Crop breeding for a changing climate in the Pannonian region: towards integration of modern phenotyping tools.

The Pannonian Plain, as the most productive region of Southeast Europe, has a long tradition of agronomic production as well as agronomic research and plant breeding. Many research institutions from the agri-food sector of this region have a significant impact on agriculture. Their well-developed and fruitful breeding programmes resulted in productive crop varieties highly adapted to the specific regional environmental conditions. Rapid climatic changes that occurred during the last decades led to even more investigations of complex interactions between plants and their environments and the creation of climate-smart and resilient crops. Plant phenotyping is an essential part of botanical, biological, agronomic, physiological, biochemical, genetic, and other omics approaches. Phenotyping tools and applied methods differ among these disciplines, but all of them are used to evaluate and measure complex traits related to growth, yield, quality, and adaptation to different environmental stresses (biotic and abiotic). During almost a century-long period of plant breeding in the Pannonian region, plant phenotyping methods have changed, from simple measurements in the field to modern plant phenotyping and high-throughput non-invasive and digital technologies. In this review, we present a short historical background and the most recent developments in the field of plant phenotyping, as well as the results accomplished so far in Croatia, Hungary, and Serbia. Current status and perspectives for further simultaneous regional development and modernization of plant phenotyping are also discussed.

Dynamics of Maize Vegetative Growth and Drought Adaptability Using Image-Based Phenotyping Under Controlled Conditions.

Changes in climate are likely to have a negative impact on water availability and soil fertility in many maize-growing agricultural areas. The development of high-throughput phenotyping platforms provides a new prospect for dissecting the dynamic complex plant traits such as abiotic stress tolerance into simple components. The growth phenotypes of 20 maize (Zea mays L.) inbred lines were monitored in a non-invasive way under control, nitrogen, and water limitation as well as under combined nitrogen and water stress using an automated phenotyping system in greenhouse conditions. Thirteen biomass-related and morphophysiological traits were extracted from RGB images acquired at 33 time points covering developmental stages from leaf count 5 at the first imaging date to leaf count 10-13 at the final harvest. For these traits, genetic differences were identified and dynamic developmental trends during different maize growth stages were analyzed. The difference between control and water stress was detectable 3-10 days after the beginning of stress depending on the genotype, while the effect of limited nitrogen supply only induced subtle phenotypic effects. Phenotypic traits showed different response dynamics as well as multiple and changing interaction patterns with stress progression. The estimated biovolume, leaf area index, and color ratios were found to be stress-responsive at different stages of drought stress progression and thereby represent valuable reference indicators in the selection of drought-adaptive genotypes. Furthermore, genotypes could be grouped according to two typical growth dynamic patterns in water stress treatments by c-means clustering analysis. Inbred lines with high drought adaptability across time and development were identified and could serve as a basis for designing novel genotypes with desired, stage-specific growth phenotypes under water stress through pyramiding. Drought recovery potential may play an equal role as drought tolerance in plant drought adaptation.

Investigations on the formation of Maillard reaction products in sweet cookies made of different cereals.

In this study, the content of Maillard reaction products from its initial, intermediate and final stage (5-hydroxymethylfurfural, α-dicarbonyl compounds, furosine, N-ε-carboxymethyllysine and N-ε-carboxyethyllysine) was measured in sweet cookies made of wholegrain flour of eight genotypes of small-grain cereals (bread wheat, durum wheat, soft wheat, hard wheat, triticale, rye, hulless barley and hulless oat) and four corn genotypes (white-, yellow- and red-colored standard seeded corn and blue-colored popping corn). Furthermore, the effect of the initial content of sugars, total proteins, free and total lysine in flour on the formation of Maillard reaction products was investigated using the principle component analysis. 3-deoxyglucosone was the predominant α-dicarbonyl compound in all cereal cookies and the highest content was measured in those made from flour of different colored corn genotypes (on average, 98.35, 151.28 and 172.85 mg/kg after baking for 7, 10 and 13 min, respectively). Heating dough at 180 °C for 7, 10 and 13 min differently affected the content of 5-hydroxymethylfurfural and α-dicarbonyl compounds in the cereal cookies. The 5-hydroxymethylfurfural content gradually increased, while a reduction in 3-deoxyglucosone content was observed in the cookies baked for 13 min except for those made from soft wheat, hulless oat, red- and blue-colored corn. After 7 min of heating, the content of furosine measured in the cereal cookies reached its maximum (from 320.9 mg/kg in yellow-colored corn-based cookies to 585.7 mg/kg in hulless oat-based cookies), while N-ε-carboxymethyllysine and N-ε-carboxyethyllysine showed the opposite trend. The highest content of advanced glycation end products was detected in cookies also made from hulless oat flour rich in proteins (16.80%) and total lysine (10670.3 mg/kg). The interrelationship analysis showed that the initial content of sugars in flour of cereals affected 5-hydroxymethylfurfural and 3-deoxyglucosone formation in the cookies. In addition, a high correlation between protein-bound Maillard reaction products in the cookies and the total proteins and the total lysine content in the flours was found.

Acrylamide formation in biscuits made of different wholegrain flours depending on their free asparagine content and baking conditions.

Due to a high content of bioactive compounds with beneficial health effects, wholegrain flours of different cereals have been extensively used in the confectionery industry. However, according to our study, cereal species and their varieties have different potential for the formation of acrylamide in biscuits. In this study, wholegrain flours of eight genotypes of small grain cereals (bread wheat, durum wheat, soft wheat, hard wheat, triticale, rye, hulless barley and hulless oat) and four genotypes of maize (white-, yellow- and red-coloured standard seeded maize, and blue-coloured popping maize) were used to prepare biscuits. The biscuits were baked at 180 °C for 7, 10 and 13 min. At 180 °C, acrylamide was detected at all baking times, reaching a final concentration of 72.3 up to 861.7 µg/kg after 13 min of baking in refined bread wheat-based biscuits and hulless oat-based biscuits, respectively. Data indicated that acrylamide in biscuits could not exactly correspond to free asparagine in flour. However, hulless oat, durum wheat and rye flour with the highest content of free asparagine of 859.8, 603.2 and 530.3 mg/kg, respectively, generated most acrylamide in biscuits baked for 13 min. The lowest content of acrylamide was found in biscuits prepared from refined bread wheat flour and wholegrain red maize flour that also contained the lowest content of free asparagine. After baking for 7, 10 and 13 min, the content of acrylamide in these samples was 17.9 and 24.4 µg/kg, 51.9 and 28.7 µg/kg and 72.3 and 95.2 µg/kg, respectively. The results suggest that the use of cereal flours low in free asparagine can be an effective strategy for acrylamide mitigation in biscuits, together with the use of lower thermal load during baking.

Image-Derived Traits Related to Mid-Season Growth Performance of Maize Under Nitrogen and Water Stress.

Phenotypic measurements under controlled cultivation conditions are essential to gain a mechanistic understanding of plant responses to environmental impacts and thus for knowledge-based improvement of their performance under natural field conditions. Twenty maize inbred lines (ILs) were phenotyped in response to two levels of water and nitrogen supply (control and stress) and combined nitrogen and water deficit. Over a course of 5 weeks (from about 4-leaf stage to the beginning of the reproductive stage), maize phenology and growth were monitored by using a high-throughput phenotyping platform for daily acquisition of images in different spectral ranges. The focus of the present study is on the measurements taken at the time of maximum water stress (for traits that reflect plant physiological properties) and at the end of the experiment (for traits that reflect plant architectural and biomass-related traits). Twenty-five phenotypic traits extracted from the digital image data that support biological interpretation of plant growth were selected for their predictive value for mid-season shoot biomass accumulation. Measured fresh and dry weights after harvest were used to calculate various indices (water-use efficiency, physiological nitrogen-use efficiency, specific plant weight) and to establish correlations with image-derived phenotypic features. Also, score indices based on dry weight were used to identify contrasting ILs in terms of productivity and tolerance to stress, and their means for image-derived and manually measured traits were compared. Color-related traits appear to be indicative of plant performance and photosystem II operating efficiency might be an importance physiological parameter of biomass accumulation, particularly under severe stress conditions. Also, genotypes showing greater leaf area may be better adapted to abiotic stress conditions.

Genetic analysis of water loss of excised leaves associated with drought tolerance in wheat.

BACKGROUND: Wheat is widely affected by drought. Low excised-leaf water loss (ELWL) has frequently been associated with improved grain yield under drought. This study dissected the genetic control of ELWL in wheat, associated physiological, morphological and anatomical leaf traits, and compared these with yield QTLs. METHODS: Ninety-four hexaploid wheat (Triticum aestivum L.) doubled haploids, mapped with over 700 markers, were tested for three years for ELWL from detached leaf 4 of glasshouse-grown plants. In one experiment, stomata per unit area and leaf thickness parameters from leaf cross-sections were measured. QTLs were identified using QTLCartographer. RESULTS: ELWL was significantly negatively correlated with leaf length, width, area and thickness. Major QTLs for ELWL during 0-3 h and 3-6 h were coincident across trials on 3A, 3B, 4B, 5B, 5D, 6B, 7A, 7B, 7D and frequently coincident (inversely) with leaf size QTLs. Yield in other trials was sometimes associated with ELWL and leaf size phenotypically and genotypically, but more frequently under non-droughted than droughted conditions. QTL coincidence showed only ELWL to be associated with drought/control yield ratio. DISCUSSION: Our results demonstrated that measures of ELWL and leaf size were equally effective predictors of yield, and both were more useful for selecting under favourable than stressed conditions.

Free asparagine and sugars profile of cereal species: the potential of cereals for acrylamide formation in foods.

Cereals-based food is one of the major source of Maillard reaction products in the diet. Free amino acids and reducing sugars are considered to be the main precursors in the formation of these heat-induced compounds. In order to determine genetic resources with reduced potential for acrylamide formation, the content of sugars as well as free asparagine were analysed in a total of 30 cultivars of 10 varieties belonging to eight species (Triticum aestivum var. lutescens, T. aestivum var. alba, T. aestivum var. compactum, T. durum, T. spelta, T. dicoccum, Secale cereale, Hordeum vulgare var. nudum, Avena sativa var. nudum, and Zea mays var. indentata) grown at the same location in the 2015 growing season. Our results provide evidence of differences in the content of sugars and asparagine between and within species of small grain cereals and maize. The highest content of glucose, fructose and asparagine was found in cultivars of rye and hull-less oat. All maize varieties examined contained significantly higher amounts of non-reducing and total sugars (on average 1.25% and 2.36%, respectively) than small cereal grain species. Principal component analysis showed a high positive correlation between monoreducing sugars and asparagine in bread wheat, durum wheat and hull-less barley.

Population structure in a wheat core collection and genomic loci associated with yield under contrasting environments.

A set of 96 winter wheat accessions sampled from a variety of geographic origins, including cultivars and breeding lines, were characterized with 46 genome-wide SSR loci for genetic diversity and population structure. The genetic diversity within these accessions was examined using a genetic distance-based and a model-based clustering method. The model-based analysis identified an underlying population structure comprising of four distinct sub-populations which corresponded well with distance-based groupings. Information on the population structure is taken into account in an association mapping study of grain yield from a 3-years field trial incorporating fully irrigated, rainfed and drought stress treatments. A total of 21 marker-grain yield associations (P < 0.01) were identified with nine SSR markers. Most associations were detected only in one to three environments (treatment/year combination), with an average R ( 2 ) value around 13 %. However, marker gwm484 (on chromosome 2D) was associated with yield in six environments, including irrigated, rainfed and drought stress treatments, suggesting it could be used to improve grain yield across a range of environments. Variation in grain yield at this locus was associated with earliness, early vigour, kernels per spikelet and harvest index. Microsatellite locus psp3200 (on chromosome 6D) was associated with yield in dry and hot environments, which was related to earliness, early vigour, productive tillering and total biomass per plant. Partial least squares regression, with nine environmental factors, showed that precipitation from tillering to maturity was the main environmental factor causing marker × environment associations for grain yield.

Genetic and association mapping study of wheat agronomic traits under contrasting water regimes.

Genetic analyses and association mapping were performed on a winter wheat core collection of 96 accessions sampled from a variety of geographic origins. Twenty-four agronomic traits were evaluated over 3 years under fully irrigated, rainfed and drought treatments. Grain yield was the most sensitive trait to water deficit and was highly correlated with above-ground biomass per plant and number of kernels per m(2). The germplasm was structured into four subpopulations. The association of 46 SSR loci distributed throughout the wheat genome with yield and agronomic traits was analyzed using a general linear model, where subpopulation information was used to control false-positive or spurious marker-trait associations (MTAs). A total of 26, 21 and 29 significant (P < 0.001) MTAs were identified in irrigated, rainfed and drought treatments, respectively. The marker effects ranged from 14.0 to 50.8%. Combined across all treatments, 34 significant (P < 0.001) MTAs were identified with nine markers, and R(2) ranged from 14.5 to 50.2%. Marker psp3200 (6DS) and particularly gwm484 (2DS) were associated with many significant MTAs in each treatment and explained the greatest proportion of phenotypic variation. Although we were not able to recognize any marker related to grain yield under drought stress, a number of MTAs associated with developmental and agronomic traits highly correlated with grain yield under drought were identified.

Seed germination and seedling vigour of italian ryegrass, cocksfoot and timothy following harvest and storage / Germinação de sementes e o vigor de plantas jovens de azevem italiano, dactilis e timóteo após a colheita e o armazenamento

During post-harvest maturation, different species vary in the length of dormancy breaking or germination increases. Seed dormancy and slow seedling development often limit establishment of forage grass stands. Seed germination and seedling vigour of Italian ryegrass (Lolium italicum A. Braun, Synonym Lolium multiflorum L.), cocksfoot (Dactylis glomerata L.) and timothy (Phleum pretense L.) were observed after harvest and storage. After harvest in June, seeds were stored under standard storage conditions and sampled every 30 days after harvest (DAH), up to 270 DAH, and then every 60 days up to 990 DAH. At each date, seeds were tested for final germination percentage and for seedling vigour traits. Timothy seeds had a maximum germination (88 percent) and the best seedlings vigour at 90 DAH, which implies that early autumn (September-October) is the best sowing period for freshly harvested seeds of timothy. Timothy seed germination was poor from 270 DAH (73 percent). The best germination and vigour of Italian ryegrass and cocksfoot seedlings were between 270 and 330 DAH, which equates to spring sowing time (March-April) in the succeeding year. Cocksfoot and Italian ryegrass seeds maintained satisfactory germination levels up to 630 DAH (81 percent) and 810 DAH (81 percent), respectively. The data can serve for the determination of a proper storage duration management between harvest and sowing of the tested species under ambient conditions of south-eastern Europe.

Durante o armazenamento, diferentes espécies comportam-se diferentemente quanto à dormência e crescimento de plântulas. A dormência o desenvolvimento lento das plântulas limitam a época do plantio das gramíneas forrageiras plurianuais. A germinação de sementes e o crescimento das plântulas de azevem italiano (Lolium italicum A. Braun sin. Lolium multiflorum L.), Dactilis (Dactilis glomerata L.) e de timoteo (Phleum pratense L.) foi pesquisada durante a maturação pós-colheita e a silagem. Após a colheita, em junho, sementes foram armazenadas em condições tradicionais e analisadas a cada 30 dias após a colheita, até 270 dias e, em seguida, a cada 60 dias até 990 dias. Nessa pesquisa, avaliou-se a germinação final e o vigor das plântulas. As sementes de timoteo obtiveram germinação máxima (88 por cento) e o melhor crescimento das plântulas após 90 dias de armazenamento, indicando que o melhor período para o plantio das sementes colhidas em junho é o inicio de outono (setembro-outubro). A germinação de sementes de timoteo reduziu nos 270 dias (73 por cento) seguintes. A melhor germinação, e crescimento da plântula do azevem italiano e do dactilis ocorreu entre 270 e 330 dias, indicando que a melhor época para plantio é na primavera (março-abril), do ano seguinte. As sementes de dactilis e azevem italiano mantiveram germinação satisfatória até 630 dias (81 por cento) e 810 dias (81 por cento), respectivamente. Essa pesquisa pode servir para determinação de gerenciamento adequado de duração de armazenamento entre a colheita e o plantio das espécies testadas em condições de armazenamento no sudeste da Europa.

Characterization of proteins from grain of different bread and durum wheat genotypes.

The classical Osborne wheat protein fractions (albumins, globulins, gliadins, and glutenins), as well as several proteins from each of the four subunits of gliadin using SDS-PAGE analyses, were determined in the grain of five bread (T. aestivum L.) and five durum wheat (T. durum Desf.) genotypes. In addition, content of tryptophan and wet gluten were analyzed. Gliadins and glutenins comprise from 58.17% to 65.27% and 56.25% to 64.48% of total proteins and as such account for both quantity and quality of the bread and durum wheat grain proteins, respectively. The ratio of gliadin/total glutenin varied from 0.49 to 1.01 and 0.57 to 1.06 among the bread and durum genotypes, respectively. According to SDS-PAGE analysis, bread wheat genotypes had a higher concentration of α + ß + γ-subunits of gliadin (on average 61.54% of extractable proteins) than durum wheat (on average 55.32% of extractable proteins). However, low concentration of ω-subunit was found in both bread (0.50% to 2.53% of extractable proteins) and durum (3.65% to 6.99% of extractable proteins) wheat genotypes. On average, durum wheat contained significantly higher amounts of tryptophan and wet gluten (0.163% dry weight (d.w.) and 26.96% d.w., respectively) than bread wheat (0.147% d.w. and 24.18% d.w., respectively).

Different levels of humoral immunoreactivity to different wheat cultivars gliadin are present in patients with celiac disease and in patients with multiple myeloma.

BACKGROUND: Immunity to food antigens (gliadin, cow's milk proteins) is in the centre of the attention of modern medicine focused on the prevention of diseases, prevention which is based on the use of appropriate restriction diet. Detection of the enhanced levels of the immune reactions to antigen(s) present in food is from this point of view of great importance because there are reports that some of health disturbances, like celiac disease (CD) and some premalignant conditions, like monoclonal gammopathy of undetermined significance (MGUS), were vanished after the appropriate restriction diets. It is well known that gliadin is toxic to small bowel mucosa of relatively small population of genetically predisposed individuals, who under this toxic action develop celiac disease (CD). As the quantity of immunogenic gliadin could vary between different wheat species, the first aim of this work was to determine the percentage of immunogenic gliadin in ten bread wheat cultivars and in three commercially grown durum wheat cultivars. The second part of the study was initiated by results of previous publication, reporting that sera of some of multiple myeloma (MM) patients showed the presence of elevated levels of anti-gliadin IgA, without the enhanced levels of anti-gliadin IgG antibodies, determined with commercial ELISA test. It was designed to assess is it possible to reveal is there any hidden, especially anti-gliadin IgG immunoreactivity, in serum of mentioned group of patients. For this purpose we tested MM patients sera, as well as celiac disease (CD) patients sera for the immunoreaction with the native gliadin isolated from wheat species used for bread and pasta making in corresponding geographic region. RESULTS: Gliadin was isolated from wheat flour by two step 60% ehanolic extraction. Its content was determined by commercial R5 Mendez Elisa using PWG gliadin as the standard. Results obtained showed that immunogenic gliadin content varies between 50.4 and 65.4 mg/g in bread wheat cultivars and between 20 and 25.6 mg/g in durum wheat cultivars. Anti-gliadin IgA and IgG immunoreactivity of patients' sera in (IU/ml) was firstly determined by commercial diagnostic Binding Site ELISA test, and then additionally by non-commercial ELISA tests, using standardized ethanol wheat extracts -gliadin as the antigen. In both patients groups IgA immunoreactivity to gliadin from different cultivars was almost homogenous and in correlation with results from commercial test (except for one patient with IgA(lambda) myeloma, they were more then five times higher). But, results for IgG immunoreactivity were more frequently inhomogeneous, and especially for few MM patients, they were more then five times higher and did not correlate with results obtained using Binding Site test. CONCLUSION: Results obtained showed different content of immunogenic gliadin epitopes in various species of wheat. They also point for new effort to elucidate is there a need to develop new standard antigen, the representative mixture of gliadin isolated from local wheat species used for bread production in corresponding geographic region for ELISA diagnostic tests.