3.4.3. Histological Analysis of Callus: Parenchymatic Cells Full of Starch Inclusions

A closer view of the small cells residing in the parenchymatic callus tissue and located very close to organogenetic centers and XLEs revealed that they contained many starch inclusions (Figure 4). This effect may be related to the high metabolic activity that is required in these zones to support the development of new organogenetic centers or XLEs. This observation further supports the findings of previous authors working on different species [98,99]. Recently, it was proven that there is a close relationship between lignin metabolic processes and the metabolism of starch and sucrose as the main factors associated with callus regeneration [100], which could be further investigated for different processes of cell differentiation.

### 3.4.4. Biochemical Analysis

By analyzing the spectra of electrophoretic peroxidases (POXs) published in 1998, we found clear differences between these two types of callus, further supporting our previous statement that N69 culture medium does not support root organogenesis [31,34]. Based on that analysis, 13 POXs' electrophoretic bands were described for a complete morphogenetic callus obtained in the MS62 culture medium (i.e., roots and shoots). The 6th electrophoretic band belonged to green tissues and the 12th belonged to roots. The 4th, 7th, 11th, and 13th appeared to mark the morphogenetic callus without visible specialized organs. The 3rd electrophoretic band appeared in all samples, and the 12th was the single band that was missing in the callus cultivated in N69 medium [31,34].

The continued development of morphogenetic processes revealed that N69 basal mineral medium was able to produce 42.56 propagules as buds and shoots per explant after 8 weeks of cultivation, while MS62 basal mineral medium induced 26.06 buds and shoots per explant. This result is consistent with the idea that the mineral composition is relevant both for increasing de novo shoot development per explant and for improving shoots' elongation.

## *3.5. Effects of the Vitamins MS62 and N69 on the De Novo Proliferation of Shoots*

Another experiment was conducted to further study the effects of the vitamins MS62 and N69 on shoot multiplication and shoot height, as well as the effects of the composition of the plant growth regulators (but using only an MS62 basal mineral composition). Data on this topic were also published in 2004 [33,34]. In this experiment, we used NAA at different concentrations (0, 0.1, 0.5, and 1 mg/L) and a constant concentration of BAP (1 mg/L). The analysis of the results revealed that the best balance of plant growth regulators for the MS62 culture medium was 0.1 mg/L NAA and 1 mg/L BAP, which produced an average of 9 shoots/explant, considering only shoots > 7 mm in height. When increasing the auxin to 1 mg/L, the shoot multiplication rate decreased significantly compared to the control (i.e., no auxin) and the culture medium supplemented with 0.1 mg/L NAA. In this case, apical dominance of the main shoot was also observed (i.e., an average of 25.5 mm). All the culture media induced the formation of both shoots and roots, as well as a healthy appearance of the shoots. Again, the results of this experiment suggest that the best formula for de novo shoot formation was that provided by the MS62 basal culture medium [34].
