*3.6. Molecular Characterization*

DAMD and ISSR primers were used for molecular characterization of in vitro propagated plants growing in the greenhouse and compared with the DNA profile of the donor plant. Five DAMD primers were selected and screened out, where all the primers generated a total of 48 bands after PCR amplification with an average of 9.6 bands per DAMD primer (Table 1). The bands were monomorphic across all in vitro propagated plants of *P. amboinicus* and 100% similar to that of donor plants. The DNA banding profiles of the *P. amboinicus* genotypes using DAMD primers HVR and 33.6 are presented in Figure 5. Similarly, all nine ISSR primers tested also produced very clear, monomorphic bands within all in vitro propagated plants with a total of 150 scorable bands. The number of bands for each ISSR primer varied from 9 to 17 with an average of 11.4 bands per primer (Table 2, Figure 6).

**Figure 5.** DAMD-PCR profiles of tissue culture and donor plants of *Plectranthus amboinicus*: (**A**) profile generated using primer HVB5 and (**B**) profile generated using primer 33.6. Lane M = Lambda DNA/EcoRI+HindIII marker; lanes T1–T5 = randomly selected regenerated plants; lane DP = donor plant.

**Figure 6.** ISSR-PCR profiles of tissue culture and donor plants of *Plectranthus amboinicus*: (**A**) profile generated using primer UBC827 and (**B**) profile generated using primer UBC855. Lane M = Lambda DNA/EcoRI+HindIII marker; lanes T1–T5 = randomly selected regenerated plants; lane DP = donor plant.

#### **4. Discussion**

The most widely used approach for multiple shoot induction and plant regeneration is in vitro axillary bud proliferation, which is also thought to be the best way to ensure that the regenerated plants will have the same genetic makeup as the donor plants. Normally, dormant axillary buds are forced to develop into multiple shoots by proper application of growth regulators. Addition of cytokinins, viz., BA, Kin, TDZ, 2iP, and m-Topolin, in the medium led to the successful formation of multiple shoots in many plant species, including *P. edulis* [37,38], *P. amboinicus* [39,40], *Ruta graveolens* [33], *Pongamia pinnata* [59], *Rumex*

*pictus* [60], *Mentha* × *piperita* [61], *Atropa acuminata* [62], *Cannabis sativa* [63], *Campomanesia xanthocarpa* [64], *Zingiber officinale* [65], *Cicer arietinum* [66,67], and *Humulus lupulus* [68].

The type of cytokinin used in the current research had a significant influence on the induction of multiple buds from nodal sections, with BA being shown to be more efficient than Kin. Apart from that, until BA concentration was optimized, both the regeneration frequency and the mean number of shoots generated per nodal sections continued to increase. Similar findings have been made for shoot organogenesis in a number of therapeutic plants in the past [69–73]. From the nodal explants of *P. amboinicus*, the concentration of BA at 5.0 μM produced the greatest number of shoots, compared to the other concentrations and PGR tested in this investigation. The current findings are comparable with those of Arumugam et al. [40] and Belete and Balcha [37], where 0.5 mg/L and 1.5 μM BA alone was proved to be more effective than kinetin for shoot induction in *P. amboinicus* and *P. edulis*, respectively. The presence of high BA in the culture medium, on the other hand, showed inhibitory effects on both shoot response and proliferation, which was consistent with the findings in *Bambusa ventricose* [74] and *Sapium sebiferum* [72].

The balance between auxin and cytokinin is critical during the whole micropropagation process, since the combination of the two regulates growth and development in a plant species. As a relatively high ratio of cytokinins is combined with a low ratio of auxins, they have a synergistic effect on cell division, resulting in a higher frequency of shoot bud induction and a greater number of shoots/explants when compared to cytokinin alone in the same experiment [75,76]. It is important to maintain a proper balance of plant growth regulators in the culture medium in combination with phytohormones produced by the plant when regenerating tissues in vitro, as this is one of the primary factors responsible for the induction, differentiation, and proliferation of shoots in the growth media. Similarly, in the present study, low auxin and high cytokinin concentrations also showed the greatest potential for inducing and multiplying shoots in *P. amboinicus*, and MS medium augmented with 5.0 μM BA in conjunction with 2.5 μM NAA was found to be the most effective treatments for direct shoot regeneration and multiplication in the microenvironment. In contrast, BA in combination with TDZ promoted a high number of shoots in *P. bourneae* [35]. The role of auxin and cytokinins in increasing axillary buds proliferation and ending apical dominance is thought to be the cause of the rise in the number of shoots observed in this investigation. The combination of auxin with cytokinin was also found to be effective for shoot multiplication and elongation in many plant species, including *P. amboinicus* [39,40], *Cassia alata* [77], *Artemisia abrotanum* [78], *Syzygium cumini* [79], *Manihot esculenta* [80], *Hildegardia populifolia* [81], *Asparagus cochinchinensis* [82], *Basella rubra* [83], and *Sapium sebiferum* [72].

The ability of microshoots to induce roots is critical because it has a direct impact on their survival in the greenhouse and their ability to adapt to their new environment. In the great majority of species, auxins are significant factors in root production because they stimulate the development of adventitious roots, which are essential for rooting [84]. The micropropagated shootlets of *P. amboinicus* were effectively rooted with IBA supplied in MS agar medium. The effects of IBA containing agar rooting media are in agreement with earlier approaches for inducing roots in *P. amboinicus* [39,40] and in several medicinal plants, including *Ruta graveolens* [33], *Hemidesmus indicus* [85], *Rauvolfia tetraphylla* [86], *Sapium sebiferum* [72], *Artemisia vulgaris* [87], and *Asystasia gangetica* [88].

Preserving the genetic consistency in tissue culture plants is critical for any micropropagation system prior to commercialization or in germplasm conservation. The PCR-based SPAR markers technique is one of the most significant approaches with increased use in the assessment of the genetic stability of tissue culture plants, since it uses only a small amount of genomic DNA, avoids the use of radioactivity and DNA blotting, and is susceptible to automation. In this study, DAMD and ISSR markers, as well flow cytometry, were used to confirm the genetic integrity of *P. amboinicus* micropropagated plants. Because micropropagation is known to induce somaclonal variation in micropropagated plants, the use of multiple markers has long been advocated for a better evaluation of genetic uniformity

of in vitro plantlets [47]. Both DMAD and ISSR analyses showed a 100% monomorphic banding pattern, indicating the absence of variability among tissue culture plantlets of *P. amboinicus*. The appraisal of genetic constancy in a variety of micropropagated medicinal plants such as *Mentha arvensis* [46], *Pittosporum eriocarpum* [47], *Bacopa monnieri* [44], *Curcuma zedoaria* [48], *Ruta chalepensis* [50], *Rauvolfia serpentina* [89], *Nepenthes khasiana* [90] and *Ficus carica* [91] has been documented.
