diversity of genes that have now become available for modifying crop plants, it is agreed internationally that there should be a risk assessment before transgenic plants are grown outside the laboratory or glasshouse. Various aspects are considered in a risk assessment including any non-target effects of the transgene, changes in plant persistence and invasiveness, and the possibility of movement of the transgenes to wild populations by cross pollination. It is generally argued that the need for risk assessment and regulation should be determined by an analysis of certain products of transformation, rather than a risk assessment being required for all plants modified by the process of transformation. A possible consequence of considering the product only, however, could be that some of the products of conventional breeding may need to be assessed by the risk assessment procedures developed for transgenic plants. There are discussions with interest groups on the use of transgenic plants in the environment and in food products. It is likely that some form of labelling will be required for certain foods containing ethically-sensitive genes. There is little doubt that transgenic plants will make a significant contribution to agriculture in the coming decades. Developments in the patenting of genes, release regulations, food labelling, consumer reaction etc., will influence the rate of progress and should be considered in the strategic planning of plant breeding programmes.     

大豆引进种质抗胞囊线虫病、抗花叶病毒病和耐盐基因型鉴定及优异等位基因聚合种质筛选

我国从美国、俄罗斯、日本等26个国家或地区共引进大豆种质3218份, 仅对部分种质进行了大豆胞囊线虫病(Soybean cyst nematode, SCN)、大豆花叶病毒病(Soybean mosaic virus, SMV)和盐敏感性的抗性鉴定, 但基因型的系统分析尚未见报道。本研究针对大豆抗胞囊线虫病3个基因(rhg1、Rhg4、SCN3-11)和耐盐基因(GmSALT3)开发KASP标记5个, 结合与大豆花叶病毒抗性相关的1个SCAR标记(SCN11), 对1489份大豆引进种质进行基因型鉴定。结果表明, 具有优异等位基因的种质共1084份; 携带3个位点优异等位基因的种质19份, 包括抗胞囊线虫病3个位点(rhg1、Rhg4、SCN3-11)叠加(Peking型)种质3份, 聚合抗胞囊线虫病基因和抗花叶病毒病标记7份, 聚合抗胞囊线虫病和耐盐基因2份, 聚合抗胞囊线虫病、抗花叶病毒病和耐盐基因7份; 携带4个位点优异等位基因的种质9份, 包括聚合抗胞囊线虫病基因和抗花叶病毒病标记6份, 聚合抗胞囊线虫病和耐盐基因2份, 聚合抗胞囊线虫病、抗花叶病毒病和耐盐7份; 携带5个位点优异等位基因8份, 聚合了抗胞囊线虫病、抗花叶病毒病和耐盐优异等位变异。在这些携带优异等位变异的种质中, 44份已由前人证明具有相应的抗性。携带3个或3个以上优异等位基因的36份种质中, 有52.78%种质的一种或两种特性已被报道。在不携带抗性优异等位变异的种质中, 93份已证明有耐盐性或对SMV3号株系抗性, 这些种质可能存在新的抗性(等位)基因。本研究利用高通量分子标记筛选出的携带抗病、抗逆优异等位基因的种质为我国大豆资源表型鉴定、抗源的快速筛选及利用提供理论依据和新思路。