3.3.3. Total Peroxidase Activity

The total peroxidase activity (TPA) was assessed at 72 h according to the method of Dana Iordăchescu and Ioan Florea Dumitru published in 1988 [71]. The size of the leaf fragments was 0.5 cm × 0.5 cm, while the petiole fragments were 1 cm in length. As a general control, unwounded shoots were used. In this case, our experimental hypothesis was based on the knowledge that wound stress is already expressed during in vitro cultivation of plants, according to various authors [72,73]. Moreover, it has long been observed that a burst of phenolic compounds is generally released at the wound sites of plants, and that this process is immediately followed by the activation of membrane-bound peroxidases that are required to initiate healing processes [65,74].

• Effects of 2,4-D:

After 72 h, the petiole fragments treated with 2,4-D exhibited a 1.84-fold increase in TPA compared to control shoots (i.e., intact shoots). In this case, the auxin and the supplemented cytokinin clearly contributed to an increase in TPA to initiate cell proliferation, as the first and most obvious event was recorded four weeks later. In the case of control petioles, we found a 1.6-fold decrease in the TPA activity compared to explants in the presence of auxin, suggesting that auxin activated peroxidase activity in the petiole fragments. In fact, upon the increase in TPA activity at the wound site, cell proliferation began as an essential process for the survival of the plant inoculum. The calli or fragments of plants can further act to continue the plants' morphogenesis [10]. In the case of leaf fragments, 2,4-D induced a 2.45-fold increase in TPA activity compared to intact leaves and a 1.25-fold increase compared to the control leaf fragments. Due to the larger leaf surface for wounding, the release of phenolic compounds [27–29] might have been responsible for the activation of enzymes with peroxidase activity, which should greatly enhance the wound stress response of leaf fragments in the presence of 2,4-D.

• Effects of salicylic acid:

For petiole fragments, SA induced a 4.26-fold increase in TPA activity compared to petioles taken from intact shoots and a 1.46-fold increase compared to the control petiole fragments. This value was more than double the effect of 2,4-D at the wound site and is consistent with previous scientific results [60]. In the case of leaf explants, SA induced a ~1.2-fold increase in TPA activity compared to the intact leaves but, conversely, a 1.6-fold decrease compared to control leaf fragments. Here, SA induced TPA activity at half the rate of 2,4-D. Moreover, the TPA value in fragmented leaves was almost equal to that in intact leaves from intact shoots. It can be concluded that the exogenously applied SA may minimize the expression of TPA in syngonium at the leaf level, but it can induce overexpression in the fragments of petioles.

• Effects of 2,4-D and salicylic acid:

When both compounds (2,4-D and SA) were exogenously applied, it appeared that the TPA activity was similar to that obtained in the fragmented petioles or leaves subjected only to 2,4-D. Thus, the effects of SA appeared to be hidden by the presence of 2,4-D.

#### 3.3.4. Callus Initiation from Fragmented Petioles and Leaves

The entire experiment was observed continuously for 8 weeks. In the first three weeks, leaf and petiole fragments subjected to 2,4-D became etiolated compared to controls and those treated with SA, which remained green. All petiole fragments subjected to SA developed hypertrophy at the cut part of the petiole following the circular cambium, which also spread along the entire length of the fragment. However, some of these samples only developed callus at the wound site—white and friable, in small quantities—and later in the cambial tissue as well. Among the two types of explants, only petiole fragments were able to produce a proliferative callus for syngonium at the basipetal part of the petiole, in a similar manner to that described for other species [62]. In contrast, based on the conditions of this experiment, it was not possible to induce callus formation at the leaf fragment level. Moreover, other authors were successful in initiating and studying callus formation based on a specific hormone balance [46,49,51–53,56,63]. However, it should be noted that a burst of phenolic compounds may also affect callus formation [75].

In another experiment conducted by Jin Cui and collaborators on the same cultivar but under different culture conditions (i.e., different plant growth regulators and different balances), the proliferation of callus was also possible at the leaf level [56].

Re-evaluating these results, it can be concluded that wound stress is also responsible for a clear sequence of events, i.e., increased peroxidase activity, followed by the expression of hypertrophy at the wound sites and, subsequently, by callus development. Hormone balance and mineral composition are the major inductors of callus formation, and different organ fragments may respond differently. The lack of callus formation in fragmented leaves under our experimental conditions may have been due to the culture medium composition, which was not supportive of the expression of hypertrophy, although it was proven to express the highest peroxidase activity. This subject should be of interest when investigating callus initiation for different purposes [46,49,51–53,56,63].
