Bioreactor-Based Liquid Culture and Production of Konjac Micro-Corm

Abstract

Konjac (Amorphophallus konjac K. Koch) has numerous health benefits, but traditional propagation is hindered by long growth periods and soil-borne diseases. This study developed a novel cell liquid culture system to directly produce micro-corms of konjac for large-scale production. The results demonstrated significant improvements in bud induction and rooting compared to solid culture. Under MS + 1.0 mg/L 6-BA + 0.5 mg/L NAA, the induced buds per culture vessel and final fresh weight were 24.87 ± 0.06 and 6.64 ± 0.12 g, respectively, 1.95 and 1.67 times higher than those in solid culture. Rooting experiments showed that 1/2 MS + 0.5 mg/L IBA + 1.0 mg/L NAA resulted in a root length of 25.23 ± 0.04 cm and 18.12 ± 0.01 roots per vessel. Using a 5 L bioreactor for micro-corm induction led to a 2.51-fold increase in fresh weight (52.67 ± 0.01 g) after 31 days, with glucomannan production reaching 0.48 g/g _{(fresh weight)}. The optimized culture system also significantly reduced the propagation time and increased the yield of healthy micro-corms. Bioreactor-based cultivation effectively enhances konjac induction efficiency and shortens breeding time, making it a promising approach for commercial production of konjac micro-corms and potentially improving the economic viability of konjac farming.

1. Introduction

Konjac (Amorphophallus konjac K. Koch), a perennial herbaceous plant of the Araceae family, is widely distributed in Asian countries such as China and Japan [1]. Its underground corms are rich in glucomannan, alkaloids, and polyphenol oxidase, giving it various pharmacological activities such as blood sugar reduction, lipid-lowering, and anti-inflammatory properties. Recently, konjac has been utilized extensively in the food industry, especially as a low-calorie food for weight loss, which has been recognized by the World Health Organization as one of the top ten health products [2,3]. With the continuous expansion of the konjac industry chain, the planting area of konjac has been increasing in recent years. However, konjac is primarily produced by its micro-corms (also named konjac seeds), which are generally sourced from field farming and exhibit typical germplasm degradation characteristics such as virus infection, yield decline, and quality degradation. These propagation methods cannot fulfill the needs of the rapid increase in the cultivation area of konjac [4]. The use of cell engineering technologies is expected to achieve an efficient technology for konjac micro-corm production, which might promote the vigorous development of the whole konjac industry chain.

Compared to traditional cell tissue culture on solid media, the pathway of liquid culture has garnered widespread attention due to its advantages such as minimal material requirements, shorter growth cycles, and higher proliferation rates. It has already been demonstrated that liquid culture technology can efficiently increase the cell density of cowpeas in a short period, emphasizing the role of precise control over culture conditions in improving production efficiency [5]. The most effective combination of plant growth regulators for somatic embryogenesis of oil palm seeds on MS (Murashige and Skoog) medium through liquid culture methods has been identified [6]. In addition, bioreactors can further scale up the cultivation of plant cells or other culture types based on liquid culture, not only promoting plant growth but also increasing the synthesis of plant secondary metabolites, which is beneficial for the mode of industrial production [7,8,9]. However, there is a scarcity of research on liquid culture technology for the propagation of konjac corms. Most studies have focused on solid culture methods or only on the liquid culture of the sprouting process, lacking advancements in large-scale liquid culture for konjac seeds [10,11]. This gap has hindered the development of effective propagation strategies for konjac plantlets. In contrast, our research pioneers liquid culture techniques for konjac micro-corms, aimed at producing high-quality konjac seeds.

The spherical bioreactor was selected for its unique advantages in the large-scale production of konjac micro-corms. Its design ensures uniform mixing and oxygen transfer, which are critical for the efficient growth and development of micro-corms in liquid culture [12]. The bubble-driven circulation mechanism promotes liquid flow and nutrient distribution, reducing the need for mechanical agitation and minimizing shear stress on delicate tissues, which is particularly important for konjac micro-corms [13]. These features collectively make the spherical bioreactor an optimal choice for plant tissue mass propagation, laying a solid foundation for the large-scale commercial production of konjac micro-corms.

The objective of this study is to establish a rapid and effective method for generating konjac micro-corms using a bioreactor-based liquid culture system (Figure 1). It is expected to determine the optimal nutritional and environmental conditions for a konjac liquid culture system, thereby optimizing the cultivation process to yield more konjac micro-corms. In this study, we mainly focused on the induction of adventitious shoots within the liquid culture system, the induction of roots, and the conditions necessary for the growth of micro-corms. By measuring the number and fresh weight of adventitious buds, the number and length of roots, and the number and fresh weight of micro-corms, we can select the most suitable hormone ratios, sucrose concentrations, and photoperiods. The optimal factors identified through this screening process will then be used to establish the cultural conditions. Bioreactors will be employed to induce the proliferation of konjac, aiming to obtain high-yielding konjac plants, and the main storage substances of these plants will be identified. This research could more efficiently provide seeds for the field cultivation of konjac plants, address the issue of seed shortage in traditional planting, and provide technical support for subsequent industrial scale-up cultivation.

2. Materials and Methods

2.1. Plant Materials

Embryogenic callus of A. konjac was induced from petiole explants of tissue-cultured konjac seedlings, following the procedures described in a previous study by Li et al. [10]. The konjac callus was then cultured on MS solid medium, supplemented with 1.2 mg/L 6-BA (6-Benzylaminopurine), 1.2 mg/L NAA (1-Naphthaleneacetic acid), and 1.0 mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid), and containing 30 g/L sucrose and 5 g/L agar at pH 5.8. The cultures were incubated in the dark at 25 °C, and the medium was refreshed every two weeks.

2.2. Liquid Culture for Adventitious Shoot Inductions

Konjac callus that exhibited loose texture without browning was selected for further liquid culture. Approximately 1 g of fresh callus was chopped and transferred to 250 mL Erlenmeyer flasks, each containing 100 mL of liquid culture medium. To achieve optimal phytohormone combination for inducing adventitious shoots in konjac, the MS-based culture medium (pH 6.2) was supplemented with varying concentrations of 6-BA (0.5–3.0 mg/L) and NAA (0.5–1.5 mg/L). The treatment with the lowest concentrations of 6-BA (0.5 mg/L) and NAA (0.5 mg/L) was used as the control group, serving as the baseline for evaluating the effects of increased hormone concentrations. Based on the above-mentioned results, single-factor experiments were conducted with different sucrose concentrations (10–50 g/L) and inoculation amounts (1–6 g). For each experimental group, 60 bottles were used and placed on a shaker set at 25 ± 1 °C, with a light intensity of 2500 Lx, a photoperiod of 12 h/d, and a rotation speed of 70 rpm. Each experiment was repeated three times. After 31 days of liquid culture, the number and rate of adventitious shoot induction were determined and are as follows:

Induction rate = (Number of explants with shoot points)/(Number of total explants) × 100%

The optimal culture media formula (MS + 2.0 mg/L 6-BA + 0.5 mg/L NAA + 30 g/L sucrose; pH 6.2) was used to compare adventitious shoot induction in liquid culture and solid culture (with the addition of 5 g/L agar). Each culture group was inoculated with approximately 2 g of callus. The liquid culture was conducted under the same conditions as previously described. The solid medium cultures were maintained at 25 ± 1 °C, with an illumination intensity of 2500 Lx for 12 h each day. The number of shoots, fresh weight, and relative growth rate (RGR) were determined at 6, 12, 18, 24, and 30 days of culture as follows:

$$RGR(\mathrm{g}/\mathrm{g})={\displaystyle \frac{\ln W_2-\ln W_1}{t-1}}$$

where t represented the culture duration; W₁ and W₂ indicated the initial and final fresh weight of konjac explants, respectively.

2.3. Liquid Culture for Adventitious Root Formation

The individual konjac bud with callus at the base was then aseptically isolated to serve as the initial experimental material for adventitious root formation. Approximately 3 g of fresh weight of these materials was transferred to 100 mL of MS-based liquid culture medium containing 30 g/L sucrose (pH 6.2). Different treatment groups with IBA (indole-3-butyric acid) (0.1–0.9 mg/L), NAA (0.5–2.5 mg/L), and inorganic salts (1/2 MS to full MS strength) were established for single-factor experiments. The treatment with the lowest concentration of IBA (0.1 mg/L), NAA (0.5 mg/L), and MS strength was designated as the control group. Based on these treatments, a three-factor, three-level orthogonal experimental design was used to evaluate the effects of IBA, NAA, and inorganic salts on adventitious root formation (

Table 1. The design of inorganic salt concentration, IBA concentration, and NAA concentration level.

Level	IBA (mg/L)	NAA (mg/L)	Inorganic Salt (mg/L)
1	0.3	1.0	1/2MS
2	0.5	1.5	3/4MS
3	0.7	2.0	MS

). The liquid culture conditions were the same as those described previously. Observations of root growth and induction were made, and the root length and number of roots were recorded after 31 days of culture.

2.4. Liquid Culture for Micro-Corm Generations

Rooted callus with adventitious shoots was collected as the experimental material for micro-corm formation. Approximately 1 g of fresh weight of the material was transferred to an MS-based liquid culture medium (pH 6.2), with various treatment groups of 6-BA (0.5–3.0 mg/L), NAA (0.5–3.0 mg/L), KT (Kinetin) (0.2–1.4 mg/L), and sucrose (10–50 g/L). The treatment with the lowest concentrations of 6-BA (0.5 mg/L), NAA (0.5 mg/L), KT (0.2 mg/L), and 30 g/L sucrose was designated as the control group. A three-factor, three-level orthogonal experiment was conducted to assess the effects of 6-BA, NAA, and KT on micro-corm formation (

Table 2. The 6-BA, NAA and KT concentration level design.

Level	6-BA (mg/L)	NAA (mg/L)	KT (mg/L)
1	1.0	0.5	0.4
2	1.5	1.0	0.6
3	2.0	1.5	0.8

). Subsequently, the optimal culture medium (MS + 2.0 mg/L 6-BA + 1.5 mg/L NAA + 0.6 mg/L KT + 40 g/L sucrose; pH 6.2) was used to conduct further single-factor experiments on micro-corm formation, including inoculation size (2–7 g), photoperiod (4–24 h/d), and shaker rotational speed (50–100 rpm). The experiment investigated the effects of nitrogen and phosphorus concentrations on the induction of micro-corms (

Table 3. Effects of nitrogen and phosphorus concentrations on micro-corm induction.

Group	Level	Factor Concentration (mmol/L)			Increase in Fresh Weight (g)	Relative Growth Rate (g/d)
		NH4NO3	KNO3	Nitrogen
Nitrogen	1	5.2	4.7	15	3.13 ± 0.02 f	0.001
	2	10.3	9.4	30	4.54 ± 0.06 d	0.0138
	3	15.5	14.1	45	5.62 ± 0.01 c	0.021
	4	20.6	18.8	60	5.98 ± 0.05 b	0.023
	5	30.9	28.2	90	6.31 ± 0.04 a	0.025
	6	41.2	37.6	120	4.03 ± 0.03 e	0.010
NO3−/NH4+	1	1:1			4.78 ± 0.02 e	0.016
	2	2:1			7.21 ± 0.03 a	0.029
	3	3:1			6.31 ± 0.12 b	0.025
	4	4:1			6.02 ± 0.16 c	0.023
	5	5:1			5.70 ± 0.04 d	0.021
	6	90:0			1.81 ± 0.09 f	−0.020
KH2PO4	1	0.25			2.19 ± 0.09 d	−0.010
	2	0.5			3.20 ± 0.03 c	0.002
	3	0.75			5.23 ± 0.05 b	0.019
	4	1.0			7.01 ± 0.27 a	0.029
	5	1.25			6.89 ± 0.15 a	0.028
	6	1.5			4.91 ± 0.31 b	0.016

Data represent the mean values ± SE. Different letters indicate significant differences (p < 0.05).

). The MS medium with standard concentrations of NH₄NO₃ (20.6 mmol/L), KNO₃ (18.8 mmol/L), and KH₂PO₄ (1.25 mmol/L) was used as the control treatment to evaluate the effects of varying concentrations of these components on the fresh weight and relative growth rate of konjac micro-corms. An orthogonal experiment on the concentrations of nitrogen and phosphorus was also conducted (

Table 4. The nitrogen and phosphorus concentration orthogonal design.

Level	NH4NO3 (mmol/L)	KNO3 (mmol/L)	KH2PO4 (mmol/L)
1	10.3	9.4	0.75
2	15.5	14.1	1.0
3	20.6	18.8	1.25

). The liquid culture conditions were the same as previously described. After 31 days of cultivation, the growth morphology was observed, and the fresh weight and number of micro-corms were determined.

2.5. Bioreactor-Based Scale-Up Culture of Micro-Corms

A 5 L spherical bioreactor was used to scale up the culture of konjac micro-corms. The culture medium was prepared with MS medium supplemented with 2.0 mg/L 6-BA, 1.5 mg/L NAA, 0.6 mg/L KT, and 40 g/L sucrose (pH 6.2). Different volumes of culture liquid (2.0–3.5 L) were added to the bioreactor. The micro-corms were divided into small corms of approximately 0.5 g each and inoculated into the culture medium at a density of 5 g/L. The aeration rate was set to 200 cm³/min. The liquid culture conditions were the same as previously described. The fresh weights of the micro-corms were measured after 31 days of cultivation, and the main bioactive compounds were identified.

2.6. Determination of Glucomannan Content

Konjac micro-corms, devoid of skins and adventitious buds, were oven-dried and ground into powder. A mixture of 1 g of micro-corm powder and 25 mL of distilled water was prepared, dissolved overnight, and continuously stirred at 35 °C in a water bath. The glucomannan content in the micro-corms was determined using the dinitrosalicylic acid (DNS) method as described by Chua [14]. The wavelength for the absorbance measurement was set at 540 nm.

2.7. Statistical Analyses

The experiment was repeated three times. Results are presented as mean values ± standard error (SE). All data were analyzed using Microsoft Excel 2013 and IBM SPSS Statistics 25. For data obtained from a one-factor experimental design, one-way analysis of variance (ANOVA) was applied to assess the significant differences in the mean values of the indicators; for data obtained from a multi-factor experimental design, multivariate analysis of variance (MANOVA) was used to determine the significant differences in the mean values of multiple factors. Post hoc comparisons were conducted using the Duncan test to identify specific differences between groups.

3. Results

3.1. The Induction of Adventitious Shoots Under Liquid Culture

The key role of phytohormones in adventitious shoot induction of Amorphophallus was first explored. As shown in Figure 2a,b and

Table 5. The impact of 6-BA and NAA concentration on bud and fresh weight in bud induction.

6-BA (mg/L)	NAA (mg/L)	Induction Rate (%)	Number of Bud (nuo)	Fresh Weight (g)	Bud Morphology
0.5		91.0 ± 0.07 e	10 ± 0.12 a	1.67 ± 0.10 c	A higher number of bud spots, tender, with no obvious micro-corm formation
1.0		98.7 ± 0.02 a	8 ± 0.02 b	2.09 ± 0.03 a	The number of bud spots is moderate, but the bud spots are generally more mature, conducive to the next step of differentiation.
1.5		93.8 ± 0.02 d	3 ± 0.04 e	1.88 ± 0.03 b	The number of bud spots is relatively low, and the growth is slow.
2.0		95.2 ± 0.04 c	2 ± 0.14 f	1.45 ± 0.02 de	Few bud spots, and the buds are immature.
2.5		96.4 ± 0.01 b	5 ± 0.05 b	1.52 ± 0.11 d	Numerous bud spots with obvious micro-corm growth, exhibiting signs of aging.
3.0		90.3 ± 0.03 f	6 ± 0.07 c	1.36 ± 0.06 e	Small and numerous bud spots, with no obvious micro-corm formation.
1	0.5	100	28.01 ± 0.05 a	4.09 ± 0.09 a	Many bud spots, and there is obvious konjac formation below the bud spots
1	1.0	100	6.00 ± 0.21 c	2.78 ± 0.15 c	Few bud spots, but the Amorphophallus are larger and more pronounced.
1	1.5	100	16.53 ± 0.16 b	3.32 ± 0.14 b	Numerous bud spots, but no obvious formation of konjac.

Data represent the mean values ± SE. Different letters indicate significant differences (p < 0.05).

, konjac adventitious shoots exhibited the maximum induction rate and fresh weight at a 6-BA concentration of 1.0 mg/L (98.7 ± 0.02% and 2.09 ± 0.03 g, respectively), with 8 ± 0.02 buds per culture vessel. Although the group treated with 0.5 mg/L 6-BA produced the most buds (10 ± 0.12 per culture vessel), its induction rate was significantly lower. Therefore, the 6-BA concentration of 1.0 mg/L was chosen as the best condition for further analysis. Combinations of hormones (1.0 mg/L 6-BA with various NAA concentrations) were also tested in experiments. Interestingly, all treatment groups exhibited a 100% induction rate of adventitious shoots (Figure 2c and Table 5). Among these, the combination of 1.0 mg/L 6-BA and 0.5 mg/L NAA resulted in pink and slightly expanded bracts and the highest bud number and fresh weight, which increased by 3.5 and 1.96 times, respectively, compared to the single application of 1.0 mg/L 6-BA (Figure 2c and Table 5).

Further research was conducted to investigate the effects of sucrose concentration and inoculation amount on adventitious shoot induction in konjac (Figure 2d–g). As sucrose concentration increased, the number of buds and fresh weight initially increased and then subsequently decreased (Figure 2f). The highest bud number and fresh weight were observed at a sucrose concentration of 30 g/L (30.06 ± 0.05 and 6.34 ± 0.02 g per culture vessel, respectively), which were 2.09 and 1.73 times higher, respectively, than those at a sucrose concentration of 10 g/L (Figure 2f). Additionally, an inoculation amount of 2 g/L was identified as the optimal condition for maximizing fresh weight (Figure 2g). In summary, the MS-based medium supplemented with 1.0 mg/L 6-BA, 0.5 mg/L NAA, and 30 g/L sucrose, with an inoculation amount of 2 g/L of callus, was the best combination for inducing adventitious shoots in konjac callus liquid culture.

To investigate the effects of different culture methods on the induction and growth of konjac adventitious shoots, their development processes in solid and liquid culture were studied further. As demonstrated in Figure 3, the relative growth rate in solid culture increased more rapidly than in liquid culture within 12 days, but then decreased drastically between 12 and 30 days. In solid culture, small bud tips with noticeable browning phenomena were detected, and no visible micro-corm development was observed (Figure 3a). However, when cultured in liquid, konjac seedling formation was found clearly on the surface of the tissue, some of which had expanded to form the body of the micro-corm, and the browning phenomenon was significantly reduced or inhibited (Figure 3a). The number (24.87 ± 0.06) and final fresh weight (6.64 ± 0.12 g) of adventitious buds per culture vessel in liquid cultures were 1.95 and 1.67 times higher, respectively, than those in solid culture (Figure 3b). These findings showed that liquid culture clearly has a positive impact on the initiation and development of konjac adventitious shoots.

3.2. The Formation of Adventitious Roots in Liquid Culture

The effects of inorganic salts (ranging from 1/2 MS to full MS strength), IBA (0.1–0.9 mg/L), and NAA (0.5–2.5 mg/L) on rooting induction were studied in konjac. As shown in Figure 4a, the optimal rooting effect was observed at the 1/2 MS inorganic salt concentration, characterized by the thickest and longest roots. Both excessively high and low levels of IBA and NAA were unfavorable for the formation of konjac roots (Figure 4b,c). For the single-factor test of IBA, the highest rooting number was observed at a concentration of 0.5 mg/L, which was 1.96 times higher than that at 0.1 mg/L IBA (Figure 4b). Although the group treated with 0.7 mg/L IBA exhibited the longest root length, the number of roots was significantly lower (Figure 4b). For the single-factor test of NAA, the maximum number of induced roots was 10.32 ± 0.09 roots per culture vessel at an NAA concentration of 1.0 mg/L, representing a 1.47-fold increase compared to that at 0.5 mg/L NAA (Figure 4c). Through orthogonal experimental design and analysis, the concentration of inorganic salts was identified as the main factor affecting rooting length, while the concentrations of IBA and NAA significantly affected the rooting number (roots per culture vessel) (

Table 6. Orthogonal table range analysis of inorganic salt concentration, IBA concentration, and NAA concentration.

Experiment Number	IBA A	NAA B	Inorganic Salt C	Root Length (cm)	Number of Roots (nuo)
1	1	1	1	21.21	20.17
2	1	2	2	15.32	15.54
3	1	3	3	6.35	10.75
4	2	1	3	8.52	18.01
5	2	2	1	24.19	16.45
6	2	3	2	17.28	12.38
7	3	1	2	13.74	14.83
8	3	2	3	4.63	8.96
9	3	3	1	20.78	9.15
Root length
K1	42.88	43.47	66.18
K2	49.99	44.14	46.32
K3	32.94	44.41	19.50
R	3.61	0.31	15.56
Primary and secondary factors			C > A > B
Optimal combination			A2B3C1
Number of roots
K1	46.46	53.01	45.77
K2	46.84	46.82	42.75
K3	42.94	32.38	37.72
R	1.3	6.88	2.68
Primary and secondary factors			B > C > A
Optimal combination			A2B1C1

and

Table 7. Variance analysis of root length and root number results of inorganic salt concentration, IBA concentration, and NAA concentration.

Group	Source	Sum of Squares	df	Mean Square	F	Sig.	Significance
Root length	A	15.906	2	7.953	35.461	0.118
	B	0.086	2	0.043	0.191	0.850
	C	236.273	2	118.136	526.767	0.031	*
	Error	0.224	1	0.224
	Total	1891.458	8
	Total corrected	344.666	7
Number of roots	A	41.794	2	20.897	176.147	0.006	**
	B	72.261	2	36.130	304.554	0.003	**
	C	11.025	2	5.512	46.466	0.021	*
	Error	0.237	2	0.119
	Total	1896.043	9
	Total corrected	125.317	8

* and ** indicate the significant (p < 0.05 and p < 0.01) relationships, respectively.

). In summary, the optimal culture condition for adventitious root formation in konjac was determined to be a 1/2 MS medium supplemented with 0.5 mg/L IBA and 1.0 mg/L NAA.

3.3. Konjac Micro-Corm Induction and Growth Under Liquid Culture

In this study, the effects of 6-BA, NAA, and KT on konjac micro-corm induction were investigated, and it was found that hormone concentration significantly affected the fresh weight and quantity of konjac micro-corms. In Figure 5a, the morphological characteristics of adventitious buds and roots are shown. At a concentration of 1.5 mg/L 6-BA, the fresh weight of micro-corms per culture vessel reached a maximum of 3.25 g, and at 1.0 mg/L, the maximum number of micro-corms per culture vessel was 6.34 ± 0.21 (Figure 5c). The increase in NAA concentration had little effect on the fresh weight of micro-corms, but when the concentration reached 2.0 mg/L, the number of micro-corms significantly decreased, being only 0.77 times that at an NAA concentration of 1.5 mg/L, indicating that high NAA concentrations inhibit micro-corm induction (Figure 5d). The maximum fresh weight of micro-corms per culture vessel induced by KT was 2.12 ± 0.16 g at 0.6 mg/L, and the maximum number of micro-corms per culture vessel was 4.8 ± 0.11 at 0.8 mg/L (Figure 5e). The orthogonal experimental design and analysis further confirmed the interactions between the hormones. The range analysis results in

Table 8. Orthogonal table range analysis of 6-BA concentration, NAA concentration, and KT concentration.

Experiment Number	6-BA A	NAA B	KT C	Fresh Weight (g)	Number of Micro-Corm (nuo)
1	1	1	1	7.31	7.23
2	1	2	2	6.28	8.79
3	1	3	3	9.52	7.09
4	2	1	3	8.67	12.41
5	2	2	1	7.90	10.53
6	2	3	2	10.54	15.87
7	3	1	2	9.32	19.90
8	3	2	3	9.65	18.16
9	3	3	1	11.42	19.66
Fresh weight
K1	23.11	25.30	26.63
K2	27.11	23.83	26.14
K3	30.39	31.48	27.84
R	2.43	2.55	0.57
Primary and secondary factors			B > A > C
Optimal combination			A3B3C3
Number of micro-corms
K1	23.11	39.54	37.42
K2	38.81	37.48	44.56
K3	57.72	42.62	37.66
R	11.53	1.71	2.38
Primary and secondary factors			A > C > B
Optimal combination			A3B3C2

showed that for fresh weight, the influence of each factor was NAA > 6-BA > KT, which differed from the single-factor experiment results due to interactions among hormones. In terms of the number of micro-corms, the influence of the factors was A > C > B, with A3B3C2 being the best combination. According to the analysis of variance of fresh weight in

Table 9. Variance analysis of fresh weight and number of micro-corms of 6-BA concentration, NAA concentration and KT concentration.

Group	Source	Sum of Squares	df	Mean Square	F	Sig.	Significance
Fresh weight	A	10.986	2	5.493	49.651	0.020	*
	B	8.862	2	4.431	40.051	0.024	*
	C	0.510	2	0.255	2.307	0.302
	Error	0.221	2	0.111
	Total	742.577	9
	Total corrected	20.580	8
Number of micro-corms	A	4.461	2	2.231	1.587	0.387
	B	200.214	2	100.107	71.214	0.014	*
	C	10.961	2	5.480	3.899	0.204
	Error	2.811	2	1.406
	Total	1808.862	9
	Total corrected	218.448	8

* Indicates the significant (p < 0.05) relationships.

, the effects of 6-BA and NAA on fresh weight were significant, while that of KT was not, indicating that factors A and B are at 3 levels. In the quantitative variance analysis of micro-corms, the effect of NAA was significant at the B3 level, while the effects of 6-BA and KT were not significant (Table 9). Considering cost-effectiveness, the best hormone combination for micro-corm induction was determined to be A3B3C2, namely 2.0 mg/L 6-BA, 1.5 mg/L NAA, and 0.6 mg/L KT. Additionally, testing the effect of different sucrose concentrations on micro-corm induction revealed that the fresh weight and quantity of micro-corms per culture vessel were optimal at a sucrose concentration of 40 g/L, with the number of micro-corms per culture vessel being 12.56 ± 0.07, which was 1.66 times higher than that at 10 g/L sucrose (Figure 5g).

This study deeply explored the key factors affecting the induction efficiency of konjac micro-corms and was devoted to optimizing the induction process through accurately adjusting the experimental conditions to achieve efficient reproduction of konjac.

As shown in Figure 6a,b, the number of induced micro-corms per culture vessel was 8.78 ± 0.02 with an inoculation amount of 3 g, which was 12.81% less than that with an inoculation amount of 7 g, but the fresh weight was 3.59 times higher; thus, an inoculation amount of 3 g was determined to be optimal. Moreover, the RGR and fresh weight of micro-corms increased initially and then decreased with increasing photoperiod, reaching their highest values at a light duration of 12 h/d (0.031 and 7.67 ± 0.12 g, respectively) (Figure 6c,d). At a light duration of 16 h/d, the RGR and fresh weight were 0.03 and 7.36 ± 0.09 g, respectively. Considering that the growth morphology under 16 h/d light was much better than that under 12 h/d, and the subculture effect was more favorable, this study determined that the optimal light duration is 16 h/d. The induction of micro-corms cannot occur without oxygen, and an appropriate oxygen concentration facilitates efficient micro-corm induction. At a rotation speed of 70 rpm, the fresh weight per culture vessel is maximized at 8.72 ± 0.03 g, and the relative growth rate is 0.036 g/d (Figure 6e,f).

As a key component of proteins and nucleic acids, nitrogen source is very important for the growth of plants. It showed that the fresh weight of induced micro-corms initially increases and then decreases with increasing nitrogen source concentration (Table 3). When the total nitrogen source concentration reaches 90 mmol/L, the fresh weight per culture vessel increase reaches 6.31 ± 0.04 g, which was 1.06 times that of the control group. Under the condition of a fixed total nitrogen source concentration at 90 mmol/L, the ratio of nitrate to ammonium nitrogen varies in the media, and then check the growth of micro-corms. The results are presented in Table 3. As the ratio of nitrate to ammonium nitrogen increases, the fresh weight of induced micro-corms initially increases and then decreases. When the ratio is 2:1, the fresh weight of micro-corms is maximized at 7.21 ± 0.03 g. Phosphorus, one of the three major nutrients essential for plant growth, primarily exists in the form of KH₂PO₄ in the culture medium. To explore whether different concentrations of phosphorus affect the induction of micro-corms, the concentration of KH₂PO₄ was modified in this study. The objective was to determine the concentration of phosphorus that would achieve the experimental purpose. As the concentration of KH₂PO₄ increases, the fresh weight of micro-corms initially increases and then decreases; when the concentration is 1.0 mmol/L, the fresh weight increase in micro-corms is 7.01 ± 0.27 g, and the relative growth rate is 0.029 g/d (Table 3). Compared to the control group, it increased by 0.12 g per vessel and 0.001 g/d, respectively.

Different concentrations of nitrogen and phosphorus had a significant impact on the induction of micro-corms. This study designed an L₉(3⁴) orthogonal experiment. The nine sets of experimental results indicate that different concentrations of nitrogen and phosphorus, when interacting, promote seedling growth to varying degrees (Figure 7). More adventitious roots were observed in experiments 3 and 4; however, the growth of seedlings in experiment 6 was found to be the best. The seedlings in experiment 8 were relatively robust, and the induced micro-corms were tender. The micro-corms in experiments 1 and 9 were smaller in size. In terms of the fresh weight of induced micro-corms, all three factors were significant, with the order of priority being KNO₃ > NH₄NO₃ > KH₂PO₄. The optimal combination was A2B2C1, which corresponds to NH₄NO₃ at 15.5 mmol/L, KNO₃ at 14.1 mmol/L, and KH₂PO₄ at 0.75 mmol/L (

Table 10. Orthogonal table range analysis of nitrogen and phosphorus.

Experiment Number	NH4NO3 A	KNO3 B	KH2PO4 C	Increase in Fresh Weight (g)
1	1	1	1	8.34
2	1	2	2	7.39
3	1	3	3	6.54
4	2	1	3	10.32
5	2	2	1	11.10
6	2	3	2	7.09
7	3	1	2	6.13
8	3	2	3	9.21
9	3	3	1	6.64
Increase in fresh weight
K1	22.27	24.79	26.08
K2	28.51	27.70	20.61
K3	21.98	20.27	26.07
R	2.18	2.48	1.82
Primary and secondary factors		B > A > C
Optimal combination		A2B2C1

and

Table 11. Variance analysis of the increase in fresh weight of nitrogen and phosphorus.

Source	Sum of Squares	df	Mean Square	F	Sig.	Significance
A	9.074	2	4.537	69.358	0.014	*
B	9.345	2	4.672	71.431	0.014	*
C	6.637	2	3.318	50.733	0.019	*
Error	0.131	2	0.065
Total	613.410	9
Total corrected	25.186	8

* Indicates the significant (p < 0.05) relationships.

).

3.4. Scale-Up Culture of Micro-Corms in Spherical Bioreactor and Identification of Main Storage Substances

Micro-corms were enlarged and cultured to explore the effects of different liquid filling volumes on induction. All experiments were carried out in spherical bioreactors (Figure 8a). When the liquid volume was 3 L, the fresh weight of micro-corms per culture vessel was the largest, approximately 52.67 ± 0.01 g, and the fresh weight increased by a factor of 5.21. Using the glucose standard curve y = 1.0514x−0.039 with R^2 = 0.9955, the glucomannan content in the harvested micro-corms was determined to be 48.26%. In the starch color development experiment, the newly induced part turned blue upon exposure to the iodine solution, while the original material part (encircled in red) showed no change in color (Figure 9).

4. Discussion

4.1. Liquid Culture of Adventitious Bud Induction

In this text, it has been found that the induction of adventitious buds is a process influenced by multiple factors, including explants, plant growth regulators, and culture media. Plant growth regulators play a significant role in regulating cell differentiation and in vitro culture. Leveraging the totipotency of plant cells, the appropriate combination of growth regulators can induce the formation of adventitious buds. This study shows that the ratio of hormones plays a key role in the induction of konjac adventitious buds, with the balance between auxin and cytokinin being crucial for bud formation. Li et al. [10] found that supplementing the MS medium with 6-BA at 2.0 mg/L, NAA at 0.5 mg/L, gibberellin at 0.1 mg/L, and sucrose at 30 g/L can induce seedling formation in one step, with an average of 6 ± 2 regenerated buds per explant. When the dosage ratio of 6-BA to NAA was 2:1, the number of buds was the highest, reaching 28.01 ± 0.05, which is consistent with the findings of Li et al. [10]. This indicates that appropriate concentrations of 6-BA and NAA are beneficial for the induction of konjac adventitious buds.

Liquid culture systems have been widely recognized for their advantages in plant tissue culture, including rapid cell proliferation, efficient nutrient utilization, and reduced issues such as browning and nutrient depletion compared to solid culture systems [15,16,17]. Previous studies have demonstrated that optimizing liquid culture conditions and bioreactor structures can significantly enhance the proliferation rate of plant cells and the synthesis efficiency of secondary metabolites, especially in medicinal plants [18]. In this study, the liquid culture system significantly improved the induction efficiency and growth quality of konjac adventitious buds, resulting in healthier morphology and less browning compared to solid culture. These improvements are likely attributed to the enhanced nutrient availability and oxygen exchange in liquid systems, which support more vigorous metabolic activity and reduce oxidative stress in explants. These findings align with previous reports on other plant species, such as Malus domestica and Musa sp., where liquid culture improved tissue growth and reduced browning issues [15,18]. Therefore, liquid culture exhibits unique advantages in addressing browning and nutrient depletion commonly observed in solid culture systems, providing technical support for the efficient propagation of konjac.

4.2. Liquid Culture for Induced Rooting

Inorganic salt strength plays a crucial role in the rooting culture process of konjac, as plant growth depends on adequate hydration and nutrition of minerals [19,20]. In this study, the rooting of konjac explants was significantly influenced by the optimization of inorganic salt strength. The results revealed that the 1/2 MS medium combined with 1.0 mg/L IBA and 0.5 mg/L NAA was most effective in promoting root induction. These findings are consistent with previous studies on other plant species, such as Pongamia pinnata and hybrid Cymbidium, where lower salt concentrations promoted better rooting performance [21,22].

High concentrations of plant growth regulators (PGRs), however, negatively impacted root length and number [23]. Studies have shown that high concentrations of auxin can disrupt the balance between cell division and elongation near the root apex in Camellia sinensis, leading to excessive cell proliferation, impaired cell elongation, and ultimately inhibiting root tip elongation [24]. In addition, high concentrations of plant growth regulators may lead to the accumulation of H₂O₂, which damages cellular structures, inhibits root cell elongation, and ultimately suppresses root growth [24]. This study found that high concentrations of IBA and NAA were detrimental to the growth of root number and length in konjac micro-corms, which is consistent with the findings of Ivanchenko et al. [25].

4.3. Liquid Culture Induced by Micro-Corms

During the in vitro culture of plantlets, organs, tissues, or cells, it is necessary and important for these cultures to absorb the nutrients from the culture medium, where sucrose serves as the most important nutrient and energy source [26]. A sucrose concentration that is too low can lead to insufficient plant growth due to inadequate nutrient absorption. An excessively high sucrose concentration will inhibit plant growth [27,28]. High sucrose concentrations can elevate the osmotic potential of the medium, reducing water uptake by tissues and hindering cell expansion and division, thereby decreasing bud numbers and fresh weight. Moreover, excessive sucrose may disrupt metabolic balance, leading to carbon overload that favors the accumulation of storage compounds rather than active growth [29]. Oxidative stress induced by high sucrose levels, due to the overproduction of reactive oxygen species (ROS), may further damage cellular structures, impair metabolic functions, and reduce tissue vitality. Through the study on the effects of different carbon sources on the biomass of Hancornia speciosa (Gomes), it was found that 30 g/L sucrose promotes the growth of Hancornia speciosa (Gomes) [29]. Furthermore, sucrose concentration also significantly influences the plant’s metabolic products. Using response surface methodology to optimize the sucrose concentration, stirring speed, and naphthalene acetic acid concentration in the callus cell liquid culture of Hibiscus cannabinus, it was found that these three factors significantly affected the biomass of callus [30]. In this study, the optimal culture conditions for inducing A. konjac were an MS medium supplemented with 2.0 mg/L 6-BA, 1.5 mg/L NAA, 0.6 mg/L KT, and 40 g/L sucrose. The optimal sucrose concentration for micro-corms in this study differs, possibly because micro-corms synthesize a large amount of glucomannan during their growth process, requiring a substantial amount of carbon sources [31,32]. However, sucrose concentrations exceeding this optimal level likely impose osmotic and oxidative stress, ultimately reducing bud numbers and fresh weight.

Light, as an environmental signal, significantly influences plant growth and development [33,34,35]. Light can influence the induction rate during plant growth, but insufficient light duration can reduce this rate [36]. Excessive light duration can lead to sunburn diseases, making it essential to select the appropriate light exposure [37]. It has been shown that long-day treatments often promote an increase in the dry weight of plants originally grown under short-day conditions [38]. In the present study, culturing with a specific dark period, compared to continuous light, resulted in greater micro-corm biomass and improved morphology. Interestingly, the optimal illumination time for inducing micro-corms under liquid culture in this study is 16 h, indicating a dependency on light for micro-corm induction, consistent with previous findings [38]. Konjac seedlings under 12 h/d and 16 h/d illumination can produce a large number of seedlings in a single subculture, indicating their ability to use photosynthesis to provide additional nutrients for konjac growth.

In a series of studies in recent years, the importance of nitrogen and phosphorus in plant tissue culture has garnered extensive attention and has been widely verified. These elements have been shown to play vital roles in plant cell proliferation, differentiation, and the synthesis of secondary metabolites [39,40]. By precisely controlling the supply of these key nutrients, the efficiency and quality of plant tissue culture can be significantly improved, thereby promoting plant growth and development and increasing the production of bioactive compounds. In Leucojum aestivum shoot culture, the production of galantamine was optimized by applying different concentrations of sucrose, NO₃⁻, NH₄⁺, and PO₄³⁻ [41]. Yang et al. [42] studied the effects of carbon and nitrogen sources on biomass accumulation and flavonoid formation in licorice callus and found that nitrate nitrogen was beneficial to the growth of licorice callus and the accumulation of flavonoids. When the NO₃⁻/NH₄⁺ concentration ratio was 2:1, the sum of the five flavonoids in the licorice callus reached its maximum value of 151.47 μg·g⁻¹. This study shows that a NO₃⁻/NH₄⁺ concentration ratio of 2:1 is most beneficial to the liquid culture of micro-corms. The results of this experiment align with those of Yang et al. [42], indicating a similar beneficial effect of the nitrate-to-ammonium ratio on plant growth and metabolism. Interestingly, the added amounts of NH₄NO₃, KNO₃, and KH₂PO₄ obtained in this experiment are all less than those in MS medium, suggesting that during the growth and development of micro-corms, large amounts of nitrogen and phosphorus elements are not required to maintain cell proliferation, differentiation, and the synthesis of secondary metabolites. This study successfully optimized the culture conditions for the induction of Amorphophallus konjac through precise control of hormone concentration, inoculum volume, light duration, shaker speed, and nitrogen–phosphorus nutrient ratio, providing a scientific basis for the efficient propagation of Amorphophallus konjac and also for its efficient reproduction. It provides an important reference for the further development of plant tissue culture technology.

4.4. Scale-Up Culture of Micro-Corms in Spherical Bioreactor and Identification of Main Storage Substances

The scarcity of seed sources has prompted the exploration of faster plant seed production methods. Bioreactors offer a potential solution to this issue, providing an optimal growth environment with a uniform nutrient and oxygen supply and controllable environmental conditions. These conditions promote plant morphology, biomass, and the synthesis of plant secondary metabolites [43,44,45]. Furthermore, their scalability supports efficient mass production and significantly increases productivity. In the micropropagation of Pennisetum × advena “Rubrum”, Pożoga et al. used a temporary immersion system with MS medium containing 1 mg/L BAP, obtaining over 90% of new plants in half the time compared to agar culture, with no signs of vitrification [46]. In this study, the fresh weight of micro-corms cultured in a spherical bioreactor increased by 1.05 times compared to that of the 3 g culture in an Erlenmeyer flask, indicating the significant role of the bioreactor in micro-corm growth.

5. Conclusions

This research is dedicated to developing konjac seed breeding technology based on bioreactors, with the aim of establishing a konjac cell engineering seed breeding system for efficient and low-cost production of seeds, thereby promoting the rapid development of the konjac industry. Through an in-depth exploration of the optimal conditions for micro-corm induction, we have completed the scale-up culture in bioreactors and identified the main storage substances. Future research can further explore the effects of hormone ratios and culture conditions on other physiological characteristics of konjac, as well as how to further improve the quality and yield of konjac seeds, potentially opening up new directions for the development of tissue culture technology for konjac and other plants.