*3.4. Review of Histological Studies on Syngonium Calli Obtained with Different Media Compositions*

In 1998, the very first scientific article dedicated to the study of the effects of medium composition on in vitro morphogenesis was published in Romania in the English language [31].

The first hypothesis of the experiment was based on the statement that morphogenesis in plants is a very complex process that is strictly controlled in time and space by its genetic and external factors [76]. In this regard, we considered that a histological study was needed to reveal morphogenetic events such as de novo morphogenesis of shoots and roots—as proposed for other species by various authors—to better describe and understand the in vitro morphogenesis of syngonium [77,78]. To this end, young shoots of 8–10 mm in height were tested by cultivating them in two types of cultivation media—MS62 [39] and Nitsch 1969 (N69) [79]—as basal mineral compositions [31,34].

The second hypothesis considered that, with *Syngonium* being a tropical plant, it would be interesting to study the effects of a completely different mineral composition in parallel to that of the MS62 culture medium. We considered the fact that essential elements would be at different concentrations in the original tropical rainforest environment. Therefore, for successful in vitro multiplication at a low cost, it was also considered relevant to test various culture media whose mineral composition could be modified. At the time, we also considered that the electrolytic strength of the MS62 culture medium should be different compared to that of N69. Furthermore, it may play an important role in organogenetic processes such as de novo shoot formation and rooting (i.e., different osmotic pressure). In the case of N69 s mineral composition, calcium was at half the usual concentration, while zinc and manganese were increased (from 8.6 to 10 mg/L and from 16.9 to 18.94 mg/L, respectively). In addition to these changes, cobalt and iodine were absent, boron was supplemented to an increased concentration (from 6.2 to 10 mg/L), and the concentrations of phosphate and nitrate were reduced (from 85 to 68 mg/L and from 825 to 720 mg/L, respectively). Later publications provided highly accurate descriptions of relevant differences in mineral changes for tropical forests [80,81].

### 3.4.1. Experimental Design and Histological Method

The basal mineral culture media (i.e., MS62 and N69) were supplemented only with MS62 vitamins and the same hormonal balance: 2,4D (0.1 mg/L), BAP (1 mg/L), and Kin (3 mg/L). The molar ratio was inclined towards cytokinin which, according to our previous experience, was needed to test the morphogenetic process. Based on the aforementioned working hypotheses for implementing this experiment, the morphogenetic callus obtained after 8 weeks of cultivation was processed for histological study.

For histological analysis, all callus samples were collected at the same time and immersed in Navashin's fixation solution at room temperature, as recommended by other authors [82], and then in liquid paraffin. After the samples solidified, they were sectioned at 8–10 μm and colored on slides with a solution of hematoxylin–eosin in order to enhance the contrast between different tissue structures [83].

The published conclusions of this experiment revealed that different medium compositions induced distinct responses in syngonium callus organogenesis: MS62 culture media induced the formation of a softer, yellowish callus expressing complete organogenesis (i.e., shoots and roots), as opposed to N69, which induced the formation of a harder, greener callus expressing only shoot formation [31]. In 1992, Scaramuzzi and collaborators in 1992 published the first histological analysis of the morphogenetic callus, the findings of which were similar to our results [46]. Later, a similar yellowish callus was also obtained and described by Jin Cui and collaborators [56].
