## 4 Discussion
### Octaploids Derived from Entopolyploid Callus Treated with TDZ  
In the present paper, we have developed an effective protocol for the regeneration of octaploid specimens of metal-tolerant _A. arenasa_. The obtained plants turned out to be fertile and produced viable seeds, which provides the opportunity to introduce them to polluted sites also (except of plantlets) in the form of propagules. Furthermore, the obtained octaploid regenerants could also be well propagated vegetatively in artificial conditions (not shown). Both pathways of propagation likely could be complementary in the field. Referring to the fertility of polyploids in _Arabidopsis_, [PERSON] et al. (2010) based on  
Figure 4: Morpho-anatomical characters of octaploid regenerants of _Arabidopsis arenasa_ and chromosome number of their progeny. Metaphase plate \(2n\approx 64\) (**A**), stomata length (marked with a red line) (**B**), flower width (marked with a yellow line) and area (marked with an orange line) (**C**), viable pollen grains stained with Alexander dye (**D**). The added lines illustrate how stomata and flowers were measured. the analysis of diploid and artificial tetraploid of _A. thaliana_, proposed that this species manifested an inherent and heritable ability to respond to increased chromosome numbers. It was associated with alterations in gene expression (DNA methylation), which are stable, nonstochastic, and developmentally specific [52]. Similarly, the autotetraploid cytotype Warschau of _A. thaliana_ was fully fertile. It set fewer seeds per silique; however, it produced more of the latter [53]. Taking all of this into account, including our results, it seems that the genus _Arabidopsis_ is a good object for any kind of genetic modification. Thidiazuron (1-Phenyl-3-(1,2,3-thidiazol-5-yl)urea) is a very effective synthetic plant growth regulator commonly used in in vitro culture for inducing organogenesis and somatic embryogenesis [54; 55]. It is normal that polyploids are obtained when administered with antimitotic agents (e.g., [56; 57; 58; 59; 60]), but it was a positive surprise that without the use of such agents, just in the presence of TDZ, endopolyploid callus of _A. arenasa_ initiated polyploids whose frequency was very high, reaching even almost 80%. The varied frequency of polyploids at different stages of their development could have resulted from non-random selection of the specimens. If, for example, octaploids had emerged earlier than tetraploids and the former ones were larger than the latter, which cannot be ruled out, then the collection of samples for measurement could not have been devoid of randomness. Overall, such a high frequency of polyploids could be explained by the immanent properties of the polyploid cells of the specific tissue (here specifically callus), which tended to develop into plants, similar to the studies of [PERSON] et al. (2022), who also obtained polyploid plants from endopolyploid protocorm and leaves of orchids [61]. Callus used for the regeneration of _A. arenasa_, both treated and non-treated with TDZ, manifested a high frequency of endopolyploidy (see Figure 3). This phenomenon could also be a result of 2,4-D action, which induced somaclonal variation (genetic or epigenetic changes that arise in vitro between clonal regenerants and their corresponding donor plants) during in vitro culture of other species [62; 63]. It was not possible to examine whether obtaining such a high frequency of polyploids was influenced by TDZ, by stimulating the proliferation of endopolyploid cells, or by just the presence of cells with increased ploidy within callus tissue which would have developed into polyploid plants anyway (even without the presence of TDZ). Simply, without TDZ, cells did not undertake regeneration into adventitious shoots; therefore, the role of TDZ here remains unresolved. The mode of action of this cytokinin and auxin-like growth regulator is still unknown [54], though it has been proved that it prolonged the cell division phase [64] and doubled cell diameter [65; 66].