Plant material and growing conditions
To observe effects of variations in environmental factors on grain composition, three localities (Čáslav, Uherský Ostroh, and Lednice) with intensive cultivation of barley specifically grown for malt production were selected. Their altitude ranges from 171 to 260 m above sea level, with differing soil conditions, air temperatures, rainfall (Table 1). Four high-quality malting varieties of spring barley (Hordeum vulgare, L.) Overture, KWS Amadora, Bojos and Laudis 550 were grown. The Bojos and Laudis 550 varieties met the requirements set out in the application for the protected geographical indication (PGI) ‘České pivo’34. Therefore, the Research Institute of Brewing and Malting recommended them for the production of beer under the PGI ‘České pivo’. The cultivation process followed common agricultural practices during the growing seasons of 2021, and 2022. The Central Institute for Supervising and Testing in Agriculture, in cooperation with the Research Institute of Brewing and Malting, oversaw the cultivation process and provided the seeds.
Data from the climatological stations of the Czech Hydrometeorological Institute36 were used to evaluate weather conditions of the years (Lednice, data from station Strážnice for Uherský Ostroh, Čáslav), as outlined in Table 2. From the perspective of the possible influence on the quality of seeds, the course of weather during the filling and ripening stage in grains has a fundamental impact. In this context, there is a significant difference between the meteorological conditions in May and June 2021 and 2022. The year 2021 was characterized by significantly lower temperatures and significantly higher precipitation totals during May. This could affect the quality of seeds during the grain filling period – including a lower content of photosynthates. In contrast, conditions in June were temperature-wise comparable in both years, with limited precipitation totals in 2021. Meteorological conditions in May 2022 (drought) and in June 2022 (higher precipitation totals) could lead to biochemical differences in the grain compared with 2021.
Determination of germination characteristics
The germination characteristics of the harvested seeds were assessed after they had broken dormancy, i.e., three months post-harvest. Selected barley seeds represented four different varieties and three localities. The first step involved sorting the seeds (in the sieving fraction over 2.5 mm)37, and then they were immersed in a 3% sodium hypochlorite solution for 10 min, followed by washing with distilled water three times.
Germination of barley seeds is an important characteristic for both seed and malting quality. While this parameter is evaluated under optimal conditions for germination (laboratory temperatures and optimal moisture), it differs from germination in the soil and malting38.
The evaluation of germination characteristics in barley, with a focus on germination under drought conditions (−0.5 MPa) and suboptimal temperature (10 °C), was carried out. The control variant underwent a standard germination test conducted according to the ISTA standard method, with no significant differences found. Drought conditions at -0.5 MPa were induced using polyethylene glycol (PEG 6000) based on the methodology of Michel and Kaufmann39. Additionally, six seeds of each variety were placed in separate 90 mm diameter plastic sterile Petri dishes, each containing 8 ml of PEG 6000 solution. The experiment involved three repetitions of each variety at each locality to ensure accuracy. This quantity of seeds was most suitable for ease of manipulation on the limited scanning area of the scanner, meeting the technical requirements. The use of PEG in the Petri dishes, without filter paper, was considered a valuable technique, as filter paper could potentially interfere with the integrity of the root structure during sample manipulation. Petri dishes were labelled and enclosed in plastic bags to prevent evaporation. Subsequently, the Petri dishes were placed in a climate-controlled chamber (in darkness conditions) set at 10 °C. After durations of seven and fourteen days, the germinated seeds from the Petri dishes were scanned and subjected to analysis using WinRHIZO (Régent Instruments Inc., Quebec, Canada), version 2020 Arabidopsis. To assess germination characteristics under drought in the Petri dishes, the following metrics were employed: Len 7, Len 14, AvgD 7, and AvgD 14 (root length (cm) and average diameter of roots (mm) after seven and fourteen days).
Preparation of the extract for TPC evaluation
Twenty grams of barley grains from each variety and locality were grounded finely on a 1 mm sieve barley grinder (each sample in three repetitions). After that, 5 g of ground barley was weighed into a 200 ml tall beaker. Then, 100 ml of 75% dimethylformamide solution was added, according to the EBC method 9.1140. Shortly afterward, the mixer head was immersed in the beaker and mixed for ninety seconds. The mixture was supposed to be left from 12 to 15 min and the whole procedure was repeated three times in each sample. After the last mixing, the solution was poured into a centrifuge tube. Samples were centrifuged at 3000 rpm for 10 min. From the centrifuged solution 25 ml of the solution was pipetted into a 100 ml volumetric flask and made up to the mark line with deionized water. The solution was poured into a centrifuge tube and for the last time samples were centrifuged at 15,000 rpm for 30 min. The pure solution has been used for the determination of total polyphenols content.
Preparation of a blank solution
Twenty ml of extract was pipetted into a 50 ml volumetric flask. After that, 0.10 ml ammonium solution was added. The solution was mixed thoroughly and made up to the mark line with deionized water. Then it was let to stand for 10 min and the clarity of the solution was observed.
Determination of total polyphenol content (TPC)
Twenty ml of extract was pipetted into a 50 ml volumetric flask, then 16 ml of ethylenediaminetetraacetic sodium hydroxide (CMC/EDTA) was added. Then, 0.10 ml of ammonium solution was added, made up to the mark line with deionized water and mixed thoroughly. Just before the measurement, a solution of ammonium ferric citrate in an amount of 0.10 ml was added and the content was shaken. The measurements were carried out with the requirement that each sample undergo three measurements within a 10-min window. This protocol was implemented to avoid potential colour changes in the sample resulting from the introduction of an ammonium ferric citrate solution over extended periods, which could potentially affect the recorded results. Consequently, it is crucial to strictly follow the measurement guidelines and complete the measurements within 10 min of introducing the ammonium ferric citrate solution. The absorbance was measured spectrophotometrically in a 10 mm cuvette at a wavelength of 525 nm against a blank solution.
Calculation and evaluation of TPC
The calculation was performed according to the formula for calculating the content of polyphenols in the original solution:
\({{\text{P}}_{\text{p}}}=\left( {{{\text{A}}_{\text{h}}} – {{\text{A}}_{\text{k}}}} \right) * 2.52\)
where Pp is a polyphenols content in the original solution (%).
Ah is the absorbance of the main solution (%).
Ak is the absorbance of the blank solution (at 525 nm wavelength).
Afterward, it was essential to perform a recalculation of the sample’s dry matter, which was carried out using the following formula:
$${{\text{P}}_{\text{s}}}=\frac{{{{\text{P}}_{\text{p}}} * 100}}{{100 – V}}$$
where Ps is a polyphenols content in dry matter of the sample (%).
V is the content of the water in barley seeds.
To obtain the water content of the barley seeds, it was necessary to determine the percentage of seeds moisture for each variety.
Statistical analyses
All statistical analyses were performed using the Python (3.10.12), using the libraries SciPy (1.12.0)41, statsmodels, scikit-learn, and matplotlib. All data were tested for normality using the Shapiro-Wilk test42. For datasets not exhibiting a normal distribution, multiple Kruskal-Wallis one-way analyses of variance were utilized43. In cases of normal distribution, a two-way MANOVA was conducted to assess the significant differences in total polyphenol content and germination characteristics across four different genotypes at three distinct localities. To identify the specific traits, varieties, and localities that demonstrated significant differences, separate ANOVAs were conducted for each parameter that exhibited a normal distribution after MANOVA. This step was essential for identifying precise differences. Subsequently, Tukey’s HSD test was used for the 2022 data which met a normal distribution criteria, and Dunn’s post-hoc test was used for the 2021 data which did not meet a normal distribution criteria with applied significance (p < 0.05) within the traits. The Tukey’s HSD test was used to assess the percentage contributions of genotype, environment, and genotype-environment interaction (G x E) to the observed relationship, with each effect’s contribution calculated using partial eta squared (η2)42,43.
Principal Component Analysis (PCA) was conducted to group and relate traits for each year. The Spearman correlation coefficient (rs) was used for datasets not exhibiting normal distribution, while the Pearson correlation coefficient (r) was applied to datasets with a normal distribution. This approach ensures accurate analysis of the relationships between traits. Graphical representations (Figs. 1, 2 and 3) were generated using the Plotly Python Graphing Library (5.19.0). Principal Component Analysis (PCA) biplot visualizes the multivariate relationships among total polyphenol content (TPC) and germination characteristics (such as root length and average root diameter) across different barley varieties and localities. The PCA biplot reduces the dimensionality of the data, making it easier to identify clustering patterns and correlations between variables. Meanwhile, the box plots represent median values across multiple categories. These box plots highlight the variations in TPC and seed germination characteristics (root length, average root diameter) among different barley varieties across localities. Box plots were chosen for their simplicity, yet they provide an effective visual representation of the results.
