*3.6. Effects of Cysteine on Callus Formation in N69 Medium*

A follow-up experiment was carried out to test the effects of cysteine. Figure 5 shows that a greener callus developed on the N69 medium in the presence of cysteine [34]. Moreover, visible root development and shoot elongation were observed in the presence of cysteine. The purpose of this experiment was to understand whether shoot regeneration in this species may be influenced by the potential release of phenolic compounds—as established in other species according to several reports [101,102]. It should also be noted that cysteine has long been recognized for its action against the release of phenolic compounds, and it has been recommended to be used for in vitro micropropagation of plant species [103,104]. Moreover, the fact that *Syngonium* produces phenols was first observed by Lynn Miller and Toshio Murashige [22]. These observations were later confirmed by several teams of scientists, proving that this ornamental species also has a high therapeutic value—mainly due to phenolic compounds with high antioxidant activity [27–29].

**Figure 5.** Different types of *Syngonium podophyllum* calli: (**a**) Control callus obtained in MS62 medium without cysteine. (**b**) Control callus obtained in N62 culture medium without cysteine. (**c**) Callus obtained in N69 culture medium supplemented with cysteine. It can be seen that the callus was much greener when it was cultivated in N69 culture medium, and that the shoots were more elongated in the presence of cysteine.

Later, in 2001, Jose Carlos Lorenzo et al. proved that the addition of cysteine reduced shoot formation and the excretion of phenols in the in vitro cultivation of sugarcane [105]. These authors also acknowledged several examples that showed contrasting results in developmental processes for other species and in vitro systems. Positive effects for micropropagation were also observed for grapevine (*Vitis vinifera*) [106], air potato (*Dioscorea bulbifera*) [107], and *Prosopis* species [108]. The elongation of shoots was also observed under cysteine treatment in *Prosopis* species [108]. In the case of the common bean (*Phaseolus vulgaris*), shoot elongation was also observed [109].

De novo shoot formation is a complex process that is highly desirable for all in vitro plant systems, especially to reduce costs for industry. *Syngonium* was investigated in vitro by various research teams before and after our publications [22,46–63]. Most of the teams obtained de novo shoots by using the same basal mineral and vitamin composition of MS62, but using different plant growth regulators as well as different balances between cytokinin and auxin.

The histological study of the callus may provide new insights regarding the stages needed for de novo development of the shoots [31,46,48,49,51–53,57,61]. Moreover, by studying the wound stress of the species, we may provide further evidence for the relevance of the hormone balance used for initiating in vitro callus development [32]. The chain of events consisting of increasing the peroxidase activity, hypertrophy of the wound sites, and callus formation starting from the cambium in petioles is already consistent with findings for other studied plant species [19,54].

Whether the callus can be well established in an in vitro culture to produce de novo shoots rests largely on the use of appropriate mineral, vitamin, and plant growth regulator balances. In this regard, the histological analysis of both types of calli that were cultivated—in MS62 mineral composition culture media and in N69 mineral composition culture media—revealed significant differences that were supported by biochemical analysis of peroxidases.

The aforementioned results raise the following question: is it possible for *Syngonium* calli cultivated in N69 media to also assist in the increased accumulation of phenolic compounds responsible for the development of XTEs? Here, we must underline the clear difference between XLEs developed for the calli cultivated in MS62 and those obtained in N69, as observed in all investigated slides. It should be noted that it has already been proven that cysteine plays a direct role in decreasing the accumulation of phenolic compounds and supporting organogenesis in the hybrid *Miscanthus* × *giganteus* [110].

It has been proven that the addition of an extra nitrogen source can have a positive influence on the development of xylematic bundles and on pigment accumulation in paperwhite (*Narcissus tazetta*) [111]. The direct relationship between lignin production and the development and maturation of tracheary elements upon apoptosis has also been well documented [112]. Additionally, it is possible that the tracheary elements observed in the tiny calli could be formed as a result of phenol accumulation under more stressful in vitro culture conditions. Moreover, it was observed that an increased thickening of the xylematic vessels of candy leaf (*Stevia rebaudiana*) took place under cold stress [113]. Previous studies also proved that the production of xylematic vessels is stimulated under different stress factors, e.g., physical and chemical [114].

In 2018, Shokoofeh Hajihashemi and Omolbanin Jahantigh put forward the hypothesis that an increase in the development of xylematic vessels may further support water transport, among other processes [111]. They also cited an earlier observation relating to the diameter and frequency of xylem vessels, which are critical determinants of water conductance [115]. In this regard, the clear and consistent positioning of the xylematic tracheary elements close to de novo shoot development areas and other meristematic structures provides rapid access to nutrients and water, as previously stated by other authors [114]. Regarding the positive effects of cysteine in supporting shoot elongation, it should be noted that it was proven to stimulate shoot elongation for *Petunia x hybrida* by acting on the gibberellic acid pathway [116,117]. Conversely, by inhibiting the synthesis of cysteine, the opposite effects were observed in cockspur (*Echinochloa crus-galli*) [118].

All of these findings are relevant to further support the improvement of the technology for industrial-scale *Syngonium* production in a more cost-efficient manner, and theoretical study of de novo shoot formation can contribute to this technological improvement. Cysteine may play an important role in decreasing the effects of phenolic compounds discharged under these stressful conditions when cultivating *Syngonium* in N69 culture medium, as well as improving de novo shoot elongation [119]. Thus, the addition of cysteine in the pre-acclimation phase can more quickly reinforce the formation of more adaptable plantlets for acclimation [120,121].
