*3.3. Effect of Light on Percentage of Somatic Embryo Formation*

The MS medium cultured vials augmented with 0.1 mg L−<sup>1</sup> Kn and 0.5 mg L−<sup>1</sup> IAA, incubated in the dark, produced more somatic embryos (77.34 ± 0.05%) than the cultured vials fortified with the same PGRs (0.1 mg L−<sup>1</sup> Kn and 0.5 mg L−<sup>1</sup> IAA) incubated in the light (56.21 ± 0.07). Likewise, other concentrations showed similar patterns of somatic embryo formation. Thus, it was demonstrated that darkness had a significant impact on somatic embryo formation in *Aconitum violaceum* (Figure 3). In all trials, cultures that were incubated in the dark for a minimum of 10–25 days may facilitate the induction of somatic embryos.

#### *3.4. Complete Plant Development from Somatic Embryos*

To regenerate complete plantlets from somatic embryos, the effects of Kn and IAA on further differentiation of somatic embryos were studied. Somatic embryos of different stages were sub-cultured on the MS basal medium augmented with various concentrations of Kn (0.05–1.0 mg L−1) and IAA (0.1–1.0 mg L−1), either individually or in combination (Figure 2I). However, somatic embryo conversion into plantlets was recorded only in 0.1 mg L−<sup>1</sup> Kn, 0.1 mg L−<sup>1</sup> Kn + 0.3 mg L−<sup>1</sup> IAA, and 0.1 mg L−<sup>1</sup> Kn + 0.5 mg L−<sup>1</sup> IAA. The highest percentage of whole plant regeneration from somatic embryos was achieved on Kn 0.1 + IAA 0.5 mg L−<sup>1</sup> with a percentage culture response of 68.00 ± 1.52%. These observations clearly illustrate that the 0.1 mg L−<sup>1</sup> Kn + 0.5 mg L−<sup>1</sup> IAA treatment had a more stimulatory effect than other treatments employed to convert somatic embryos into normal plantlets (Table 3, Figures 2J and 4F,G). The healthy plantlets were transferred into earthen/plastic pots consisting of a mixture of garden soil, peat moss, and vermicompost in a ratio of (1:1:1) and kept in a plant growth chamber (Figures 2L and 4H–J). These findings clearly demonstrate that complete plant regeneration via somatic embryogenesis could be possible.

**Figure 3.** Influence of light conditions and concentrations of Kn and IAA (mg L−1) on somatic embryo formation from the embryogenic callus of *Aconitum violaceum*. Mean value ± standard error mean followed by different letters in each column represents that the observations were substantially different according to DMRT (one-way ANOVA) at *p* ≤ 0.05. Abbreviations: Kn: kinetin. IAA: indole-3-acetic acid.

**Table 3.** Effect of Kn and IAA on whole plant regeneration from somatic embryos and in vitro rooting in *Aconitum violaceum*.


The data were evaluated for up to 8 weeks. The results were displayed as mean value ± SEM. The same letters within each column represent that data are statistically different at *p* ≤ 0.05 according to DMRT (one-way ANOVA). Abbreviations: MS: Murashige and Skoog. PGRs: plant growth regulators. Kn: kinetin. IAA: indole-3-acetic acid.

**Figure 4.** Callus development and plant regeneration from the seeds of *Aconitum violaceum* in MS basal medium enriched with various concentrations of PGRs, either alone or in combination: (**A**) callus induction in MS medium + 0.5 mg L−<sup>1</sup> 2,4-D; (**B**) callus induction in MS medium + 0.1 mg L−<sup>1</sup> Kn; (**C**) multi-shoot formation in MS medium + 0.1 mg L−<sup>1</sup> Kn; (**D**) root induction when sub-culture in MS medium is augmented with 0.1 mg L−<sup>1</sup> Kn + 0.5 mg L−<sup>1</sup> IAA; (**E**) embryogenic callus production and root induction in MS medium fortified with 0.1 mg L−<sup>1</sup> Kn; (**F**,**G**) multiple shoot and root development from embryogenic callus when sub-cultured in MS medium enriched with 0.1 mg L−<sup>1</sup> Kn + 0.5 mg L−<sup>1</sup> IAA; (**H**,**I**) hardened plants in the pots containing loamy soil, coco-peat, and vermicompost (1:1:1 *v*/*v*); (**J**) plant acclimatized in green house. Scale bar (**A**–**G**) represents 5 mm; (**H**–**J**) represents 1 cm.

#### *3.5. In Vitro Rooting*

The in vitro developed plantlets, through various methods (i.e., somatic embryogenesis and callusing), developed roots through sub-culturing in the MS basal medium enriched with the same concentrations (concentrations at which somatic embryos were developed) or different concentrations of Kn and IAA (Table 3, Figure 4D–G). The multi-shoot plantlets from sub-culturing in the MS medium, enriched with rooting hormone, developed roots within four weeks of inoculation. In most cases, more than three roots were formed per shoot in each treatment. The highest proportion of root formation occurred on the MS

basal medium fortified with 0.1 mg L−<sup>1</sup> Kn + 0.5 mg L−<sup>1</sup> IAA and 30 g L−<sup>1</sup> sucrose, with a percentage response of 68.00 ± 1.52% and average root length 4.66 ± 0.23, followed by 0.1 mg L−<sup>1</sup> Kn + 0.3 mg L−<sup>1</sup> IAA with a percentage response of 56.33 ± 4.91% and average root length of 5.66 ± 0.88. Among all the PGRs tested, the Kn and IAA combination was proven to be most effective for healthy root development and formation of maximum root–shoot ratio. The root induction ratio (number of roots formed per shoot) is further enhanced by dim light or complete darkness. Several cultured vials were wrapped in aluminum foil to reduce the intensity of light; these vials developed roots rapidly compared to vials that were kept in light (16/8-h photoperiod). Further root induction in *A. violaceum* was facilitated by a low concentration of PGRs. Increasing the concentration of PGRs promotes further callusing on the developed roots.

#### *3.6. Hardening and Acclimatization of In Vitro Plantlets*

The plantlets were then transferred into jiffy pots, filled with a mixture of sterilized garden soil/loamy soil, coco-peat/peat moss, and vermicompost (1:1:1). The plantlets in the jiffy pots were enclosed in transparent polybags for two weeks to ensure adequate humidity and were kept in the growth chamber. After the third week, the hardened plantlets were transferred into the greenhouse for further acclimatization. Healthy and well-established plantlets were then transplanted into the Kashmir University Botanical Garden (KUBG) and kept in the shade with occasional watering. After three to five weeks, well-established plantlets produced three to five leaves (Figure 5B). Transplanted plantlets attained a maximum height of 25.33 ± 1.76 after eight weeks (Table 4; Figure 5D). Overall, 55% of the transplanted plantlets survived and reached the budding stage (Figure 5D).

**Figure 5.** Hardening and acclimatization of in vitro raised plantlets of *Aconitum violaceum* Jacq. ex Stapf: (**A**,**B**) three to four week old hardened plantlets; (**C**) five to six week old acclimatized plants; (**D**) eight week old acclimatized plants. Scale bar represents 1 cm.

**Table 4.** Morphological data of tissue culture-raised plantlets of *Aconitum violaceum* after acclimatization in the field.


Data represented as mean value ± SEM (standard error mean). The same letters in each column represent that the data were not considerably different according to DMRT (one-way ANOVA) at *p* ≤ 0.05.

#### **4. Discussion**

*Aconitum* species are primarily harvested on a large scale for their rhizome, which has excellent pharmacological properties and is widely used in indigenous medicines, indirectly resulting in the decline of several *Aconitum* species from their native habitat [31]. Seed-based propagation is the most efficient, affordable, and practicable technique for most species to be cultivated on a large scale for commercial purposes [31]. *Aconitum* species are mostly wild-growing at high elevations, so their propagation through seeds at low altitudes is constrained by ecological factors such as soil fertility, soil textures, pH, humidity, temperature, phytosociology, nature of vegetation, and dormancy of seeds [4,32,33]. Thus, providing pre-sowing treatments (chilling, water soaking, hot water treatment, or with different chemicals etc.) can enhance its cultivation at lower elevations [32]. Soaking seeds in cold water or hot water is often used in seed germination of *Aconitum* spp [33] and *Iris* spp [34]. Cold scarification is the most efficient strategy for breaking dormancy in Ranunculaceae [35–37]. The seeds stratified at 4 ◦C for 4–10 days enhanced the germination rate in *A. chasmanthum* and *A. nagarum* when cultured on the MS basal medium [22,25]. Similarly, *A. violaceum* seeds also require chilling treatment for successful germination. *A. violaceum* seeds were cultured in different seasons to determine the organogenic response. However, the seeds sown in winter and early spring showed a better germination rate of >77% and gave peculiar organogenic responses (i.e., non-embryogenic and embryogenic callus formation, somatic embryos development, multiple shooting and rooting, and direct seed germination) on the MS basal medium supplemented by different PGRS (auxins and cytokinins), either alone or in combination. Similar results were achieved by Deb and Lunghu with *A. nagarum* [25]. Among the different PGRs used, Kn and IAA were found to be best for direct seed germination, embryogenic callus induction, and multiple shoot regeneration in *A. violaceum*. Seeds stored at lower temperatures (1 ◦C to 6 ◦C) showed a better rate of germination during an in vitro culture. Similarly, seeds from *A. heterophyllum* that have chilled for 25–40 days showed a germination rate of 60% [38]. These studies suggest that cold storage and cold-frame sowing or culturing of *A. violaceum* seeds showed a high frequency of germination. Other studies have also suggested that a low temperature is the most significant factor in the germination and in vitro propagation of *Aconitum* spp, which was also verified in the current study. The proposed in vitro regeneration will be helpful in the domestication of understudied plant species at lower altitudes. In its natural habitat, *A. violaceum* regenerates with rhizomes and the new stocks grow healthy and vigorous. The seed also has the potential to germinate into new seedlings; however, the plantlets of germinated seeds are delicate and susceptible to environmental fluctuation and rarely reach maturity stage. Most of the seeds face juvenile mortality at early stages due to extreme diurnal climatic fluctuation in their habitat.

The key rationale is that plants reproduced from direct somatic embryogenesis are commonly more uniform than plants regenerated indirectly by callus tissues [39]. Secondary embryogenesis approaches allow for the rapid production of enormous populations in a short period of time [40]. Secondary somatic embryos could also be developed from the surfaces of somatic embryos [41]. In the current study, somatic embryos were generated from immature and mature seed cultures of *A. violaceum.* Prior to culture on the growth medium, all seeds were stored at a low temperature (1 ◦C to 6 ◦C). The seeds cultured in winter and early spring produced a nodular mass of embryogenic potential callus on the MS basal medium enriched with various concentrations of auxins and cytokinins. Similar findings were observed by Vandelook et al. [42] in *Aconitum lycoctonum,* where a low temperature (below 10 ◦C) was suitable for the growth and germination of embryos. Giri et al. [24] developed complete plantlets from somatic embryos of leaf and petiole explants of *Aconitum heterophyllum* on the MS basal media augmented with 1 mg L−<sup>1</sup> 2,4-D and 0.5 mg L−<sup>1</sup> Kn, or 5 mg L−<sup>1</sup> NAA and 1 mg L−<sup>1</sup> BAP. Among the various growth regulators studied, Kn and IAA were found to be the most effective for the direct production of an embryogenic callus from immature and mature seed cultures. At lower concentrations, Kn alone was able to induce a nodular mass of embryogenic callus, but the same was

insufficient for the conversion of somatic embryos into complete plantlets. However, Kn in combination with IAA converts embryogenic calluses into somatic embryos and facilitates the regeneration of complete plantlets. Similarly, Kn also enhanced embryogenic callus induction in *Drimiopsis kirkii* [43], and in *Iris* species such as *Iris sanguinea* [44,45]. The addition of IAA in the range of 0.1–1.0 mg L−<sup>1</sup> accelerated the rate of somatic embryo germination, which eventually reached to 68.00 ± 1.52%. The dark conditions promote somatic embryo formation and development of roots in in *A. violaceum*. Similar results were reported by Xu Kd [28] in *Ranunculus sceleratus*, where darkness enhanced the frequency of somatic embryo formation. Cold storage can enhance somatic embryo conversion frequency, potentially caused by epigenetic changes triggered by temperature stress [46]. Moreover, in vitro culturing of seeds on the MS basal medium fortified with various concentrations of 2,4-D induced callusing. Increasing the concentration of 2,4-D to >1.0 mg L−<sup>1</sup> resulted in a decrease in the percentage of callus induction, which is consistent with the findings of Li et al. [47,48], who found that increasing the concentration of 2,4-D from 11.3 to 18 μM decreased callus induction in *Rosa hybrida*. Lower concentrations of PGRs and a low temperature promotes optimal growth and development of in vitro culture of *A. violaceum*. From the current investigation, we observed that the optimal ambient temperature in the growth chamber should be maintained at 8 ◦C–10 ◦C, and relative humidity of 50–55% should be maintained for complete regeneration of plantlets through somatic embryogenesis. Moreover, pre-soaking in cold water for 96 h enhances the rate of seed germination, embryogenic callus production, and other organogenic responses. Optimal application of Kn along with IAA is sufficient for direct seed germination, multiple shoot induction, somatic embryo induction, and complete plant regeneration from somatic embryos in a short time. In the tissue culture-raised plantlets, the mortality rate was highest in the juvenile stage. The acclimatized plantlets required slightly acidic, porous, and loamy soil for successful establishment. The plantlets grew well in semi-shaded places with temperature fluctuations ranging from 10 ◦C to 20 ◦C. A further rise in temperature would not be feasible for the survival of *A. violaceum.*

#### **5. Conclusions**

The present study is the first to report on the development of an in vitro propagation protocol for seed germination and somatic embryo formation from seeds of the threatened endemic plant species, *A. violaceum*. Furthermore, the study revealed that seeds are suitable explants for efficient multiplication and restoration of *A. violaceum* within a short period of time (approximately three to five months), starting from the initiation of seed germination or somatic embryo development to final tissue culture-raised plantlets. The regeneration protocols established here could be useful for mass multiplication and conservation of this important economic plant species. In addition, this work may be useful in the discovery of physiologically active secondary metabolites from in vitro-derived plantlets under controlled circumstances and their commercial utilization.

**Author Contributions:** Conceptualization, A.H. and S.S.; methodology, A.H., S.S., S.R. and I.A.N.; software, A.H., S.S., I.A.N., S.R. and N.A.W.; validation, A.H. and S.S.; formal analysis, A.H., S.S. and S.R; investigation, A.H., S.S. and S.R.; resources, E.A.M., D.O.E.-A., H.S., R.C., K.Y. and H.O.E.; data curation, writing—original draft preparation, A.H., S.S., I.A.N. and N.A.W.; writing—review and editing, A.H., S.S., I.A.N. and S.R.; funding acquisition, A.H., S.S., I.A.N., N.A.W., E.A.M., D.O.E.-A., H.S., R.C., K.Y. and H.O.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** King Saud University (RSP-2021/118).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The first author acknowledges the Council of Scientific and Industrial Research (CSIR), Pusa, New Delhi, for providing financial assistance as SRF during this study. The authors extend their deep appreciation to the Researchers Supporting Project (RSP-2021/118), King Saud University, Riyadh, Saudi Arabia.

**Conflicts of Interest:** The authors declare no conflict of interest.
