*2.9. Experimental Design and Statistical Analysis*

The data were evaluated using a factorial design that was completely randomized (CRD). Substantial variations between each treatment were assessed using Duncan's multiple range test (DMRT) of one-way ANOVA using SPSS statistical software version 23 (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, version 23.0. Armonk, NY, USA: IBM Corp.). Results were shown as mean value ± SEM (standard error mean) for each experiment. Graphs were prepared in origin pro (version 9, 2021; developed by originLab corporation, Northampton, MA, USA).

#### **3. Results**

#### *3.1. Seed Germination*

Seed germination rate was enhanced when the seeds were immersed in tap water for 96 h in winter and early spring, prior to culture. Among the various PGRs used, the highest rate of in vitro seed germination was recorded in winter and spring on the MS basal medium enriched with 0.5 mg L−<sup>1</sup> Kn and 1.0 mg L−<sup>1</sup> Kn with a MGT of 27.22 ± 0.70 and 26.88 ± 0.16 days, and percentage germination of 77.32 ± 0.38 and 75.33 ± 0.14. The number of shoot buds was also recorded as maximum (2.0 ± 0) in this treatment. The shoot length of each seed was recorded highest in the MS medium augmented with 1.0 mg L−<sup>1</sup> Kn with an average length of 4.7 ± 0.11 cm (Table 1, Figure 1). Kn at low concentrations (<0.01 mg L<sup>−</sup>1) and high concentrations (>1.5 mg L−1) reduced seed germination percentage. Likewise, high concentrations of IAA (>1.5 mg L<sup>−</sup>1) also reduced the rate of seed germination. Thus, concentrations of Kn and IAA ranging between 0.1 and 1.5 mg L−<sup>1</sup> were proven to be ideal for seed germination in *A. violaceum*. None of the seeds germinated in the control condition (MS basal) without PGRs (plant growth regulators). The data were recorded for up to eight weeks, which was represented by mean value ± SEM (standard error mean).

**Figure 1.** Direct in vitro seed germination of *Aconitum violaceum* Jacq. ex Stapf on the MS basal medium enriched with different concentrations of PGRs, either individually or in combination: (**A**) 0.1 mg L−<sup>1</sup> Kn; (**B**) 0.35 mg L−<sup>1</sup> Kn; (**C**) 0.5 mg L−<sup>1</sup> Kn; (**D**) 1.0 mg L−<sup>1</sup> Kn; (**E**) 1.5 mg L−<sup>1</sup> Kn; (**F**) 0.1 mg L−<sup>1</sup> IAA; (**G**) 0.5 mg L−<sup>1</sup> IAA; (**H**) 1.0 mg L−<sup>1</sup> IAA; (**I**) 0.1 mg L−<sup>1</sup> BAP; (**J**,**K**) germinated seeds with well-formed roots before hardening; and (**L**) hardened plantlets in the jiffy pots composing a mixture of loamy soil, coco-peat, and vermicompost (1:1:1 *v*/*v*). Scale bar (**A**–**I**) represents 5 mm; (**J**–**L**) represents 1 cm.


germination

 of

wild-growing

*Aconitum violaceum*

**Table 1.** Effects of plant growth regulators (IAA, Kn, BAP, and NAA) on direct in vitro seed

growth regulators. IAA:

standard error mean.

indole-3-acetic

 acid. Kn: kinetin. BAP:

6-benzylaminopurine.

 NAA:

1-napthaleneacetic

 acid. MGT: mean germination

 time. SEM:
