Establishment of an In Vitro Micropropagation System for Cannabis sativa ‘Cheungsam’

Abstract

Cannabis has been cultivated for thousands of years for a variety of purposes, including fiber, seeds, oil, and medicinal compounds. The cannabis industry is growing rapidly because several countries have recently legalized the use of cannabis. In these countries, the industry related to cannabinoid ingredients such as cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC) is steadily increasing every year. High concentrations of cannabinoids are mainly produced in unfertilized female flowers. Maintaining plants with high cannabinoid content is essential for producing uniform substances in large quantities. This study established an in vitro micropropagation protocol that can maintain the mother plant characteristics of Cannabis sativa ‘Cheungsam’. As a result of this experiment, the shoot tips and nodes of Cannabis sativa ‘Cheungsam’ at various concentrations (0, 0.25, 0.5, 1.0 mg/L) of 2iP, BA, and mT plant growth regulators (PGRs), and all concentrations of 2iP showed better results compared to two other hormones. However, the cut surfaces turned black, and excessive hyperhydricity occurred. Based on these symptoms, activated charcoal was added to the medium with the assumption that necrosis and hyperhydricity occur due to the accumulation of reactive oxygen species (ROS). When treated with 0.5 g/L charcoal, hyperhydricity was not overcome, and there was no difference compared to the control. As a new alternative, we divided the experiments into MS (Murashige and Skoog) and DKW (Driver and Kuniyuki Walnut) medium, which were commercially available. As a result, the rate of hyperhydricity was reduced, the cut surface did not turn black, and the growth conditions were also improved. Subsequently, ½ MS medium and ½ DKW medium were treated with various concentrations of IBA alone and with combinations of IBA and NAA for rooting. As a result, ½ DKW with IBA 0.5 mg/L showed the highest rooting rate and the best root condition for Cheungsam. After 4 weeks, when considering rooted plants with a height above 5 cm that were acclimatized, the acclimatization rate reached 100%. In conclusion, the Cannabis sativa ‘Cheungsam’ plants used in this study produced healthy shoots on DKW medium containing 1.0 mg/L 2iP and 0.5 mg/L of IBA in ½ DKW medium showed the best rooting rate.

1. Introduction

Cannabis sativa, commonly known as hemp, is an annual herbaceous plant in the Cannabaceae family [1,2,3]. It has been cultivated for thousands of years for various purposes, including fiber, seed, oil, and medicinal compounds [4].

The global cannabis industry is growing rapidly and with the legalization of medical and industrial cannabis worldwide, the potential of cannabis is being recognized in various applications. As of 2024, Canada, Uruguay, and 18 U.S. states have legalized both medical and recreational cannabis. Additionally, several European countries including Germany and the UK, as well as New Zealand and Thailand, have legalized medical cannabis. In Korea, since the amendment of the Narcotics Control Act in 2019, research and industrial efforts related to medical cannabis have been increasing [5].

Cannabis can be broadly classified into marijuana and hemp. Marijuana, derived from Cannabis sativa subsp. sativa or Cannabis sativa subsp. indica plants, contains high levels of THC, a psychoactive compound classified as a narcotic in many countries [3]. Some countries use marijuana for both recreational and medical purposes. Hemp, a variant of Cannabis sativa subsp. sativa, contains very low levels of THC (typically less than 0.3% on a dry weight) and is primarily used for medical and industrial purposes [6]. Hemp is characterized by its high CBD content, another major cannabinoid found in cannabis, which has various medical benefits such as treating epilepsy syndromes in medications like Epidiolex^®, alleviating anxiety and depression, as well as having anti-inflammatory and analgesic properties, without the psychoactive effects of THC [2,7,8,9]. High concentrations of cannabinoids are primarily produced in young leaves and unfertilized female flowers. The value of the crop is determined by cannabinoid content in the flowers.

The increasing demand for clean propagated cannabis material necessitates the development of scalable production systems, such as vertical farms, plant factories, and smart farms [10]. Micropropagation is a tissue culture technique that produces numerous identical clone plants from tissue sections of a mother plant. This allows for the mass production of genetically identical plants in a shorter time than traditional propagation methods [11]. Plants are cultured in bottles and grown in a culture room equipped with LED lighting, enabling the maintenance of a large number of plants in a small space. Additionally, cultivation in sterile media allows for the production and maintenance of disease-free plants.

Cannabis is a typical dioecious plant and anemophilous, relying on wind for pollination. Thus, it is difficult to maintain genetically identical plants to the mother plant using traditional propagation methods. High concentrations of cannabinoids are primarily produced in young leaves and unfertilized female flowers. However, cannabinoid production decreases when pollination occurs through wind [12]. Moreover, infections such as Hops Latent viroid (HLVd), Fusarium oxysporum, and Powdery Mildew, as well as pests like spider mites and aphids, can reduce quality and yield in traditional propagation methods [13,14].

‘Cheungsam’ is a F₁ hybrid cultivar developed by crossing IH3, a low-narcotic genetic resource among 44 introduced germplasms from CPRO in the Netherlands in 1997, with a Korean native strain. ‘Cheungsam’ is characterized by its low narcotic content, with a THC content of 0.34% and a CBD content of 1.34%. In Korea, ‘Cheungsam’ is the most widely cultivated outdoor cultivar due to strict regulations on the use of cannabis and its derived ingredients and products. It was primarily cultivated in Korea for fiber production, but the industrial use of its by-products, such as roots and stems, has recently been growing.

A previous study screened various conditions to optimize in vitro clonal propagation and ex vitro rooting and acclimatization of fiber hemp via shoot tips. However, low rooting rates of 74.6% were reported [15]. Another study investigated benefit of using DKW medium instead of MS. DKW basal salts improved micropropagation as demonstrated by the canopy area, multiplication, and callogenesis in five different cultivars (BA-21, BA-41, BA-49, BA-61, and BA-71) [16]. Another study tested the impacts of different compositions of basal media with PGRs on hemp cultivar TJ’s CBD during initiation, multiplication, in vitro rooting, ex vitro rooting, and acclimatization [17]. Despite the studies above, there has been no studies on the development of optimized systems of micropropagation for ‘Cheungsam’, which is widely cultivated in the Republic of Korea for various industrial purposes.

Therefore, this study selected micropropagation as a method to maintain the traits of the mother cannabis plant. We addressed the issues at each stage and established a protocol tailored to Cannabis sativa ‘Cheungsam’ to obtain normal plantlets.

2. Materials and Methods

2.1. Seed Germination

The cannabis used in this study was provided by the cultivation farmland of the Korea Hemp Industry Association, an incorporated association located in Imha-myeon, Andong-si Gyeongsangbuk-do, Republic of Korea.

Seeds of Cannabis sativa ‘Cheungsam’ were surface sterilized in 75% ethanol for 2 min 30 s followed by rinsing with distilled water. They were then sterilized with 10% sodium hypochlorite (NaClO, Daejung, Siheung, Republic of Korea) solution for 10 min, before being rinsed with sterile distilled water eight times. The seeds were then subjected to sonication for 10 min. After sonication, the seeds were primed with 1% hydrogen peroxide (H₂O₂, Daejung, Siheung, Republic of Korea) solution cultivated in darkness (25 ± 2 °C) for 3 days. The seeds were germinated in Phytohealth plant culturing containers (lid size: 96.65 mm [diameter] × 41.00 mm [height], dish size: 91.02 mm [diameter] × 81.60 mm [height], SPL Life Sciences, Pocheon, Republic of Korea) containing 50 mL MS medium. The MS medium contained full MS, including a Gamborg B5 vitamin mixture, 3% sucrose (MB cell, Seoul, Republic of Korea), and 0.8% plant agar (Duchefa Biochemie, The Netherlands). The pH was adjusted to 5.8 with 1N NaOH or HCl. Medium and culture vessels were autoclaved at 121 °C for 15 min. The culture plate was cultured in a culture room set at a temperature at 25 ± 2 °C under a 16/8 h (light/dark) photoperiod with a light intensity of 120.25 µmol m⁻² s⁻¹ LED daylight (Figure 1).

2.2. Multiplication

Sections of 0.5–1 cm length of explants (shoot tip and node) were cut from two-week-old in vitro seedlings and used as the explants. The explants were cultured on MS or DKW medium supplemented with various concentration of 2iP, mT, and BA, with or without charcoal. After four weeks of culture, the plant growth parameters (plant height, number of leaves, leaf chlorosis rate, and number of shoots) were assessed in the resulting cannabis plants. All cultures were placed in a culture room set at a temperature at 25 ± 2 °C under a 16/8 h (light/dark) photoperiod with a light intensity of 120.25 µmol m⁻² s⁻¹ LED daylight. Each measurement included ten bottles with three replications.

2.3. Effect of Plant Growth Regulators (PGRs) and Activated Charcoal on In Vitro Plant Multiplication of the Cannabis sativa ‘Cheungsam’

Two-week-old hemp plants were cultured in vitro on MS media supplemented with different concentrations of 6-(γ,γ-Dimethylallylamino)purine (2iP; PhytoTech Labs, Lenexa, KS, USA), meta-Topoline (mT; Duchefa Biochemie, The Netherlands), and 6-Benzylaminopourine (BA; Sigma-Aldrich, St. Louis, MO, USA). Furthermore, charcoal (Daejung, Siheung, Republic of Korea) was added for plant growth and suppression of hyperhydricity to some samples. Each bottle contained five plants, and there were ten bottles per treatment with three replications.

2.4. Effect of Medium Formulation on In Vitro Plant Multiplication of the Cannabis sativa ‘Cheungsam’

Several commercial basal nutrient formulations were tested for their effect on in vitro cannabis explant growth. In this study, we used MS or DKW medium. Each medium was evaluated at their recommended concentrations of 4.4 and 5.22 g/L, respectively. Each bottle contained five plants, and there were ten bottles per treatment with three replications.

2.5. Effect of Explants Type on In Vitro Plant Multiplication of the Cannabis sativa ‘Cheungsam’

Shoot tip and nodal segments were compared as explant types for their effect on in vitro propagation. Each bottle contained five plants, and there were ten bottles per treatment with three replications.

2.6. Rooting of Shoots

Shoots grown to over 5 cm were cultured for four weeks on a half-strength MS or DKW medium supplemented with various concentration of Indole-3-butyric acid (IBA; Sigma-Aldrich, St. Louis, MO, USA) and 1-Naphthaleneacetic acid (NAA; MB cell, Seoul, Republic of Korea) for root formation. Each bottle contained five plants, and there were five bottles per treatment with three replications.

2.7. Acclimatization

The plantlets with well-developed roots were removed from the culture vessels and washed several times with running tap water. The plantlets were then transplanted into pots containing mixed soil (cocopeat:peatmoss:perlite = 4:4:2, w/w/w) and acclimatized for two weeks in clean plastic trays. Then, the plastic trays of successfully adapted plants were removed. Following one month, the plant survival rate was assessed by outer appearance.

2.8. Statistical Analysis

The statistical analysis of data was performed using GraphPad Prism version 5.03 (La Jolla, CA, USA) and presented as mean ± standard errors of three replicates. The significance of differences between means was determined using Tukey’s multiple range test and the least significant difference test, with a significance level of p < 0.05

3. Results

3.1. Effect of PGRs on In Vitro Shoot Induction

Shoot tip and node explants of Cannabis sativa ‘Cheungsam’ cultured on MS medium without PGRs struggled to maintain a healthy state for up to four weeks. When explants were cultured on media containing mT (0.25 or 0.5 mg/L) and BA (0.25 or 0.5 mg/L), their condition was better compared to those without PGRs, but the results were not as favorable as those observed with 2iP (0.25, 0.5 or 1.0 mg/L). In contrast, healthy shoot formation occurred in media containing 2iP regardless of the concentration. However, the maximum number of shoots per explant did not show significant differences among all treatments (

Table 1. Comparison of plant growth of various PGRs concentration on shoot induction from shoot tip explants of Cannabis sativa ‘Cheungsam’ after four weeks of culture.

Explant	PGRs (mg/L)			No. of Shoot/Explant	Plant Height (cm)	No. of Leaf	Leaf Chlorosis Rate (%)	Hyperhydricity
	2IP	mT	BA
Shoot tip	-	-	-	1.12 ± 0.12 b	1.30 ± 0.30 d	4.94 ± 0.65 c	23.17 ± 7.13 bc	+
	0.25	-	-	1.05 ± 0.05 b	3.64 ± 0.48 a	7.81 ± 0.60 b	10.49 ± 3.00 d	-
	0.50	-	-	1.18 ± 0.13 b	3.31 ± 0.30 ab	8.94 ± 0.57 ab	26.55 ± 5.86 bc	-
	1.00	-	-	1.00 ± 0.00 b	3.46 ± 0.36 ab	8.18 ±0.57 b	6.36 ± 2.51 d	-
	-	0.25	-	1.26 ± 0.06 ab	2.73 ± 0.21 abc	5.54 ± 0.13 bc	36.11 ±13.60 b	+
	-	0.50	-	1.59 ± 0.23 ab	2.99 ± 0.20 abc	5.82 ± 0.23 bc	48.99 ± 4.18 a	+
	-	-	0.25	1.77 ± 0.10 ab	1.80 ± 0.16 d	8.60 ± 0.38 ab	32.52 ± 2.31 b	+
	-	-	0.50	2.13 ± 0.12 a	1.44 ± 0.21 d	5.47 ± 0.39 c	30.13 ± 2.46 b	+

These data represent the means of three replicates with standard error. Means with the same letters are not significantly different (LSDT, p < 0.05). -: normal plant; +: mild hyperhydricity symptoms.

and

Table 2. Comparison of plant growth of various PGRs concentration on shoot induction from node explants of Cannabis sativa ‘Cheungsam’ after four weeks of culture.

Explant	PGRs (mg/L)			No. of Shoot/Explant	Plant Height (cm)	No. of Leaf	Leaf Chlorosis Rate (%)	Hyperhydricity
	2IP	mT	BA
Node	-	-	-	1.94 ± 0.06 a	1.60 ± 0.12 bc	8.22 ± 0.53 a	45.13 ± 5.58 ab	++
	0.25	-	-	1.90 ± 0.07 a	2.77 ± 0.24 a	9.05 ± 0.60 a	39.67 ± 4.53 b	++
	0.50	-	-	1.80 ± 0.20 a	2.92 ± 0.81 a	8.00 ± 1.41 a	24.83 ± 6.87 bc	++
	1.00	-	-	2.00 ± 0.00 a	3.10 ± 0.48 a	9.78 ± 1.28 a	36.81 ± 7.01 b	++
	-	0.25	-	1.78 ± 0.08 a	2.33 ± 0.12 ab	3.95 ± 0.06 c	51.27 ± 0.31 a	++
	-	0.50	-	1.86 ± 0.14 a	1.87 ± 0.18 b	3.80 ± 0.07 c	31.99 ± 6.57 a	++
	-	-	0.25	1.47 ± 0.09 ab	0.76 ± 0.07 d	4.47 ± 0.30 c	31.42 ± 3.30 bc	++
	-	-	0.50	1.55 ± 0.10 ab	0.73 ±0.06 d	5.32 ± 0.33 b	25.41 ± 2.87 bc	++

These data represent the means of three replicates with standard error. Means with the same letters are not significantly different (LSDT, p < 0.05). -: normal plant; ++: hard hyperhydricity symptoms.

). Unfortunately, the shoots developed from some treatments (mT and BA), shoot tips, and all treatments nodes exhibited symptoms of hyperhydricity, such as a glassy appearance, thin stems, and translucent leaves (Table 2). When these plants were subcultured in the same composition media, all of them died. Therefore, among the three PGRs (2iP, mT, BA), 2iP was confirmed to be the most suitable hormone, and subsequent experiments were conducted using only the 2iP (0.25, 0.5, and 1.0 mg/L).

3.2. Effect of Activated Charcoal on In Vitro Shoot Induction and Overcome Hyperhydricity

Activated charcoal is known to help plant growth and development. Thus, activated charcoal (0.5 g/L) was added to the medium to overcome hyperhydricity and prevent the cut surface from blackening. However, almost all characteristics were significantly better without charcoal across all treatments (Figure 2A–H and Figure 3A–H). In the case of the leaf chlorosis rate, an increase was observed (

Table 3. Comparison of the impact of activated charcoal on plant growth and shoot induction from shoot tip explants of Cannabis sativa ‘Cheungsam’ after four weeks of culture.

Explant	PGRs (mg/L)	Charcoal (g/L)	No. of Shoot/Explant	Plant Height (cm)	No. of Leaf	Leaf Chlorosis Rate (%)	Hyperhydricity
	2IP
Shoot tip	-	-	1.17 ± 0.17 a	1.53 ± 0.40 b	5.33 ± 0.87 c	13.38 ± 5.97 ab	+
	-	0.5	1.00 ± 0.00 a	2.38 ± 0.28 ab	6.50 ± 0.34 c	21.83 ± 7.04 ab	-
	0.25	-	1.06 ± 0.06 a	3.69 ± 0.54 a	7.94 ± 0.76 b	6.73 ± 2.77 b	-
	0.25	0.5	1.06± 0.20 a	1.82 ± 0.41 b	5.00 ± 0.45 cd	48.11 ± 6.83 a	-
	0.50	-	1.25 ± 0.18 a	3.41 ± 0.28 a	9.17 ± 0.58 a	22.61 ± 6.98 ab	-
	0.50	0.5	1.17 ± 0.20 a	2.32 ± 0.34 ab	7.60 ± 0.83 bc	26.25 ± 5.69 ab	-
	1.00	-	1.00 ± 0.00 a	3.54 ± 0.29 a	8.38 ± 0.84 ab	6.36 ± 2.51 b	-
	1.00	0.5	1.00 ± 0.00 a	1.14 ±0.25 b	4.60 ± 0.40 d	24.00 ± 7.97 ab	-

These data represent the means of three replicates with standard error. Means with the same letters are not significantly different (LSDT, p < 0.05). -: normal plant; +: mild hyperhydricity symptoms.

and

Table 4. Comparison of the impact of activated charcoal on plant growth and shoot induction from node explants of Cannabis sativa ‘Cheungsam’ after four weeks of culture.

Explant	PGRs (mg/L)	Charcoal (g/L)	No. of Shoot/Explant	Plant Height (cm)	No. of Leaf	Leaf Chlorosis Rate (%)	Hyperhydricity
	2IP
Node	-	-	2.00 ± 0.00 a	1.64 ± 0.50 a	8.22 ± 0.53 ab	42.43 ± 6.69 a	++
	-	0.5	1.80 ± 0.13 a	2.70 ± 0.72 a	7.90 ± 0.57 ab	48.92 ± 4.94 a	++
	0.25	-	1.87 ± 0.09 a	2.77 ± 0.32 a	9.05 ± 0.60 ab	35.56 ± 5.61 a	++
	0.25	0.5	1.90 ± 0.10 a	1.39 ± 0.20 a	6.70 ± 0.63 b	47.58 ± 6.20 a	++
	0.5	-	1.80 ± 0.20 a	2.92 ± 0.81 a	8.00 ± 1.41 ab	24.83 ± 6.87 a	++
	0.5	0.5	1.90 ± 0.10 a	2.22 ± 0.28 a	10.80 ± 0.68 a	47.42 ± 4.17 a	++
	1.0	-	2.00 ± 0.00 a	2.08 ± 0.36 a	9.78 ± 1.28 ab	35.55 ± 7.01 a	++
	1.0	0.5	1.88 ± 0.13 a	1.99 ± 0.39 a	6.50 ± 0.93 b	55.54 ± 9.44 a	++

These data represent the means of three replicates with standard error. Means with the same letters are not significantly different (LSDT, p < 0.05). -: normal plant; ++: hard hyperhydricity symptoms.

). Additionally, hyperhydricity was not attenuated and was remained at similar levels (Table 4). Therefore, it was concluded that charcoal is not a suitable component for overcoming hyperhydricity.

3.3. Effect of Media Formulation on In Vitro Shoot Induction

To determine the optimal medium composition for multiplication, shoot tips and nodes were cultured on MS or DKW media containing various concentration of 2iP and observed after four weeks.

For shoot tips, there were no significant difference in the number of shoots per explant across all treatment. This is likely due to the presence of apical meristems in shoot tips, which generally produce only one shoot. The plant height was smaller in media without 2iP compared to media with 2iP, indicating that 2iP, a type of cytokinin, plays a role in promoting plant elongation. The number of leaves was highest in MS and DKW media containing 0.5 mg/L 2iP, while leaf chlorosis was least observed in MS and DKW media containing 1.0 mg/L of 2iP (

Table 5. Comparison of the impact of two different media formulation on plant growth and shoot induction from shoot tip explants of Cannabis sativa ‘Cheungsam’ after four weeks of culture.

Explant	Media Type	2IP	No. of Shoot/Explant	Plant Height (cm)	No. of Leaf	Leaf Chlorosis Rate (%)	Hyperhydricity
Shoot tip	MS	-	1.12 ± 0.12 a	1.30 ± 0.30 a	4.94 ± 0.65 b	23.17 ± 7.13 ab	-
	MS	0.25	1.05 ± 0.05 a	3.64 ± 0.48 d	7.81 ± 0.60 a	10.49 ± 3.00 b	-
	MS	0.50	1.18 ± 0.13 a	3.31 ± 0.30 cd	8.94 ± 0.57 a	26.55 ± 5.86 a	-
	MS	1.00	1.00 ± 0.00 a	3.46 ± 0.36 cd	8.18 ±0.57 a	6.36 ± 2.51 bc	-
	DKW	-	1.00 ± 0.00 a	1.53 ± 0.11 a	5.06 ± 0.25 b	11.89 ± 2.60 b	-
	DKW	0.25	1.03 ± 0.03 a	2.39 ± 0.17 bc	7.14 ± 0.27 a	10.47 ± 1.47 b	-
	DKW	0.50	1.11 ± 0.06 a	2.82 ± 0.17 cd	8.29 ± 0.32 a	6.61 ± 1.21 bc	-
	DKW	1.00	1.09 ± 0.05 a	2.69 ± 0.19 cd	7.95 ± 0.35 a	7.65 ± 1.22 bc	-

These data represent the means of three replicates with standard error. Means with the same letters are not significantly different (LSDT, p < 0.05). -: normal plant.

). This suggests that higher concentrations of 2iP have a positive effect on shoot formation compared to lower concentrations. No significant differences were observed between MS and DKW media (Figure 4A–H). Therefore, for shoot tip multiplication, the optimal protocol is to culture shoot tips in MS or DKW media containing high concentration (1.0 mg/L) of 2iP.

Similarly for nodes, there were no significant differences in the number of shoots per explant across all treatments. However, compared to shoot tips, approximately twice as many adventitious shoots were formed. This is likely due to the axillary buds present in the nodes. In terms of plant height, as with shoot tips, both MS and DKW media containing 2iP showed greater growth compared to media without 2iP (Figure 5A–H). For the number of leaves, DKW media produced statistically significantly more leaves compared to MS media, regardless of 2iP concentration (0.25, 0.5 and 1.0 mg/L). In terms of leaf chlorosis rate, the MS control showed statistically significant higher chlorosis, while DKW with 2iP 1.0 mg/L treatment exhibited the lowest chlorosis. There were no statistically significant differences when comparing MS with 2iP 0.25 mg/L and DKW with 2iP (0, 0.25, 0.5 mg/L). Regarding hyperhydricity, the DKW media had lower occurrence rates compared to MS media, regardless of the 2iP concentration (

Table 6. Comparison of the impact of two different media formulation on plant growth and shoot induction from node explants of Cannabis sativa ‘Cheungsam’ after four weeks of culture.

Explant	Media Type	2IP	No. of Shoot/Explant	Plant Height (cm)	No. of Leaf	Leaf Chlorosis Rate (%)	Hyperhydricity
Node	MS	-	1.94 ± 0.06 a	1.60 ± 0.12 c	8.22 ± 0.53 b	45.13 ± 5.58 a	++
	MS	0.25	1.90 ± 0.07 a	2.77 ± 0.24 ab	9.05 ± 0.60 b	39.67 ± 4.53 a	++
	MS	0.50	1.80 ± 0.20 a	2.92 ± 0.81 ab	8.00 ± 1.41 b	24.83 ± 6.87 ab	++
	MS	1.00	2.00 ± 0.00 a	3.10 ± 0.48 a	9.78 ± 1.28 b	36.81 ± 7.01 ab	++
	DKW	-	1.96 ± 0.03 a	1.27 ± 0.09 c	9.18 ± 0.52 b	19.68 ± 2.85 b	+
	DKW	0.25	1.92 ± 0.04 a	2.69 ± 0.16 ab	12.64 ± 0.47 a	22.79 ± 1.94 b	-
	DKW	0.50	1.90 ± 0.04 a	2.86 ± 0.14 ab	13.23 ± 0.41 a	21.23 ± 1.84 b	-
	DKW	1.00	1.92 ± 0.04 a	2.57 ± 0.15 b	13.22 ± 0.48 a	17.79 ± 2.03 b	-

These data represent the means of three replicates with standard error. Means with the same letters are not significantly different (LSDT, p < 0.05). -: normal plant; +: mild hyperhydricity symptoms; ++: hard hyperhydricity symptoms.

). Therefore, the optimal medium composition for node multiplication was determined to be DKW with 2iP 1.0 mg/L.

3.4. Effect of Explants Type on In Vitro Shoot Induction

Shoot tip and node explants were compared for their effect on propagation productivity. After four weeks of culture, comparing the shoot tip and node revealed that the shoot tip exhibited relatively longer shoot length and lower chlorosis rates, while the node produced a higher number of shoots per explant and number of leaves. The best results for the shoot tip were observed in MS medium with 1.0 mg/L 2iP, achieving the greatest plant height of 3.46 cm and the lowest chlorosis rate of 6.36% (Table 5). The highest number of shoots per explant was observed in nodes cultured in MS medium with 1.0 mg/L 2iP, producing an average of 2.0 shoots. The greatest number of leaves was formed in nodes cultured in DKW medium with 0.5 mg/L 2iP with an average of 13.23 leaves (Table 6).

However, all node treatments cultured in MS medium exhibited hyperhydricity, while no hyperhydricity occurred in the shoot tips (Figure 4 and Figure 5). Therefore, while it is difficult to definitively determine the superior tissue, nodes, which produced more shoots per explant, are deemed more suitable for the purpose of mass propagation in micropropagation.

3.5. Rooting of Shoots

The derived shoots were cultured in MS or DKW media supplemented with 2iP and then transferred to media containing IBA (0.25 and 0.5 mg/L) for four weeks. As a result, rooting did not occur in most media, and the plants became necrotic. Consequently, a new solution was implemented by reducing the concentration of MS and DKW media to half. This adjustment resulted in a higher rooting rate compared to full-concentration media. When comparing MS and DKW media, DKW showed a higher rooting rate at all IBA concentrations (0.1, 0.25, and 0.5 mg/L). Specifically, compared to MS medium, DKW media with IBA 0.25 mg/L exhibited over four times higher rooting rate than MS with IBA 0.25 mg/L, and DKW with IBA 0.5 mg/L showed approximately twice the rooting rate of MS with IBA 0.5 mg/L (

Table 7. Comparison of various PGRs concentrations on root induction from Cannabis sativa ‘Cheungsam’ after four weeks of culture.

Media Type	PGRs (mg/L)		Rooting Rate (%)
	IBA	NAA
MS	-	-	18.52 ± 10.36 c
MS	0.1	-	76.92 ± 6.35 ab
MS	0.25	-	19.51 ± 6.00 c
MS	0.5	-	48.15 ± 6.29 bc
MS	0.25	0.05	4.92 ± 7.50 d
DKW	-	-	61.54 ± 4.60 b
DKW	0.1	-	78.95 ± 6.59 ab
DKW	0.25	-	85.37 ± 6.14 a
DKW	0.5	-	91.49 ± 5.26 a

These data represent the means of three replicates with standard error. Means with the same letters are not significantly different (LSDT, p < 0.05).

).

Next, the rooting rates were compared between IBA alone and combination of IBA and NAA. In all concentrations, IBA alone resulted in better rooting rates when compared to the combination of IBA and NAA (Figure 6A–E). In fact, the IBA and NAA combination had lower rooting rates than treatments without PGRs. Based on these results, the combination of NAA with IBA was not used in the DKW media with IBA treatments. Among the IBA-alone treatments, DKW with IBA 0.5 mg/L showed the best result, with no statistically significant difference when compared to DKW with IBA 0.25 mg/L (Figure 6A–I). However, upon visual inspection, root condition, including root length and root thickness, appeared to be better in DKW with IBA 0.5 mg/L (Figure 6H,I). Therefore, the optimal medium composition for rooting was determined to be DKW with IBA 0.5 mg/L.

3.6. Acclimatization

After culture on rooting media for four weeks, in vitro grown plants with heights of over 5 cm were acclimatized. During acclimatization, the relative humidity was gradually decreased. As a result, all plants were successfully acclimatized (Figure 7A). Two weeks after acclimatization, the plants were transplanted into larger pots (Figure 7B). The survival rate was assessed again six weeks later, showing a 100% acclimatization success rate.

4. Discussion

Previous studies have reported successful micropropagation of different cannabis cultivars [15,16,17]. In this study, we developed a protocol for micropropagation from shoot tip and node of cannabis ‘Cheungsam’ for industrial development purposes.

To determine the optimal multiplication medium of ‘Cheungsam’, we tested with various PGRs, such as 2iP, mT, and BA at concentration of 0.25, 0.5, and 1.0 mg/L. Shoot tips cultured on PGR-free media exhibited lower height and fewer leaves. Media containing 2iP and mT, regardless of concentration, showed statistically significant taller plant heights. All treatments with 2iP, mT, and BA resulted in a significant number of leaves. In the case of nodes, PGR-free treatments exhibited better growth than mT and BA, but had a higher leaf chlorosis rate. Among the cytokinin tested, media containing 2iP, irrespective of concentration, resulted in the tallest plant height, the highest number of leaves, and the lowest leaf chlorosis rate. BA, a common cytokinin, is used for multiplication of micropropagation in various crops (Cannabis, Quercus, Tea Tree, Ginger, Stevia) [18,19,20,21,22]. However, in our study, BA-treated plants showed similar plant heights and higher chlorosis rates compared to those without PGRs treatment. Although mT has been recommended as an excellent cytokinin for multiplication in other cannabis micropropagation studies [23,24,25,26], our results for mT-treated plants were not favorable in terms of plant height, number of leaves, and leaf chlorosis rate, similar to the results from BA-treated plants. Conversely, 2iP-treated shoot tip and node explants displayed the greatest plant height, greatest number of leaves, and lowest leaf chlorosis rate across all concentrations. While higher concentrations of 2iP produced better results, the differences were not statistically significant. This accords with the findings of Stephen et al. [17], who reported that 2iP at 5.0 µM yielded the best quality shoots of Cannabis sativa ‘TJ’s CBD’. Therefore, we concluded that 2iP is the most suitable PGRs for the multiplication phase of Cannabis.

However, all explants exhibited blackened cut surfaces regardless of hormone type and concentration, and node explants showed severe hyperhydricity. Hyperhydricity is a common issue in plant tissue culture where plants absorb excessive water, leading to softened tissues and physiological abnormalities [27]. Typical symptoms include translucent, fragile, and twisted leaves that are easily damaged, shortened internodes, and thickened stems [28,29]. Causes of hyperhydricity include high humidity in the culture vessel, excessive cytokinin use, poor vessel ventilation, influence of nitrogen ions, and accumulation of ethylene and ROS. We hypothesized that the hyperhydricity in our experiment was due to the accumulation of phenolic compounds in the culture vessel, leading to high ROS levels. ROS affects ethylene metabolism [30], and ethylene in turn increases ROS accumulation [31]. Thus, explants were likely due to phenolic compound accumulation. To address these issues, we added 0.5 g/L charcoal to the media.

Activated charcoal is composed of carbon arranged in a semi-graphitic form with small particle sizes. It removes non-carbon impurities and oxidizes the carbon surface. Activated charcoal is used in plant tissue culture primarily to adsorb growth-inhibiting substances (such as phenolic compounds) in the culture medium, thereby promoting plant growth [32]. According to a study by Lee and Chang [33], the addition of 200 mg/L of activated charcoal during the multiplication phase in micropropagation improved shoot length and prevented the regeneration of abnormal shoots. In other cannabis varieties, culturing shoot tip and node explants in a medium with 1 g/L of activated charcoal resulted in the formation of long shoots in some genotypes [34]. However, in this study, the addition of activated charcoal to all treatments resulted in inhibited plant growth compared to treatments without charcoal. Komalaballi and Rao [35], reported a decrease in the number of shoots when activated charcoal was added to the micropropagation medium of Gymnema sylvestre [35], and Tivarekar and Eapen [36], found that the addition of activated charcoal to the regeneration medium of mung beans inhibited shoot growth. These results suggest that activated charcoal not only adsorbs growth-inhibiting substances but also essential nutrients necessary for plant growth, leading to reduced growth. Therefore, it is highly suspected that the observed inhibition of the plant growth in our study also caused by the absorption of essential nutrients by charcoal.

When cultured on MS medium, plant cut surfaces turned black and necrotic, and node explants were hyperhydricity, causing growth problems. To solve this problem, experiments were conducted using different commercial media formulations. As a result, the shoot tip grew well in both MS and DKW media. In the case of nodes, DKW medium showed better results than MS medium in all growth indicators including height and number of leaves. In particular, severe hyperhydricity occurred when cultured on MS medium, but no hyperhydricity occurred when cultured on DKW medium. This suggests that the components in the medium have a negative effect on the plants when cultured on MS medium. MS medium is the most widely used medium in plant tissue culture and is effective for most plants, but there are some plants that do not grow well in MS medium. For this reason, a variety of commercial media formulations exists and was developed for specific plants. Compared to MS medium, DKW has a similar ammonium-to-nitrate ratio but lower total amount of nitrogen in the medium [37,38]. El-Dawayati and Zayed [39] reported that hyperhydricity was overcome during the culture process and redifferentiation was successful by reducing the nitrogen ratio of the culture medium. In Phillips and Garda [38], it was reported that excessive ammonium in MS medium can cause growth problems, and Liu et al. [40] showed that when garlic was cultured in MS medium, growth was slow and hyperhydricity occurred. DKW medium also contains significantly higher levels of sulfur (~7×), calcium (~3×) and copper (10×) [16]. However, no clear research has been conducted on which of these factors are important for micropropagation of Cannabis sativa ‘Cheungsam’. Therefore, this is an area that requires further research.

In this study, auxin such as IBA and NAA were added to ½ MS and ½ DKW media to induce rooting. The rooting rate was better in half-concentration media than in full-concentration media. In the case of PGRs, treatment with IBA alone showed a higher rooting rate than treatment without PGRs or with a combination of NAA and IBA. In Stephen et al. [17], the result was contrary to the result that the control group and 0.05 µM IBA were the best. However, according to the study in Lata et al. [41], when 2.5 µM of IBA was applied to the rooting medium of the Cannabis sativa ‘MX’, a 95% rooting rate was achieved. This is the same IBA concentration as the best treatment in this study: ½ DKW with IBA 0.5 mg/L. It is believed that the concentration of hormones required varies depending on the cultivar. Through this study, it was confirmed that IBA induces high rooting in Cannabis sativa, although the cultivars are different. The fact that the DKW medium showed a better rooting rate than the MS medium is presumed to be due to the same factors affecting shoot induction. Therefore, further research is needed on the factors of DKW medium that affect rooting rate of Cannabis sativa ‘Cheungsam’.

5. Conclusions

Cannabis exists as many genetically diverse cultivars. To establish the optimal in vitro micropropagation protocol, a series of experiments were conducted focusing on ‘Cheungsam’ cultivars. In conclusion, for proliferation, the best shoots were obtained in DKW medium containing 1.0 mg/L 2iP, and shoot elongation was achieved when subcultured in the same medium. Subsequently, when cultured in ½ DKW medium containing 0.5 mg/L IBA, the rooting rate was highest, and healthy roots were obtained. When the rooted plants were acclimatized, the acclimatization rate was 100%. Therefore, this protocol can increase the rate at which stable populations can be maintained through in vitro mass production of Cannabis sativa ‘Cheungsam’.