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The development of automated, image-based, high-throughput plant phenotyping enabled the simultaneous measurement of many plant traits. Big and complex phenotypic datasets require advanced statistical methods which enable the extraction of the most valuable traits when combined with other measurements, interpretation, and understanding of their (eco)physiological background. Nutrient deficiency in plants causes specific symptoms that can be easily detected by multispectral imaging, 3D scanning, and chlorophyll fluorescence measurements. Screening of numerous image-based phenotypic traits of common bean plants grown in nutrient-deficient solutions was conducted to optimize phenotyping and select the most valuable phenotypic traits related to the specific nutrient deficit. Discriminant analysis was used to compare the efficiency of groups of traits obtained by high-throughput phenotyping techniques (chlorophyll fluorescence, multispectral traits, and morphological traits) in discrimination between nutrients [nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), and iron (Fe)] at early and prolonged deficiency. Furthermore, a recursive partitioning analysis was used to select variables within each group of traits that show the highest accuracy for assigning plants to the respective nutrient deficit treatment. Using the entire set of measured traits, the highest classification success by discriminant function was achieved using multispectral traits. In the subsequent measurements, chlorophyll fluorescence and multispectral traits achieved comparably high classification success. Recursive partitioning analysis was able to intrinsically identify variables within each group of traits and their threshold values that best separate the observations from different nutrient deficiency groups. Again, the highest success in assigning plants into their respective groups was achieved based on selected multispectral traits. Selected chlorophyll fluorescence traits also showed high accuracy for assigning plants into control, Fe, Mg, and P deficit but could not correctly assign K and N deficit plants. This study has shown the usefulness of combining high-throughput phenotyping techniques with advanced data analysis to determine and differentiate nutrient deficiency stress.

Drought is a significant constraint in bean production. In this study, we used high-throughput phenotyping methods (chlorophyll fluorescence imaging, multispectral imaging, 3D multispectral scanning) to monitor the development of drought-induced morphological and physiological symptoms at an early stage of development of the common bean. This study aimed to select the plant phenotypic traits which were most sensitive to drought. Plants were grown in an irrigated control (C) and under three drought treatments: D70, D50, and D30 (irrigated with 70, 50, and 30 mL distilled water, respectively). Measurements were performed on five consecutive days, starting on the first day after the onset of treatments (1 DAT–5 DAT), with an additional measurement taken on the eighth day (8 DAT) after the onset of treatments. Earliest detected changes were found at 3 DAT when compared to the control. D30 caused a decrease in leaf area index (of 40%), total leaf area (28%), reflectance in specific green (13%), saturation (9%), and green leaf index (9%), and an increase in the anthocyanin index (23%) and reflectance in blue (7%). The selected phenotypic traits could be used to monitor drought stress and to screen for tolerant genotypes in breeding programs.

Viruses are responsible for more than 50% of annual potato tuber yield losses and cause great economic damage. The traditional Croatian potato cultivar ‘Brinjak’ is important for local growers because of its economically profitable production and as a gene pool for future breeding programs. However, the full genetic potential of the cultivar cannot be exploited due to virus infection. In this study, we attempted to eliminate potato virus M (PVM) and potato virus S (PVS) from potato cultivar ‘Brinjak’ and to evaluate the effects on physiological parameters and yield. Shoot apices were isolated from PVM + PVS-infected sprouts and cultivated for six weeks on MS medium with the addition of 50 or 100 mg L−1 ribavirin. The surviving shoot apices were micropropagated. The in vitro post-eradication period lasted 200 days. DAS-ELISA and RT-PCR were performed on R0 and R1 plants 90 days after acclimatization to determine the sanitary status of the plants. Chlorophyll fluorescence and multispectral imaging were performed on the R0 plants at the same time. The success of PVS elimination was 33% at both ribavirin concentrations. However, neither concentration was successful in eliminating PVM. Plants with mixed infection (PVM + PVS) had more severe disease symptoms compared to PVM-infected plants, affecting photochemistry and multispectral parameters and, consequently, yield. PVM + PVS plants had significantly lower number and weight of tubers per plant and lower average tuber weight than plants with single PVM infection in most of the generations studied. The results indicate a strong negative impact of PVS in mixed infections with PVM and show the importance of its elimination from potato plants.

Drought negatively affects plants by altering morphological, physiological and metabolic processes and ultimately reducing yields. Garlic (Allium sativum L.), an important member of the Alliaceae family, is also sensitive to drought and maximizing the yield of garlic bulbs is largely dependent on water availability. The objective of this study was to determine the effects of drought stress on morphological and physiological characteristics, as well as on phenolic, sugar, inulin and free amino acid content and antioxidant activity in two Croatian garlic ecotypes, ‘Istarski crveni’ (IC) and Istarski bijeli (IB). Drought was induced by using polyethylene glycol 8000 (PEG) solution (−0.6 MPa) starting 21 days after clove planting and lasted for 20 days. Drought reduced plant height, number of leaves and plant weight, but increased root length in both ecotypes compared to the control treatment. Among the physiological parameters, significant differences were observed between the two ecotypes studied in the spectral characteristics of the leaves, namely reflection in red, green and blue, VAL, values of the vegetation indices related to the chlorophyll content (CHI, GI), and the anthocyanin content (ARI). Ecotype IC showed higher antioxidant activity in the control treatment due to higher total phenolic content (TPC), but under drought conditions higher DPPH radical scavenging activity was determined in ecotype IB and higher values of FRAP in IC. Sucrose and glucose generally decreased under drought, while inulin increased in IB but decreased in IC. Total free amino acid content increased under drought in both ecotypes. In conclusion, drought tolerance of IB might be associated with increased accumulation of inulin and higher levels of amino acids, especially those shown to contribute to drought resistance. In IC, drought tolerance is associated with an increase in some amino acid compounds and better root growth in depth, probably due to a more efficient translocation of sucrose to the underground part of the plant.

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.

Potato (Solanum tuberosum L.) is vulnerable to high temperatures, which are expected to increase in frequency and duration due to climate change. Nondestructive phenotyping techniques represent a promising technology for helping the adaptation of agriculture to climate change. In this study, three potato cultivars (Agria, Bellarosa and Desiree) were grown under four temperature treatments: 20/15 °C (T1), 25/20 °C (T2), 30/25 °C (T3), and 35/30 °C (T4). Multispectral and chlorophyll fluorescence imaging, 3D multispectral scanning, and gas exchange analysis were used to study the effect of moderate heat stress on potato morphology and physiology and select phenotypic traits most responsive to increased temperatures. The most responsive morphological traits to increased temperatures are related to decreased leaf area, which were detected already at T2. Increased temperatures (already T2) also changed leaf spectral characteristics, indicated by increased red, green, and blue reflectance and decreased far-red reflectance and anthocyanin index (ARI). Regarding chlorophyll fluorescence, increasing temperatures (T2) caused an increase in minimal fluorescence of both dark-adapted (F0) and light-adapted (F0’) plants. Stomatal conductance, transpiration rate, photosynthetic rate, instantaneous water use efficiency (WUE), and intrinsic water use efficiency increased from T1 to T3 and decreased again in T4. Using recursive partitioning analysis, the most responsive potato phenotypic traits to increased temperature were leaf area projected (LAP), ARI, F0, and WUE. These traits could be considered marker traits for further studying potato responses to increased temperatures.

Basil is one of the most widespread aromatic and medicinal plants, which is often grown in drought- and salinity-prone regions. Often co-occurrence of drought and salinity stresses in agroecosystems and similarities of symptoms which they cause on plants complicates the differentiation among them. Development of automated phenotyping techniques with integrative and simultaneous quantification of multiple morphological and physiological traits enables early detection and quantification of different stresses on a whole plant basis. In this study, we have used different phenotyping techniques including chlorophyll fluorescence imaging, multispectral imaging, and 3D multispectral scanning, aiming to quantify changes in basil phenotypic traits under early and prolonged drought and salinity stress and to determine traits which could differentiate among drought and salinity stressed basil plants. Ocimum basilicum “Genovese” was grown in a growth chamber under well-watered control [45–50% volumetric water content (VWC)], moderate salinity stress (100 mM NaCl), severe salinity stress (200 mM NaCl), moderate drought stress (25–30% VWC), and severe drought stress (15–20% VWC). Phenotypic traits were measured for 3 weeks in 7-day intervals. Automated phenotyping techniques were able to detect basil responses to early and prolonged salinity and drought stress. In addition, several phenotypic traits were able to differentiate among salinity and drought. At early stages, low anthocyanin index (ARI), chlorophyll index (CHI), and hue (HUE2D), and higher reflectance in red (RRed), reflectance in green (RGreen), and leaf inclination (LINC) indicated drought stress. At later stress stages, maximum fluorescence (Fm), HUE2D, normalized difference vegetation index (NDVI), and LINC contribute the most to the differentiation among drought and non-stressed as well as among drought and salinity stressed plants. ARI and electron transport rate (ETR) were best for differentiation of salinity stressed plants from non-stressed plants both at early and prolonged stress.

Micronutrient malnutrition is one of the main public health problems in many parts of the world. This problem raises the attention of all valuable sources of micronutrients for the human diet, such as common bean (Phaseolus vulgarisL.). In this research, a panel of 174 accessions representing Croatian common bean landraces was phenotyped for seed content of eight nutrients (N, P, K, Ca, Mg, Fe, Zn, and Mn), and genotyped using 6,311 high-quality DArTseq-derived SNP markers. A genome-wide association study (GWAS) was then performed to identify new genetic sources for improving seed mineral content. Twenty-two quantitative trait nucleotides (QTN) associated with seed nitrogen content were discovered on chromosomes Pv01, Pv02, Pv03, Pv05, Pv07, Pv08, and Pv10. Five QTNs were associated with seed phosphorus content, four on chromosome Pv07, and one on Pv08. A single significant QTN was found for seed calcium content on chromosome Pv09 and for seed magnesium content on Pv08. Finally, two QTNs associated with seed zinc content were identified on Pv06 while no QTNs were found to be associated with seed potassium, iron, or manganese content. Our results demonstrate the utility of GWAS for understanding the genetic architecture of seed nutritional traits in common bean and have utility for future enrichment of seed with macro– and micronutrients through genomics-assisted breeding.

Basil is one of the most widespread aromatic and medicinal plants, which is often grown in drought- and salinity-prone regions. Often co-occurrence of drought and salinity stresses in agroecosystems and similarities of symptoms which they cause on plants complicates the differentiation among them. Development of automated phenotyping techniques with integrative and simultaneous quantification of multiple morphological and physiological traits enables early detection and quantification of different stresses on a whole plant basis. In this study, we have used different phenotyping techniques including chlorophyll fluorescence imaging, multispectral imaging, and 3D multispectral scanning, aiming to quantify changes in basil phenotypic traits under early and prolonged drought and salinity stress and to determine traits which could differentiate among drought and salinity stressed basil plants. Ocimum basilicum “Genovese” was grown in a growth chamber under well-watered control [45–50% volumetric water content (VWC)], moderate salinity stress (100 mM NaCl), severe salinity stress (200 mM NaCl), moderate drought stress (25–30% VWC), and severe drought stress (15–20% VWC). Phenotypic traits were measured for 3 weeks in 7-day intervals. Automated phenotyping techniques were able to detect basil responses to early and prolonged salinity and drought stress. In addition, several phenotypic traits were able to differentiate among salinity and drought. At early stages, low anthocyanin index (ARI), chlorophyll index (CHI), and hue (HUE2D), and higher reflectance in red (RRed), reflectance in green (RGreen), and leaf inclination (LINC) indicated drought stress. At later stress stages, maximum fluorescence (Fm), HUE2D, normalized difference vegetation index (NDVI), and LINC contribute the most to the differentiation among drought and non-stressed as well as among drought and salinity stressed plants. ARI and electron transport rate (ETR) were best for differentiation of salinity stressed plants from non-stressed plants both at early and prolonged stress.

Terminal drought i.e. drought during grain-filling phase is the most devastating environmental stress to wheat production. In present study the effect of terminal drought on physiological traits and its influence on yield and yield components in two winter wheat varieties (Kuna and Karla) were investigated. Terminal drought stress was applied from the beginning of anthesis by installing mobile plastic roof above the crops. Leaf gas exchange parameters, chlorophyll content index (CCI), relative water content (RWC), and nitrogen (N) content were measured three times during grain-filling phase, at early milk maturity (EMM), late milk maturity (LMM), and at early wax maturity (EWM). Grain yield and 1000 grain weight were measured by harvesting of each plot at crop maturity. Terminal drought enhanced leaf senescence and caused reduction of RWC, CCI, net photosynthetic rate (A), stomatal conductance (gs), and transpiration rate (E) as well as, grain yield and all measured yield components. However, grain yield and grain weight per ear were less affected in Karla indicating enhanced tolerance to terminal drought compared to Kuna variety. Higher tolerance to terminal drought in Karla is based on stay-green strategy. Stay-green strategy in Karla was characterized by retention of CCI at early wax maturity, which contributed to higher E and lower intercellular CO2 concentration compared to Kuna under terminal drought. Stay-green strategy as trait that enhanced terminal drought tolerance in Karla should be used in breeding programs and utilized to ensure maximum economic yields under terminal drought conditions.

Arbuscular mycorrhizas (AM) can improve phosphorus (P) nutrition and could serve as an environmentally friendly approach for sustainable crop production under P-limiting conditions. The objectives of this study were to assess the effect of AM on different physiological traits and to quantify the responsiveness of different basil (Ocimum basilicum L.) cultivars to AM under low P availability. The basil cultivars &lsquo, Genovese&rsquo, &lsquo, Sweet Basil&rsquo, Dark Opal&rsquo, and &lsquo, Erevanskii&rsquo, were inoculated (AMI) using Rhizophagus irregularis. Photochemical efficiency and gas exchange were measured on AMI and non-inoculated (AMC) plants and, at harvest, the shoot biomass, shoot P concentration, root morphological traits, frequency of mycorrhizas in the roots (F%), and extent of root colonization (M%) were determined. Significant differences in F% and M% were found among the examined cultivars, with the highest found in &lsquo, Dark Opal&lsquo, and the lowest in &lsquo, Erevanskii&lsquo, AMI reduced the shoot biomass and increased the shoot P concentration as well as other examined root traits in &lsquo, whereas it did not affect those traits in &lsquo, indicating differences in responsiveness to AM. AMI positively affected the gas-exchange parameters in all examined cultivars, probably due to the increased sink capacity of a bigger root system and/or AM structures within the roots.

In Croatia common bean is a traditional grain legume, grown by small farmers in low input agriculture systems. The majority of the production is still based on local landraces adapted to the specific growing conditions and agro-environments and showing a great morphological diversity. Local landraces are in danger of genetic erosion caused by complex socio-economic changes in rural communities. The low profitability of farms and their small size, the advanced age of farmers and the replacement of traditional landraces with modern bean cultivars and/or other more profitable crops have been identified as the major factors affecting genetic erosion. Three hundred accessions belonging to seven most widely used morphotypes (landraces) were genotyped by microsatellite markers in order to assess the genetic diversity and population structure of Croatian common bean germplasm. PCR-based phaseolin type analysis was used to trace the origin of accessions to Mesoamerican and Andean domestication centres. One-third of accessions showed phaseolin type I ("S"), predominant in germplasm of Mesoamerican origin while the rest of accessions belonged to Andean domestication centre characterized by the phaseolin type II ("H" or "C" ; 22%) or III ("T" ; 47%). A model-based structure analysis based on microsatellite markers revealed the presence of three clusters, for the most part, in congruence with the results of phaseolin type analysis.

The research aim was to determine the influence of different organic and mineral fertilization treatments and post-harvest treatments on the content of nitrogen and crude proteins in the edible part of beetroot (Beta vulgaris var. conditiva). A field trial (2003-2005) was set up in a hilly part of Croatia according to the Latin square method with four types of fertilization (control, 50 t ha-1 stable manure, 500 and 1000 kg ha-1 NPK 5-20-30), while treatments involved harvested fresh beetroot and stored fresh beetroot. The highest dry weight (DW) content was determined in climatologically favourable 2004 (average 14.8% DW) and in the treatment with 1000 kg ha-1 NPK 5-20-30 (15.6% DW) in harvested beetroot. In 2004 and 2005, the highest levels of nitrogen and crude proteins in harvested beetroot were determined in the treatment with 1000 kg ha-1 NPK 5-20-30 (2.41 and 2.43 g N kg-1 in fresh weight and 15.07 and 15.21 g crude proteins kg-1 in fresh weight, respectively). Regardless of fertilization treatment or studied year, nitrogen and crude protein contents were higher in stored than in harvested beetroot, by 12% on average. The lowest crude protein content was determined in treatment with stable manure what confirmed that protein content decreased by organic fertilization. It can be concluded that beetroot lost some water during the storage period, which increased its content of nitrogen and crude proteins in fresh weight and thus increased the nutritional quality of beetroot as a functional food.