*3.2. Effects of Kn, IAA, and 2, 4-D on Embryogenic and Non-Embryogenic Callus Formation*

The impacts of Kn and IAA on embryogenic callus production from immature and mature seed cultures were explored. Various concentrations of Kn, either individually or in combination with IAA, were found to be efficient in inducing a nodular mass of embryogenic callus. However, the highest proportion of embryogenic callus production was achieved on the MS basal medium supplemented with 0.1 mg L−<sup>1</sup> Kn + 0.5 mg L−<sup>1</sup> IAA, with a percent culture response of 51.0 ± 1.0% and a mean initiation time of 31.0 ± 0.57 days, followed by 0.1 mg L−<sup>1</sup> Kn alone (35.53 ± 2.23). At concentrations ranging between 0.1 and 1.5 mg L−<sup>1</sup> Kn and IAA, the proportion of somatic embryo formation is high. Raising the concentrations further dramatically reduces somatic embryo development and promotes callus induction. The MS media enriched with 2,4-D at concentrations 0.1 to 0.5 mg L−<sup>1</sup> produced only non-embryogenic calluses (Figure 4A). Establishment of embryogenic callus induction was examined in this study (Table 2, Figure 2A–L). When the immature seeds were incubated on the MS medium augmented with various doses of Kn and IAA (0.1 mg L−<sup>1</sup> to 0.5 mg L−1), either individually or in combination, two different types of calluses were generated simultaneously. Light green transparent calluses developed from immature and mature seeds after four to six weeks of culture, whereas creamy nodular calluses were mainly produced after six to eight weeks of incubation (Figure 2A,B). During callus proliferation, nodular calluses developed into many globular staged embryos (Figure 2C,D) after eight weeks of culture. Globular stage somatic embryos further transformed into subsequently staged embryos (Figure 2E,H).

**Figure 2.** Direct somatic embryogenesis and complete plant regeneration from immature and mature seed cultures of *Aconitum violaceum* in the MS medium fortified with various concentrations of PGRs, either separately or in combination: (**A**,**B**) embryogenic and non-embryogenic callus formation from the immature and mature seed cultures, enriched with 0.1 mg L−<sup>1</sup> Kn + 0.5 mg L−<sup>1</sup> IAA and 0.1 mg L−<sup>1</sup> Kn; (**C**,**D**) globular-shaped somatic embryo development; (**E**–**H**) torpedo and subsequent stages of somatic embryo formation; (**I**) direct germination of somatic embryos on the MS medium, augmented with Kn 0.1 + IAA 0.5 mg L<sup>−</sup>1; (**J**) multiple shoot and root development from the cultured somatic embryos; (**K**) well-developed rooted plantlets before hardening; (**L**) hardening of plantlet in jiffy pots. Scale bar (**A**–**I**) represents 5 mm; (**J**–**L**) represents 1 cm.


**Table 2.** Effects of Kn, IAA, and 2, 4-D on embryogenic and non-embryogenic callus formation from the immature and mature seeds of *Aconitum violaceum*.

Data are expressed as mean value ± SEM. The same letters within each column represent that the data were not considerably different at *p* ≤ 0.05 according to DMRT (one-way ANOVA). Each treatment is represented by 10 replicates (one vial is considered one replicate) in three repetitions. Abbreviations: MS: Murashige and Skoog. PGRs: plant growth regulators. Kn: kinetin. IAA: indole-3-acetic acid. 2,4-D: 2,4-dichlorophenoxyacetic acid. SEM: standard error mean.
