**1. Introduction**

One of the most important abiotic stress factors is drought. Drought periods during the growing season are becoming more common due to climate change and global warming [1]. Stress caused by water deficit is even greater in areas free of permanent precipitation or where an adequate irrigation system is not available [2].

Water deficit affects almost all plant growth and developmental processes [3], can induce several morphological, physiological and biochemical changes [4], and can result in large yield losses of up to 50–70% in crop production [5].

Improving the drought tolerance and water use efficiency of varieties by using breeding methods can play an important role in reducing drought-related crop losses and thus contribute to a secure food supply for the growing population. This means that demand for drought-tolerant plant species and especially cultivars that are able to adapt to drought conditions is constantly increasing [6,7].

Potato (*Solanum tuberosum* L.) is one of the most important crops worldwide, because of its high productivity and nutritional values [8]. However, the potato crop often suffers from stress because it is sensitive to both drought [9,10] and high temperature [11]. Even though potato has difficulty tolerating water shortages due to its shallow roots [12,13], it basically manages water well.

**Citation:** Hanász, A.; Dobránszki, J.; Mendler-Drienyovszki, N.; Zsombik, L.; Magyar-Tábori, K. Responses of Potato (*Solanum tuberosum* L.) Breeding Lines to Osmotic Stress Induced in In Vitro Shoot Culture. *Horticulturae* **2022**, *8*, 591. https:// doi.org/10.3390/horticulturae8070591

Academic Editor: Sergio Ruffo Roberto

Received: 26 May 2022 Accepted: 29 June 2022 Published: 1 July 2022

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By changes in physiology and morphology, plants are able to respond adaptively to altered environmental conditions, preceded and accompanied by cellular and molecular changes [14], so many processes are involved in the development of drought tolerance. This fact suggests that there may be large differences between species and cultivars in drought stress responses, and even variability has been found in the susceptibility of potato cultivars [15,16]. Several experiments were performed to identify traits that could be related to drought stress and to select genotypes with the desired characters in several crops [4,17] including potatoes [18–20]. Some of these traits can also be detected at the cellular and/or tissue level, such as the adjustment of osmotic pressure, which can be tested/modelled under laboratory conditions [21] or even in vitro [22,23]. Micropropagation is a commonly used process to produce virus- and disease-free plant propagating material [24], and in vitro conditions provide good opportunities to study different physiological processes and interactions and to select appropriate genotypes [25].

Osmotically active compounds such as the inert and non-penetrating polyethylene glycol (PEG) with various molecular sizes or the sugar alcohols such as D-mannitol and sorbitol are widely used to induce osmotic stress in plants [25]. Although sugars and their derivatives can significantly increase the osmotic pressure of the medium used, it must also be taken into account that they can be taken up and metabolized by plants. In addition, a high sugar content (8%) can induce tuber development in potatoes [26].

PEG is a neutral polymer, and its high molecular weight makes it suitable to imitate water deficit when added to the medium, because it cannot pass through the cell wall of the plant [27–30]. D-mannitol can be produced and metabolized by certain plant species [31]; however, it has been used efficiently in many osmotic stress tolerance experiments [25].

Numerous studies on osmotic stress tolerance of potato have been conducted in recent years. Gopal and Iwama [6] studied the effect of PEG and sorbitol on the morphological development of potato in vitro shoot cultures, and they found that in vitro tests gave results similar to those obtained under field conditions.

In addition to shoot culture, callus culture was also suitable for osmotic stress tolerance testing of genotypes, and results were well related to their field performance during drought [32]. The in vitro model provided a good basis for assessing the drought tolerance of genotypes also in other experiments [33,34]. Changes in morphological and physiological traits affected by osmotic stress are similar under in vivo conditions to those observed in in vitro tissue cultures. Survival rate, shoot length, fresh and dry weight, and number and length of roots are the most commonly affected characteristics [35–38]. Despite the fact that the growth parameters of the plantlets were strongly influenced by osmotic stress during the stress resistance experiments—in fact, they were most often inhibited—it was possible to differentiate the genotypes according to their stress tolerance only if a stress index was formed based on the measured growth parameters [32,39,40].

Although a stress index based on morpho-physiological characters alone could be used to distinguish potato genotypes [32,35,41–43], identification of quantitative trait loci (QTLs) for morphological characters [1,42] and using molecular and biochemical markers as a tool for drought tolerance study are becoming more and more widespread [1]. Our experiments were focused on the investigation of the osmotic stress tolerance of 27 potato breeding lines under in vitro conditions to reveal whether there were significant differences in SI counted from simple morphological parameters compared to the drought-tolerant referent lines. In addition, different types of osmotic agents were applied at three levels to find the effective method(s) to distinguish our breeding lines according to their osmotic stress tolerance and to select varieties that should be included in further studies. Targeted trait-specific selection started under in vitro conditions can significantly reduce the time required for development of a new variety, so the results of laboratory tests prior to the field experiment may provide a good basis for further stages of plant breeding.
