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Plant micropropagation has been adapted in the fields of agriculture, horticulture, forestry, and other related fields for large-scale production of elite plants. The use of liquid media and adoption of bioreactors have escalated the production of healthy plants. Several liquid-phase, gas-phase, temporary immersion, and other modified bioreactors have been used for plant propagation. The design, principle, operational mode, merits, and demerits of various bioreactors used for the regeneration of propagules, such as bulblets, cormlets, rhizomes, microtubers, shoots (subsequent rooting), and somatic embryos, are discussed here. In addition, various parameters that affect plant regeneration are discussed with suitable examples.

The purpose of the present study was to evaluate the growth potential and some rarely reported bioactivities (antioxidant, thrombolytic, anticoagulant, and anthelmintic) of Panax ginseng C.A. Meyer adventitious roots. To demonstrate the growth, shake flask and laboratory-scale bioreactor cultures have been employed. The obtained biomass was dried and extracted with water, ethanol, and methanol. The growth ratio (12.62 ± 1.03) observed in the bioreactor was significantly higher than in the shake flask culture. The presence of 10 different phytochemical classes, including carbohydrates, saponins, glycosides, and terpenoids were detected in qualitative estimation. Significant quantities of phenolics, flavonoids, proteins, and tannins were determined. Dose-dependent antioxidant activities were observed, and the IC50 values of methanolic and ethanolic extracts were very similar to the standard. The highest (29.26 ± 5.31%) thrombolytic potential was shown by the methanolic extract. The ethanolic extract significantly extended the coagulation times up to 2.5 fold. The highest anthelmintic properties in terms of paralyzing (2.21 ± 0.31 min) and killing (3.69 ± 0.41 min) of the parasitic worms were displayed by the aqueous extract. The in vitro root growth implies the commercial feasibility of ginseng production in Bangladesh and the demonstration of potential bioactivities strengthens medicinal implications and also offering new research areas.

Eleutherococcus senticosus (Rupr. & Maxim.) Maxim., popularly known as ‘Siberian ginseng’, is an important medicinal plant. Pharmacologically active compounds of this plant are called eleutheroside...

In this study, we aimed to verify the effect of nutritional factors on the accretion of secondary metabolites in the adventitious root (AR) cultures of Malaysian ginseng (Eurycoma longifolia Jack) grown in small-scale bioreactors. AR were induced from leaf explants and cultured in different types of media including Murashige and Skoog (MS) medium, Driver Kuniyuki Walnut (DKW) medium, Gamborg’s B5 medium, Woody Plant Medium (WPM), and ¾ MS medium. Among these media, the MS and Gamborg’s B5 media induced lateral root development from initial inoculum, which accounted for the increase in AR biomass accretion. By contrast, the DKW and WPM media did not induce lateral root formation from the cultured explants. The ¾ MS medium was optimal for the growth of AR and accretion of secondary metabolites, after 7 weeks of culture, the biomass of AR increased by 8.6-fold in ¾ MS medium, and the total phenolic and flavonoid contents reached 5.23 and 2 mg g−1 of tissue dry weight, respectively. Analysis of mineral elements in the spent medium revealed that ¾ MS medium was most suitable for nutrient supply to developing AR. LC–MS analysis showed the accretion of eurycomanone, a therapeutically useful metabolite, in the AR of Malaysian ginseng.

The Rubia genus includes major groups of medicinal plants such as Rubia cordifolia, Rubia tinctorum, and Rubia akane. They contain anthraquinones (AQs), particularly alizarin and purpurin, which have pharmacological effects that are anti-inflammatory, antioxidant, anticancer, hemostatic, antibacterial, and more. Alizarin and purpurin have been utilized as natural dyes for cotton, silk, and wool fabrics since the dawn of time. These substances have been used in the cosmetics and food industries to color products. The amount of AQs in different Rubia species is minimal. In order to produce these compounds, researchers have established cell and organ cultures. Investigations have been conducted into numerous chemical and physical parameters that affect the biomass and accumulation of secondary metabolites in a cell, callus, hairy root, and adventitious root suspension cultures. This article offers numerous techniques and approaches used to produce biomass and secondary metabolites from the Rubia species. Additionally, it has been emphasized that cells can be grown in bioreactor cultures to produce AQs.

Paphiopedilum spp. is one of the most commercially popular orchids because of its variety of shapes, sizes, and colors. However, it is at risk for extinction because of its exploitation. Regeneration of orchid plants using internode segments is extremely difficult. In this study, young P. callosum plants (1.5 cm) were exposed to eight dark–light cycles (14 days of dark and 1 day of light) for stem elongation to increase the number of nodes to obtain internode tissues. After 75 days of culture, the highest callogenesis (31.25%) was achieved when internode tissue was cultured on liquid Schenk and Hildebrandt (SH) medium containing 30 g·L−1 sucrose, 1.0 mg·L−1 Thidiazuron (TDZ), 1.0 mg·L−1 2,4-Dichlorophenoxyacetic acid (2,4-D), and cotton wool as the support matrix. The optimal media for induction of protocorm-like bodies (PLBs) were the same compositions as previously mentioned and were supplemented with 9 g·L−1 Bacto agar as the gelling agent. PLB clumps (5–6 PLBs/clump) produced the best shoots on medium containing 0.5 mg·L−1 α-Naphthaleneacetic acid (NAA) and 0.3 mg·L−1 TDZ. Among the organic substances tested, 200 g·L−1 potato homogenate (PH) added to Hyponex N016 medium supplemented with 1.0 mg·L−1 NAA, 30 g·L−1 sucrose, 170 mg·L−1 NaH2PO4, 1.0 g·L−1 peptone, and 9 g·L−1 Bacto agar resulted in the best rooting. The rooted plantlets with four to five leaves were acclimatized and had a 100% survival rate. The method presented in this research provides a strategy for the development of highly effective propagation of Paphiopedilum species using ex vitro explants for both conservation and horticultural purposes.

Mountain ginseng (Panax ginseng Mayer) is known as the most valuable herb around the world. Several biotechnological tools have been developed to enhance the production of secondary metabolites in medicinal plants, especially mountain ginseng. Mutagenesis is used to create desirable mutations at the genetic level. Among several energy sources used in mutant breeding programs, gamma rays are considered the most common and efficient tool for mutagenesis. Gamma rays are preferred over other energy sources because of convenient operation, short cycle, and high mutation frequency. In this study, gamma rays were used to generate mutant adventitious root lines of P. ginseng with high biomass and ginsenoside content. Callus and adventitious root cultures of P. ginseng, which were in culture short-term (1 year) and long-term (20 years), were irradiated with various doses of gamma rays. Exposure to higher doses of gamma rays caused necrosis in long-term explants. Four mutant lines of adventitious roots were identified with unique RAPD markers using eight primers. The genetic similarity among mutant lines ranged from 0.67 to 0.96. We also analyzed the total ginsenoside content and levels of 12 individual ginsenosides using HPLC analysis. The total ginsenoside content was 4.2-fold higher in mutant lines than in the control. The mutant line 1G-20-19 showed the highest ginsenoside content compared with the untreated control and other mutant lines. Overall, mutant lines of adventitious roots by gamma ray treatments in this study represent good candidates for commercial production of ginsenosides.

In this study, we compared two in vitro culture systems, dedifferentiated cell suspension culture and differentiated adventitious root culture, using inoculum from long-term (20-year culture period) and short-term (1-year culture period) cultures, for the production of ginsenosides in wild ginseng (Panax ginseng). Increases in biomass and ginsenoside content were monitored in a 3-L bioreactor. The biomass in short-term cell suspension and adventitious root cultures increased at a faster rate than that in the long-term cultures, reaching a peak after 4 and 8 weeks of culture, respectively. In long term cultured materials, the biomass of cell suspension and adventitious root cultures decreased by 1.67- and 1.52-fold, respectively. Although the amount of ginsenosides in cell suspension and adventitious root cultures varied during the culture periods, the total ginsenoside content remained constant. These data suggest that both culture systems are advantageous for the production of different ginsenosides for pharmaceutical purposes.

Dioscorea deltoidea is a medicinal plant valued for its high content of steroidal glycosides (SG) – bioactive compounds with cardioprotective and immunomodulation actions, also used to treat reproductive system disorders. To overcome the limitations of natural resources of this species, a suspension cell culture of D. deltoidea was developed as a renewable and ecologically sustainable source of raw biomass and SG. Cell culture demonstrated stable and intensive growth in the laboratory (20 L) and industrial (630 L) bioreactors operated under a semi-continuous regime (specific growth rate 0.11–1.12 day−1, growth index 3.5–3.7). Maximum dry weight accumulation (8.5–8.8 g/L) and SG content (47–57 mg/g DW) were recorded during the stationary phase. Bioreactor-produced cell biomass contained inorganic macro (К, Ca, Mg, Na) and micro (Zn, Mn, Fe, B, Al, Cu, Cr, Se, Co, Ni) elements in concentrations within the safe range of dietary recommendations. Acute toxicity test showed no or insignificant changes in organ weight, hematological panel and blood biochemistry of laboratory animals fed with 2000 and 5000 mg/kg dry biomass. The results suggest that cell culture of D. deltoidea grown in bioreactors has great potential to be used as functional foods and a component of specialized dietary supplements in complex therapy of reproductive system disorders and mineral deficiency.

Andrographis paniculata (AP) is popularly known as ‘King of bitters’, and it is an important medicinal plant which is cultivated in Southeast Asia and other tropical parts of the world. AP contains monoterpenoids, ent-labdane diterpenoids, flavonoids, quinic acids and xanthones. Andrographolide and its derivatives isolated from AP are reported to have a wide range of pharmacological effects including anti-inflammatory, antimalarial, antidiabetic, antileukemia, and anticancer activities. In this review, we trace domestication of AP by considering phytogeographical and historical evidences. These lines of evidences suggest that AP was domesticated in South India. Further, AP was involved in diversification through natural selection when it was introduced to new environmental and ecological conditions.

Orchids are considered to be the most highly differentiated and horticulturally important plants. Additionally, orchids have been used as traditional medicines in many countries since ancient times. Different organs of orchid plants, such as leaves, stems, and bulbs, contain various biologically active substances such as alkaloids, phenolics, terpenoids, and derivatives thereof. These bioactive compounds are secondary metabolites synthesized from primary metabolites of plants. To improve the utility of orchids, it is important to identify the pharmacological function of these plants. Moreover, the establishment of technologies for the large-scale production of a biomass of orchid plants using field cultivation or biotechnological methods is needed to prevent the overaccumulation of these plants in the natural state, which would ultimately result in these plants being enlisted as endangered species. Among various factors affecting the in vitro culture of medicinal orchids, growth regulators, light, sugar and activated charcoal are the most important. To establish a successful mass production system, it is necessary to determine the optimal concentration at which these factors maximize the production of biomass and bioactive compounds. In this chapter, we provide an overview of medicinal orchids and review recent studies on the in vitro production of biomass and bioactive compounds from these plants.

In the present study, the effect of light quality on endoreduplication and growth in Phalaenopsis ‘Spring Dancer’ plantlets was studied. The response of protocorm-like body (PLB)-derived plantlets subjected to monochromatic red (R60), blue (B60), and various combinations of both lights was investigated. Flow cytometry was used to investigate the effect of light on endocycle and growth, cell division, and endopolyploidy levels. In addition, the activities of stress-related enzymes such as catalase (CAT) and peroxidase (POD) were analyzed from leaves and roots. After 8 weeks, the leaf area of plants grown under monochromatic R60 and B60 light was found to be higher than that of plants grown under other wavelengths of light, except the control plants (fluorescent light). These results revealed monochrome blue (B60) light increased the ratio of endoreduplicated cells (4C–8C). CAT activity was highest in leaves grown under R60; however, the oxidized phenol concentration in the culture medium was lowest under R60 while it was the highest under B60 and fluorescent light (F). This indicates that plantlets were less stressed under R60 than B60 or F. The results of this study reveal that stress induced by monochromatic light stimulates endopolyploidy in leaves, which may subsequently increase Phalaenopsis leaf size.

Biotic elicitation is an important biotechnological strategy for triggering the accumulation of secondary metabolites in adventitious root cultures. These biotic elicitors can be obtained from safe, economically important strains of bacteria found in the rhizosphere and fermented foods. Here, we assayed the effects of filtered cultures of five nitrogen-fixing bacteria and four types of fermentation bacteria on mutant adventitious Panax ginseng root cultures induced in a previous study by colchicine treatment. The biomass, pH, and electrical conductivity (EC) of the culture medium were altered at 5 days after treatment with bacteria. The saponin content was highest in root cultures treated with Mesorhizobium amorphae (GS3037), with a concentration of 105.58 mg g-1 dry weight saponin present in these cultures versus 74.48 mg g-1 dry weight in untreated root cultures. The accumulation of the ginsenosides Rb2 and Rb3 dramatically increased (19.4- and 4.4-fold, and 18.8- and 4.8-fold) 5 days after treatment with M. amorphae (GS3037) and Mesorhizobium amorphae (GS336), respectively. Compound K production increased 1.7-fold after treatment with M. amorphae (GS3037) compared with untreated root cultures. These results suggest that treating mutant adventitious root cultures with biotic elicitors represents an effective strategy for increasing ginsenoside production in Panax ginseng.

We examined the effects of abiotic (methyl jasmonate [MeJA] and salicylic acid [SA]) and biotic (yeast extract and chitosan) elicitors for improvement of bioactive compounds production on adventitious root cultures in Polygonum multiflorum. The application of yeast extract resulted in significantly (p ≤ 0.05) higher dry root biomass (9.98 g/L) and relative growth rate versus the control. Cultures treated with abiotic elicitors showed higher percentage of dry weight than the other samples. Low concentrations of all elicitors (50 μM MeJA and SA, and 50 mg/L yeast extract) improved secondary metabolite production except for chitosan, whose performance was worse than that of the control. HPLC analysis of various bioactive compounds revealed significantly higher elicitation efficiency for MeJA than for the other treatments, with an approximately 2-fold increase in root dry weight (22.08 mg/g DW) under 50 μM MeJA treatment versus the control (10.35 mg/g DW). We also investigated the feasibility of scaling up the production process by comparing shake flask cultures with 3- and 5-L balloon type bubble bioreactors (BTBB) using 50 μM MeJA as an elicitor. Growth and metabolite accumulation increased in BTBB compared with shake flask cultures. We detected a non-significant difference in biomass productivity between 3 and 5-L BTBB, but the efficiency of bioactive compound accumulation decreased with increasing volume. These findings will be useful for developing a pilot-scale P. multiflorum adventitious root cultivation process for high biomass and bioactive compound production to meet the demands for natural ingredients by the pharmaceutical and cosmetic industries without affecting the natural habitat of this plant.

Panax ginseng and Panax quinquefolius of Panax genus are valuable as health foods as well as pharmaceuticals for the treatment of cancer, diabetes and ageing as these plants possess saponins. In the current study, Cell and adventitious root cultures of P. ginseng and P. quinquefolius were investigated for the biomass, cell division, saponin content and ginsenosides profile from four lines namely P. quinquefolius (AM), P. ginseng mountain (Mt.) Baekdu line, P. ginseng Cheong-sol line (CS) and P. ginseng CBN line (CBN) with the objective of comparing cell and adventitious root systems to check their efficacy for the production of ginseng saponins. Additionally, genes related to ginsenoside biosynthesis were also analyzed concerning to cell and adventitious root lines. The results indicated that various cell lines were better in multiplication and growth compared to adventitious root lines. However, adventitious root lines showed higher accumulation of dry biomass (1.5–2 fold) than that of cell lines. CS adventitious root line showed higher saponin content and ginsenoside productivity (10.48 mg·g−1 DW, 12.88 mg·L−1, respectively) than that of CS cell line (9.50 mg·g−1 DW, 2.39 mg·L−1, respectively). Especially, Rd ginsenoside productivity of CS adventitious root line recorded fourfold higher than CS cell line. Genes which are related to ginsenoside biosynthesis such as P. ginseng squalene synthase (PgSS2), P. ginseng squalene epoxidase (PgSE2), P. ginseng protopanaxadial synthase (PgPPDS) and P. ginseng protopanaxatriol synthase (PgPPTS) were analyzed by real time quantitative polymerase chain reaction to support ginsenoside production. The adventitious root culture system described in this study is useful system for biomass and ginsenoside production.

Using endophytic fungi elicitors to enhance the yield of valuable secondary metabolites in plant tissue culture is an effective biotechnology strategy. In this study, we isolated an endophytic fungus Chaetomium sp. from cultivated Panax ginseng root and detected a high concentration of Rg3 in its mycelium. By comparing the effects of different treatments of Chaetomium sp. on ginsenoside production in Panax ginseng adventitious roots (PGARs), the immobilized spores of Chaetomium sp. (ISC) were proved to be the best elicitor. An initial pH of 6.5 and cocultivation time of 24 h yielded the highest ginsenosides content (56.29 ± 0.62 mg/g) in ISC-treated PGARs, increasing ginsenosides production by 3.2-fold compared with the blank group (17.56 ± 0.49 mg/g). Moreover, we show that ISC could stimulate the temporary accumulation of defense-related signal molecules, intensify the expression of defense genes in PGARs, and upregulate biosynthetic genes involved in the biosynthetic pathway of ginsenosides. Besides, the expression of the biosynthetic gene FPS (farnesyl diphosphate synthase) was deemed the most relevant factor for ginsenosides production during the elicitation period, and the L -aspartic acid secreted by Chaetomium sp. was proven to play a key role in the elicitation process. In conclusion, this study provides an accessible way of improving pharmacologically active compounds production in medicinal plants.

Polygonum multiflorum Thunb. is an important medicinal plant that synthesizes an array of phenolic compounds. Its roots are used in a variety of pharmacological and cosmetic formulations, notably as hair dye. In the present study, the inoculum density (3–15 g/L) and culture period (1–7 weeks) were optimized in a 3 L bioreactor. High root biomass (14.18 g/L dry weight (DW)) was recorded with an inoculum of 7 g/L (p ≤ 0.05), which is consistent with the results for 5 and 10 g/L. However, significantly higher yield of bioactive compounds (53.87 mg/g DW total phenolics and 27.96 mg/g DW total flavonoids) with high free radical scavenging activity was obtained in root samples from 5 g/L inoculum density. A 4 week culture period was sufficient for optimum root growth and metabolite production. The optimized conditions were used for large-scale (5 and 20 L) and pilot-scale (500 L) studies. Considering that the continuous aeration of root cultures may lead to oxidative stress, antioxidant enzyme activity and lipid peroxidation also were studied. The results revealed high catalase (CAT) and guaiacol peroxidase (G-POD) activities, and low malondialdehyde (MDA) production, with increasing culture scale (20 and 500 L), which may indicate low-level oxidative damage to the cultures. An optimal yield of 4.01 kg dry root biomass with 287.12 mg/L of total phenolic productivity was achieved in a 500 L pilot-scale bioreactor. This work can pave the way for commercial production of biomass and secondary metabolites at the industrial level, and meet the rising demand for natural ingredients, especially in the pharmaceutical and cosmetic industries.

In the current study, we studied somaclonal variants of Phalaenopsis ‘Wedding Promenade’ (WP) to identify the factors that determine variation in this ornamental orchid. We grouped somaclonal variants of Phalaenopsis ‘Wedding Promenade’ based on their flower morphology (WP-N, WP-V1, WP-V2, and WP-V3). Morphological characteristics and histological studies were conducted in addition to flow cytometry and gene expression analyses of the flowers, leaves, and flower stalks. Morphological changes were observed in all WP groups; particularly, the width of the flower and the thickness of each organ were significantly increased when compared with those of normal flowers. The ploidy level of WP-V2 and WP-V3 was doubled in all organs, resulting in hexaploidy, while WP-N and WP-V1 were triploids. A high level of endoreduplication (3C-48C) was detected in the petals, leaves, and flower stalks of the WP Phalaenopsis variants. The expression patterns of HIGH PLOIDY 2 (HPY2) and MADS box transcription factor (PMADS4) isolated from Phalaenopsis were analyzed by real time quantitative PCR. HPY2 expression was the highest in the leaves of WP-V1, whereas its expression in flowers was similar in WP-V1 and normal flowers. Among the various flower organs analyzed, HPY2 expression was the highest in petals and lips of WP-V2. PMADS4 expression was not affected in the floral organs of the Phalaenopsis WP variants. The data identify a simple way to detect somaclonal variation in the Phalaenopsis WP variants and also provide insight into the causes of these variations.

A vast array of plant-based compounds has enriched red biotechnology to serve the human health and food. A peculiar medicinal plant which was an element of traditional Chinese medicine for centuries as a liver and kidney tonic, for life longevity and hair blackening, is Polygonum multiflorum Thunb. (PM) which is popularly known as “He shou wu” or “Fo-ti” and is rich in chemical components like stilbenes, quinones, and flavonoids which have been used as anti-aging, anti-alopecia, anti-cancer, anti-oxidative, anti-bacterial, anti-hyperlipidemia, anti-atherosclerosis, and immunomodulating and hepatoprotective agents in the modern medicine. The health benefits from PM are attained since long through commercial products such as PM root powder, extract, capsules, tincture, shampoo, and body sprays in the market. Currently, the production of these pharmaceuticals and functional foods possessing stilbenes, quinones, and flavonoids is through cell and organ cultures to meet the commercial demand. However, hepatotoxic effects of PM-based products are the stumbling blocks for its long-term usage. The current review encompasses a comprehensive account of bioactive compounds of PM roots, their biological activities as well as efficacy and toxicity issues of PM ingredients and future perspectives.

Systematics plays an important role in identifying medicinal plants and searching for new medicinal plant sources. This chapter introduces a theoretical basis and development and research methods for medicinal plant systematics. Research methods of medicinal plant systematics contain morphological taxonomy, anatomy taxonomy, embryo taxonomy, cell taxonomy, chemical taxonomy, numerical taxonomy and branch taxonomy. We also introduce the methods and application of molecular systematics, which can be used to identify and protect medicinal plants, as well as search for new medicinal plants.

Leguminous plants include many of the most important food crops and are also cultivated for forage, used as a rich fiber source, and are important in the manufacture of pharmaceutical compounds. Their ability to biologically fix atmospheric nitrogen enhances soil fertility. Beyond conventional propagation methods, modern in vitro technology offers new means for propagating these plants. Significant progress has been achieved in somatic embryogenesis and embryo rescue, despite legumes’ notorious recalcitrance to transformation and regeneration of legumes. The establishment of stable and reproducible regeneration protocols has resulted in commercially successful propagation of some legume species. Still, regeneration protocols for many other legumes have not been successful, primarily due to poor in vitro rooting during regeneration. This review addresses the factors constraining rooting, describes alternative methods to promote in vitro rooting, and provides a summary of in vitro rooting studies performed across a large number of leguminous species.

Somatic embryogenesis represents a powerful in vitro plant culture system and provides high potential for the production of biomass in woody plants. Rosa rugosa is known not only for its ornamental value, but also as a source of antioxidants with the potential to be used as ingredients for industrial products such as cosmetics. The present study was conducted to investigate the effects of hormonal concentration on embryogenic callus (EC) induction from petal and leaf segments of this species. The effect of selected cell lines on the production of total phenolics and flavonoids in R. rugosa was also studied. The highest percentage of EC (14.1 ± 3.9%) and root formation (20.3 ± 6.9%) was observed in the presence of 45.0 μM of 2,4-D. Among the three studied lines, the highest total phenolics and flavonoid levels were observed in H-1 (17.71 mg·g-1 DW), while H-18 contained the lowest. Histological studies of the H-1 and H-18 cell lines revealed differences in polyphenol accumulation and polysaccharides. The effect of developmental stage on growth, shoot, and root regeneration, as well as on phenolic and flavonoid content, was also studied in H-18. The present study indicates that selection of the proper type and concentration of hormone is essential for EC induction from petals in R. rugosa. Moreover, the accumulation of bioactive compounds was found to greatly depend on the genotype. The results of this study are useful for the production of selected antioxidants by somatic embryos from R. rugosa petals.

As the effective elicitors, methyl jasmonate (MeJA) and salicylic acid (SA) have been widely used in plant cell and organ culture for improvement of bioactive compound accumulation. The protocorm-like bodies (PLBs) of Dendrobium candidum were stimulated with different amounts of methyl jasmonate (MeJA) and salicylic acid (SA) during suspension culture to improve the production of bioactive compounds in PLBs. The optimal concentration of MeJA and SA varied among the types of bioactive compounds. Treatment with 75 μM MeJA enhanced the production of alkaloids, polysaccharides, and flavonoids, whereas 100 μM MeJA improved phenolic production. An elicitation effect was also exhibited in SA, such that 75 μM SA yielded the highest accumulation of alkaloids and polysaccharides, whereas 100 μM SA generated the highest amount of flavonoids. Moreover, SA concentration did not affect phenolic production. Furthermore, MeJA was used to treat PLBs after 30 days of bioreactor culture to investigate the effect on large-scale applications. The highest amounts of alkaloids and polysaccharides were determined after 4 days of MeJA elicitation, and those of phenolics and flavonoids were detected after 10 days of MeJA treatment during PLB bioreactor culture. Bioactive compound content and antioxidant activity in bioreactor-cultured PLBs were compared with those in whole plants. The contents of alkaloids, polysaccharides, phenolics, and flavonoids in the PLBs were higher than those in the plants. Protocorm-like bodies treated with MeJA also contained the maximum amount of bioactive compounds. The antioxidant activity of MeJA-treated PLBs was also evaluated using the following parameters: 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, reducing power, and ferrous ion (Fe2+) chelating activity, which were higher than those in the extracts of PLBs without MeJA treatment and plants. This study reveals that MeJA as an efficent elicitor can be used for the mass production of bioactive compounds by PLB culture using bioreactor culture systems and the obtained PLBs can be provided to the commercial production of D. candidum products.

The genus Cypripedium, known as lady's slipper orchid, has been considered to have a considerable marketability as a potted and garden plant with beautiful flowers; however, this species is becoming endangered and, in some places extinct, due to habitat destruction and illegal collections. As such, an optimized artificial propagation system is necessary for its conservation and horticultural cultivation. For the establishment of the in vitro proliferation of the endangered Cypripedium macranthos Sw., native to Korea, the effects of medium strength and sucrose concentrations on germination, protocorm formation and seedling growth were investigated through asymbiotic seed culture. The highest germination rate and protocorm formation rate were obtained with a 1/4 MS medium; higher MS medium strengths did not generate the favorable conditions required for germination and protocorm growth. The overall growth characteristics of roots and buds developed from protocorms were best in cultures of 1/4 MS medium. On this medium containing 10 g·L -1 sucrose, both the germination rate and protocorm formation rate increased significantly. The general growth properties of seedlings developed on the medium with 10 g·L -1 sucrose were best, showing the highest bud formation rate and root number. Our results demonstrate that the 1/4 MS medium, supplemented with 10 g·L -1 sucrose, could improve in vitro germination, and facilitate the growth of seedlings developed from the protocorms of C. macranthos Sw.

To find the optimal propagation condition for endangered Cypripedium macranthos Sw., also known as lady's slipper orchid, the effect of various organic additives on in vitro germination, protocorm formation and seedling growth was investigated during asymbiotic seed culture. When 100 ml·L -1 coconut water was added to the basal medium, the highest germination rate and protocorm formation rate were achieved, with 70.8% and 74.2% respectively. Supplementation of phloem sap from birch tree or maple tree also showed a facilitating effect to improve the germination and protocorm development. With 100 ml·L -1 birch sap or maple sap, both the germination and protocorm formation rates were roughly more than 65% and 68%. The roots and buds of the seedlings grew vigorously in the medium containing 100 ml·L -1 coconut water or phloem sap, in particular, their bud formation rates increased by more than 70%. Addition of banana powder and peptone could not create a more significantly favorable culture condition, and non-addition had the worst results. Our results demonstrated that proper organic amendments such as coconut water and phloem sap might be preferred to in vitro germination and the growth of seedlings developed from the protocorm of C. macranthos Sw. during asymbiotic seed culture.

Rosa rugosa is a medicinal, ornamental, and edible plant native to Eastern Asian countries, including Korea, Japan, and China. The aim of this study was to establish a system for biomass production and secondary metabolite accumulation during in vitro culture and acclimatization of Rosa rugosa. The highest rate of multiple shoot proliferation was achieved with benzyladenine (BA) (83.3%). However, the number of shoots (14.4 per explant) at BA was higher than that at BA. Compared to BA, a combination of thidiazuron (TDZ) and indole butyric acid (IBA) exhibited significantly lower shoot induction, with only 50.0~79.2% and 4.2~16.7% relative shoot formation, respectively. During acclimatization, shoots were sampled every week and their total phenolic contents were analyzed. Among various growth factors, fresh weight showed the most dramatic increase from the 3rd week (88.0 mg/plant) to 4th week (132.7 mg/plant). Total phenolics and flavonoids contents were the highest at week of acclimatization. Depending on developmental stages, total phenolics and flavonoids contents were higher in 1-yr-old shoots grown ex vitro than in those of older field-grown or in vitro-grown plants. Amongst different ages of field grown plants, 6-year-old plants, the oldest in this study, showed the lowest content in total phenolics.

A micropropagation system is essential for obtaining elite clones of Phalaenopsis in the floricultural industry; however, somaclonal variation frequently occurs during long-term micropropagation, which reduces the uniformity and quality of clonally propagated plants. To detect the factors influencing this variation and thus obtain high-quality, uniform Phalaenopsis plants, we analyzed somaclonal variants in the Phalaenopsis ‘Spring Dancer’ (SD) variety collected from an orchid farm, which were grouped into three types according to flower morphology (SD-N, SD-V1, SD-V2). Morphological differences were observed between all SD groups; in particular, the diameter of the petal and thickness of each organ were significantly larger in the variants than SD-N (normal) plants. Both SD variant groups showed the same ploidy level as that of SD-N. The degree of endoreduplication of the petals and flower stalks was higher in the variants (4C–16C) than in SD-N (2C–8C). Cell area was increased and the shape of epidermal cells was changed in both variants. Expression levels of MADS box transcription factor (MADS4) of the flowers were higher than those of the leaves in the variants. Among the three flower organs, MADS4 expression was the highest in the petals of SD-V2. These results revealed that MADS4 is involved in the modification of flower morphology in somaclonal SD variants. High ploidy 2 (HPY2) expression was highest in the flowers of SD-V1, whereas its expression in the leaf was similar to that of SD-N. Among the various flower organs, HPY2 expression was highest in the lips of SD-V1.

Rosa rugosa Thunb. is a popular ornamental and medicinal plant native to eastern Asia. In this study, a successful bioreactor culture system was established for the production of secondary metabolites of rugosa roses. We tested different concentrations and combinations of plant hormones in growth media for maximum shoot proliferation and production of bioactive compounds, different bioreactor systems for maximum biomass production and production of bioactive compounds, and different ratios of nitrogen sources for maximum shoot growth and accumulation of bioactive compounds. For multiple shoot proliferation, Murashige and Skoog (MS) medium was used, supplemented with different concentrations and combinations of plant hormones: 6-benzylaminopurine (BA; 0-13.2 µM), thidiazuron (TDZ; 0-13.5 µM), and indole butyric acid (IBA) at 2.5 µM, used alone or in a combination of IBA with BA or TDZ. Rapid micropropagation of multiple shoots of rugosa roses was successfully achieved using shoot tips explanted in semisolid MS medium supplemented with 4.4 µM BA. The average number of shoots grown was 15.6 per explant and the maximum shoot length was 2.7 cm at 8 weeks of culture. To investigate the effect of nitrogen sources on shoot growth and bioactive compound accumulation, shoots were treated with different ratios of nitrogen sources (NH4 +:NO3 -) for 1 week after 7 weeks of shoot culture. Next, to scale up biomass production for the generation of useful phytochemicals, multiple-shoot cultures were developed in large-scale bioreactors. Four bioreactor systems were used: continuous immersion bioreactor (CIB), continuous immersion bioreactor with net (CIB-N), temporary immersion bioreactor (TIB), and temporary immersion bioreactor with net (TIB-N). Solid and liquid media were used as controls. Of the different bioreactor types, the CIB system produced the highest biomass, followed by the TIB system. Multiple shoots grown in the CIB system resulted in the accumulation of 39.21 mg·g-1 dry weight (DW) of total phenolics and 13.28 mg·g-1 DW of total flavonoids. The productivity of total phenolics and flavonoids was highest in the shoots harvested from the CIB system. The results of this study suggest that multiple shoots of rugosa roses can be used in commercial-scale bioreactors to produce useful bioactive compounds for the pharmaceutical and cosmetic industries.

Ginsenosides are important metabolites synthesized by the traditional Chinese medicinal plant, Panax ginseng. The market demand for ginsenosides is increasing. Fungal elicitor is effective for improving ginsenosides biosynthesis. Here, we isolated an effective fungal elicitor, Chaetomium globosum from Panax notoginseng. Total saponins content in adventitious roots of P. ginseng was 3.94 times higher than that in control under C. globosum treatment. Correspondingly, the antioxidant activity of C. globosum-stressed roots is significantly increased than control adventitious roots and cultivated ginseng roots. In this study, PgWRKY4X, a novel pathogen-related gene encoding WRKY transcription factor from P. ginseng was isolated and functionally characterized. PgWRKY4X was responsive to the treatment of C. globosum. Subcellular localization assay indicated PgWRKY4X located in the nucleus. Electrophoretic mobility shift assay showed PgWRKY4X binds to the W-box of squalene epoxidase (PgSE) promoter. The interaction between PgWRKY4X and PgSE was discovered by glutathione S-transferase pull-down assay. Homology modeling and molecular docking was also performed to reveal the putative spatial interaction between PgWRKY4X and PgSE. Overexpression of PgWRKY4X in P. ginseng transgenic cells could significantly enhance ginsenosides accumulation by comprehensively upregulating ginsenosides biosynthetic genes, especially PgSE. Our study suggested that PgWRKY4X may be a potential target for metabolic engineering of ginsenosides biosynthesis in P. ginseng.

Ginseng (Panax ginseng C. A. Meyer, Family Araliaceae) is one of the major medicinal and nutraceutical plants, which is native to oriental region. It is used worldwide as a popular herbal medicine because of its pharmacological effects like anti-oxidative, anti aging, anti-cancer, adaptogenic, and other health-improving activities. Chief components of ginseng identified till date are ginsenosides, a group of saponins with triterpenoid structure. Ginseng is cultivated under controlled conditions, and for harvesting of fully grown roots of the plant, the cultivation takes long duration of about 5–7 years and cultivated ginseng roots are inferior in quality and ginsenoside content. Wild Mountain ginseng is superior in quality and ginsenoside content but is scarce in nature. Therefore, for obtaining the useful compounds of this plant at commercial scale, cell and organ cultures especially adventitious roots have been established by using superior clones of wild mountain ginseng, ginseng biomass is produced by applying large scale bioreactors. In this paper, an effort has been made to shed light on the scientific literature and to decipher the evidences for quality, safety, and efficacy of ginseng adventitious roots produced from in vitro cultures.

Plants are the reservoirs of various valuable secondary metabolites like camptothecin, podophyllotoxin, and ginseng saponins which are used as anticancer agents. Plant cell and organ cultures are competent to synthesize and accumulate many of these compounds and can be used as a source for extraction of such compounds. Different strategies have been applied for overaccumulation of secondary metabolites in plant cell cultures which include screening and selection of high yielding cell lines, optimization of nutrient media, and elicitation. In the present chapter, some of the anticancer compounds derived from plant cell and organ cultures are described, and various strategies for improving the accumulation of useful secondary metabolites in cell and organ cultures and methods for the large-scale production using bioreactors are highlighted.