Establishment of an In Vitro Regeneration System Using Shoot Tips of Iris setosa

Abstract

Iris setosa is a characteristic perennial wild herbaceous flower in the Changbai Mountain region of China, boasting significant ornamental and medicinal values. Given the increasing scarcity of its wild resources, this study developed an efficient in vitro regeneration system using shoot tips as explants via the direct organogenesis pathway. The optimal surface sterilization protocol was achieved with a treatment of 0.1% HgCl₂ for 8 min, resulting in an explant survival rate of 57.78%. The highest multiple shoot induction rate (88.89%) of shoot tips was achieved on MS medium supplemented with 2.0 mg·L⁻¹ 6-benzylaminopurine (6-BA), 0.5 mg·L⁻¹ naphthalene acetic acid (NAA), and 1.0 mg·L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). The optimal shoot differentiation and proliferation medium was MS + 2.0 mg·L⁻¹ 6-BA + 0.3 mg·L⁻¹ NAA, achieving a proliferation coefficient of 3.37. The optimal medium for rooting was confirmed to be 1/2 MS + 0.5 mg·L⁻¹ indole-3-butyric acid (IBA), exhibiting a high rooting rate reached 98.33%. During transplantation, plantlets exhibited high survival rates (over 90%) and vigorous growth across all three tested substrates, with no significant differences in survival rates among substrates. The key advance of this study lies in the development of a highly efficient and stable regeneration protocol for I. setosa derived from shoot tip explants, providing critical technical backing for the conservation and sustainable exploitation of its wild-type germplasm.

1. Introduction

Iris setosa is a perennial herbaceous species of the Iridaceae family, widely distributed across China, Japan, Korea, Russia, and North America [1]. This plant is characterized by its compact growth habit, with basal leaves that are sword-shaped or broadly linear, and vibrant blue-purple flowers measuring 7–8 cm in diameter. Blooming typically in July, I. setosa possesses high ornamental value. Additionally, its rhizomes and extracts are recognized for their medicinal properties [2,3]. The species predominantly inhabits moist environments such as subalpine meadows and swamps, demonstrating strong adaptability to cold, drought, and dry heat conditions. However, due to threats like wetland development and excessive harvesting, it has been classified as an EN B2ab(iii) endangered species in Korea [4]. In China, I. setosa is found primarily in the wild in the Changbai Mountain region of Jilin Province, where the systematic research and utilization of its germplasm resources remain at a preliminary stage.

Similarly to other species within the genus Iris, the conventional propagation methods for I. setosa are seed propagation and division. However, seed propagation requires cold stratification to break dormancy, resulting in a prolonged germination cycle, while division suffers from low proliferation rates and the need for periodic separation, making it unsuitable for large-scale production [5]. Consequently, tissue culture-based rapid propagation techniques, which offer advantages such as high efficiency and shorter cycles, have emerged as a vital research direction for germplasm conservation and efficient breeding in Iris.

To date, efficient in vitro regeneration systems have been successfully established for numerous Iris species, primarily via two pathways: somatic embryogenesis and organogenesis [6]. Organogenesis, being more widely applied, has enabled highly efficient adventitious shoot induction and plantlet regeneration in species such as I. laevigata, I. dichotoma, I. sanguinea, I. kirkwoodiae, and I. ensata [7,8,9,10,11]. This was achieved through precise modulation of cytokinin (e.g., BAP) and auxin (e.g., NAA, 2,4-D) ratios in the culture medium, utilizing various explants including leaf bases, root segments, flower stems, hypocotyls, and immature embryos. Regarding somatic embryogenesis, plant regeneration has been accomplished in I. halophila and I. sibirica using mature zygotic embryos as explants [12], while I. germanica has been regenerated via multiple pathways, including protoplast culture, using explants such as shoot apices, leaf bases, and suspension cells [13,14,15].

In contrast, research on the in vitro regeneration of I. setosa remains limited and faces significant constraints. Although studies have achieved plant regeneration via organogenesis using immature ovaries and perianth tubes as explants induced with groups of 2,4-D, BA, and kinetin, the established systems exhibit pronounced genotype dependence, and their regeneration efficiency and stability require substantial improvement [16,17]. To address this limitation, the present study developed an optimized and stable in vitro regeneration method for I. setosa using shoot tips as explants—an explant type that has not been applied in I. setosa regeneration studies yet—through the systematic optimization of the ratios of three key phytohormones: 6-BA, NAA, and 2,4-D. This technical system delivers essential technical backing for the preservation and exploitation of the germplasm resources of wild I. setosa.

2. Materials and Experimental Procedures

2.1. Experimental Plant Materials and Media Constituents

Shoot tips were excised from healthy four-year-old I. setosa plants maintained in the Plant Germplasm Resource Garden (Figure 1) of Jilin Agricultural University to serve as explants. The plant species was identified as I. setosa by the authors based on its morphological characteristics with reference to the Flora of China. All cultured materials were kept at 25 °C under a 16 h photoperiod, with an illuminance of approximately 2000 Lux. For the basal medium, Murashige and Skoog salts (MS, coolaber, Beijing, China) were amended with 24 g/L sucrose (coolaber, Beijing, China) and 7 g/L agar (coolaber, Beijing, China) (pH 5.8–6.0). For root induction, 1/2 MS medium was amended with 24 g/L sucrose (coolaber, Beijing, China) and 7 g/L agar (coolaber, Beijing, China) (pH 5.8–6.0). All media were sterilized by autoclaving at 121 °C for 20 min.

2.2. Disinfection of Explant Surfaces

Healthy plants of I. setosa were selected. After removal of leaves and roots, the shoots were thoroughly rinsed under running tap water. Shoot tips were excised and subjected to a 10 min wash in sterile water containing 1% Tween 20, followed by a 30 min rinse under flowing tap water. The samples were subsequently moved to a laminar flow cabinet for surface disinfection, surface-sterilized by a 30 s immersion in 75% ethanol and three washes with autoclaved distilled water. Then, the explants were treated with 0.1% (w/v) HgCl₂ for durations of 6, 7, 8, and 9 min, respectively [18], and fully rinsed five times using sterile distilled water. The oxidized basal tissues resulting from HgCl₂ treatment were aseptically trimmed, the 7–8 mm long shoot tip explants were transferred to MS basal medium (80 mL) for inoculation in vented cap vessels. For each treatment, 30 explants were used per replicate with three replicates. After 7 days of culture, the contamination rate, browning rate, and survival rate were calculated as follows: Contamination rate (%) = (number of contaminated explants/total inoculated explants) × 100; Browning rate (%) = (number of browned and non-viable explants/total number of explants inoculated) × 100; Survival rate (%) = (number of surviving explants/total number of explants inoculated) × 100 [19].

2.3. Induction of Multiple Shoots

Surface-sterilized shoot tips of I. setosa served as explants to promote the induction of multiple shoots. A three-factor, three-level orthogonal design was employed using the standard L₉(3⁴) array, with the factors and their levels set as follows: 6-BA (coolaber, Beijing, China) (1.0, 2.0, and 3.0 mg·L⁻¹), NAA (coolaber, Beijing, China) (0.3, 0.5, and 1.0 mg·L⁻¹), and 2,4-D (coolaber, Beijing, China) (0, 0.5, and 1.0 mg·L⁻¹). These three factors were assigned to three columns of the array, while the remaining column was left blank to serve as the error term in the subsequent Analysis of Variance (ANOVA). Each treatment included 30 replicates with one explant per vessel, with three independent repetitions of the experiment performed. Following 30 days of culture, the induction rate of shoot tips was calculated in the following manner: Induction rate (%) = (number of explants with shoot cluster formation/total number of inoculated explants) × 100% [19]. The induced multiple shoots from each treatment group were subjected to qualitative assessment. Through visual observation, key attributes including shoot thickness (e.g., slender or thick), color, growth vigor, and morphological normality were described and compared to evaluate the quality of shoots across different treatments.

2.4. Proliferation Culture

When the multiple shoots reached 2–3 cm in height, robust and contamination-free shoots were selected, they were divided into individual shoots and trimmed to 1–2 cm in length before being introduced into the proliferation culture medium. The MS basal medium was added with 6-BA (1.0, 2.0, and 3.0 mg·L⁻¹) and NAA (0, 0.3, and 0.5 mg·L⁻¹) in arbitrary combinations, forming nine treatment groups: Group 1: 1.0 mg·L⁻¹ 6-BA + 0 mg·L⁻¹ NAA; Group 2: 1.0 mg·L⁻¹ 6-BA + 0.3 mg·L⁻¹ NAA; Group 3: 1.0 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA; Group 4: 2.0 mg·L⁻¹ 6-BA + 0 mg·L⁻¹ NAA; Group 5: 2.0 mg·L⁻¹ 6-BA + 0.3 mg·L⁻¹ NAA; Group 6: 2.0 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA; Group 7: 3.0 mg·L⁻¹ 6-BA + 0 mg·L⁻¹ NAA; Group 8: 3.0 mg·L⁻¹ 6-BA + 0.3 mg·L⁻¹ NAA; Group 9: 3.0 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA. Each treatment included ten bottles inoculated with one explant per bottle, and all experiments were performed with three biological replicates. After 20 days of culture, the proliferation coefficient and growth status of the regenerated shoots were recorded. The proliferation coefficient was calculated as follows: proliferation coefficient = total number of shoots after proliferation/number of inoculated shoots [19]. After the proliferation cycle, shoot clusters were assessed visually. Key qualitative descriptors included shoot thickness, growth rate, the abundance of differentiated shoots, and overall color, providing a comparative profile of shoot quality across treatments.

2.5. Induction of Adventitious Root

When the shoot clusters grew to 3–5 cm in height, robust and contamination-free shoots were selected. Individual shoots were excised and transferred to 1/2 MS-based rooting media supplemented with IBA or NAA at concentrations of 0.1, 0.3, or 0.5 mg·L⁻¹ each. This constituted six independent treatments as follows: Group 1: 0.1 mg·L⁻¹ IBA; Group 2: 0.3 mg·L⁻¹ IBA; Group 3: 0.5 mg·L⁻¹ IBA; Group 4: 0.1 mg·L⁻¹ NAA; Group 5: 0.3 mg·L⁻¹ NAA; Group 6: 0.5 mg·L⁻¹ NAA. Twenty plantlets per treatment were used, and the experiment was conducted with three independent biological replicates. After 30 days of culture, the rooting rate, average root number, average root length, and root morphology were evaluated. The rooting rate was calculated as (number of rooted plantlets/total number of inoculated plantlets) × 100%. The average number of roots was determined by dividing the total root number by the number of rooted plantlets, while the average root length was measured by dividing the total length of all viable adventitious roots by their total quantity [19]. The morphology of induced adventitious roots was assessed visually, focusing on primary root sturdiness and thickness, lateral root density and development, as well as overall growth vigor.

2.6. Acclimatization

Well-rooted tissue-cultured plantlets of I. setosa were transferred to acclimatization conditions. The caps of the culture vessels were removed at room temperature, and the plantlets were carefully extracted using forceps, followed by washing away of the residual medium attached to the root surface. The plantlets were then transplanted into individual pots (8 cm in diameter × 10 cm in height) containing three different substrate formulations: loam soil alone, loam soil:perlite (2:1), and loam soil:perlite (1:1). The loam soil was locally sourced from a horticultural market. Each treatment contained 20 pots (plantlets), with three independent replicates. After transplanting, the substrates were watered thoroughly. After 20 days of acclimation, the survival rate was documented and computed as follows: Survival rate (%) = (number of surviving plantlets/total number of transplanted plantlets) × 100% [19].

2.7. Statistical Analysis

Statistical analyses were carried out via SPSS 27.0.1, and all experimental data are presented as the mean ± standard error (SE) of three independent replicates (IBM, Armonk, NY, USA). To examine notable differences between treatment groups (across all experiments), one-way ANOVA was adopted, with subsequent Duncan’s multiple range test as a post hoc procedure, with the threshold for significance defined as p < 0.05.

3. Results

3.1. Disinfection of Explant Surfaces

Shoot tips of I. setosa underwent surface sterilization using 75% ethanol for a 30 s period, and were subsequently immersed into 0.1% HgCl₂ with durations of 6, 7, 8, and 9 min, respectively. As shown in

Table 1. Effects of HgCl₂ treatment duration (6, 7, 8, 9 min) on surface sterilization of explants.

HgCl2 Treatment Time	Contamination Rate %	Browning Rate %	Survival Rate %
6 min	34.44 ± 0.05 a	7.78 ± 0.02 c	52.22 ± 0.02 a
7 min	25.56 ± 0.02 b	10.00 ± 0.03 c	55.56 ± 0.05 a
8 min	20.00 ± 0.03 bc	18.89 ± 0.04 b	57.78 ± 0.04 a
9 min	15.55 ± 0.04 d	34.44 ± 0.05 a	41.11 ± 0.04 b

Note: Significant differences (p < 0.05) between treatments are indicated by different letters within a column.

, extending the HgCl₂ treatment from 6 min to 9 min resulted in a significant decrease in the contamination rate from 34.44% to 15.55%, whereas the browning rate increased markedly from 7.78% to 34.44%. Correspondingly, the survival rate of explants exhibited an initial increase followed by a decline, reaching a maximum of 57.78% after 8 min of disinfection. Based on these findings, it was determined that the best sterilization method involved 75% ethanol for 30 s, subsequently treated with 0.1% HgCl₂ for 8 min.

3.2. Induction of Multiple Shoots

For the purpose of screening the optimum culture medium for multiple shoots induction, an L₉(3⁴) orthogonal test design was adopted with MS as the basal medium to examine the impacts of three growth regulators at different concentration levels: 6-BA (1, 2, and 3 mg·L⁻¹), NAA (0.3, 0.5, and 1.0 mg·L⁻¹), and 2,4-D (0, 0.5, and 1.0 mg·L⁻¹). A total of nine hormone groups were tested. Following a 30-day culture period, the growth status and induction rates were documented. The growth performance of multiple shoots varied among the nine treatment groups (Figure 2). In group 5 (Figure 2e), shoots were dense, robust, bright green, and exhibited vigorous growth. However, some abnormal shoots were observed in groups 3 (Figure 2c) and 9 (Figure 2i). Shoots induced in groups 1 (Figure 2a) and 4 (Figure 2d) appeared slender and weak. Although most other groups produced healthy shoots, statistical analysis of the induction rates revealed significant differences among the treatments (

Table 2. Effects of different plant growth regulator combinations on multiple shoots induction rate and growth state.

Groups	6-BA (mg·L−1)	NAA (mg·L−1)	2,4-D (mg·L−1)	Induction (%)	Growth State of Multiple Shoot
1	1.00	0.30	0	56.67 ± 1.93 de	Slender, green
2	1.00	0.50	0.50	82.22 ± 2.94 ab	Thick, green, and fast growing
3	1.00	1.00	1.00	51.11 ± 1.11 e	Slender, green, and partially abnormal
4	2.00	0.30	0.50	77.78 ± 2.94 b	Slender, green, and fast growing
5	2.00	0.50	1.00	88.89 ± 1.11 a	Thick, green, and fast growing
6	2.00	1.00	0	67.78 ± 1.11 c	Thick, dark green
7	3.00	0.30	1.00	52.22 ± 4.01 e	Thick, dark green, and slow growing
8	3.00	0.50	0	61.11 ± 4.01 cd	Thick, green
9	3.00	1.00	0.50	50.00 ± 1.92 e	Thick, partially abnormal

Means followed by the different letters in rows are significantly different at p < 0.05.

), with values ranging from 50.00% to 88.89%. The maximum induction rate was attained by group 5, comprising 2.00 mg·L⁻¹ 6-BA, 0.50 mg·L⁻¹ NAA, and 1.00 mg·L⁻¹ 2,4-D.

The concentrations tested for each factor were: 6-BA at 1.0, 2.0, and 3.0 mg·L⁻¹; NAA at 0.3, 0.5, and 1.0 mg·L⁻¹; and 2,4-D at 0, 0.5, and 1.0 mg·L⁻¹. The K-value analysis (Figure 3), which reflects the mean induction rate at each concentration level, showed that the highest induction efficiency for each factor was achieved at the K2 level. This corresponds to 2.0 mg·L⁻¹ for 6-BA, 0.5 mg·L⁻¹ for NAA, and 0.5 mg·L⁻¹ for 2,4-D. Analysis of variance (ANOVA) results (Table S1) indicated that both 6-BA and NAA significantly influenced the induction rate of multiple shoots (p < 0.05), whereas 2,4-D had no significant effect (p > 0.05). Taking into account both the induction rate and the growth performance of the induced shoots (Table 2), the optimal plant growth regulator combination for multiple shoot induction in I. setosa was determined to be: 2.0 mg·L⁻¹ 6-BA, 0.5 mg·L⁻¹ NAA, and 1.0 mg·L⁻¹ 2,4-D. This combination was selected based on the overall outcome of the orthogonal experiment as the optimal treatment, even though the single-factor effect of 2,4-D was not statistically significant.

3.3. Proliferation of Shoots

To achieve the research objective of establishing an efficient in vitro propagation protocol for I. setosa, two critical response variables were evaluated to optimize the shoot proliferation stage: the proliferation coefficient and the growth state of shoots. The effects of different concentrations of 6-BA (1.0, 2.0, and 3.0 mg·L⁻¹) and NAA (0, 0.3, and 0.5 mg·L⁻¹) on these parameters are shown in

Table 3. Effects of different 6-BA and NAA concentrations on the proliferation of multiple shoots.

Groups	6-BA (mg·L−1)	NAA (mg·L−1)	Proliferation Coefficient	Growth State of Shoots
1	1.00	0.00	2.17 ± 0.08 bcd	Thick, fast growing
2	1.00	0.30	2.37 ± 0.05 bc	Thick, green, and many differentiated shoots
3	1.00	0.50	1.07 ± 0.03 f	Slow growing, few differentiated shoots
4	2.00	0.00	2.53 ± 0.14 b	Thick, fast growing and many differentiated shoots
5	2.00	0.30	3.37 ± 0.15 a	Thick, fast growing and many differentiated shoots
6	2.00	0.50	2.20 ± 0.19 bc	Thick, fast growing and many differentiated shoots
7	3.00	0.00	1.77 ± 0.14 de	Slow growing, few differentiated shoots
8	3.00	0.30	1.97 ± 0.18 cd	Slow growing, few differentiated shoots
9	3.00	0.50	1.47 ± 0.12 e	few differentiated shoots, yellow at the base

Values in one row suffixed with different letters differ significantly (p < 0.05).

and Figure 4.

As shown in Table 3, with the NAA concentration maintained at a fixed level, the proliferation coefficient increased initially followed by a decrease with the elevation of 6-BA concentrations. Furthermore, at a fixed 6-BA concentration, the addition of 0.3 mg·L⁻¹ NAA significantly enhanced the proliferation coefficient, whereas increasing the NAA concentration to 0.5 mg·L⁻¹ markedly suppressed shoot differentiation and reduced the proliferation coefficient.

Consequently, the lowest proliferation coefficients were observed in treatment groups 3 and 9 (Figure 4a), which were associated with higher NAA (0.5 mg·L⁻¹) or high 6-BA (3.0 mg·L⁻¹) concentrations and produced slender, slow-growing, or basally yellowed shoots, indicating a suboptimal hormonal balance. While treatment groups 1, 2, 4, 6, 7, and 8 showed intermediate coefficients (1.77 to 2.53) and produced thick, green shoots, they did not reach the maximum proliferation rate, representing a compromise. In contrast, the ideal medium was identified as Group 5 (2.0 mg·L⁻¹ 6-BA and 0.3 mg·L⁻¹ NAA), which achieved the highest proliferation coefficient (3.37) and consistently produced vigorous, fast-growing shoots with many differentiated shoots (Table 3, Figure 4c). Therefore, this combination was selected as optimal for fulfilling the dual requirement of maximizing the proliferation rate while ensuring shoot vigor, which is critical for developing an efficient propagation system.

3.4. Induction of Adventitious Roots

Among the tested auxin treatments, increasing the concentration of NAA consistently led to unfavorable rooting responses (

Table 4. Effects of IBA and NAA concentrations on rooting percentage, adventitious root number, and root length in I. setosa cultured in vitro.

Groups	IBA (mg·L−1)	NAA (mg·L−1)	Rooting (%)	Adventitious Roots Number	Average Root Length (cm)	Growth Condition of Roots
1	0.10	0	76.67 ± 0.03 bc	4.17 ± 0.13 c	4.27 ± 0.27 bc	Thin and weak primary root, dense lateral roots.
2	0.30	0	91.67 ± 0.02 a	4.41 ± 0.12 c	5.30 ± 0.12 a	thick primary root and dense well-developed lateral roots
3	0.50	0	98.33 ± 0.02 a	5.23 ± 0.17 b	5.53 ± 0.28 a	thick primary root and dense well-developed lateral roots
4	0	0.10	88.33 ± 0.03 ab	6.14 ± 0.16 a	4.90 ± 0.12 ab	thick primary root, Slow growing and sparse lateral roots.
5	0	0.30	68.33 ± 0.07 c	5.89 ± 0.09 a	4.47 ± 0.23 bc	Thin and weak primary root, Slow growing and sparse lateral roots.
6	0	0.50	51.67 ± 0.04 d	5.05 ± 0.08 b	3.90 ± 0.17 c	Thin and weak primary root, Slow growing and sparse lateral roots.

Note: Values in one row suffixed with different lowercase letters are significantly different (p < 0.05).

). Specifically, the rooting rate declined significantly from 88.33% at 0.1 mg·L⁻¹ NAA to 51.67% at higher concentrations, indicating a clear inhibitory effect on root induction. In contrast, elevating the concentration of IBA progressively improved rooting performance. The rooting rate rose from 76.67% at 0.1 mg·L⁻¹ IBA to 91.67% at 0.3 mg·L⁻¹, and reached a maximum of 98.33% at 0.5 mg·L⁻¹ IBA. Notably, even the intermediate IBA concentration (0.3 mg·L⁻¹) yielded a significantly higher rooting rate (91.67%) than the best-performing NAA treatment (0.1 mg·L⁻¹, 88.33%). Thus, the optimal rooting response was achieved with 0.5 mg·L⁻¹ IBA, which produced the highest rooting rate observed in this study.

Regarding root number (Table 4 and Figure 5), a clear differential response was observed between the two auxin treatments. Overall, NAA consistently induced a higher average number of adventitious roots per plantlet compared to IBA across the tested concentration range. The most pronounced difference was evident at the lower concentration of 0.1 mg·L⁻¹: shoots treated with NAA at this level produced a robust average of 6.14 roots, a value significantly greater than the maximum average of 5.23 roots achieved by any IBA treatment (specifically at 0.5 mg·L⁻¹). This trend underscores the superior efficacy of NAA over IBA in promoting the initiation of a greater number of root primordia under the established in vitro conditions. In terms of root length, however, the average values of 5.30 cm and 5.53 cm obtained with 0.3 mg·L⁻¹ and 0.5 mg·L⁻¹ IBA, respectively, were significantly greater than those observed at all three NAA concentrations. It was also noted that root initiation occurred earlier in IBA-containing media, and IBA promoted the formation of more lateral roots, while NAA induced few or no lateral roots (Figure 5). In conclusion, 1/2MS medium supplemented by 0.5 mg·L⁻¹ IBA was emerged as the optimal option for inducing root formation.

3.5. Acclimatization

I. setosa tissue-cultured plantlets were transplanted into three substrate formulations: loam soil:perlite (1:1), loam soil:perlite (2:1), and loam soil. After 20 days of cultivation, the survival rates of plantlets in all substrates exceeded 90% (Table S2). Furthermore, the plantlets exhibited vigorous and uniform growth across all substrate types (Figure 6). These results demonstrate that I. setosa tissue-cultured plantlets possess broad adaptability to substrate composition, indicating that any of the tested formulations can be flexibly selected for practical transplantation based on specific conditions.

4. Discussion

I. setosa possesses significant ornamental and medicinal value; however, traditional propagation methods within the Iris genus are generally inefficient, limiting its utilization and germplasm conservation [20]. Existing tissue culture systems for I. setosa primarily rely on floral explants and remain constrained by genotype-dependent regeneration efficiency [17]. Therefore, this study established an efficient micropropagation protocol using shoot tips as explants via direct organogenesis.

In the establishment of an efficient regeneration system for I. setosa, surface sterilization of explants constitutes a critical initial step to ensure successful aseptic culture. This study demonstrates that treatment with 0.1% HgCl₂ for 8 min effectively sterilizes shoot-tip explants while maintaining a relatively high survival rate. However, given the relatively high cytotoxicity of HgCl₂, exposure time must be precisely controlled to avoid excessive damage to explant tissues [21]. Through a time-gradient experiment, this study identified 8 min as the optimal duration for shoot-tip sterilization in I. setosa. This result is consistent with a report on another monocot, Lilium longiflorum, where a 7 min treatment with 0.1% HgCl₂ was determined to be optimal for shoot-tip disinfection [18]. The establishment of this sterilization protocol provides a reliable foundation of sterile starting material for the subsequent stable construction of a shoot-tip-based regeneration system for I. setosa.

The multiple shoots induction rate and the propagation coefficient are subject to the influence of several variables, most notably the growth medium and the types and plant growth regulators applied [22]. Using germinated I. sanguinea seedlings as explants, it was found that MS medium amended with 0.5 mg·L⁻¹ 6-BA, 0.2 mg·L⁻¹ NAA, and 1.0 mg·L⁻¹ KT was the optimal formulation for inducing adventitious buds and promoting their proliferation, achieving a 93.3% induction rate along with a 5.3 propagation coefficient [23]. The combination of 0.5 mg·L⁻¹ 2,4-D and 1.0 mg·L⁻¹ 6-BA significantly enhanced the induction rate of adventitious shoot in I. dichotoma Pall [7]. During the multiple shoots formation and proliferation phases, 2,4-D showed no significant effect on shoot induction, though it promoted callus formation, supporting the tendency of I. setosa shoot tips toward direct organogenesis rather than indirect regeneration via callus [24]. As essential triggers of organogenesis, auxin actions are modulated by endogenous cytokinin levels [25]. Here, the optimal 6-BA concentration for both shoot induction and proliferation was 2 mg·L⁻¹, while the optimal NAA levels were 0.5 mg·L⁻¹ and 0.3 mg·L⁻¹, respectively. The corresponding 6-BA/NAA ratios—4:1 during induction and 6.7:1 during proliferation—agree with the requirement for a relatively high cytokinin-to-auxin ratio to promote shoot differentiation and proliferation [26]. In summary, this optimal hormone ratio serves as the decisive factor for the successful induction and propagation of shoots from shoot-tip explants, establishing a reproducible and efficient regulatory foundation for the in vitro regeneration system of I. setosa.

Auxin type and concentration were equally crucial during adventitious root induction. Although IBA and NAA promote rooting in many plants, their effectiveness depends strongly on species, explant type, and culture conditions [27]. The application of mg·L⁻¹ IBA alone resulted in the highest adventitious root induction rate in both I. sari and I. schachtii, which was significantly higher than that achieved by combinations of IBA and NAA [28]. The optimal rooting medium for the two Hosta cultivars, ‘Blue Mouse Ears’ and ‘Lemon Lime’, was 1/2 MS supplemented with 1.0 mg·L⁻¹ IAA, with an induction rate exceeding 90% for both [29]. In I. setosa, the rooting rate increased with IBA concentration (0.1–0.5 mg·L⁻¹) but decreased with increasing NAA. Root system architecture also differed markedly: 0.1 mg·L⁻¹ NAA generated more roots (6.14 on average), but these were mainly primary roots with few laterals, whereas 0.5 mg·L⁻¹ IBA gave fewer roots (5.23 on average) but induced a more branched lateral system, longer roots, and faster rooting. These results are consistent with Yusnita [30], where NAA induced numerous but poorly branched roots in Malay apple, while IBA promoted a highly branched, complex root system. In contrast, a study in sugarcane found NAA more effective than IBA, accelerating root initiation and increasing the proportion of primary roots [31]. Collectively, these findings confirm the species-specific nature of auxin responses and establish 0.5 mg·L⁻¹ IBA as the optimal choice for adventitious root induction in I. setosa. The optimal hormone combination for adventitious root induction established in this study (0.5 mg·L⁻¹ IBA) represents a critical step in obtaining complete, transplantable plantlets within the shoot tip based in vitro regeneration system of I. setosa, marking the final accomplishment of transitioning from shoot proliferation to whole plant regeneration.

During the acclimatization stage, a mixed substrate of loam soil:perlite (2:1) demonstrated optimal performance, achieving a plant survival rate of 95%. This formulation effectively balanced water retention, aeration, and nutrient availability, thereby significantly promoting the adaptation of tissue-cultured seedlings to soil conditions. This result indicates the successful establishment of a complete technical pathway—from sterile proliferation and rooting to final transplant survival—within the shoot-tip-based regeneration system of I. setosa, providing a reliable foundation for subsequent germplasm conservation and field establishment.

5. Conclusions

The optimal sterilization method for I. setosa shoot tips was achieved by surface-sterilizing with 75% ethanol (30 s) and 0.1% HgCl₂ (8 min). The suitable medium for multiple shoot induction was MS + 2.0 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA + 1.0 mg·L⁻¹ 2,4-D, with an induction rate of 88.89%. The optimal medium for shoot proliferation was MS + 2.0 mg·L⁻¹ 6-BA + 0.30 mg·L⁻¹ NAA, yielding a proliferation coefficient of 3.37. For root induction, the most effective medium was 1/2 MS + 0.5 mg·L⁻¹ IBA, achieving a rooting rate of 98.33%. When plantlets were transplanted into a substrate mixture of loam soil:perlite (2:1), the highest survival rate after 20 days reached 95.00%. The efficient in vitro regeneration system established for I. setosa in this study not only provides key technical support for the conservation of this endangered species, but also offers valuable insights for the genetic improvement and production practices of related species within the same family. The development of this technical system facilitates both in situ and ex situ conservation of wild Iris germplasm resources. Through the establishment of sterile germplasm repositories and the reintroduction of regenerated plants into natural habitats, it enhances the population recovery capacity of endangered species and contributes positively to environmental restoration.