Establishment of an Efficient System for Rhizome Proliferation and In Vitro Flowering Induction from Protocorm Explants in Cymbidium goeringii

Abstract

Unlike other orchids in the Orchidaceae family, Cymbidium goeringii presents significant challenges for in vitro flowering. In this study, through the screening of different basal media, hormone combinations, and other conditions, we developed efficient rhizome regeneration (micropropagation) and in vitro flowering induction systems from protocorm explants of C. goeringii hybrids. To obtain protocorm explants, seeds were pretreated with either NaOH or NaOCl. Our results indicated that NaOH pretreatment enhanced seed germination more effectively than NaOCl, and Knudson C medium proved more suitable for protocorm induction. The resulting protocorms were then used as primary explants for efficient rhizome micropropagation. An orthogonal design identified the optimal combination for rhizome proliferation: 9.0 mg/L 6-BA, 9.0 mg/L NAA, 3.0 mg/L IBA, and 0.1 g/L activated charcoal (Treatment 9), which achieved a proliferation rate of 35.17%. For rhizome differentiation, MS medium supplemented with 10 mg/L 6-BA, 0.1 mg/L NAA, and 0.1 mg/L AgNO₃ (Treatment 6) achieved a 100% differentiation rate and produced 3.93 buds per explant. Building on this optimized micropropagation system, in vitro flowering was induced directly from rhizomes. The most effective medium was MS (1/3N, 3P) supplemented with 9.0 mg/L 6-BA, 0.1 mg/L NAA, and 0.1–0.3 mg/L TDZ (Treatment 6), resulting in a 36% flower bud induction rate and a 16% normal flower bud formation rate. Orthogonal analysis and ANOVA confirmed that 6-BA was the most significant factor influencing floral transition, with the low-nitrogen and high-phosphorus MS (1/3N, 3P) medium also being a key contributor. Consequently, our study has established an efficient rhizome micropropagation system that enables in vitro flowering induction in C. goeringii hybrids within just six months. This represents a substantial 60–80% reduction in the flowering time (from 6–7 years to 1–2 years), compared to the traditional 6–7-year cultivation period. Future work will focus on ex vitro acclimatization, detailed floral-trait validation, and hormone-regime refinement for fast-tracking breeding programs.

1. Introduction

Cymbidium goeringii, commonly known as the spring orchid, is a culturally significant orchid species in China and a member of the Orchidaceae family. Its wide geographic distribution and high phenotypic diversity have led to its widespread cultivation and given it considerable cultural and economic value. This species is renowned for its numerous prestigious varieties, characterized by their pure fragrance, elegant petals, delicate flower shapes, graceful plant structures, and a harmonious blend of texture, color, and lively spirit. These qualities have earned it the esteemed title “Queen of Orchids” [1].

For many years, extensive research in Japan and South Korea has focused on the hybridization and breeding of Cymbidium species [2]. Numerous intra-specific hybrid combinations within C. goeringii and interspecific hybridization between C. goeringii and several other Cymbidium species (including C. kanran, C. sinense, C. serratum, and C. ensifolium), as well as with Phalaenopsis aphrodite and large-flowered C. hybridum, have been conducted [3,4]. Notably, the traditional spring orchid cultivar ‘Lüyun’ (C. goeringii) has produced variegated mutants with both floral and foliar marginal variegation (Fukurin) in South Korea, significantly influencing the Chinese orchid community [5]. In recent years, China has also made significant progress in orchid breeding, developing new cultivars such as ‘Flying Fairy’ [6], ‘Xi Zi Niao Niao’ [7], ‘Xian Mei Ren’ [8], and ‘Cai Hong He’ [9], as well as new cultivars with spring orchid as a parent, such as ‘Huangyi’ [10].

Despite these advances, challenges persist, including long breeding cycles and low market acceptance. Establishing an efficient tissue culture system for spring orchid seeds can overcome the natural difficulties in germination caused by the lack of endosperm. By simulating endogenous nutrient supply through artificial media, this approach significantly improves the propagation efficiency and speed while maintaining superior parental characteristics. This provides technical support for the conservation and utilization of endangered species [2]. Furthermore, complete in vitro flowering initiation can significantly shorten the breeding cycle. It allows for the direct observation of trait segregation in hybrid progeny under controlled conditions, accelerating the screening process for superior traits and the rapid development of new cultivars that meet commercial demands [11]. Therefore, developing an efficient micropropagation system for hybrid spring orchids and promoting in vitro flowering to accelerate breeding are critical issues.

Although progress has been made in orchid in vitro flowering induction techniques for several Dendrobium species and hybrids (e.g., D. candidum, D. huoshanase, D. moniliforme, D. primulinum, D. officinale, D. nobile Lindl., and D. strongylanthum), the explant and culture conditions varied significantly, leading to different durations of in vitro flowering induction [11,12]. Despite these developments in orchid in vitro flowering, inducing in vitro flowering in C. goeringii remains challenging due to its stringent vernalization and carbohydrate metabolism constraints.

Considering that the protocorm is considered a natural early stage during orchid embryo development, and it is reasonable to expect a certain degree of influence by hormones during the early development phases of protocorms, we successfully established a rhizome regeneration system in this study. This system achieved in vitro flowering induction from seed-derived protocorm explants of a C. goeringii hybrid germplasm. This was accomplished through the induction of seed-derived protocorms, followed by rhizome proliferation and rhizome bud differentiation into seedlings. Building on this, we established an efficient rhizome micropropagation system for C. goeringii hybrids that achieves in vitro flowering initiation in only six months. This dramatically reduces the time to flowering by 60–80% compared to the traditional 3–5 years, providing important technologies for the breeding, maintenance, and amplification of C. goeringii.

2. Materials and Methods

2.1. Capsule (Seed) Materials

The experimental material consisted of hybrid seeds of C. goeringii, resulting from a cross between the paternal parent ‘Kuaiji Hongxia’ (a bicolor cultivar, Figure 1a) and the maternal parent ‘Songmei’ (Figure 1b). The capsules were harvested eight months post-artificial pollination, while the fruits were still green and before dehiscence (splitting open to release seeds). Plump and healthy capsules were selected for aseptic sowing. Capsules are dry fruit that develops from a flower and contains multiple seeds. In the case of orchids, these capsules (also known as seed pods) are typically elongated structures that house numerous microscopic seeds. Thus, this study first collected capsules to obtain the seed materials.

2.2. Capsule Disinfection

The harvested green capsules were trimmed with a scalpel to remove any excessively long pedicels and debris from the anterior end. Subsequently, the capsules underwent surface sterilization by immersion in 70% ethanol for 30 s, followed by three rinses with sterile water. Further disinfection involved treatment with a 0.1% (w/v) mercuric chloride (HgCl₂) solution for 5 min, with three subsequent rinses using sterile water.

2.3. Seed Pretreatment

Following the opening of the disinfected capsules, the seeds were subjected to a 10 min pretreatment using either NaOH or NaOCl solutions. Concentration gradients of 0 (control), 0.5%, 2%, 5%, and 10% (w/v, effective chlorine for NaOCl) were tested for each solution. Approximately 200 seeds were used per concentration, with three replicates for each treatment. After pretreatment, the seeds were rinsed three times with sterile water and then uniformly inoculated onto Knudson C (KC) medium. This medium was supplemented with 0.5 mg/L naphthaleneacetic acid (NAA), 1.5 mg/L 6-benzylaminopurine (6-BA), 1.0 g/L activated charcoal (AC), and 30 g/L sucrose, with the pH adjusted to 5.5.

In this study, AC was included to adsorb phenolic compounds and other exudates released by C. goeringii explants (e.g., from the wound part during tissue culture or during the growth process), which can otherwise cause medium browning and inhibit rhizome proliferation. While AC does adsorb some medium components, including plant growth regulators (PGRs), we compensated for this adsorption by using elevated hormone concentrations in the medium.

2.4. Protocorm Induction

The seeds pretreated with the optimal 2% NaOH solution, as determined in the previous step, were inoculated onto four different basal nutrient media: Vacin and Went (VW), KC, MS, and ½ MS. All media were adjusted to pH 5.5. Approximately 50 seeds were used per treatment, with five replicates established (10 seeds per replicate). Germination, indicated by protocorm formation, was assessed at various intervals over a 120-day culture period. The additional supplements and their concentrations remained consistent with those used in the seed pretreatment stage (NAA 0.5 mg/L, 6-BA 1.5 mg/L, AC 1.0 g/L, and sucrose 30 g/L).

2.5. Rhizome Proliferation

Protocorms developed from seed culture served as the initial explants for rhizome proliferation studies. Using ½ MS medium as the basal component, an L9(3⁴) orthogonal experimental design (four factors, three levels) was employed to optimize the concentrations of the following supplements: 6-BA (0.1, 3.0, and 9.0 mg/L), indole-3-butyric acid (IBA) (0.1, 3.0, and 9.0 mg/L), NAA (0.1, 3.0, and 9.0 mg/L), and AC (0.1, 0.5, and 2.5 g/L). All treatments were supplemented with 30.0 g/L sucrose, 5.2 g/L agar, and 5% (v/v) coconut water, with the pH adjusted to 5.5. For each medium combination, 60 rhizomes of approximately 1.5 cm in length were inoculated, with three replicates per treatment.

Rhizomes (180-day-old post protocorm differentiation) were sourced from synchronized protocorm cultures cultivated under axenic conditions. The explants were inoculated into 300 mL glass culture vessels containing 50 mL medium/vessel, with 5 explants per vessel (density: 16.7 explants/L; headspace ratio 5:1 vol:gas). This configuration resulted in 12 vessels per treatment group, systematically allocated as 3 biological replicates × 4 vessels each. The cultures were maintained under scattered light with a 12 h photoperiod and a temperature of 26 ± 2 °C for 120 days.

Following this period, the proliferation rate and rhizome reproduction coefficient were calculated using the following formulas, and the resulting values were used for subsequent statistical analysis:

Proliferation rate (%) = (Rhizome mass after proliferation − Rhizome mass at inoculation)/Rhizome mass at inoculation × 100%;

Coefficient of proliferation = Number of new lateral branches on inoculated rhizomes/Number of inoculated rhizomes (new rhizome length > 0.5 cm).

2.6. Rhizome Differentiation

To investigate the effects of basal media (MS, ½ MS, Hyponex No. 2), plant growth regulators (PGRs) such as 6-BA (1.0, 5.0, and 10.0 mg/L), NAA (0.1, 0.3, and 0.9 mg/L), and the exogenous additive AgNO₃ (0.02, 0.1, and 0.5 mg/L) on the differentiation of C. goeringii rhizomes, media were formulated based on an L9(3⁴) orthogonal design. Culture vessels, rhizome segment length (approximately 1.5 cm), and explant density (5 explants per vessel) followed the same protocols as rhizome proliferation (Section 2.5). For each treatment combination, 10 replicate vessels were inoculated. The explants were obtained from rhizomes subcultured for 120 days under proliferation conditions.

All treatments were supplemented with 30.0 g/L sucrose, 5.2 g/L agar, and 5% (v/v) coconut water (pH 5.5). Material selection (rhizomes) and general culture conditions were consistent with those described for rhizome proliferation. After 120 days, we calculated the differentiation rate and the number of induced buds using the following formulas for subsequent statistical analysis:

Differentiation rate (%) = (Number of differentiated rhizomes/Numbers of inoculated rhizomes) × 100%;

Number of induced buds = Total number of induced buds/Numbers of differentiated rhizomes.

2.7. In Vitro Flowering Induction from Rhizomes

An L9(3⁴) orthogonal experiment was conducted to determine the optimal conditions for in vitro flowering from rhizomes. The tested factors included different MS formulations (1/3N, 3P and 1/5N, 5P), 6-BA (1.0, 3.0, and 9.0 mg/L), thidiazuron (TDZ) (0.1, 0.3, and 0.9 mg/L), and NAA (0.1, 0.3, and 0.9 mg/L). All media were supplemented with 50.0 g/L sucrose, 5.5 g/L agar, 0.02 mg/L AC, and 5% (v/v) coconut water and adjusted to pH 5.8. The rhizome selection and culture conditions were consistent with those used for rhizome propagation. After 180 days of culture, the growth responses were recorded. The flower bud induction rate and normal flower bud formation rate (defined as buds with complete floral organs and normal development) were calculated using the following formulas, and the resulting values were used for subsequent statistical analysis:

Flower bud induction rate (%) = (Number of induced flower buds/Number of inoculated samples) × 100%;

Normal flower bud formation rate (%) = (Number of normal flowers/Numbers of inoculated samples) × 100%.

The assessment of normal flower buds was performed by visual examination of the fully opened flowers, with the normality determined by intact organ morphology and typical development according to Cymbidium horticultural standards.

2.8. Data Analysis

The experimental data from the rhizome proliferation, differentiation, and flower bud induction experiments were analyzed using orthogonal range analysis and one-way ANOVA to evaluate the influence of various factors. A Taguchi L9 (3⁴) orthogonal array design (https://en.wikipedia.org/wiki/Taguchi_methods (accessed on 13 June 2025)) was employed to assess the effects of four independent variables: 6-BA, NAA, IBA, and AC in the rhizome proliferation and proliferation coefficient experiments; 6-BA, NAA, and AgNO₃ in the rhizome differentiation and bud induction experiments; and the basal medium, 6-BA, NAA, and TDZ in the in vitro flower bud induction and normal flower bud formation experiments.

Each factor was tested at three concentration levels. One-way ANOVAs were conducted to determine the statistical significance of each factor on the corresponding response variables. The statistical significance was set at p < 0.05, and F values greater than 1 were considered indicative of a meaningful contribution to variation. Both orthogonal range analysis and one-way ANOVA were performed using SPSS Statistics version 28.0.

3. Results

3.1. Effect of Pretreatment on Seed Germination

To investigate the optimal pretreatment for seed germination, we compared five concentration gradients of NaOH (0%, 0.5%, 2%, 5%, and 10%) and NaOCl (0%, 0.5%, 2%, 5%, and 10%). As shown in Figure 2a, a 2% NaOH solution applied for 10 min was the most effective pretreatment, significantly improving the seed germination. The promoting effect of NaOH on seed germination exhibited a concentration-dependent pattern, initially increasing and then decreasing, with the optimum concentration at 2% resulting in 72.3 germinated seeds. Similarly, NaOCl showed a concentration-dependent effect, with an optimum concentration of 5% yielding 35.7 germinated seeds, which was significantly lower than that achieved with the optimum NaOH treatment. When the NaOCl concentration exceeded 5%, the germination rate tended to decrease.

3.2. Protocorm Induction

We compared four basal media (KC, MS, VW, and ½ MS) for their efficacy in protocorm induction. The results (Figure 2b) indicated that the KC medium was the most effective, achieving a protocorm induction rate of approximately 70% in pretreated samples. Among the tested media, MS medium ranked second with an induction efficiency of 34%, while ½ MS medium achieved only 12% efficiency under the same conditions. Notably, the VW medium failed to induce significant protocorm formation, suggesting its limited efficacy in this process. Figure 3 illustrates the germination of C. goeringii hybrid seeds (Figure 3a,b) and subsequent protocorm induction (Figure 3c,d) across different media.

3.3. Proliferation of Rhizomes

To optimize the medium for rhizome proliferation, we employed an orthogonal experimental design with four factors at three levels. As shown in

Table 1. Range analysis of the orthogonal experiment on rhizome proliferation and proliferation coefficient from protocorm explants of Cymbidium goeringii hybrids after 120 days of treatment. The experiment followed an L9 (3⁴) orthogonal design to evaluate the effects of four factors: 6-BA, NAA, IBA, and activated charcoal (AC), each at three concentration levels.

Treatment	Orthogonal Factors				Proliferation Rate (%)	Proliferation Coefficient
	6-BA (mg/L)	NAA (mg/L)	IBA (mg/L)	AC (g/L)
1	0.1	0.1	0.1	0.1	10.24	2.85
2	0.1	3.0	3.0	0.5	16.02	1.95
3	0.1	9.0	9.0	2.5	8.79	1.65
4	3.0	0.1	3.0	2.5	12.23	2.53
5	3.0	3.0	9.0	0.1	17.23	2.9
6	3.0	9.0	0.1	0.5	15.46	3.2
7	9.0	0.1	9.0	0.5	17.51	2.9
8	9.0	3.0	0.1	2.5	9.41	2.9
9	9.0	9.0	3.0	0.1	35.17	1.7
Proliferation rate (%) summary
Factors	Level 1	Level 2	Level 3	Range (R)	Best level
6-BA	11.683	14.973	20.697 *	9.014	Level 3 (9.0 mg/L)
NAA	13.327	14.22	19.807 *	6.48	Level 3 (9.0 mg/L)
IBA	11.703	21.14 *	14.51	9.437	Level 2 (3.0 mg/L)
AC	20.88*	16.33	10.143	10.737	Level 1 (0.1 g/L)
Proliferation coefficient summary
Factors	Level 1	Level 2	Level 3	Range (R)	Best level
6-BA	2.15	2.877 *	2.5	0.727	Level 2 (3.0 mg/L)
NAA	2.76 *	2.583	2.183	0.577	Level 1 (0.1 mg/L)
IBA	2.983 *	2.06	2.483	0.923	Level 1 (0.1 mg/L)
AC	2.483	2.683 *	2.36	0.323	Level 2 (0.5 g/L)

Note: An asterisk (*) indicates the best-performing value among the three tested levels for each factor. “Best level” refers to the factor level that yields the highest rhizome proliferation rate or proliferation coefficient. “Range (R)” reflects the degree of influence of each factor; a larger R value indicates that the differences among the levels of that factor had a stronger effect and contributed more to the variation, whereas a smaller R value suggests a weaker or less influential effect.

, treatment 9 achieved the highest proliferation rate (35.17%), although its proliferation coefficient (1.70) was only marginally higher than that of treatment 3. Treatment 9, with the highest auxin and cytokinin concentrations, induced both root and shoot differentiation, which is consistent with the known synergistic effects of these PGRs. In contrast, treatment 6 exhibited the highest proliferation coefficient (3.20) but a proliferation rate (15.46%) less than half that of treatment 9. Treatment 3 performed the poorest, with a proliferation rate of 8.79% and a coefficient of 1.65.

The orthogonal analysis identified 9.0 mg/L 6-BA (Level 3), 9.0 mg/L NAA (Level 3), 3.0 mg/L IBA (Level 2), and 0.1 g/L AC (Level 1) as the optimal formulation for rhizome proliferation (Table 1), which corresponded to treatment 9. In contrast, the optimal combination for the proliferation coefficient was 3.0 mg/L 6-BA (Level 2), 0.1 mg/L NAA (Level 1), 0.1 mg/L IBA (Level 1), and 0.5 g/L AC (Level 2). Notably, this combination did not match any of the nine tested treatments, suggesting that an additional experimental combination, outside of the original L9 array, may be required to optimize this parameter further.

Furthermore, the ANOVA results revealed that the AC had the most significant influence on the rhizome proliferation rate, followed by NAA, 6-BA, and IBA, all of which reached statistical significance (p < 0.05) (

Table 2. One-way ANOVA results showing the effects of 6-BA, NAA, IBA, and activated charcoal (AC) on the rhizome proliferation rate and proliferation coefficient in Cymbidium goeringii hybrids after 120 days of treatment.

Rhizome Proliferation Rate (%)
Source of Variance	Sum of Squares	Degree of Freedom	Mean Square	F Value	p Value
6-BA	468.548	2	234.274	6.431	0.006 *
NAA	492.136	2	246.068	6.755	0.005 *
IBA	266.104	2	133.052	3.653	0.041 *
AC	623.413	2	311.706	8.557	0.002 *
Error	874.243	24	36.427	-	-
Rhizome proliferation coefficient
Source of variance	Sum of squares	Degree of freedom	Mean square	F value	p value
6-BA	3.181	2	1.591	1.58	0.227
NAA	2.508	2	1.254	1.246	0.306
IBA	1.91	2	0.955	0.949	0.401
AC	1.619	2	0.809	0.804	0.459
Error	24.157	24	1.007	-	-

Note: An asterisk (*) indicates a statistically significant effect (p < 0.05) on the rhizome proliferation rate. None of the treatments showed a statistically significant effect on the rhizome proliferation coefficient (p > 0.05). A higher F value (greater than 1) suggests that the factor has a stronger effect relative to random variation (error).

). In contrast, none of the factors had a statistically significant effect on the rhizome proliferation coefficient (Table 2).

3.4. Rhizome Differentiation

To optimize the medium for rhizome differentiation, we established an orthogonal experimental design with four factors at three levels. As shown in

Table 3. Range analysis of the orthogonal experiment on rhizome differentiation and bud induction in Cymbidium goeringii hybrids after 120 days of treatment. The experiment followed an L9 (3⁴) orthogonal design to evaluate the effects of four factors: basal medium, 6-BA, NAA, and AgNO₃, each at three concentration levels.

Treatment	Orthogonal Factors				Differentiation Rate (%)	Average Number of Induced Buds
	Basal Medium	6-BA (mg/L)	NAA (mg/L)	AgNO3 (mg/L)
1	1/2MS	1.0	0.1	0.02	94	1.46
2	1/2MS	5.0	0.3	0.1	84	2.04
3	1/2MS	10	0.9	0.5	91	2.53
4	MS	1.0	0.3	0.5	100	2.68
5	MS	5.0	0.9	0.02	98	2.31
6	MS	10	0.1	0.1	100	3.93
7	Hyponex 2	1.0	0.9	0.1	9	0.09
8	Hyponex 2	5.0	0.1	0.5	15	0.3
9	Hyponex 2	10	0.3	0.02	3	0.07
Differentiation rate (%) summary
Factors	Level 1	Level 2	Level 3	Range (R)	Best level
Basal medium	0.897	0.993 *	0.09	0.903	Level 2 (MS)
6-BA	0.677 *	0.657	0.647	0.03	Level 1 (1.0 mg/L)
NAA	0.697 *	0.623	0.66	0.074	Level 1 (0.1 mg/L)
AgNO3	0.65	0.643	0.687 *	0.044	Level 3 (0.5 mg/L)
Summary of average number of induced buds
Factors	Level 1	Level 2	Level 3	Range (R)	Best level
Basal medium	2.01	2.973 *	0.153	2082	Level 2 (MS)
6-BA	1.41	1.55	2.177 *	0.767	Level 3 (10 mg/L)
NAA	1.897 *	1.597	1.643	0.3	Level 1 (0.1 mg/L)
AgNO3	1.28	2.02 *	1.837	0.74	Level 2 (0.1 mg/L)

Note: An asterisk (*) indicates the best-performing value among the three tested levels for each factor. “Best level” denotes the concentration level that resulted in the highest rhizome differentiation rate or average number of induced buds. “Range (R)” reflects the degree of influence of each factor; a larger R value indicates that the differences among the levels of that factor had a stronger effect and contributed more to variation, whereas a smaller R value suggests a weaker or less influential effect.

, treatment 6 (MS medium supplemented with 10 mg/L 6-BA, 0.1 mg/L NAA, and 0.1 mg/L AgNO₃) exhibited the highest performance, achieving a differentiation rate of 100% and an average of 3.93 induced buds per explant. In contrast, treatment 9 showed the lowest differentiation efficiency, with a differentiation rate of 3% and only 0.07 induced buds per explant. Notably, treatment 2 developed robust white roots during the same cultivation period, representing a significant morphological trait for rhizome development.

The orthogonal analysis identified MS (Level 2), 1.0 mg/L 6-BA (Level 1), 0.1 mg/L NAA (Level 1), and 0.5 mg/L AgNO₃ (Level 3) as optimal for the differentiation rate (Table 3). In contrast, the optimal bud induction combination (MS, 10 mg/L 6-BA, 0.1 mg/L NAA, 0.1 mg/L AgNO₃) matched treatment 6, indicating divergent hormonal requirements. Statistical validation via ANOVA (

Table 4. One-way ANOVA results showing the effects of basal medium, 6-BA, NAA, and AgNO₃ on rhizome differentiation and bud induction in Cymbidium goeringii hybrids after 120 days of treatment.

Rhizome Differentiation Rate
Source of Variance	Sum of Squares	Degree of Freedom	Mean Square	F Value	p Value
6-BA	0.008	2	0.004	0.174	0.841
NAA	0.043	2	0.022	0.962	0.388
AgNO3	0.021	2	0.011	0.467	0.629
Basal medium	10.344	2	5.172	228.932	0 *
Error	1.333	59	0.023	-	-
Bud induction rate
Source of variance	Sum of squares	Degree of freedom	Mean square	F value	p value
6-BA	5.617	2	2.808	6.062	0.004 *
NAA	0.913	2	0.456	0.985	0.38
AgNO3	6.228	2	3.114	6.722	0.002 *
Basal medium	77.97	2	38.985	84.154	0 *
Error	27.332	59	0.463	-	-

Note: An asterisk (*) indicates a statistically significant effect (p < 0.05) on either rhizome differentiation rate or bud induction rate. A higher F value (greater than 1) suggests that the factor has a stronger effect relative to random variation (error).

) confirmed the basal medium as the most influential factor for both responses (p = 0.000), with 6-BA (p = 0.004) and AgNO₃ (p = 0.002) significantly affecting bud induction.

3.5. In Vitro Flowering from Rhizomes

To explore the regulation of in vitro flowering from rhizomes, we implemented an orthogonal experimental design with four factors (basal medium, 6-BA, NAA, and TDZ) at three levels (

Table 5. Range analysis of the orthogonal experiment on in vitro flower bud induction rate from rhizomes and normal flower bud formation in Cymbidium goeringii hybrids after 180 days of treatment. The experiment followed an L9 (3⁴) orthogonal design to evaluate the effects of four factors: basal medium, 6-BA, NAA, and TDZ, each at three concentration levels.

Treatment	Orthogonal Factors				Flower Bud Induction Rate (%)	Normal Flower Bud Formation Rate (%)
	Basal Medium	6-BA (mg/L)	NAA (mg/L)	TDZ (mg/L)
1	MS	1.0	0.1	0.1	10	3
2	MS	3.0	0.3	0.3	8	4
3	MS	9.0	0.9	0.9	28	6.7
4	MS (1/3N, 3P)	1.0	0.3	0.9	8	0
5	MS (1/3N, 3P)	3.0	0.9	0.1	15	5
6	MS (1/3N, 3P)	9.0	0.1	0.3	36	16
7	MS (1/5N, 5P)	1.0	0.9	0.3	4	0
8	MS (1/5N, 5P)	3.0	0.1	0.9	8	0
9	MS (1/5N, 5P)	9.0	0.3	0.1	24	8
Summary ofin vitroflower bud induction rate
Factors	Level 1	Level 2	Level 3	Range (R)	Best level
Basal medium	0.153	0.197 *	0.12	0.077	2 MS (1/3N, 3P)
6-BA	0.073	0.103	0.293 *	0.22	3 (9.0 mg/L)
NAA	0.18 *	0.133	0.157	0.047	1 (0.1 mg/L)
TDZ	0.163 *	0.16	0.147	0.016	1 (0.1 mg/L)
Summary of normal flower bud formation rate
Factors	Level 1	Level 2	Level 3	Range (R)	Best level
Basal medium	0.047	0.07 *	0.027	0.043	2 MS (1/3N, 3P)
6-BA	0.011	0.03	0.102 *	0.091	3 (9.0 mg/L)
NAA	0.064 *	0.04	0.039	0.025	1 (0.1 mg/L)
TDZ	0.054	0.067 *	0.022	0.045	2 (0.3 mg/L)

Note: An asterisk (*) indicates the best-performing value among the three tested levels for each factor. “Best level” refers to the concentration level that resulted in the highest in vitro flower bud induction rate or normal flower bud formation rate. “Range (R)” reflects the degree of influence of each factor; a larger R value indicates that the differences among the levels of that factor had a stronger effect and contributed more to variation, whereas a smaller R value suggests a weaker or less influential effect.

). Successful induction and differentiation of flower buds, leading to in vitro flowering, were achieved (Figure 4).

Treatment 6 (MS (1/3N, 3P) medium supplemented with 9.0 mg/L 6-BA, 0.1 mg/L NAA, and 0.3 mg/L TDZ) resulted in the highest in vitro flower bud induction rate (36%), with a normal flower bud formation rate of 16% (Figure 4e). In contrast, treatment 7 exhibited the lowest flower bud induction rate (4%) and no normal flower bud formation (0.00), with induced buds either arresting during development or exhibiting morphological abnormalities (Figure 4d,f).

According to the orthogonal analysis, the optimal combination for in vitro flower bud induction was MS (1/3N, 3P) basal medium (Level 2), 9.0 mg/L 6-BA (Level 3), 0.1 mg/L NAA (Level 1), and 0.1 mg/L TDZ (Level 1). This combination closely aligns with the composition of treatment 6, although treatment 6 used 0.3 mg/L TDZ, confirming its efficacy. For normal flower bud formation, the optimal combination was again MS (1/3N, 3P) basal medium (Level 2), 9.0 mg/L 6-BA (Level 3), 0.1 mg/L NAA (Level 1), and 0.3 mg/L TDZ (Level 2), which also corresponds to treatment 6 (Table 5). These results strongly suggest that treatment 6 is the most effective for promoting both flower bud induction and normal flower bud formation from rhizomes (Figure 4e).

Moreover, the ANOVA results (

Table 6. One-way ANOVA results showing the effects of basal medium, 6-BA, NAA, and TDZ on in vitro flower bud induction rate from rhizomes and normal flower bud formation in Cymbidium goeringii hybrids after 180 days of treatment.

In Vitro Flower Bud Induction Rate
Source of Variance	Sum of Squares	Degree of Freedom	Mean Square	F Value	p Value
Basal medium	0.046	2	0.023	1.416	0.256
6-BA	0.382	2	0.191	11.72	0 *
TDZ	0.01	2	0.005	0.298	0.744
NAA	0.019	2	0.01	0.595	0.557
Error	0.603	37	0.016	-	-
Normal flower bud formation rate
Source of variance	Sum of squares	Degree of freedom	Mean square	F value	p value
Basal medium	0.014	2	0.007	0.811	0.452
6-BA	0.073	2	0.036	4.362	0.02 *
TDZ	0.016	2	0.008	0.98	0.385
NAA	0.006	2	0.003	0.387	0.681
Error	0.309	37	0.008	-	-

Note: An asterisk (*) indicates a statistically significant effect (p < 0.05) on either the in vitro flower bud induction rate or the normal flower bud formation rate. A higher F value (greater than 1) suggests that the factor has a stronger effect relative to random variation (error).

) indicated that among the four tested factors, only 6-BA had a statistically significant effect on both in vitro flower bud induction (p = 0.000) and normal flower bud formation (p = 0.02). In contrast, the effects of the basal medium, TDZ, and NAA were not statistically significant (p > 0.05). These findings emphasize the central role of 6-BA in regulating floral transition and organogenesis under in vitro conditions in C. goeringii hybrids.

4. Discussion

Basic media, hormones, and other exogenous additives are fundamental to plant tissue culture, influencing growth and development. While their applications are widespread, specific requirements for different explants, even within the same species and across varieties or developmental stages, remain complex, and their regulatory mechanisms are often unclear. This underscores the continued importance of research into plant tissue culture. This study specifically addressed the challenges associated with C. goeringii by investigating and successfully establishing an efficient system for both rhizome proliferation and, notably, direct in vitro flowering induction from protocorm-derived rhizomes. The protocol’s success highlights the potential for bypassing conventional developmental stages, offering a novel model for accelerating orchid breeding.

4.1. Seed Germination and Protocorm Induction

Pretreatment strategies for Cymbidium seeds exhibit genotype-dependent efficacy. Zeng reported accelerated germination with 10% NaOCl (10–20 min) [12], while Sun and Wang favored NaOH [2,13]. Contradictory optima—low-concentration NaOCl (0.5%, 4–6 min) vs. NaOH (0.1 mol/L, 20 min) [14,15]—likely reflect differential seed coat responsiveness among hybrids. Media suitability for Cymbidium protocorm induction is species-specific: 1/2 MS is optimal for C. sinense [16], C. hybridum [17], and C. ensifolium [18]; KC/modified MS for C. faberi, C. finlaysonianum, and C. mastersii [19,20,21,22]; and M/PM-medium for C. giganteum [23]. Our data corroborate KC’s superiority for C. goeringii, reinforcing the need for taxon-tailored formulations.

4.2. Rhizome Proliferation and Differentiation

Rhizome proliferation in C. goeringii is optimally driven by balanced auxin/cytokinin ratios, contrasting with reports that high auxin (>1:1 NAA:6-BA) promotes proliferation in other orchids [24,25,26,27,28]. Genotypic variation is critical, as evidenced by C. finlaysoniamum’s preference for low NAA:6-BA (1:2) [29]. Notably, activated charcoal (AC) enhanced rhizome proliferation while suppressing differentiation, a trade-off underscoring AC’s role in modulating morphogenic fate [30,31,32]. This optimized proliferation system provides uniform biomass essential for downstream flowering induction.

Rhizome differentiation in C. goeringii requires synergistic cytokinin dominance and ethylene suppression. MS basal medium exerted stronger effects than growth regulators alone. While 2,4-D or TDZ can independently induce buds in some orchids [33,34], 6-BA caused abnormal growth in prior Cymbidium studies [35,36]. Our protocol, combining high 6-BA (10 mg/L), low NAA (0.1 mg/L), and AgNO₃ (0.1–0.5 mg/L), achieved 100% differentiation (3.93 buds/explant), validating AgNO₃’s role as an ethylene inhibitor in bud maturation [37,38].

4.3. In Vitro Flowering Induction from Protocorm-Derived Rhizomes

This study pioneers direct in vitro flowering from C. goeringii rhizomes, circumventing conventional juvenile phases. Hormonal synergy (6-BA + TDZ) and modified MS (1/3N, 3P) were pivotal, with 6-BA confirmed as the primary driver of floral initiation (ANOVA, p < 0.05) [37,39,40]. Reduced nitrogen and elevated phosphorus promoted floral transition, aligning with Cymbidium’s flowering physiology [40,41]. This system reduces the flowering time by 60–80% (~6 months), enabling rapid trait screening. Future work should refine the nutrient ratios and investigate ammonium nitrogen’s role [40] to improve flower development rates and decipher the underlying molecular mechanisms.

4.4. Limitations of the Study

While our study successfully established a proof of concept for in vitro flowering initiation in C. goeringii, we acknowledge certain limitations. First, we used high PGR concentrations (up to 9 mg/L 6-BA). These levels are unusual for plant tissue culture and can lead to somaclonal variation. We found these elevated levels necessary to overcome C. goeringii’s notorious recalcitrance to in vitro flowering, a requirement similarly observed in C. niveo-marginatum (10 mg/L 6-BA) [42]. Future investigations must include comprehensive genetic stability assessments using genomics, transcriptomics, metabolomics, and hormone-profiling to refine the hormone regimes, balancing the induction efficiency with developmental normalcy. Second, our study currently lacks comparative physiological data between in vitro and ex vitro flowers. While direct physiological comparisons are challenging given the multi-year large-scale nature of conventional crosses (6–7 years), our primary aim was to demonstrate a 60–80% reduction in flowering time (from 6–7 years to 1–2 years) and establish a rapid screening platform. Future work will focus on ex vitro acclimatization, detailed floral-trait validation, and hormone-regime refinement to address this.

5. Conclusions

This study successfully established an efficient in vitro propagation system for C. goeringii hybrids, encompassing optimized protocols for seed germination, protocorm induction, and, notably, rapid rhizome proliferation. Building upon this, we achieved a significant breakthrough by developing a novel in vitro flowering induction system directly from protocorm-derived rhizomes. The optimized conditions, particularly the use of MS medium with a modified nitrogen–phosphorus ratio and specific hormone combinations (especially 6-BA and TDZ), enabled in vitro flowering within six months. This represents a substantial 60–80% reduction in the traditional breeding cycle of C. goeringii. The established system provides a valuable tool for accelerated trait screening, germplasm conservation, and the efficient development of new cultivars in this horticulturally important orchid species. Future research should focus on further enhancing the efficiency of normal in vitro flower development and elucidating the underlying physiological and molecular mechanisms governing flowering induction in C. goeringii rhizomes.