1.1. Shoot Multiplication and Rooting
The medium to multiplication phase was prepared using 0.35 mg·L− 1 BA and 0.35 mg·L− 1 IBA were added to the Murashige and Skoog (1962) basal media with 30 mg·L− 1 of sucrose, gelled with supplementation of 8 g·L− 1 agar, the pH was adjusted to 5.8 before autoclaving. Media with PGRs was autoclaved for 20 min. at 12oC and 110 kPa. The microshoots produced in the multiplication stage were suitable for rooting. After 6 weeks multiplied shoots were transferred to the rooting MS medium supplemented with 0.5 mg·L− 1 IBA and 1 mg·L− 1 activated charcoal (Sigma Aldrich) with 30 mg·L− 1 of sucrose and 8 g·L− 1 agar, pH = 5.8 before autoclaving. Plantlets regenerated in the rooting medium for approx. 6 weeks (Fig. 1). During both phases, the glass jars containing explants were kept in a growth phytotron with 23 ± 1℃, 16 h day with light at 35 µmol m− 2 s− 1 photosynthetic photon flux density, provided by fluorescent lamps.
1.2. Acclimation phase experimental rationale and design
After 6 weeks of regeneration in growth phytotron, the rooted plantlets were removed from the culture jars and the roots were washed using distilled water to remove traces of agar in the surface of roots. Next, microplants were planted in plastic boxes filled with peat (pH = 5.0–6.0) and perlite 1:1 (v/v). Then plantlets were sprayed separately with 200 mL water solution of: ABA in concentration 0.001 mL·L− 1, 0.001 mL·L− 1 BL, 0.001 mL·L− 1 24-epiBL and 0.3 mL·L− 1 biostimulant Goteo (Table 1). The concentrations were selected after preliminary experiments. Control treatment was distilled water. Each treatment were tested in a thrice replicated experiment with 10 plantlets. After spraying, plants were covered with transparent lids and kept in growing room in temp. 25oC, the photoperiod was established 16 h day (50 µmol·m2·s1). After 4 weeks of growing in phytotron, plants were transferred to the greenhouse (Fig. 1).
Table 1
A list of treatments in the experiment.
No. of treatment | Treatment | Concentration |
---|
1. | Control “0” | distilled water |
2. | ABA | 0.001 ml·L− 1 |
3. | BL | 0.001 ml·L− 1 |
4. | 24-epiBL | 0.001 ml·L− 1 |
5. | Goteo | 0.3 ml·L− 1 |
1.3. Evaluation
1.3.1. Stomatal conductance measurements
Stomatal conductance (Gs) of leaves in plantlets was measured from in vitro plants (0 term), at the beginning of ex vitro acclimation (1st term), at the end of growing in phytotrone (2nd term) and at the end of growing in the greenhouse (3rd term). During acclimation using a portable SC − 1 leaf porometer (Geomor Technik). Device consists in the principle of measuring the static change of humidity inside a closed measuring head with a leaf placed inside. Gs was expressed as mmol H2O m− 2s− 1.
1.3.2. Biochemical analyses
The samples for biochemical analyses (leaves) were picked in the same terms as Gs was measured. The material was chopped, mixed, and samples were collected for each analysis. Three extractions were done for each analysis and 3 measurements were made for each extract. The absorbance was measured in spectrophotometer UV-1601 PC (Shimadzu, Columbia, MD, USA).
1.3.2.1. Total chlorophyll and carotenoids content
The total contents of chl and carotenoids were assessed using the methods described by Lichtenthaler and Wellburn (1983). Plant material was ground in a mortar with a slight amount of quartz sand and 2.5 mL of 80% acetone. The extracts were subsequently filtered through filter paper into 25 mL volumetric flasks and filled up with acetone. The absorbance of filtrates was measured at: 470, 646, 652, and 663 nm.
1.3.2.2. Preparation of extracts to H2O2, catalase, soluble proteins
0.25 g samples were homogenized in 2.5 mL K-phosphate buffer. The extracts were centrifuged by 20 min. at 20.000 xg at 4°C. Next, the extract was transferred to tubes.
1.3.2.3. Hydrogen peroxide content
H2O2 content was measured using Pick and Keisari (1980) method. Reaction mixture was composed by 0.5 mL extract, 0.5 mL 0.1 M potassium phosphate buffer (pH = 7.0) and 1 mL of 1 M potassium iodide. Samples were incubated by 1 h in the darkness at room temperature and their absorbance were measured at 390 nm.
1.3.2.4. Catalase activity
The analysis of catalase activity was conducted following the methodology outlined in the work by Goth (1991). The tubes were divided into two sets, each receiving 0.05 mL of the extract along with 0.45 ml of K-phosphate buffer. In tubes labeled 'A', 1 mL of 0.1 M K-phosphate buffer was added, while tubes labeled 'B' received 1 mL of H2O2 in buffer (at a concentration of 65 µM). A control sample, denoted as 'K', was prepared by adding 1.5 mL of 0.1 M K-phosphate buffer, while sample 'C' consisted of 0.5 mL of 0.1 M K-phosphate buffer mixed with 1 mL of H2O2 in buffer (at a concentration of 65 µM). All tubes were then placed in darkness for incubation for 10 minutes. Following incubation, 1 mL of 32.5 mM ammonium molybdate was added to each tube and thoroughly mixed. Subsequently, absorbance was measured at 405 nm.
1.3.2.5. MDA content
MDA determination was based on the method described by Heath and Packer (1968). Leaf samples weighing 0.25 g were homogenized in 2.5 ml of trichloroacetic acid (TCA) (w/v). The resulting extract was centrifuged for 10 minutes at 10,000 xg and 4°C. The reaction mixture consisted of 1.0 mL of supernatant and 4.0 mL of 20% thiobarbituric acid (TBA) in 0.5% TCA (w/v). Subsequently, the samples were incubated for 30 minutes at 95°C, followed by stopping the reaction on ice for 10 minutes. Afterward, the samples underwent another centrifugation at 10,000 xg for 10 minutes at 4°C. Absorbance was measured at 532 nm and 600 nm to account for nonspecific absorbance using an extinction coefficient (ε) of 155 mM− 1cm− 1.
1.3.2.6. Free amino acids
The procedure for determining the content of free amino acids was adapted from the Rosen (1957) method. Specifically, 0.2 ml of extract was combined with 0.5 mL of 0.2 mM sodium cyanide in acetate buffer (pH = 5.3–5.4) and 0.5 mL of ninhydrin. After thorough mixing, the solutions were incubated in a water bath at 100°C for 15 minutes. Subsequently, the tubes were taken out, and 5 mL of isopropyl alcohol was added to each warm tube. The mixtures were then allowed to cool. Absorbance readings were taken at 570 nm against a standard curve for leucine.
1.3.2.7. Total soluble proteins
The concentration of total soluble proteins was measured using Bradford's method (1976). The reaction mixture comprised 0.1 mL of the extract and 5.0 mL of Bradford's reagent. The mixture was incubated in the dark for 5 minutes, and the absorbance was then measured at 595 nm.
1.3.2.8. Total sugars content
Total sugars were measured by the colorimetric method of Dubois et al. (1956). The samples were homogenized in hot 80% ethanol. The extract was centrifuged for 20 minutes at 20.000 xg at 4°C. Then, the supernatant was transferred into resealable tubes and adjust to 25 mL with 80% ethanol. The reaction mixture comprised 100 µl of extract, 1 mL of 5% phenol, and 5 ml of sulphuric acid (H2SO4, 96%) and mixed thoughtfully. The solutions incubated for 20 minutes. The absorbance was measured against a standard curve for glucose at 490 nm.
1.3.2.9. Reduced sugars content
Reduced sugars content was determined by Nelson (1944) method. The samples were homogenized in hot 80% ethanol. The extract was centrifuged for 20 minutes at 20.000 xg at 4°C. Then, the supernatant was transferred into resealable tubes and adjust to 25 mL with 80% ethanol. 100 µL of extract were taken. Then, 1 mL of copper reagent was added. The mixture was incubated for 20 minutes in a water bath at 100°C. Afterward, it was cooled in cold water. 1 mL of molybdenum-arsenic reagent was added and mixed. Finally, distilled water was added to make up the volume to 5 mL. The absorbance was measured against a standard curve for glucose at 520 nm.
1.3.3. Morphological parameters
Biometric data (plant height, number of leaves, average length and width of 3 leaves, number of roots, length of roots) were recorded at the 4th week of acclimation stage. Mortality of the plantlets were calculated using following equation:
$$\:mortality\:\left[\%\right]=\frac{number\:of\:dead\:plants}{total\:number\:of\:plantlets}*100$$
1.4. Data analysis
Statistical analysis, data and graphical presentation were performed with Statistica Version 13.1 (STATSoft, Tulsa, OK, USA) and Microsoft Excel 365 (Version 2023). Experimental data were subjected to one way and two-way analysis of variance and then to Tukey’s multiple range test to separate the means at the significance level of p ≤ 0.05.