**2. Materials and Methods**

## *2.1. Location of the Experiments and Plant Material*

The study was conducted at the Centre for Agricultural Genomics and Biotechnology, Faculty of the Agricultural and Food Science and Environmental Management, University of Debrecen, Hungary. This study was carried out on 29 potato (*Solanum tuberosum* L.) genotypes including 27 breeding lines (C2, C3, C4, C5, C6, C8, C9, C10, C11, C12, C14, C17, C19, C20, C21, C22, C26, C28, C30, C32, C35, C37, C41, C42, C57, C58 and C63) and two drought-tolerant referent lines (C103 and C107). Selection of referent genotypes was based on their response to drought conditions in previous in vivo (field and greenhouse) experiments, in which they were proven to be drought-tolerant. C103 parental line belongs to the mid-early maturity group; its tuber is round oval, its skin is red, and the tuber flesh is yellow. C107 parental line is a late genotype with oblong, purple pink skinned tubers and white flesh. They are tolerant to late blight (*Phytophthora infestans*). All of the breeding lines tested originated from crossings of referent lines. In vitro culture establishment of breeding lines was initiated from seeds; they were surface sterilized by 3% NaOCl for 4 min, then they were washed with sterile distilled water three times. The seeds were placed onto medium containing Murashige and Skoog [44] (MS) salts and vitamins, 3% sucrose (VWR Chemicals, Radnor, PA, USA) and 0.7% agar-agar (Sigma-Aldrich, A1296, St. Louis, MO, USA) and incubated in dark at 24 ◦C for 5 days. Then, they were transferred to a growing room (at 22/15 ± 2 ◦C day/night temperature and 16 h daily illumination by 65 μmol m−<sup>2</sup> s−<sup>1</sup> PPF). In vitro shoot cultures of both referent lines and breeding lines were subcultured every 4 weeks on the same MS medium mentioned above.

## *2.2. Explant, Treatments and Culture Conditions*

Single nodal cuttings, each containing an axillary bud from 4-week-old in vitro shoot cultures, were used as explant, and the shoot apex and basal part of the shoots were discarded. Explants were placed onto the basal MS medium supplemented with 3% sucrose and 0.7% agar and with polyethylene glycol (Alfa Aesar, Haverhill, MA, USA) with different molecular weights either in concentrations of 2.5, 5.0 and 7.5% or 5.0, 7.5 and 10% for PEG 600 and PEG 6000, respectively. Moreover, 0.1, 0.2 and 0.3 M D-mannitol (Merck, Burlington, VT, USA) were also applied in order to induce the osmotic stress, while the control medium was free of osmotic agent. Twenty explants per jar (450 mL, cylindrical shaped) were placed onto 50 mL of medium. Treatments consisted of five repetitions (five jars); thus, a total of 100 plantlets were observed. Experiments were repeated twice. Shoot cultures were grown under controlled conditions, in a culture room at 22/15 ± 2 ◦C day/night temperature and 16 h daily illumination by 65 μmol m−<sup>2</sup> s−<sup>1</sup> PPF for 4 weeks. At the end of experiments, the rate of survival (SR) was observed and the shoot length (SL) and the number and length of roots (RN and RL, respectively) on surviving explants were measured. Before analysis, results were expressed as percentages of the results obtained on the medium without stress agents (SI, stress index; [45]), to compare the responses to the different levels of osmotic stress in the breeding lines and referent lines.

SISL = Shoot length of treated shoots (mm)/shoot length of control shoots (mm) × 100

SIRL = The longest root length of treated shoots (mm)/the longest root length of control shoots (mm) × 100

SIRN = Root number of treated shoots/root number of control shoots × 100

SISR = Survival rate of treated shoots (per jar)/survival rate of control shoots (per jar) × 100

## *2.3. Statistical Analysis*

SI calculated from morphological data (shoot and root parameters) were analyzed statistically by ANOVA followed by LSD and Tukey-B tests, using SPSS Statistics 27.0 (IBM, New York, NY, USA).

#### **3. Results**

Osmotic stress tolerance of 27 breeding lines and 2 drought-tolerant referent genotypes and their responses were compared to each other after SI values were calculated. We found that all morpho-physiological parameters were affected by treatments (Table 1), and differences were found between genotypes. Significant interactions between genotypes and type and concentration of osmotic agent were also detected.


**Table 1.** The main effect of osmotic agents calculated from SI values for morpho-physiological traits on the average of all potato breeding lines at different osmotic stress levels.

Levels for D-mannitol: 1: 0.1 M, 2: 0.2 M, 3: 0.3 M; for PEG 600: 1: 2.5%, 2: 5%, 3: 7.5%; for PEG 6000: 1: 5%, 2: 7.5%, 3: 10%.

#### *3.1. Effect of Osmotic Stress Induced by PEG 6000 on In Vitro Shoot Cultures*

3.1.1. Changes in the SI of Survival Rate (SISR)

SI values of survival rates (SISR) were significantly decreased or most frequently not affected by 5% PEG 6000 (Table 2, Figure S1a). Some breeding lines showed significantly lower SISR values than the referent lines. The raised level (7.5%) of PEG 6000 decreased significantly the SISR values very rarely, while other responses were similar to those observed at the level of 5% PEG (Table 2, Figure S1b). Increasing the PEG 6000 level to 10% resulted in significantly decreased SISR values for seven lines (C2, C17, C20, C28, C41, C42 and C57) compared to those observed at 7.5% (Table 2, Figure S1c). Although the C103 referent line showed the best SISR results along with two breeding lines (C8, C30), very similar results were found in eight breeding lines. However, the SISR value of the C107 referent line was significantly reduced, resulting in a number of breeding lines ahead of it.


**Table 2.** Survival rate SI (SISR) values of potato genotypes under osmotic stress induced by PEG 6000 added to the medium at levels of 5%, 7.5% and 10%.

Symbols mark significant differences from the C107 referent line (-), or from the C103 referent line () according to LSD test; the small letters indicate significantly different means between the breeding lines within a treatment, and the capital letters indicate significantly different means between treatment levels within a breeding line according to Tukey-B test.

3.1.2. Changes in the SI of Shoot Length (SISL)

Each breeding line and both referent lines responded with decreased shoot length to the presence of PEG 6000 at all concentrations (Table 3, Figure S2a–c). In general, inhibition of shoot growth increased with increasing concentration of PEG 6000 to 7.5%, but higher concentrations did not result in further significant decrease. The best result (SISL 76.8) was obtained in C9 breeding line, although SISL values of some breeding lines were very similar (C2, C22, C30 and C41) (Table 3, Figure S2a). At raised PEG 6000 concentration (7.5%), five breeding lines (C2, C9, C12, C30 and C32) showed significantly higher SISL values

compared to both referent lines (Table 3, Figure S2b). At the highest level of PEG 6000 (10%), only one breeding line (C22) reached significantly higher SISL values compared to both referent lines, but most of the breeding lines performed better than the C107 referent line (Table 3, Figure S2c).



Symbols mark significant differences from the C107 referent line (-), or from the C103 referent line () according to LSD test; the small letters indicate significantly different means between the breeding lines within a treatment, and the capital letters indicate significantly different means between treatment levels within a breeding line according to Tukey-B test.
