Abstract
Panax vietnamensis Ha et Grushv., commonly known as Vietnamese ginseng or Ngoc Linh ginseng, is a notable species within the Panax genus. In Vietnamese traditional medicine, Ngoc Linh ginseng is revered as a ‘miraculous medicine,’ famed for enhancing physical strength and treating a variety of health conditions. The application of in vitro tissue culture techniques to medicinal plants is acknowledged as a strategic method for generating bioactive specialized metabolites. This comprehensive review highlights the properties of Vietnamese ginseng and emphasizes recent progress in various in vitro tissue culture techniques, including callus induction, somatic embryo induction, cell cultivation in suspension, hairy root cultivation, and adventitious root cultivation. Moreover, the review addresses the challenges and potential future developments related to these techniques. The exploration of these in vitro practices has laid the groundwork for the commercial exploitation of Vietnamese ginseng, facilitating the swift and effective production of both ginseng biomass and its bioactive compounds.
Introduction
In the 1970s, Vietnam introduced a ginseng variety now recognized as one of the top five globally for its unparalleled medicinal properties. This ginseng, known as Ngoc Linh ginseng or “ginseng from the Ngoc Linh mountains”, has become a national treasure.1,2 Despite its esteemed status, there has been a notable lack of robust scientific data regarding its health impacts. Since 1978, Ngoc Linh ginseng has been included in the Vietnamese pharmacopoeia, and its usage has surged due to its efficacy in addressing cardiovascular and neurological disorders, cancer, aging, and stress. Notably, research indicates that this variant of Vietnamese ginseng boasts a higher concentration of ginseng saponins than other species within the Panax genus, including types like protopanaxadiol, protopanaxatriol, ocotillol, and dammarane. These saponins, along with other active elements, contribute to substantiating the health benefits attributed to ginseng. Consequently, Ngoc Linh ginseng is considered one of the most precious Panax species worldwide. 3
Ngoc Linh ginseng is an endemic native plant of the Ngoc Linh mountain and only grows naturally in nine communes surrounding the Ngoc Linh mountain peak, located in the Quang Nam and Kon Tum provinces. 4 Despite this significant cultivation effort, the levels of ginseng consumption and processing have yet to fully exploit its potential. Challenges such as extended growth cycles, diseases necessitating replanting, limited arable land, and labor-intensive farming practices have hindered farmers’ ability to meet market demands. 5 Furthermore, the high cost of using pesticides and unpredictable environmental changes attributed to global warming have spurred researchers to explore alternative approaches to fulfill global demands. 6
Plant tissue culture, which includes tissue, organ, and cell cultures, has emerged as an innovative solution in this scenario. This technique, conducted in a controlled environment, allows for the enhancement of specific medicinal metabolites, offering a safe, sustainable, and efficient production method consistent with good manufacturing practices. It also facilitates the extraction of desired compounds. 7
Currently, research on the tissue culture of Ngoc Linh ginseng is limited, primarily concentrating on adventitious roots, hairy roots, clonal embryos, and cell suspensions. Each of these methods presents unique advantages and disadvantages. This mini-review endeavors to outline some characteristics and encapsulate the in vitro tissue culture approaches applied to Vietnamese ginseng, considering both the most commonly employed tissue culture techniques and the factors that influence them.
Characteristics of Vietnamese ginseng
Characteristics of Botany
Ngoc Linh ginseng is a perennial herb that typically grows to a height of approximately 0.5 meters. The tuberous roots are fleshy and branched, measuring 2–3 cm in diameter, often horizontally oriented and protruding above the soil. The plant exhibits large, spherical, or top-shaped tuberous roots, reaching diameters of 5–7 cm. The stem, with a diameter of 0.3–0.6 cm, is smooth and sheds in winter, leaving circular scars on the rootstock. The leaves are palmate, with 3–4 (occasionally 5 or 6) leaflets arranged in a whorl; each leaflet is elongated, measuring 10–14 cm long and 3–5 cm wide, with pointed tips, serrated margins, and stiff hairs on both sides. The umbellate inflorescence emerges at the apex, with a stalk 15–30 cm long, surpassing the leaves by 1.5–2 times. The floral canopy spans 2.5–5 cm, bearing 50 to 140 flowers. Flowers are small, off-white, or bluish-white, with 5 bell-shaped sepals topped with small triangular lobes and fused at the base (1-1.5 mm high); the petals are broadly triangular. The stamens are interspersed among the petals; the ovary is bilocular with a bifurcated stigma, and the pollen sacs are ovoid and dorsally attached. The fruit is a slightly flattened sphere, measuring 0.6–1.0 cm in diameter, with a stigma, turning bright red when ripening, often with black spots at the apex. It contains 1 or 2 seeds, white or off-white, measuring 6–7 mm in length, 5–6 mm in width, and 2 mm in thickness, with a rough surface marked by bumps and indentations.8–10
Natural Conditions in the Distribution Area of Ngoc Linh ginseng
Ngoc Linh ginseng is distributed in the high mountainous region of the Ngoc Linh massif, located within the territories of the provinces of Kon Tum and Quang Nam. The elevation in this area ranges from 1200 m to 2500 m, and the forest cover density exceeds 70%. This region is characterized by marked isolation, steep slopes, and numerous narrow, deep valleys. The climate transitions from humid tropical to subtropical, conducive to ginseng cultivation, with an average annual rainfall between 2500 mm and 3400 mm. Rainfall is concentrated from June to September on the western slope and from September to November on the eastern slope. 1
Ngoc Linh ginseng is primarily found within the tropical belt of the Northern Hemisphere. However, due to its occurrence at altitudes above 1800 meters, the average temperature in this region is notably lower than in lower elevations. Air temperature reaches its lowest point during December and January, while it peaks in April and May. The average annual temperature varies from 14 °C to 20.5 °C, with diurnal temperature fluctuations of 8 °C to 9 °C. Average annual evaporation rates range from 670 mm to 870 mm, and the annual average humidity lies between 84% and 87.5%. 1
The Ngoc Linh ginseng habitat is within the pristine forests of Kon Tum and Quang Nam provinces, featuring an extremely diverse vegetation community, with a coverage rate exceeding 70% and a vegetative layer thickness of over 15 cm. Ginseng grows in gray soils, formed in situ, across various terrains. Soil fertility characteristics include a mechanical composition ranging from sandy loam to clay, with sand content between 47.21% and 69.51%, silt from 6.46% to 23.22%, and clay from 17.66% to 32.71%. The soils are acidic, with pH values ranging from 3.72 to 4.81 for H2O and 3.09 to 4.07 for KCl. The cation exchange capacity (CEC) spans from 7.62 to 21.69 meq/100 g of soil, and the total exchangeable base saturation ranges from 0.22 to 3.62 meq/100 g of soil. Exchangeable acidity varies from 0.24 to 2.22 meq/100 g of soil. High levels of organic carbon and total nitrogen are present, with concentrations ranging from 2.40% to 10.07% for organic carbon (OC) and 0.08% to 0.57% for nitrogen (N). 1
Chemical Constituents of Ngoc Linh ginseng
Vietnamese ginseng stands out as a unique and highly value species within the Panax genus. Its rich and diverse chemical composition contributes to its remarkable therapeutic benefits. Previous phytochemical studies have primarily focused on the root and rhizome, which are the most commonly used parts of Vietnamese ginseng. 11
Attele et al (1999) highlighted its health benefits, emphasizing the presence of both ginseng saponins and ocotillol-type saponins. 12 Notably, the ocotillo-type saponins is an unique feature, which constitutes over 50% of the total saponin content in Vietnamese ginseng, and it has not found in any other Panax species such as Panax ginseng, Panax quinquefolius, or Panax notoginseng, respectively.3,13
Yamasaki (2020) identified 37 saponins in Panax vietnamensis, including Notoginsenoside R1, various Ginsenosides (Rg1, Re, Rb1, Rc, Rb2, Rd), Majonoside R1, Ginsenoside Rh1, Quinquenoside R1, and several Vina-ginsenosides (R1, R2, R12, R14), among others. Particularly noteworthy is Majonoside R2, accounting for 5.29% of the saponins in Vietnamese ginseng but is absent in Panax ginseng. 14
In total, 52 dammarane-type triterpene saponins have been characterized in Vietnamese ginseng, including 26 new saponins such as vina-ginsenosides R1–R25 and 20-O-methylginsenoside Rh1. 15 The substantial presence of ocotillol-type saponins, especially majonoside R2, distinguishes Vietnamese ginseng from other ginseng species. Notable saponins include majonoside R1, majonoside R2, protopanaxatriol oxide II, pseudo-ginsenoside F11, pseudo-ginsenoside RT4, ginsenoside Rh4, majoroside F1, and various ginsenosides (Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rh1), as well as gypenosides IX and XVII, 20-glucoginsenoside Rf, notoginsenosides Fa, R1, R6, pseudo-ginsenosides RC1, RS1, quiquenoside R1, ginsenoside Ro, and hemsloside Ma3. 15
Vietnamese ginseng also boasts the highest volatile oil content compared to other Panax species. Since the initial report in 1993, these saponins have been categorized into protopanaxadiol, protopanaxatriol, and ocotillol subtypes. 16 Notably, ginsenoside Ro and hemsloside Ma3 are exceptions, being oleanane-type saponins. Major saponins in Vietnamese ginseng include ginsenosides Rb1, Rb2, Rd, Re, Rg1, Majonoside R1, MR2, and Notoginsenoside R1, along with Vinaginsenosides R1, R2, and R11. The raw material of Vietnamese ginseng exhibits higher and more diverse saponin content compared to Asian and Korean ginseng. Processing methods, such as steaming, modify the saponin profile increasing less polar ginsenosides (eg, Rg3, Rg5, Rk1, Rk3, Rh4) while decreasing polar ones. Interestingly, ocotillol-type saponins remain stable during steaming.13,14
The distinctive chemical profile of Vietnamese ginseng not only sets it apart within the Panax genus but also enhances its value in traditional and modern medicine. Its unique composition of saponins and other bioactive compounds supports a variety of health benefits, including anti-inflammatory, antioxidant, and adaptogenic effects, which are highly regarded in both traditional and contemporary therapeutic practices.
In Vitro Tissue Culture Approach
The in vitro tissue culture approach encompasses several advanced techniques, including callus induction, somatic embryo induction, cell cultivation in suspension, hairy root cultivation, and adventitious root cultivation. Each technique offers unique benefits, from mass production and genetic uniformity to enhanced medicinal compound accumulation. Table 1 provided a detailed overview of these tissue culture types, including the types of explants used, culture media compositions, specific conditions, significant findings, and references, illustrating the versatility and effectiveness of in vitro culture techniques in advancing the cultivation and research of this valuable medicinal plant.
An Overview of Tissue Culture Types in Panax vietnamensis.
Callus Induction of Vietnamese ginseng
Inducing callus and promoting dedifferentiation are pivotal for the propagation of ginseng seedlings through tissue culture. Successful callus cultures of P. ginseng have been established in both solid and liquid media.39,40 It has been acknowledged that ginseng callus can synthesize secondary metabolites comparable to those found in naturally grown plants. 41 Furthermore, successful development of callus cultures has been achieved for P. quinquefolius and P. sikkimensis, aiming to produce ginsenosides and anthocyanins, respectively. 6 Factors such as the morphology and texture of the callus significantly affect its division rate and differentiation potential, which in turn impacts the formation of somatic embryos and seedlings. Optimizing culture environments for callus induction is thus crucial to ensure both the quantity and quality of the callus.42,43 Common explants for inducing Ngoc Linh ginseng callus include buds, petioles, stems, and leaves. The Murashige and Skoog (MS) basic medium is frequently used for this purpose, along with the Schenk and Hildebrandt (SH) medium. 44
Nhut et al (2009) initiated the culture of leaf and petiole explants of Vietnamese ginseng with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L TDZ (Thidiazuron), under a 16-h photoperiod. Interestingly, smaller calluses measuring 0.5 × 0.5 cm demonstrated superior growth compared to larger counterparts. Subsequent culturing on MS medium containing 1.0 mg/L BA (6-benzylaminopurine) and 1.0 mg/L NAA (α-naphthaleneacetic acid) led to enhanced shoot regeneration from the calluses. For root induction, calluses were transferred to MS/2 medium containing 3.0 mg/L NAA. Remarkably, robust root proliferation was observed on MS/2 medium containing 5.0 mg/L NAA. Thin-layer chromatography results for saponin qualification revealed the presence of ginsenoside-Rg1 and majonoside-R2 in calluses, shoots, and roots derived from the cultures. Ginsenoside-Rb1 was exclusively found in the roots. Although the exact quantities of these compounds were not determined, the intensity of the visualized colors suggests that majonoside-R2 is the most abundant, followed by ginsenoside-Rg1, and then ginsenoside-Rb1 in adventitious roots. Additionally, some bands exhibited positions similar to those of standard samples (Korean and Vietnamese ginseng), indicating the potential presence of other saponins at low concentrations in the cultured Ngoc Linh ginseng callus. 17
Nhut et al (2011) employed Ngoc Linh ginseng leaves as explants for callus induction. By comparing various media and combinations of plant growth regulators, they observed that callus formation occurred in both media supplemented with 2,4-D alone or combined with TDZ. The highest frequency of callus induction was achieved with 1.0 mg/L 2,4-D and 0.2 mg/L TDZ. Additionally, they identified SH medium containing 0.2 mg/L TDZ and 1.0 mg/L 2,4-D as the optimal medium for callus proliferation. Notably, the maximum number of callus-derived shoots was achieved on SH medium enriched with 50 g/L sucrose in conjunction with 2.0 mg/L BA. For effective root regeneration from adventitious shoots, SH medium containing 1.0 mg/L NAA proved successful. Furthermore, the researchers successfully acclimatized plantlets on Ngoc Linh mountain without the need for a growth chamber, achieving an 85% survival rate after two months. Interestingly, they also observed a substantial increase in root length. Overall, this study presented a highly effective in vitro regeneration method for P. vietnamensis, which holds promise for large-scale cultivation and propagation of this significant medicinal herb. 18
Nhut et al (2012) investigated the induction of somatic embryos, callus, shoots, and adventitious roots from longitudinal thin cell layers (lTCLs) of P. vietnamensis petioles using the Thin Cell Layer (TCL) technique. lTCL explants from three-month-old in vitro plants were cultured on semi-solid MS medium supplemented with various hormones. After eight weeks, cultures on MS medium with 1.0 mg/l 2,4-D and 0.1 mg/l TDZ in the dark, 2.0 mg/l NAA in the dark, and 1.0 mg/l 2,4-D under light resulted in a 100% callogenesis rate, 53.3% embryogenesis, 100% adventitious root formation, and 26.7% shoot formation, respectively. Metabolite extracts from both naturally grown Vietnamese ginseng tissue and callus biomass indicated that the callus biomass contained MR2, G-Rb1, and G-Rg1. Additionally, the metabolites extracted from the callus biomass exhibited bands similar to those found in extracts from naturally grown plants, indicating a similar chemical profile between petiole lTCL-derived calli and their natural counterparts. Additionally, the analysis of saponins from the in vitro P. vietnamensis callus revealed that all three key saponins present in the petiole lTCL-derived callus were present, with the following abundances: MR2 at 0.424%, G-Rg1 at 0.061%, and G-Rb1 at 0.087%. This study indicates that the lTCL morphogenesis protocol developed is effective for in vitro cultivation of Vietnamese ginseng. Additionally, there is no noticeable difference in the saponin profile between the callus produced and the plants grown in their natural environment. 19
Trieu et al (2013) utilized buds, petioles, and stems from five-year-old Vietnamese ginseng to induce callus using 1.0 mg/L 2,4-D and 0.2 mg/L TDZ. Remarkably, this medium also proved to be the most effective for callus proliferation. After an 8-week period, the callus was transferred to SH medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA to induce somatic embryo formation and shoot development from the embryogenic callus. 20
More recently, Nhut et al (2017) investigated the impact of various LED lighting conditions on callus formation, subsequent somatic embryo development, plantlet formation, and saponin accumulation. Their findings revealed significant differences in growth and development, with distinct lighting conditions being optimal for different stages. Notably, callus cultivated under yellow LEDs exhibited the maximum fresh weight (1197 mg) and dry weight (91.7 mg) after 3 months of culture. Optimal plantlet formation from embryogenic calli occurred with a combination of 60% red LED and 40% blue LED. The research further confirmed the influence of LEDs on saponin content accumulation. Notably, the combination of red LED (20%) and blue LED (80%) yielded the highest MR2 content (0.52%). Conversely, the highest levels of Rg1 (0.41%) and Rb1 (1.18%) were detected under fluorescent light conditions. Overall, these results underscore the significance of using LEDs to enhance the micropropagation of P. vietnamensis. 21
Induction of Somatic Embryos from Vietnamese ginseng
Somatic embryogenesis is the process by which entire plants are generated from somatic plant cells (or other non-sexual cells). Unlike organogenesis, where aerial structures and roots develop directly from the somatic embryo, somatic embryogenesis is a polar process. It can occur either directly or indirectly. The process involves four fundamental stages: (1) Callus induction: Initiating the formation of undifferentiated tissue (callus) from somatic cells. (2) Embryo formation and proliferation: Developing embryogenic structures within the callus. (3) Embryo maturation: Progressing through various developmental stages, including globular, heart, torpedo, and cotyledonary forms. (4) Embryo germination: Facilitating the transition from the embryonic stage to a fully developed plant. This intricate process holds promise for the large-scale propagation of important medicinal plants.45,46 For many plant species, somatic embryogenesis serves as a crucial and reliable regeneration technique for creating true-to-type clones. In the case of Vietnamese ginseng, the induction of somatic embryogenesis has become a common method for in vitro propagation.
Truong et al (2013) detailed their research on the generation and expansion of embryogenic calli within a liquid environment, alongside the initiation of somatic embryos, and the development of shoots and roots from these embryos during the culture process. They also reported on the enhancement of somatic embryo tissues in a liquid medium. Specifically, leaf sections were cultivated in an MS medium containing 2 mg/L 2,4-D to stimulate callus formation. For the generation of somatic embryo tissues, the calluses derived from these leaf sections were further cultured in an MS medium containing 1 mg/L of 2,4-D, 1 mg/L of NAA, 0.2 mg/L of kinetin, and 10% coconut water. The growth of these embryogenic calluses was observed in a half-strength MS liquid medium containing 0.5 mg/L 2,4-D and 0.5 mg/L NAA. 22
Hien et al (2014) explored the potential of direct somatic embryogenesis in P. vietnamensis using the thin cell layer technique. They cultured various explants, including leaves, petioles, and main roots from 3-month-old in vitro plantlets, in an MS medium. This medium was supplemented with NAA and 2,4-D at concentrations ranging from 0.1 to 2.0 mg/L. After 10 weeks, they observed that leaf explants were the most conducive to direct somatic embryogenesis. Specifically, leaves in an MS medium containing 2 mg/L NAA demonstrated the most significant propensity for this process. 23
Tung et al (2020) explored sterilization and embryogenic callus induction in Ngoc Linh ginseng leaf explants using nanosilver. They discovered that treating the explants with 0.5 g/L nanosilver for 15 min resulted in the lowest contamination rate of 20%. Conversely, 0.2 g/L nanosilver for 20 min was the most suitable for embryogenic callus induction, with a success rate of 72.22% and a fresh weight of 0.77 g. The researchers also found that an MS medium enriched with 1.0 mg/L 2,4-D, 0.5 mg/L NAA, 0.2 mg/L Kinetin, and 1.6 mg/L nanosilver significantly increased the frequency of somatic embryogenesis and germination. After an 8-week culture period, the somatic embryos from nanosilver-treated leaves were twice as numerous as those from untreated explants. Moreover, the addition of 1.0 mg/L NAA and 1.2 mg/L nanosilver to the medium yielded the highest shoot and root growth, number of roots, and fresh and dry weight of the plantlets. This study suggests that nanosilver pretreatment and supplementation in the culture medium can enhance embryogenesis and plantlet production in Ngoc Linh ginseng cultured in vitro. 24
Cell Cultivation in Suspension of Vietnamese ginseng
Plant cell suspension culture is currently considered the most effective approach for producing secondary medicinal metabolites on an industrial scale, typically derived from natural plants. 43 This method is ideal for large-scale operations, utilizing specially designed bioreactors for mass production. 47 The technology behind plant cell suspension culture has become increasingly favored for laboratory or industrial biosynthesis of specific metabolites, thanks to its uniform growth patterns, reduced production time, and streamlined bioreactor design. 21 In the realm of ginseng cell suspension culture, research is concentrated on factors influencing cell growth and metabolite synthesis, such as selecting high-quality cell lines, applying elicitors, and optimizing environmental and chemical conditions. 43 However, studies on Ngoc Linh ginseng cell suspension culture are still in their infancy, focusing mainly on identifying the best sources for initiating callus suspension and analyzing the growth dynamics of the cell suspension. 26
Titova et al (2024) developed six suspension cell culture lines of P. vietnamensis and assessed their growth, cytological characteristics, and ginsenoside profiles. All lines exhibited robust growth, with dry weight productivity ranging from 0.30 to 0.83 grams per liter per day and maximum biomass accumulation between 10 and 22 grams per liter. The cell biomass extracts contained ginsenosides from the protopanaxadiol group (Rb1, Rb2/Rb3, malonyl-Rb1, malonyl-Rb2/Rb3), the oleanolic acid group (R0 and chikusetsusaponin IV), and the ocotillol group (vinaginsenoside R1). Notably, this was the first time chikusetsusaponin IV was found in P. vietnamensis cell cultures. These findings highlight the potential of Vietnamese ginseng suspension cultures for the biotechnological production of ginsenoside-rich biomass, particularly those containing oleanolic acid and ocotillol groups. 25
Thai et al (2019) investigated how different sucrose levels and container sizes affect the development of cell suspensions. They found that cell suspensions grew most effectively in an MS medium enhanced with 1.5 mg/L NAA and 50 g/L sucrose, achieving a fresh weight of 37.4 mg/mL and a dry weight of 3.6 mg/mL. When grown in bioreactors of 3L, 5L, and 10L capacities, the cell suspensions expanded by 2.1 to 2.3 times the original callus biomass within four weeks. Additionally, cell suspensions cultured in 500 mL flasks on a shaker set to 120 rpm demonstrated significant growth, with the highest recorded yield being 50.2 mg/mL for fresh weight and 3.2 mg/mL for dry weight. 26
Trong et al (2017) investigated the effects of various plant growth regulators on biomass accumulation in Vietnamese ginseng cell suspension cultures. They discovered that the highest cell induction occurred with 2.0 mg/L kinetin in MS media after 24 days. The maximum biomass was obtained with 1.0 mg/L BA and 1.5 mg/L NAA after the same period. Additionally, yeast extract and casein hydrolysate also promoted biomass growth, with yeast extract increasing it by 1.4–2.4 times and casein hydrolysate by 1.8–2.6 times. The highest biomass was achieved with 1.0 g/L yeast extract and 2.0 g/L casein hydrolysate on day 21, with growth peaking at 24 days. 27
Ket et al (2017) explored the effects of macro-element concentration variations on the in vitro callus culture of P. vietnamensis, finding that optimal callus growth occurred in media with 0.5 to 1.0-strength KNO3 and CaCl2, along with 1.0-strength NH4H2PO4 and MgSO4. They also discovered that a medium replenishment strategy significantly promoted cell suspension growth, with the most substantial increase achieved by supplementing with 1/4 MS towards the end of the third week of cultivation. 28
Cuong et al. (2016) investigated the effects of various factors on cell cultivation in the suspension culture of P. vietnamensis. They explored the influence of growth regulators, formulations of mineral salts, and cultural environments. The study also included growth curve analysis and examined the impact of plant growth regulators on the regeneration of P. vietnamensis cell suspension. Notably, the optimal growth conditions for P.vietnamensis cell suspension were achieved using a MS/2 medium enriched with 1.5 mg/L NAA and 50 g/L sucrose, with a pH of 6.3. Additionally, subculturing during the stationary phase (around the 14th-16th day of cultivation) led to the strongest growth, and somatic embryos were induced by transferring the cell suspension to a fresh medium containing 3.0 mg/L NAA after 30 days of cultivation. 29
Truong et al (2014) reported on the successful initiation and proliferation of somatic embryos of Ngoc Linh ginseng in liquid culture using leaf explants. Initially, friable callus was generated on MS agar medium enhanced with 1 mg/L 2,4-D and 0.2 mg/L kinetin. This callus was then transferred to an MS liquid medium with the same concentrations of 2,4-D and kinetin, plus an addition of 500 mg/L casein hydrolysate, to establish a cell suspension. For the development of globular somatic embryos, this suspension was cultivated in B5 liquid medium containing 3 mg/L IBA (Indole-3-butyric acid). To induce secondary embryogenic callus and somatic embryos, embryonic cotyledon explants and intact immature somatic embryos were cultivated on MS agar medium with 10% coconut water, with or without 0.2 mg/L IBA. Further cultivation of these embryogenic structures was performed in liquid media, both with and without plant growth regulators. This research has been instrumental in advancing methods for large-scale micropropagation and the synthesis of secondary metabolites in plants. 30
Thanh et al (2008) highlighted the importance of optimizing macroelement concentrations to enhance both the growth of Ngoc Linh ginseng cells and the production of valuable ginsenosides. The findings revealed that the biomass of the ginseng cells expanded most significantly when the culture medium contained half the standard concentration of NH4NO3, leading to the highest recorded ginsenoside accumulation of 6.5 mg/g dry weight (DW). The optimal cell growth occurred in a medium with the standard concentration of KNO3, while doubling this concentration to 2.0 strength resulted in a notable ginsenoside content of 6.1 mg/g DW. A higher concentration of MgSO4 proved to be beneficial for both the growth of the cells and the accumulation of ginsenosides, with a peak value reaching 5.5 mg/g DW. Increasing the concentration of CaCl2 in the medium correlated with an increase in both cell growth and ginsenoside content, achieving the maximum ginsenoside concentration of 5.7 mg/g DW at 1.5 times the standard strength. 31
Hairy Root Culture of Vietnamese ginseng
Previous studies have shown that these hairy roots grow more swiftly and yield higher amounts of ginsenosides compared to both suspended cells and adventitious roots. 48 The ability of hairy roots to produce multiple metabolites suggests their potential for cost-effective mass production. The in vitro generation of secondary metabolites has been extensively explored using transformed hairy roots across various plant species. These specialized root lines are advantageous for the consistent and standardized extraction of plant-based valuable compounds. Moreover, the growth pattern of hairy root cultures is unique, and the production of metabolites may not always coincide with the growth phases of the hairy roots. 49
Ha et al (2016) developed hairy root cultures of P. vietnamensis using three Rhizobium rhizogenes strains. R. rhizogenes ATCC 15834 was the most effective, inducing roots at 14.1% of the wounding sites after 28 days. From 350 roots induced by ATCC 15834, 31 lines produced lateral roots on a hormone-free SH medium. Hairy roots were grown in 100 mL flasks with 30 mL SH liquid medium, 30 g/L sucrose, and pH 5.8, incubated in darkness at 25 ± 1 °C with shaking at 100 rpm. After 1.5 years, the roots increased approximately 20-fold in 2–3 months. Analysis revealed saponins including protopanaxadiol (0.57%), protopanaxatriol (0.028%), and ocotillol (4.3%). Rhizomes had higher levels of these ginsenosides: protopanaxadiol (2.29%), protopanaxatriol (1.81%), and ocotillol (3.59%). Hairy roots showed high ocotillol levels and trace amounts of MR2, along with significant pseudoginsenoside F11 (0.013% and 0.26%) and vinaginsenoside R1 (0.14% and 3.8%). 32
According to Loan et al (2014), the rol gene was introduced into Vietnamese ginseng via Agrobacterium rhizogenes to foster the development of hairy roots. The experiment was conducted on two tissue types from in vitro seedlings: petioles and leaves, chosen as the vectors for gene transfer. The study aimed to initiate hairy root formation by transferring the rol gene into these specific tissues. The experimental procedure involved infecting and then incubating petioles and leaves of in vitro plantlets for 30 min, followed by a 72-h culture period. The outcomes demonstrated a more successful induction of hairy roots in petiole tissues, with a 14.8% induction rate and an average of 3.3 roots per explant, compared to a 2.8% induction rate and 1.6 roots per explant in leaf tissues. The induction process took 5 weeks for petioles and 6 weeks for leaves. The presence of the rolC gene in the newly formed hairy roots was confirmed through PCR analysis. Furthermore, the secondary metabolite profile of the transgenic plants revealed the presence of MR2, Rb1, and Rg1—compounds typically found in P.vietnamensis. The study also found that liquid culture with agitation provided the optimal conditions for the development of hairy roots. The successful enhancement of root formation in this research presents a promising avenue for the industrial-scale production of saponins, leveraging the potential of hairy root cultures. 33
Hoang et al (2014) evaluated the effects of various medium components on the growth of transgenic Ngoc Linh ginseng hairy roots induced by Agrobacterium rhizogenes ATCC 15834. Factors examined included mineral content, sugar content, culture substrates, pH, temperature, and activated charcoal content. After 2 months, optimal induction, growth, and development occurred in the dark at 25 °C on SH medium with 8 g/l agar, 50 g/l sucrose, 1 g/l activated charcoal, and a pH of 5.8 to 6.1. This setup resulted in the highest number of new roots, as well as the greatest fresh and dry weights. Furthermore, culturing the hairy root lines in a liquid shaking system significantly enhanced root proliferation compared to the traditional solid agar medium. 34
Adventitious Root Culture of Vietnamese ginseng
In recent developments, Panax ginseng has witnessed successful induction of adventitious root cultures. The parameters affecting both the biomass yield and the accumulation of ginsenosides have been effectively standardized. Furthermore, the application of bioreactor and bioprocess technologies has been refined to facilitate the production of ginsenosides from these adventitious root cultures. 50
According to Lieu et al (2011), the impact of various culture media and auxin levels on the initiation and development of adventitious roots in Vietnamese ginseng was explored. The research identified optimal conditions for maximal callus formation, which was achieved after two months in an MS medium enriched with 50 g of sucrose, 8 g of agar, and 1 mg/L 2-4D. Additionally, mature explants were transferred to a Gamborg (B5) solid medium containing the same amounts of sucrose and agar, but with a higher auxin concentration of 5 mg/L IBA, and were cultivated in continuous darkness. This method resulted in a significantly high number of roots from the mature explants. The findings suggest that adjusting key organic nutrients in the culture medium can enhance both the biomass and growth of P. vietnamensis cultures. 35
Ket et al (2012) found that initial sucrose concentrations significantly influenced the growth of adventitious roots in Ngoc Linh ginseng cell suspensions, with a 5% sucrose concentration yielding the highest growth. An inoculum density of 2% was deemed optimal, as higher densities decreased growth rates. The study also reported the successful development of adventitious roots in 5-liter bioreactors with 2 liters of SH medium, enhanced with 5.0 mg/L IBA and 0.02 mg/L BA, resulting in a 5.6-fold increase in root growth over 45 days. 36
Linh et al (2015) investigated the advancement of secondary root formation and growth from individual adventitious roots. The study found that adventitious roots originating from petioles (measuring 2 cm and with the root tip excised) were most effective in initiating secondary root formation, achieving a 94.33% success rate. These roots exhibited rapid growth and high-quality development after 48 days in an MS medium enriched with 7.0 mg/L IBA and 0.5 mg/L BA. Growth indicators were twice as high as those in B5 medium, and the dry weight exceeded that in SH medium. The NH4+/NO3− ratio of 7.2 and 18.5 in the modified MS medium was found to be optimal for the proliferation of lateral roots. Remarkably, the dry weight of the secondary roots significantly increased after 56 days under 80% dark conditions. The roots cultivated in this manner have the potential to serve as starting material for scaled-up culture in bioreactors. 37
Recently, Linh et al (2018) optimized P. vietnamensis adventitious root growth by adjusting the modified MS medium with 7 mg/L IBA and 0.5 mg/L BA. Over 84 days, Rg1 and MR2 content increased, with MR2 showing a sharp rise from day 42 to 70. Although root development and Rb1 content declined after 56 days, MR2 and Rg1 continued to increase. The highest Rg1 content occurred at day 42, and saponin productivity peaked at day 56. Elicitation using three plant-hormone elicitors (jasmonic acid, abscisic acid, and salicylic acid) and two cell-wall elicitors (yeast extract and chitosan) significantly boosted saponin accumulation. Specifically, 0.48 mM jasmonic acid led to the highest Rg1 content (0.32% g/DW), while 0.24 mM jasmonic acid resulted in the highest Rb1 content (0.85% g/DW). Additionally, adventitious root cultures treated with 0.96 mM jasmonic acid exhibited the highest MR2 content (2.83%). Among the elicitors, 150 mg/L yeast extract yielded optimal root biomass and total saponin productivity. These findings suggest that P. vietnamensis adventitious root biomass effectively develops in the modified MS medium after 56 days and can be scaled up successfully in a 20 L bioreactor. 38
Challenges and Perspectives
Plant tissue culture encompasses a broad range of techniques including the cultivation of tissues (such as meristems and leaf tissues), organs (like adventitious and hairy roots), and cells, and is increasingly seen as a viable alternative for pharmaceutical production. This method is advantageous due to its controlled conditions and the ability to tailor processes to enhance specific medicinal compounds. 51 Recognized for its safety, consistency, adherence to good manufacturing practices, and simplified extraction processes, plant tissue culture is a proven method for generating substantial amounts of metabolites for research and extraction. Crucially, this in vitro production is not affected by seasonal variations, climate, or environmental factors, and the absence of pathogens in cultured cells reduces processing costs. 52
For ginseng production through tissue culture to be economically feasible, certain criteria must be fulfilled. These include the high value of the end product, eco-friendly practices, avoidance of harmful chemicals, a clearly defined manufacturing process, quick and efficient cultivation, independence from external environmental influences, consistent plant growth, and an increase in the desired metabolites’ yield. These factors are essential for the commercial success of ginseng cultivation via tissue culture. 6
While biotechnology has historically been instrumental in advancing ginseng cultivation, several challenges hinder its full potential in crop improvement. Among these, microbial contamination from bacteria, fungi, yeasts, and insects poses a significant threat, leading to tissue loss and potential financial setbacks, especially if undetected in the early stages. This issue is exacerbated in large-scale automated systems like bioreactors, where vast quantities of plant tissue are at risk. 53
Furthermore, the phenomenon of hyperhydricity, characterized by excessive water retention and abnormal plant development, poses a concern in liquid media cultures. While some plant tissues thrive in liquid media, others are adversely affected, leading to hyperhydricity due to prolonged contact with the liquid and other environmental factors. Moreover, the genetic stability and overall performance of plants cultivated in automated systems remain largely unexplored, with few studies addressing the genetic implications of such cultivation methods. The influence of liquid media on genetic anomalies has yet to be thoroughly investigated. 54
Conclusion
Ngoc Linh ginseng stands out as a particularly valuable medicinal plant that has been heavily exploited. Today, the supply of ginseng relies mainly on field cultivation, as wild Ngoc Linh ginseng is scarce. The plant grows slowly, taking at least 4 years to reach commercial viability, and requires specific environmental conditions such as height, humidity, and lighting. Moreover, to ensure quality and productivity, farmers must continually establish fresh ginseng fields for subsequent seasons. However, little has changed in field cultivation techniques to date.
While biotechnology has shown high efficiency in plant regeneration, the industrialization of Vietnamese ginseng, especially Ngoc Linh ginseng, lags behind that of other countries like China, Japan, and South Korea. Research on Ngoc Linh ginseng, compared to other species within the Panax genus, has been fragmented and less comprehensive. This paper provides an updated overview of the in vitro culture process for Ngoc Linh ginseng, highlighting researchers’ efforts to optimize and standardize culture conditions to enhance biomass and ginsenoside content. Notably, in vitro culture methods can yield significant biomass in a fraction of the time needed for traditional cultivation methods.
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
