Curcumin-rich plant material production through a robust and reproducible micropropagation protocol

Abstract

Curcuma longa Linné is a widely valued spice due to its nutritional and therapeutic properties. Its production methods, vulnerability to diseases, and increasing anthropogenic pressure contribute to the limited availability of planting materials. This study aims to develop a micropropagation protocol that ensures the production of curcumin-rich planting stocks. Rhizomes were either pre-treated or not with Copper hydroxide (8%), disinfected with Mercuric chloride (HgCl₂ at 0.5%, 1%, or 2%), then treated with Ethanol (70%), followed by disinfection with Sodium hypochlorite (NaClO at 30%, 40%, or 50%). Buds from primary and secondary rhizomes were cultured on Murashige and Skoog medium supplemented with 6-benzylaminopurine (2 mg/L or 4 mg/L), Kinetin (4 mg/L), and Magnesium chloride (0.75 g/L). Data were analyzed using ANOVA, and a linear model was applied to determine the relationships between parameters and treatments. The optimal disinfection protocol that minimized contamination and necrosis while promoting successful in vitro establishment consisted of HgCl₂ (0.1%), NaClO (30%), and Ampicillin (0.1%). The addition of Magnesium chloride (MgCl₂) to the culture medium enhanced organogenesis and proliferation, with significant improvement when combined with BAP (4 mg/L), resulting in increased shoot proliferation (15.4 shoots per plant), root number (12.1 ± 0.15), leaf development (11.3 ± 0.08), as well as improved shoot elongation (12.38 ± 1.4 cm) and root elongation (5.18 ± 0.5 cm). During acclimatization, the “Terreau Semis Pouss’activR U” (TSP) substrate ensured optimal survival (100%) and better shoot and leaf growth compared to the local substrate composed of sand and compost (1:1) and the SAH substrate based on moderately decomposed sphagnum peat and black peat. This optimized protocol supports large-scale of curcumin-rich plant material production and enhanced valorization of curcuma.

Introduction

Curcuma longa L. is a perennial, herbaceous, and rhizomatous plant [1] belonging to the Zingiberaceae family [2]. Its annual global production reaches approximately 1.1 million tons [3]. Its rhizome, highly valued [4], provides curcumin, which is well known for its therapeutic properties in traditional Indian and Chinese medicine [5]. Its importance for human health is largely attributed to this bioactive compound with significant pharmacological effects [6]. Considered a major spice capable of treating a wide range of ailments [2], curcuma is used in West Africa to color various products, including textiles and foods [1, 7]. Its rhizomes are also used as cosmetic products in Africa and Asia [5]. In Benin, where more than 80% of the population relies on traditional medicine, curcuma plays an essential role because of the curcumin-rich of the rhizome [8]. It is used as a food spice and holds cultural and symbolic importance in traditional ceremonies and rituals. It is notably used for body painting during rites associated with birth, marriage, and death [8]. Medicinally, it serves as a remedy for digestive disorders, inflammation,

wounds, and infections [9]. Curcuma cultivation also provides income for local communities, supports the local economy, promotes agricultural diversification, and contributes to biodiversity conservation while reducing dependency on major crops. However, cultivation techniques for this species remain insufficiently developed . The plant is sterile, as flowering is rare and does not result in seed production [4, 10]. Traditional propagation through underground rhizomes—the only available means of multiplication—produces a maximum of only eight plants from a single rhizome [3], and this method favors the spread of pathogens, making the species vulnerable to diseases [7]. Additionally, a large portion of rhizomes that would normally serve various purposes is diverted for use as planting material to expand cultivation . To enhance the production and valorization of Curcuma longa, given its multiple benefits, it is essential to integrate modern biotechnologies, particularly micropropagation. This technique helps overcome shortages of healthy planting material while preserving genotypes under variable agro-climatic conditions [11]. Furthermore, tissue culture plant improves the yield potential of Curcuma [4]. In Benin, the lack of optimized micropropagation protocols limits turmeric production due to the unavailability of healthy planting material for large-scale cultivation. Moreover, the success of micropropagation depends on the selection of an appropriate explant, while the disinfection procedure of the explant is a critical step to ensure its establishment and survival in vitro. Thus, explants of Curcuma longa derived from rhizomes require specific disinfection procedures [12, 13]. Most previous studies on in vitro disinfection of Curcuma longa have used either Mercuric chloride (HgCl₂) [14] or Sodium hypochlorite [15], yet explants often face severe contamination and necrosis. Mercuric chloride has historically been used for the surface sterilization of explants in in vitro culture because of its broad-spectrum activity against bacteria and fungi. However, the dosage used requires careful attention and prior studies in order to determine a non-lethal concentration for the explant and to ensure no adverse effects on the operator. Moreover, several studies reported the use of relatively high concentrations of sodium hypochlorite for surface sterilization. For instance, Abd-Alla et al. [16] used Clorox^® (containing 5.25% active chlorine) at a concentration of 20%, while Yildiz and Er [17] evaluated surface sterilization using 40%, 60%, and 80% dilutions of commercial sodium hypochlorite (5% of active chlorine). It is therefore important to establish a disinfection protocol adapted to Curcuma grown in Benin, taking into account the characteristics of the mother plants , and incorporating pre-disinfection with Copper hydroxide followed by Ampicillin treatment (0.1%) during in vitro culture. Our preliminary trials have established the concentration of 8% of copper as efficient dose for Curcuma pre-disinfection. The use of HgCl₂ at concentrations of 0.05–1% has been reported on plant explants [18, 19], with significant effects on both microbial cells and plant tissues depending on the exposure duration and applied dose [20, 21]. Explants of turmeric derived from soil-grown rhizomes require a higher disinfectant dose due to the high microbial load surrounding the explant. It is therefore necessary to test the dose-response effect of HgCl₂ to determine the thresholds at which sterilization efficacy outweighs turmeric phytotoxicity for doses exceeding 1%. Regarding in vitro proliferation, aside from the work of Elsa et al. [7] involving different concentrations of 6-benzylaminopurine (BAP) in combination with Naphthalene Acetic Acid (NAA), few studies have examined the effects of Kinetin— a natural hormone [22] —which may influence organogenesis and in vitro proliferation of curcuma. To optimize photosynthesis and stimulate bud proliferation, Magnesium chloride (MgCl₂) was also tested. MgCl₂ in the concentration of 0.75 g/L have been already established for our routing protocol for multiplication of ginger [23]. It is also used for Gossipium hirsutum explant regeneration in vitro [24]. It is important to evaluate the effect of this concentration of MgCl₂ on the development et physiological parameters of Curcuma longa. Finally, concerning the adaptation of plantlets to natural conditions, numerous authors have reported the influence of substrate type on acclimatization success for certain cultivars such as Dioscorea spp [25], Manihot sp. [12], Colocasia esculenta [13], and Zingiber officinale [26]. These three taxa share common morphophysiological traits with Curcuma longa, notably the presence of underground storage organs and similar responses during the acclimatization phase [13]. In particular, ginger and turmeric exhibit rhizomatous growth habits as well as very comparable root development patterns [26]. The acclimatization of these taxa requires substrates providing adequate aeration, good drainage, and appropriate water retention capacity [12], conditions that are also favorable for turmeric, which belongs to the same botanical family as ginger (Zingiberaceae) [26]. For instance, up to 90–100% survival rates have been reported during acclimatization of Manihot sp. and Colocasia esculenta plantlets [12, 13]. Thus, results obtained in these crops support the use of well-structured substrates to improve the survival and growth of Curcuma longa plantlets during acclimatization. Comparisons between commercial substrates and locally produced low-cost substrates are often overlooked. This study was therefore conducted to establish an efficient micropropagation protocol for Curcuma longa L. to provide curcumin-rich planting materials for large-scale production. Specifically, it aims to (1) optimize the disinfection efficiency of Mercuric chloride, Sodium hypochlorite, and Ampicillin on the in vitro survival, sprouting, and regeneration of Curcuma longa explants; (2) evaluate the effects of Benzylaminopurine, Kinetin, and Magnesium chloride on organogenesis and in vitro proliferation of Curcuma longa; and (3) assess the impact of different substrates on the survival and growth of Curcuma longa plantlets during acclimatization.

Materials and methods

Sources and collection of plant material

Curcuma longa is widely cultivated at the Songhaï Center in Benin, located in Porto-Novo which is an agro-pastoral center with national and sub-regional recognition. The farm named “Le Prince de la Paix”, located in Hounkpotanou in the township of Athiémé, sourced Curcuma rhizomes of the cultivar “Lakadong” from the Songhaï Center for conventional multiplication, where the Laboratory of Plant Biotechnology and Crop Improvement of the University of Abomey-Calavi obtained rhizomes to facilitate in vitro multiplication, in accordance with national regulations governing research on plant genetic resources in Benin. This cultivar of Curcuma longa “Lakadong” originate from India commonly called “Atalè-vivè” in south and Centre Benin is very appreciated by the users of Benin because of its high content in Curcumin. Other researchers have also reported that the cultivar “Lakadong” of Curmuma longa is rich in curcumin [27, 28]. The identification of Curcuma longa rhizomes and emerging shoots was confirmed by a team from the National Herbarium of the University of Abomey-Calavi to ensure the authenticity of the in vitro-regenerated plantlets.

Site of experiment

The experiment conducted on the proliferation, direct organogenesis, and acclimatization of curcuma was carried out at the Laboratory of Plant Biotechnology and Crop Improvement of the University of Abomey-Calavi, Benin, from 20 December 2023 to 20 February 2025.

Methods

Disinfection of explants and culture establishment

Mature secondary rhizomes at the end of the vegetative cycle (ten months), and mother rhizomes obtained six months after greenhouse cultivation of the secondary rhizomes, were washed with tap water and then with soapy water [15], followed by rinsing with distilled water. The rhizomes (secondary rhizomes and mother rhizomes) were divided into two parts. The first part was immersed separately in an antifungal solution of copper hydroxide (8%) under agitation for 6 h, then rinsed three consecutive times for 5 min with sterile distilled water. The second part was immersed in an ampicillin solution (0.1%) for 10 min, followed by three consecutive rinses with sterile distilled water for 5 min each. A portion of buds excised from rhizomes treated with copper hydroxide (8%) as surface sterilant was subsequently disinfected in a mercuric chloride solution at different concentrations (0.1%, 0.5%, 1%, and 2%) for 10 min, followed by three successive rinses with sterile distilled water for 5 min each. The remaining buds were treated with a commercial sodium hypochlorite solution containing 2.6% active chlorine at different concentrations (30%, 40%, and 50%) for 10 min. Buds excised from rhizomes treated with ampicillin solution (0.1%) were consecutively immersed in 0.1% mercuric chloride and 30% sodium hypochlorite solutions, followed by three successive rinses with sterile distilled water. The buds were then cultured on Murashige and Skoog [29] (MS) basal medium supplemented with sucrose (30 g/L), MgCl₂ (0.75 g/L), NAA (0.5 mg/L), and BAP (2 mg/L), used as the initiation medium [30].

Shoot proliferation and organogenesis

After the establishment phase, the shoots were separated and cultured on MS basal medium supplemented with sucrose (30 g/L), MgCl₂ (0.75 g/L), and combinations of growth regulators—Naphthalene Acetic Acid (0.5 mg/L) and either Benzyladenine or Kinetin (4 mg/L) [23] (Table 1).

Table 1.

Composition of the different culture media tested

Phases	Designations	Composition
Initiation [30]	M0	MS basal medium + 0.5 mg/L NAA + 2 mg/L BAP
Organogenesis and Proliferation	M1	MS basal medium + 0.5 mg/L NAA + 4 mg/L BAP + 0.75 g/L MgCl₂ [21]
	M2	MS basal medium + 0.5 mg/L NAA + 4 mg/L BAP
	M3	MS basal medium + 0.5 mg/L NAA + 4 mg/L kinetin + 0.75 g/L MgCl₂ [21]
	M4	MS basal medium + 0.5 mg/L NAA + 4 mg/L kinetin

Acclimatization

The plantlets were selected based on a well-developed root system consisting of at least 11 roots measuring approximately 3 cm in length and a minimum of three leaves. These plantlets were carefully removed from the culture jars, gently rinsed with distilled water to remove residual culture medium, and transplanted into trays containing the different substrates. Three types of substrates were used: Terreau Semis Pouss’activR U (TSP); Klasmann TS3 substrate composed of moderately decomposed sphagnum peat and black peat (SAH Technology) [31]; and a local substrate made of sand and compost (1:1) (SBL), all previously moistened with distilled water. The experiment was conducted with three replicates per treatment, including fifteen plantlets per replicate. During acclimatization, plantlets were irrigated every two days with distilled water. Murashige and Skoog salts was diluted in 1/4 and applying each week to support plantlet establishment. After transplanting, trays were placed in a controlled chamber (27 ± 1 °C; RH: 98%) for one month before being transferred to the greenhouse (30 ± 1 °C; RH: 75%).

Evaluated parameters and data analysis

A completely randomized design was adopted. During the disinfection phase of each experiment, ten buds per disinfectant type and per explant type were used. For shoot organogenesis and proliferation phase, twenty explants were used per treatment. The experiment was repeated five times. After three weeks of culture, data on contamination, necrosis, survival, sprouting, and explant regeneration were collected each week during eleven weeks before subjected to binary logistic regression. After 14 weeks of culture, number of leaves, shoots, roots, root length, shoot height, and leaf area were collected each week for twelve weeks and pooled in excel before subjected to Analysis of variance (ANOVA), one way, to identify differences among treatments. Additionally, a generalized linear model (Poisson family) was applied to assess the relationships between numbers of leaves, shoots, and roots, and the different combinations of Cytokinins with MgCl₂, as well as the substrate type. The Student-Newman-Keuls (SNK) pairwise comparison test at the 5% significance level was used for mean separation. Statistical Analyses were performed using XLSTAT v14.

Results

Effects of disinfectants on the in vitro survival of Curcuma longa L. explants

Disinfectants had a highly significant effect (p < 0.0001) on contamination and necrosis rates, and a significant effect (p = 0.0084) on the survival rate of buds (Table 2). Likewise, the type of explant had a highly significant effect on necrosis rate (p < 0.0001), but showed no significant influence on either contamination or survival rates (p > 0.05). Concerning the interaction between disinfectant type and explant type, a highly significant difference was observed for necrosis and survival rates (p < 0.0001), as well as a significant influence on contamination rate (p = 0.0051). Despite the highly significant determination coefficients, they were moderate for contamination rate (41.86%), high for necrosis rate (69.34%), and low for survival rate (27.9%) (Table 2). Analysis of Fig. 1 shows that the lowest contamination rate (30%) was found with HgCl₂ (0.1%) combined with NaClO (30%) and Ampicilin (0.1%) treatment for buds derived from mother rhizomes whereas the highest was 100% was found with Copper hydroxide (8%) + mercuric chloride (1%) for buds derived from secondary rhizomes. These buds exhibited contamination rates of 90%, 50%, and 40% when treated with Copper hydroxide (8%) combined with NaClO (30%), NaClO (40%), and NaClO (50%), respectively. Same buds treated with Copper hydroxide (8%) combined with 0.5% or 2% HgCl₂ showed a contamination rate of 0%. Buds derived from mother rhizomes exhibited contamination rates varying from 30% to 80%. Combination of HgCl₂ (0.1%), NaClO (30%), and Ampicillin (0.1%) resulted in a contamination rate of 30%, whereas Copper hydroxide (8%) combined with NaClO (30% or 40%) generated contamination rates of 80%. A contamination rate of 50% was observed when these buds were treated with Copper hydroxide (8%) combined with HgCl₂ (0.5% or 1%), as well as with Copper hydroxide (8%) combined with NaClO (50%). When treated with Copper hydroxide (8%) combined with HgCl₂ (2%), contamination was completely absent (Fig. 1). Necrosis rates for buds from secondary rhizomes ranged from 50%, with the treatment containing HgCl₂ (0.1%), NaClO (30%), and Ampicillin (0.1%), to 100% for Copper hydroxide (8%) combined with HgCl₂ (0.5% or 2%). Necrosis was absent in treatments involving HgCl₂ (1%) combined with NaClO (30%) and Ampicillin (0.1%), as well as Copper hydroxide (8%) (Fig. 1). Regarding survival rate, buds derived from secondary rhizomes showed values ranging from 10% to 50% for disinfectant solutions composed of Copper hydroxide (8%) with NaClO (30% or 40%). Survival was null for the three disinfectant solutions combining Copper hydroxide (8%) with HgCl₂, as well as for the solution combining Copper hydroxide (8%) with NaClO (50%). The disinfectant solution containing HgCl₂ (0.1%), NaClO (30%), and Ampicillin (0.1%) generated a survival rate of 30%. For buds derived from mother rhizomes, survival rate reached 20% for disinfectant solutions consisting of Copper hydroxide (8%) combined with NaClO (30%, 40%, or 50%), and 70% for the treatment containing HgCl₂ (0.1%), NaClO (30%), and Ampicillin (0.1%). Survival rates of 40%, 50%, and 60% were recorded for treatments including Copper hydroxide (8%) combined with HgCl₂ at 0.5%, 1%, and 2%, respectively (Fig. 1).

Table 2.

Analysis of variance showing the effect of disinfectants on explant survival, contamination, and necrosis rates

Source	DF	CER	NER	SER
Disinfectants	6	< 0.0001***	< 0.0001***	0.0084**
Explants	1	0.2077ns	< 0.0001***	0.0969ns
Disinfectants * Explants	6	0.0051**	< 0.0001***	0.0003***
Coefficient of determination R² (%)		41.86***	69.34***	27.9***

ns non-significant difference at the 5% level, DF Degree of freedom, CER Contaminated explant rate, NER Necrotic explant rate, SER Surviving explant rate

***highly significant difference at the 0.1% level; **significant difference at the 1% level

Fig. 1.

Variation in explant survival, contamination, and necrosis rates as a function of the disinfection treatments. BRM: Buds derived from the mother rhizome; BRS: Buds derived from the secondary rhizome; CM: Mercuric chloride; FEJ: Copper hydroxide (8%) + bleach; FCM: Copper hydroxide (8%) + Mercuric chloride; EJ: Bleach

Variability of sprouting and in vitro regeneration rates of Curcuma longa L. explants according to disinfectants

Disinfectant solutions had a highly significant effect (p < 0.0001) on both sprouting and regeneration rates, with respective coefficients of determination of 16.14% and 5.84%, which are very low (Table 3). The type of explant had a highly significant effect (p < 0.0001) on sprouting and regeneration, with coefficients of determination of 52.25% and 84.69%, respectively, which are above average. Regarding the interaction between disinfectants and explant type, a highly significant difference was observed for sprouting (p < 0.0001) with a determination coefficient of 12.85%, while regeneration was significantly influenced (p < 0.05) with a coefficient of determination of 2.37% (Table 3). Results indicated that pre-treatment of buds from secondary rhizomes with Copper hydroxide (8%) followed by NaClO (30%) yielded the lowest sprouting rate (10%), while 38.33% sprouting was obtained with pre-treatment using Copper hydroxide (8%) followed by NaClO (50%). Additionally, these buds showed sprouting rates of 20%, 10%, and 33.33% for treatment with HgCl₂ (0.1%) combined with NaClO (30%) and Ampicillin (0.1%), and for Copper hydroxide (8%) combined with NaClO (30% and 40%), respectively. No sprouting was observed in secondary rhizome buds pre-treated with Copper hydroxide (8%) followed by HgCl₂ (0.5%, 1%, and 2%). For buds derived from mother rhizomes, the lowest sprouting rate (20%) was observed with pre-treatment using Copper hydroxide (8%) followed by NaClO (40% and 50%), whereas the highest sprouting rate (80%) was obtained with HgCl₂ (0.1%) followed by NaClO (30%) and Ampicillin (0.1%). A 30% sprouting rate was obtained with Copper hydroxide (8%) combined with HgCl₂ (0.5%). Sprouting rates of 26.67% and 28.33% were obtained with pre-treatment using Copper hydroxide (8%) combined with HgCl₂ (2%) or NaClO (50%), respectively (Fig. 2).

Table 3.

Analysis of variance showing the effect of disinfectants on sprouting and regeneration rates of explants

Source	Statistic	SERP	RER
Disinfectants	R² (%)	16.14	5.84
	Pr > F	< 0.0001***	< 0.0001***
Explants	R² (%)	52.25	84.69
	Pr > F	< 0.0001***	< 0.0001***
Disinfectants * Explants	R² (%)	12.85	2.37
	Pr > F	< 0.0001***	0.028*

SERP Sprouting explant rate, RER Regeneration explant rate

*** highly significant difference at the 0.1% level* significant difference at the 5% level

R² = Coefficient of determination

Fig. 2.

Variation of sprouting and in vitro regeneration rates of Curcuma longa L. explants according to disinfectants. BRM: Buds derived from the mother rhizome; BRS: Buds derived from the secondary rhizome; CM: Mercuric chloride; FEJ: Copper hydroxide (8%) + bleach; FCM: Copper hydroxide (8%) + Mercuric chloride; EJ: Bleach; error bars represent SE

Regarding regeneration of buds from secondary rhizomes, it was 8.33% for the disinfectant combination HgCl₂ (0.1%), NaClO (30%), and Ampicillin (0.1%). No regeneration was observed in secondary rhizome buds treated with Copper hydroxide (8%) combined with HgCl₂ (0.5%, 1%, and 2%) (Fig. 2). A regeneration rate of 10% was observed for buds pre-treated with Copper hydroxide (8%) followed by NaClO (40%). For buds derived from mother rhizomes, the lowest regeneration rate (13.33%) was observed for the combination Copper hydroxide (8%) + HgCl₂ (1%), whereas the highest regeneration rate (46.67%) was obtained with HgCl₂ (0.1%), NaClO (30%), and Ampicillin (0.1%). Regeneration rates of 21.67% and 23.33% were recorded for combinations including HgCl₂ (2%) and NaClO (50%), respectively. Finally, regeneration rates of 16.67%, 30%, 20%, and 25% were obtained with pre-treatments of Copper hydroxide (8%) combined with HgCl₂ (0.5%) and NaClO (30%, 40%, and 50%), respectively (Fig. 2).

Effects of benzyl-aminopurine, kinetin, and magnesium chloride on organogenesis of plantlets

Number of leaves, number of roots, shoot length, and root length

The results show a highly significant effect (P < 0.0001) of Benzylaminopurine (BAP), Kinetin (Kin), and Magnesium chloride (MgCl₂) on caulogenesis and rhizogenesis in Curcuma plantlets (Table 4). BAP (4 mg/L) or Kinetin (4 mg/L) added to MS + NAA (0.5 mg/L) medium promoted mean shoot heights of 6.64 ± 1.4 cm and 4.35 ± 1.4 cm, respectively (Figs. 3g2, g3). In the presence of MgCl₂ added to the basal MS medium, BAP (4 mg/L) or Kinetin (4 mg/L) produced plants with mean shoot heights of 12.38 ± 1.4 cm and 6.12 ± 1.4 cm, respectively. Basal MS + NAA (0.5 mg/L) medium supplemented with BAP (4 mg/L) generated an average of 4.5 ± 0.08 leaves per plant, whereas Kinetin (4 mg/L) produced 2.2 ± 0.08 leaves per plant. In combination with MgCl₂ (0.75 g/L), BAP (4 mg/L) or Kinetin (4 mg/L) increased the mean number of leaves to 4.5 ± 0.2 and 2.57 ± 0.2, respectively (Figs. 3f, g1, g2, g3 ). Regarding rhizogenesis, 3.22 ± 2.1 roots per plant with an average length of 1.83 ± 0.07 cm were produced when BAP (4 mg/L) was added alone to MS + NAA (0.5 mg/L) medium, whereas Kinetin (4 mg/L) produced no roots. The combination of each growth regulator (BAP 4 mg/L or Kinetin 4 mg/L) with MgCl₂ (0.75 g/L) in MS + NAA (0.5 mg/L) medium resulted in 9.7 ± 2.7 roots per plant and 1.86 ± 0.2 roots per plant, with mean root lengths of 2.2 ± 0.07 cm and 0.26 ± 0.07 cm, respectively (Figs. 3i1, i2).

Table 4.

Variation in growth parameters according to Benzylaminopurine and Kinetin alone or combined with Magnesium chloride

Culture Medium	NL	NR	SL (cm)	RL (cm)
MB + BAP + MgCl₂	11.3 ± 0.08 a	12.1 ± 0.15 a	12.38 ± 1.4 a	5.18 ± 0.5 a
MB + BAP	4.5 ± 0.08 b	6.1 ± 0.15 b	6.64 ± 1.4 b	2.49 ± 0.5 b
MB + Kin + MgCl₂	5.8 ± 0.08 b	5.3 ± 0.15 b	6.12 ± 1.4 b	1.82 ± 0.5 bc
MB + Kin	2.2 ± 0.08 bc	3.1 ± 0.15 b	4.35 ± 1.4 b	0.52 ± 0.5 c
R² (%)	70.44	38.94	49.45	55.16
Pr > F	< 0.0001***	0.0004**	< 0.0001***	< 0.0001***

Means followed by the same letter are not significantly different at the 5% level

MB Basal medium, BAP Benzylaminopurine, Kin Kinetin, MgCl₂  Magnesium chloride, NL Number of leaves, NR Number of roots, SL Shoot length, RL Root length, cm centimeter

*** highly significant difference at 0.1% level; **significant difference at the 1% level

R² = Coefficient of determination

Fig. 3.

Effect of Benzylaminopurine and Kinetin, alone or combined with Magnesium chloride, on Curcuma longa organogenesis, proliferation, development, and rooting. a Curcuma longa buds initiation; b, c, d and e) Curcuma longa in vitro organogenesis; f, g1, g2, and g3) Curcuma longa shoot proliferation and development; i1 and i2 Curcuma longa plantlet rooting

Leaf area

Regarding leaf area, a highly significant variability was observed between the basal medium supplemented with BAP (4 mg/L) and MgCl₂ (0.75 g/L) and the basal medium supplemented with Kinetin (4 mg/L). The supplementation of the basal medium with benzylaminopurine (BAP) or kinetin (Kin) alone resulted in the lowest mean leaf area per plant, with values of 4.37 ± 0.17 cm² and 2.7 ± 0.17 cm², respectively. In contrast, magnesium chloride (MgCl₂) supplementation markedly influenced leaf expansion when combined with cytokinins. The addition of MgCl₂ to kinetin increased the mean leaf area to 3.33 ± 0.17 cm², whereas its combination with BAP significantly enhanced leaf development, reaching 7.21 ± 0.17 cm² per plant (Fig. 4). On the other hand, a significant difference was observed between the basal medium supplemented with BAP (4 mg/L) and MgCl₂ (0.75 g/L) and both the basal medium supplemented with Kinetin (4 mg/L) and MgCl₂ (0.75 g/L) on one hand, and the basal medium supplemented with BAP (4 mg/L) alone on the other (Table 5). No significant difference was noted between the basal media supplemented with BAP (4 mg/L) and Kinetin (4 mg/L), whether alone or combined with MgCl₂ (0.75 g/L), or between the basal medium supplemented with Kinetin (4 mg/L) alone and the basal medium supplemented with Kinetin (4 mg/L) combined with MgCl₂ (0.75 g/L) (Table 5).

Fig. 4.

Variation of mean leaf area according to culture media in Curcuma longa plantlets. **: highly significant difference at 0.01%; values followed by the same letter are not significantly different at 5%; R²: coefficient of determination

Table 5.

Comparison of differences between culture media with a 95% confidence interval

Contrast	DS	VC	Pr > Diff
MB + BAP + MgCl₂ vs. MB + Kin	3.5017	2.6933	0.0066**
MB + BAP + MgCl₂ vs. MB + Kin + MgCl₂	3.0137	2.4445	0.0128*
MB + BAP + MgCl₂ vs. MB + BAP	2.2048	2.0281	0.0340*
MB + BAP vs. MB + Kin	1.2969	2.4445	0.4061ns
MB + BAP vs. MB + Kin + MgCl₂	0.8090	2.0281	0.4238ns
MB + Kin + MgCl₂ vs. MB + Kin	0.4879	2.0281	0.6286ns

MB Basal medium, BAP Benzylaminopurine, Kin Kinetin, MgCl₂ Magnesium chloride, DS Difference of means, VC Standard error, Pr > Diff Probability of difference, ns: Not significant

** Highly significant at 1%, * Significant at 5% 

Shoot proliferation

Benzylaminopurine (BAP) and kinetin (Kin), applied either individually or in combination with magnesium chloride (MgCl₂), exerted a highly significant effect on shoot proliferation (p < 0.0002), explaining 90.29% of the observed variability (R² = 0.9029; Fig. 5). When supplemented alone to the basal medium, BAP induced a mean of 2.0 shoots per explant (Fig. 5), whereas kinetin resulted in a lower regenerative response, with an average of 1.2 shoots per explant (Fig. 5). Notably, the incorporation of MgCl₂ markedly enhanced shoot induction in the presence of both cytokinins. The combination of kinetin and MgCl₂ increased the mean shoot number to 4.2 per explant (Fig. 6), while the synergistic effect between BAP and MgCl₂ produced the highest proliferation rate, reaching 15.4 shoots per explant (Fig. 5).

Fig. 5.

Average number shoots following cytokinins (BAP or Kin) and magnesium chloride (MgCl₂). R²: Coefficient of determination; BM: Basal medium; BAP: Benzylaminopurine; Kin: Kinetin; MgCl₂: Magnesium chloride; Means followed by the same letter are not significantly different at the 5% level.

Fig. 6.

Survival rate of plantlets according to the tested substrates. TSP: Terreau Semis Pouss’activR U: mixture of peat, sand, basic amendment, organic fertilizer, and a microbial preparation; TS3: SAH substrate based on moderately decomposed sphagnum peat and black peat (Klasmann TS3); SBL: Local substrate based on sand and compost (1:1)

Effect of substrate on survival, growth, and development of plantlets during acclimatization

The substrates ‘’Terreau Semis Pouss’activR U’’ (TSP) and Klasmann TS3 showed the highest survival rates of plantlets (100%), compared to 90% on the local substrate (SBL). The tested substrates did not significantly influence plantlet survival (Pr > 0.05) with a very low coefficient of determination (6.9%) (Fig. 6).

Regarding growth, results from Chi-square test probabilities showed that the substrates had a significant effect (P < 0.05) on both height gain and leaf number (Table 6). Height gain ranged from 1.61 ± 0.03 cm on the Klasmann TS3 substrate to 3.175 ± 0.5 cm on the TSP substrate. The number of leaves ranged from 1.5 ± 0.1 on TS3 to 3.1 ± 0.19 on TSP. The TSP substrate allowed better growth (3.175 ± 0.5 cm) and a higher number of leaves (3.1 ± 0.19) in Curcuma plantlets during acclimatization (Fig. 7c), followed by the local control substrate (SBL) with an average height gain of 2.7 ± 0.5 cm and an average leaf number of 2.2 ± 0.19 (Fig. 7a). Furthermore, it was noted that plantlets, regardless of the substrate used, exhibited highly variable capacities in terms of height gain and leaf number, with low coefficients of determination of 25.45% and 25.8%, respectively, for height gain and leaf number (Table 6).

Table 6.

Variation in height gain and leaf number according to the different substrates tested during plantlet acclimatization

Substrate	Height Gain (HG, cm)	Number of Leaves (NL)
TSP	3.175 ± 0.5 a	3.1 ± 0.19 a
SBL	2.7 ± 0.5 a	2.2 ± 0.19 ab
TS3	1.61 ± 0.03 b	1.5 ± 0.1 b
R² (%)	25.45	25.8
Pr > F	0.019 *	0.017 *

U: mixture of peat, sand, basic amendment, organic fertilizer, and a microbial preparation; TS3 = SAH substrate composed of moderately decomposed sphagnum peat and black peat (Klasmann TS3); SBL = local substrate composed of sand and compost (1:1); Means followed by the same letter are not significantly different at the 5% significance level

NL number of leaves, HG height gain, TSP Terreau Semis Pouss’activR

* significant difference at the 5% level

R² = coefficient of determination

Fig. 7.

Effect of acclimatization substrates on Curcuma longa plantlets. a plantlets development on SBL substrate; b plantlets development on TS3 substrate; c plantlets development on TSP substrate

Discussion

The success of micropropagation, a proven method for the rapid and large-scale multiplication of selected or elite varieties, depends on proper disinfection, the suitability of the culture medium [3, 25], and the choice of appropriate acclimatization conditions [32, 33]. The results revealed a significant interaction between the type of disinfectant and the type of explant. This indicates that explant effect is not constant, but depends on the disinfectant effect. In other words, explant effect exists, but only when the disinfectant takes certain values. Buds derived from secondary rhizomes and those from mother rhizomes exhibited greater resistance to infection and necrosis when treated with Copper hydroxide combined with other disinfectants (Mercuric chloride followed by Sodium hypochlorite). In contrast, buds treated directly with disinfectants alone were unable to resist infections and necrosis effectively. Copper hydroxide (8%) proved efficient in eliminating fungal contamination from the explants, serving as a replacement for carbendazim [14] or benlate [15] previously used on the same species. The Copper content in Copper hydroxide effectively reduced infection and necrosis in both types of Curcuma longa buds cultivated in Benin.

Furthermore, regardless of the type of bud used (secondary or mother rhizome), treatment with 2% Mercuric chloride for 10 min effectively eliminated infections, with only 40% necrosis observed in mother rhizome buds (BRM). Secondary rhizome buds may possess an internal structure that retains the disinfectant for longer, favoring disinfection but potentially causing cell death. Maximum survival (70%) was achieved with treatment comprising Ampicillin (0.1%) followed by Sodium hypochlorite (30%) and Mercuric chloride (0.1%). Therefore, high doses of Mercuric chloride are not the main determinant of survival; rather, survival depends largely on the type of pre-disinfectant. In this study, although Copper plays an essential role in activating various enzymes involved in electron transport, protein and carbohydrate biosynthesis, and phenolic metabolism—contributing to accelerated cell proliferation and explant regeneration [25]—its combination with Mercuric chloride induced explant death. This result may also suggest a possible antagonistic chemical interaction between Copper hydroxide and Mercuric chloride, which could reduce the disinfectant efficiency of HgCl₂ and consequently lead to persistent contamination. Thus, while Mercuric chloride is an effective disinfectant for plant tissues [14], it can potentially be toxic when combined with Copper hydroxide, leading to cell death. Similarly, treatment of buds with Copper hydroxide combined with various concentrations of Sodium hypochlorite resulted in low or null survival rates, particularly at 50% NaClO in secondary buds due to high infection rates and excessive tissue damage, which contrasts with the findings of Kone et al. [23] on Zingiber officinale, where Sodium hypochlorite alone effectively eliminated contaminations, showing the influence of the plant species, explant type and disinfectant combinations. In contrast, Akplogan et al. [13], showed that apical bud explants of taro (Colocasia esculenta) treated with a higher dose (0.15%) of HgCl₂ for 5 min achieved 100% survival, suggesting that optimized HgCl₂ treatments can be effective when applied under controlled conditions. Moreover, treatment of buds with 0.1% Mercuric chloride combined with 30% Sodium hypochlorite and 0.1% Ampicillin not only provided higher survival rates but also better sprouting and regeneration, particularly for mother rhizome buds, compared with treatments involving Copper hydroxide. In addition to the effects of Mercuric chloride and Sodium hypochlorite, ampicillin likely provided a favorable environment for bud survival and growth by reducing pathogenic or contaminating bacteria. The disinfection efficiency of Mercuric chloride and Sodium hypochlorite varies depending on both the disinfectant concentration and the type of explant. HgCl₂ has been shown to be effective for surface sterilization, but even in small amounts, it can be toxic to plants and animals [34], making it less suitable for tissue culture [35]. However, increasing the concentration of disinfectants can negatively affect the survival of plant cells by causing explant necrosis. Nevertheless, even if the initial regeneration rate is low, multiplication becomes exponential from the regenerated plantlets. Developing an efficient disinfection protocol that does not rely on toxic chemicals would also be ideal for turmeric sterilization, although it is very challenging. Organogenesis results showed that 4 mg/L BAP combined with 0.75 g/L MgCl₂ significantly enhanced shoot proliferation (15.4 shoots per explant), caulogenesis (11.3 ± 0.0 leaves), rhizogenesis (12.1 ± 0.15 roots), and elongation of shoots (12.38 ± 1.4 cm) and roots (5.18 ± 0.5 cm) compared with 4 mg/L Kinetin. This effect may be attributed to the adequate availability of Mg²⁺ in the culture medium, which enhances cell division, tissue growth, and metabolic activity in vitro plantlets. MgCl₂ plays a direct role in meristematic proliferation and shoot development [36]. BAP alone also performed better than Kinetin, which aligns with previous studies by Swarnathilaka and Nilantha [37], who reported that 4 mg/L BAP + 0.5 mg/L NAA induced higher shoot proliferation in Curcuma longa. Similarly, Bhanuprakash et al. [2] demonstrated that BAP alone is more effective in inducing a large number of shoots than various concentrations of Kinetin. These results can be explained by the synthetic nature of BAP, which more rapidly promotes cell division in shoots and roots than the natural hormone Kinetin [22]. Furthermore, the presence of MgCl₂ in the culture medium, combined with either BAP or kinetin, further stimulated organogenesis and proliferation, corroborating the findings of Kone et al. [23] in ginger micropropagation. MgCl₂ plays a key role in proliferation and organogenesis by efficiently capturing light energy, converting it into chemical energy, enhancing the uptake of essential nutrients, and participating to chlorophyl biosynthesis in plants. MgCl₂ plays also the role of enzyme cofactor to facilitate cell metabolite of the plants. In other species, such as Manihot esculenta, Kinetin at 0.2–4 mg/L combined with NAA induced more roots than 4 mg/L BAP combined with NAA [12, 25], suggesting that 4 mg/L Kinetin may be too high for optimal rhizogenesis in Curcuma longa explants. Overall, BAP demonstrated stronger proliferative effects than Kinetin. The superior results obtained with 4 mg/L BAP compared with 2 mg/L in combination with MgCl₂ may be explained by the fact that higher concentrations of BAP stimulate meristem multiplication and shoot regeneration. BAP also plays an important role in cell division and bud initiation. The analysis of variance revealed a statistically significant effect of the hormonal combination on in vitro leaf development (Pr > F = 0.006). Although the coefficient of determination was moderate (R² = 28.56%), this value is consistent with the inherent biological variability associated with in vitro morphogenetic responses. Indeed, organogenesis is influenced by multiple physiological and micro-environmental factors, and therefore a single experimental factor is not expected to account for the majority of the observed variance. The R² value indicates that approximately 29% of the total variation in leaf development can be attributed to the hormonal treatment, reflecting a substantial and biologically meaningful contribution despite the multifactorial nature of the system. In acclimatization, the highest survival rates (100%) obtained with TSP and TS3 (SAH) may be explained by the stability of nutrient and the beneficial biological properties of the substrate, which reduce plantlets stress and promote root establishment compared with the local substrate (SBL). The superior performance observed with the peat-based substrates compared with the sand–compost mixture may be attributed to their higher water-holding capacity, improved aeration, and better nutrient retention. Peat-based substrates generally provide more favorable physicochemical properties for plantlet establishment, including slightly acidic pH, and higher water-holding capacity. The TS3 substrate has previously achieved 93.6% survival in Manihot esculenta genotypes in acclimatation due to its nutrient content and the beneficial biological properties [31]. Studies on pineapple (Ananas comosus) seedlings grown in Klasmann TS3 also reported higher survival rates compared with other substrates, highlighting the positive impact of SAH substrates [38]. The superior survival rates obtained with TS3 in this study, exceeding the 80% reported by Kajibwami et al. [39], may be attributed to genotype variations and growth conditions [38]. The lower survival on the local substrate is consistent with its physical properties, which can lead to poor root development, instability, and vulnerability to stress. Differences in growth and development between TS3 and SBL may also be due to the presence of microbial preparation and other nutrients in TSP not found in TS3. Ultimately, TSP allows Curcuma plants to adapt better to field conditions after transplanting. The successful acclimatization of tissue-cultured Curcuma plantlets depend on the nature and composition of the substrate [40, 41].

Conclusion

This study on Curcuma micropropagation protocol established efficient disinfection protocols for buds from different rhizomes and identified optimal growth regulator combinations for organogenesis, proliferation, rooting, and pre-acclimatization. The combination of 0.1% Mercuric chloride, 30% Sodium hypochlorite, and 0.1% Ampicillin was most effective for in vitro establishment of initiated buds. Furthermore, MS medium supplemented with 4 mg/L BAP and 0.75 g/L MgCl₂ yielded the best proliferation, growth, and root system development. For pre-acclimatization, TSP substrate provided the highest survival, growth, and development of Curcuma seedlings, making it the most suitable option for large-scale propagation. Beyond its biological efficiency, the protocol demonstrates strong potential for scalability, cost-effectiveness, and practical feasibility within the Beninese context. It is based on widely available disinfectants, commonly used plant growth regulators, and locally accessible substrates, which collectively reduce technical constraints and production costs. Consequently, this protocol can be adopted as a rapid multiplication strategy for Curcuma longa, facilitating large-scale availability and production of high-quality planting material. Its implementation would significantly contribute to the sustainable intensification and commercialization of Curcuma longa.

Abbreviations

HgCl₂

Mercuric chloride

NaClO

Sodium hypochlorite

ANOVA

Analysis of variance

MgCl₂

Magnesium chloride

TSP

Terreau Semis Pouss’activR U”

MS

Murashige and Skoog

SBL

Local substrate

BAP

Benzylaminopurine

NAA

Naphthalene Acetic Acid

Authors' contributions

Original draft preparation by GHTC; Conduct the work by BVAO, SSH, JRUK, JAH, GHTC; validation, analysis, and visualization by SSH; Review and editing by JAH; revision by GHTC, JAH; Supervision by GHTC and CA. All the authors have read and agreed to the submitted version of the manuscript.

Funding

No funding applicable.

Data availability

Data presented in this study will be available on a fair request to the corresponding author.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.