Fig 1.
Schematic overview of the experimental setup for DWC hydroponics (A), and the photograph of the 7-day-old seedlings (B) grown in control conditions using that system (Hoagland’s medium, pH = 6).
A plastic opaque container with capacity of 10 L (1), a plastic opaque lid with 20 openings (2), air distributor with 12 outlets (3) and an air pipe with a non-return valve attached to the air pump (4).
Table 1.
AlCl3 concentrations selected for the treatment of barley seedlings grown in DWC hydroponics.
The calculations of available Al3+ concentrations in the full Hoagland’s nutrient solution, pH = 4 were calculated using GEOCHEM-EZ software.
Fig 2.
The total root length of barley seedlings cv. ‘Sebastian’ treated with different concentrations of Al for 7 days.
Tested Al3+ doses included 5; 10; 20; 40; 60 μM AlCl3, in full Hoagland’s medium, pH = 4 (treatments), pH = 6 (control). The measurements were carried out using WinRHIZO system.
Fig 3.
The kinetics of root growth of barley seedlings cv. ‘Sebastian’ treated with different concentrations of Al.
The error bars represent the standard deviations of the mean. Tested Al doses consisted of: 5; 10; 20; 40; 60 μM AlCl3, in full Hoagland’s medium, pH = 4 (treatments), pH = 6 (control). The total root length was analysed at selected time points of continuous Al-treatment: 0 days; 2 days; 4 days; 6 days. The measurements were carried out using WinRHIZO system.
Fig 4.
The effect of the Al-treatment on root system parameters evaluated after 1- day and 7 days of the exposition.
Tested Al3+ doses consisted of: 10; 20; 30 μM AlCl3, in full Hoagland’s medium, pH = 4 (treatments), pH = 6 (control). The measured parameters include: total root length (A), total root area (B), total root volume (C), average root diameter (D). Different letters above the bars indicate statistically significant differences (ANOVA; p < 0.05) between combinations. The percentages above the bars indicate the reduction/ increase of the value in comparison to the control.
Fig 5.
The root apices of barley seedlings (cv. ‘Sebastian’) grown in DWC hydroponics in full Hoagland’s control medium (A), and exposed to 40 μM AlCl3 (B).
The photographs were taken using Delta Optical SZ-630T stereomicroscope, scale bar = 0.5 mm.
Fig 6.
Cytological effects of Al in root cells of barley seedlings cv. ‘Sebastian’.
(A, A`) mitotic activity of control cells (A) and cells after Al3+ treatment (A`), (B) interphase nucleus with micronucleus, (C) fragmented interphase nuclei. Bars represent 20 μm.
Fig 7.
The mitotic activity of root cells of barley seedlings cv. ‘Sebastian’—(control and Al treated).
The error bars represent the standard deviations of the mean. The significant differences (P < 0.05) between the treated combinations and the control within the same treatment day, are indicated by a capital letter A (1-day treatment) or B (7-day treatment).
Fig 8.
The frequency of root cells of barley cv. ‘Sebastian’ with micronuclei and damaged nuclei in control and after Al treatment.
The error bars represent the standard deviations of the mean. The significant differences (P < 0.05) between the treated combinations and the control within the same treatment day are indicated by capital letters: A, B, D, E.
Fig 9.
Results of TUNEL test in root cells of barley seedlings cv. ‘Sebastian’.
DAPI stained nuclei (A-H), with or without green fluorescence as a result of TUNEL reaction (A`-H`). (A, A`) control nuclei showing weak green fluorescence, (B, B`) positive control (DNAse solution was used to induce DNA strand breaks), (C, C`) negative control (nucleotide solution, without terminal transferase was used), (D, D`) not-treated cells, (E, E`) after treatment with 30 μM Al (nuclei with and without green fluorescence are shown), (F, F`) after treatment with 40 μM Al (nuclei with and without green fluorescence are shown) (G, G`) fragmented nucleus after treatment with 30 μM Al with green fluorescence, (H, H`) nuclei with micronucleus, both show green fluorescence. Bars represent 20 μm.
Fig 10.
The frequency of labelled nuclei in root cells of barley cv. ‘Sebastian’ in TUNEL test in control and after Al treatment.
The error bars represent the standard deviations of the mean. The significant differences (P < 0.05) between the treated combinations and the control within the same treatment day are indicated by a capital letter A (1-day treatment) or B (7-day treatment).
Fig 11.
Flow cytometric analysis of cell cycle in roots of barley cv. ‘Sebastian’.
(A–D) control roots, (E, F) Al treated roots.
Fig 12.
Examples of the detection of S-phase cells using EdU incorporation in roots of barley cv. ‘Sebastian’ seedlings treated with different concentrations of Al for 1 and 7 days.
(A-C) DAPI staining (A`-C`) Alexa Fluor 647 fluorescence (red) in S-phase cells detected after EdU incorporation. Bars represent 20 μm.
Fig 13.
The frequency of root meristematic cells of barley cv. ‘Sebastian’ at S-phase in control and Al-treated roots.
The error bars represent the standard deviations of the mean. The significant differences (P < 0.05) between the treated combinations and the controls (pH 4 and pH 6) within the same treatment day are indicated by a capital letter A (1-day treatment) or B (7-day treatment). The frequency of S-phase cells is not significantly different (P < 0.05) within the same treatment day for different pH.