3.4.2. Histological Analysis of Callus: Xylematic Elements and Protocorms

The histological analysis of calli obtained in MS62 and N69 culture media revealed some interesting peculiarities that should be further discussed. In 1998, we underlined the significant differences that appeared for organogenetic responses in relation to "xylematiclike structures" (XLSs) detected by our team in the structures of the calli [31]. As we now have full access to additional scientific articles, we felt it important to investigate the scientific meaning of the "xylematic-like structures" once more. We found that, in 1995, such structures had already been described as procambial cells for *A. thaliana* by Simona Baima et al. [84]. The authors proved at the time that the auxin IAA involved in vascular development modulates the expression of *Athb-8*—a gene responsible for the regulation of vascular development in *A. thaliana*. In that period, the term "vascular bundle" was well established, having also been coined early in 1920 for histological studies of callus by Robert John Harvey-Gibson and Elsie Horsman in Liverpool (UK) [85].

The scientific quest to understand how vascular bundles or strands are formed yielded more results after 1980, and yet some 20 years ago the mechanism was still largely unknown [86]. In 1987, Harry Klee and collaborators stated that auxins are able to induce differentiation of xylem tracheary elements (XTEs) in suspension cultures of certain species [87]. This is also consistent with our observations of syngonium, as we also used the term xylematic-like elements (XLEs) for tracheary elements observed on histological slides, as a precautionary step. However, the fact that auxins trigger vascular bundle formation was scientifically established in 2000 [88]. The potential role of XLEs had not been discussed previously, but in trying to explain the hard consistency of the callus obtained in N69 culture medium (mineral composition), we consider that this revision is needed.

A closer analysis of the slides for the softer calli obtained in the MS62 culture medium (see Figure 2) clearly reveals that they are of smaller dimensions and constantly appear in the vicinity of organogenetic polarized structures and embedded in the larger parenchymatic cells. The XLEs may be considered to be protoxylem elements—a topic that had not been discussed previously—but it is obvious that they appear constantly in the vicinity of de novo shoots and roots, as well as that of the nodular-like structures. This constant positioning of the XLEs may be due to their potential role in supporting the development of new organogenetic structures. Their high density around the polarized organogenetic structures may support this hypothesis, as they are not observed in callus zones where organogenesis is not occurring. Moreover, this is consistent with the study by Thomas Berleth and collaborators published in 2000 [88], as well as considering the role that auxin may play in the establishment of plant cells' polarity and oriented differentiation which, in turn, are needed for aligning vascular differentiation, among other functions. Following this logic, XLEs develop in the parenchymatic tissue of the callus to support the further development of organogenetic centers in order to accomplish their final morphogenetic objective: whole-plant formation. This hypothesis is supported by recent results that are based on the analysis of molecular markers, supporting the idea that a callus is a

group of pluripotent cells where—under appropriate conditions—the promotion of auxin self-production and the enhancement of cytokinin sensitivity are also required for organogenesis [89]. In our case, culture conditions ensured the availability of auxins as well as that of cytokinin. The left-hand image in Figure 2 presents a cross-section of the upper part of the shoot tips, as well as other organogenetic areas, in circular shapes that may occur at the basal parts of different organogenetic areas of the callus. Similar images were published by Scaramuzzi [46], further substantiating our results.

**Figure 2.** Histological analysis of slide images taken for *S. podophyllum* callus obtained in the MS62 culture medium. This was a full organogenetic callus producing roots and shoots. Nodular-like structures where organogenesis is taking place can be observed (in the tips of the right-hand shoot formation and in a transversal section of the upper part of the left-hand shoot formation; black arrows). Xylematic-like elements or bundles can be observed in the vicinity of all organogenetic centers (see red arrows; magnification ×270 (left) and ×135 (right); bars = 100 μm).

By changing the basic mineral formula, e.g., for the N69 mineral composition, the harder greener callus revealed the formation of well-developed XTEs during the histological study (see Figures 3 and 4). By analyzing different histological images taken at the time, we further observed that these XTEs were constantly arranged in areas positioned at the bases of meristematic domes, as well as those of developing leaves or shoots. Small XLSs could also be observed close to areas comprising polarized nodular-like shapes recognized for their relevance in the initiation of organogenesis.

A re-evaluation of old slides revealed the presence of protocorms (see Figure 3) that were very well differentiated in a transversal section, which were not mentioned previously [31]. Different-sized images of such protocorms were frequently seen in the slides. It should be noted that protocorms were also described by other authors in different culture conditions [56,60].

**Figure 3.** Histological analysis of slide images taken for *S. podophyllum* callus obtained in the N69 culture medium. Shoots and protocorms forming callus were observed. Nodular-like structures could also be observed (lower-left image), where organogenesis was taking place. The black arrows indicate very well-developed XTEs in the vicinity of a meristematic dome and de novo shoot formation. The meristematic dome is very well expressed, with well-developed, asymmetric, primordial leaves. In the upper- and lower-right images, protocorms can be observed in transversal section (magnification ×135 (upper and lower left) and ×270 (upper right), bars = 100 μm).

In the left-hand images (Figure 3), a transversal section of a meristematic dome is very clear, and the asymmetry of its contour reveals the initial stages of leaf formation. Again, XTEs are well developed nearby and entering the meristematic dome. Beneath the meristematic area, there are certain callus zones where XTEs reside among parenchymatic cells. No visible root formation was observed for the N69 mineral composition in 1998. Based on the reassessment of these images, it appears that the same auxin–cytokine ratio and the same vitamin composition but different mineral composition can change the pluripotency and totipotency of callus cells. While the MS62 mineral composition supported separate and complete organogenesis (i.e., shoots and roots), rooting was no longer observed when changing to the N69 mineral composition. Instead, protocorms were very well developed and expressed at a high density, appearing in almost half of the investigated images.

**Figure 4.** Histological analysis of slide images taken for callus cells of *S. podophyllum*. The small cells in the vicinity of the organogenetic areas present many starch inclusions (magnification ×45,000, bar = 10 μm).

Another easily noticed difference when we analyzed the XLEs is that they were less developed in MS62 compared to the N69 mineral composition. Therefore, it might be possible that the lack of or decrease in the concentration of certain mineral elements is responsible for this type of organogenesis, as was already stated some 47 years ago for *Antirrhinum majus* [90]. In another study, the increased concentration of boron appeared to influence shoot regeneration rather than contributing to increasing the number of shoots/explants, taking into account the results of recent studies on date palm [91]. The high expression of XTEs can also be supported by recent findings [92] and, among others, by the recognized toxicity of boron to plants [93]. The Lewis theory regarding boron's regulatory role in lignin synthesis [94]—highly supported by other scientists [95]—seems to be consistent with our findings regarding the cultivation of *Syngonium* callus in N69 culture medium. However, there are also too many other variables related to the changes in the compositions of other minerals, as mentioned previously. The lack of or decrease in the concentration of cobalt in N69 compared to MS62 was recently associated with decreased shooting in *Cucumis sativus* [96]. An interesting experiment was published by Renata Garcia and collaborators in 2011, where they used a mineral composition that was different from the basal MS62 and similar to N69 for callus cultivation of *Passiflora suberosa* [97]. They also observed the formation of a more compact callus and a significant decrease in shoot formation per explant. However, rooting was not impaired for this species.
