Water-deficit stress induces prenylated stilbenoid production and affects biomass in peanut hairy roots: Exploring the role of stilbenoid prenyltransferase downregulation.

The peanut plant is one of the most economically important crops around the world. Abiotic stress, such as drought, causes over five hundred million dollars in losses in peanut production per year. Peanuts are known to produce prenylated stilbenoids to counteract biotic stress. However, their role in abiotic stress tolerance has not been elucidated. To address this issue, hairy roots with the capacity to produce prenylated stilbenoids were established. An RNA-interference (RNAi) molecular construct targeting the stilbenoid-specific prenyltransferase AhR4DT-1 was designed and expressed via Agrobacterium rhizogenes-mediated transformation in hairy roots of peanut cultivar Georgia Green. Two transgenic hairy roots with the RNAi molecular construct were established, and the downregulation of AhR4DT-1 was validated using reverse transcriptase quantitative PCR. To determine the efficacy of the RNAi-approach in modifying the levels of prenylated stilbenoids, the hairy roots were co-treated with methyl jasmonate, hydrogen peroxide, cyclodextrin, and magnesium chloride to induce the production of stilbenoids and then the stilbenoids were analyzed in extracts of the culture medium. Highly reduced levels of prenylated stilbenoids were observed in the RNAi hairy roots. Furthermore, the hairy roots were evaluated in a polyethylene glycol (PEG) assay to assess the role of prenylated stilbenoids on water-deficit stress. Upon PEG treatment, stilbenoids were induced and secreted into the culture medium of RNAi and wild-type hairy roots. Additionally, the biomass of the RNAi hairy roots decreased by a higher amount as compared to the wild-type hairy roots suggesting that prenylated stilbenoids might play a role against water-deficit stress.

Glycyrrhizin Production in Licorice Hairy Roots Based on Metabolic Redirection of Triterpenoid Biosynthetic Pathway by Genome Editing.

Glycyrrhizin, a type of the triterpenoid saponin, is a major active ingredient contained in the roots of the medicinal plant licorice (Glycyrrhiza uralensis, G. glabra and G. inflata), and is used worldwide in diverse applications, such as herbal medicines and sweeteners. The growing demand for licorice threatens wild resources and therefore a sustainable method of supplying glycyrrhizin is required. With the goal of establishing an alternative glycyrrhizin supply method not dependent on wild plants, we attempted to produce glycyrrhizin using hairy root culture. We tried to promote glycyrrhizin production by blocking competing pathways using CRISPR/Cas9-based gene editing. CYP93E3 CYP72A566 double-knockout (KO) and CYP93E3 CYP72A566 CYP716A179 LUS1 quadruple-KO variants were generated, and a substantial amount of glycyrrhizin accumulation was confirmed in both types of hairy root. Furthermore, we evaluated the potential for promoting further glycyrrhizin production by simultaneous CYP93E3 CYP72A566 double-KO and CYP88D6-overexpression. This strategy resulted in a 3-fold increase (∼1.4 mg/g) in glycyrrhizin accumulation in double-KO/CYP88D6-overexpression hairy roots, on average, compared with that of double-KO hairy roots. These findings demonstrate that the combination of blocking competing pathways and overexpression of the biosynthetic gene is important for enhancing glycyrrhizin production in G. uralensis hairy roots. Our findings provide the foundation for sustainable glycyrrhizin production using hairy root culture. Given the widespread use of genome editing technology in hairy roots, this combined with gene knockout and overexpression could be widely applied to the production of valuable substances contained in various plant roots.

Antimicrobial and antioxidant activity of Panax ginseng and Hedysarum neglectum root crop extracts

Abstract In order to ensure the timely and uninterrupted supply of medicinal plant raw materials, the methods of cultivation of plant cell cultures, namely, the production of plant root cultures, are relevant. In this paper, the geroprotective potential of Hedysarum neglectum Ledeb and Panax ginseng C. A. Mey root cultures is studied. They were cultured under in vitro conditions by transforming the rhizome (H. neglectum) and seed seedlings (P. ginseng) with Agrobacterium rhizogenes 15834 Swiss. To identify the geroprotective potential, the antimicrobial disc-diffusion method and the antioxidant activity were analyzed by titration of KMnO4 extracts of plant root cultures. The qualitative and quantitative composition was analyzed using high-performance liquid chromatography, thin-layer chromatography, and gas chromatography with mass spectrometry. In the course of the work, the presence of antimicrobial and antioxidant activity of plant root culture extracts was established. Biologically active substances contained in extracts of Hedysarum neglectum Ledeb root crops and Panax ginseng C. A. Mey are characterized by geroprotective potential, so they can act as a source of natural antioxidants in the functional nutrition of the geroprotective orientation.

Resumo Para garantir o abastecimento em tempo e ininterrupto de matérias-primas de plantas medicinais, são relevantes os métodos de cultivo de culturas de células vegetais, nomeadamente a produção de culturas de raízes vegetais. Neste trabalho, foi estudado o potencial geroprotetor de culturas de raízes de Hedysarum neglectum Ledeb e Panax ginseng C. A. Mey. Eles foram cultivados em condições in vitro pela transformação do rizoma (H. neglectum) e mudas de sementes (P. ginseng) com Agrobacterium rhizogenes 15834 Swiss. Para identificar o potencial geroprotetor, o método antimicrobiano de difusão em disco e a atividade antioxidante foram analisados por titulação de extratos de KMnO4 de raízes de plantas. A composição qualitativa e quantitativa foi analisada por cromatografia líquida de alta eficiência, cromatografia em camada delgada e cromatografia gasosa com espectrometria de massa. No decorrer do trabalho, foi constatada a presença de atividade antimicrobiana e antioxidante dos extratos de raízes de plantas. Substâncias biologicamente ativas contidas em extratos de raízes de H. neglectum Ledeb e P. ginseng C. A. Mey são caracterizadas pelo potencial geroprotetor, podendo atuar como fonte de antioxidantes naturais na nutrição funcional da orientação geroprotetora.

Antimicrobial and antioxidant activity of Panax ginseng and Hedysarum neglectum root crop extracts / Atividade antimicrobiana e antioxidante de extratos de raízes de Panax ginseng e Hedysarum neglectum

In order to ensure the timely and uninterrupted supply of medicinal plant raw materials, the methods of cultivation of plant cell cultures, namely, the production of plant root cultures, are relevant. In this paper, the geroprotective potential of Hedysarum neglectum Ledeb and Panax ginseng C. A. Mey root cultures is studied. They were cultured under in vitro conditions by transforming the rhizome (H. neglectum) and seed seedlings (P. ginseng) with Agrobacterium rhizogenes 15834 Swiss. To identify the geroprotective potential, the antimicrobial disc-diffusion method and the antioxidant activity were analyzed by titration of KMnO4 extracts of plant root cultures. The qualitative and quantitative composition was analyzed using high-performance liquid chromatography, thin-layer chromatography, and gas chromatography with mass spectrometry. In the course of the work, the presence of antimicrobial and antioxidant activity of plant root culture extracts was established. Biologically active substances contained in extracts of Hedysarum neglectum Ledeb root crops and Panax ginseng C. A. Mey are characterized by geroprotective potential, so they can act as a source of natural antioxidants in the functional nutrition of the geroprotective orientation.

Para garantir o abastecimento em tempo e ininterrupto de matérias-primas de plantas medicinais, são relevantes os métodos de cultivo de culturas de células vegetais, nomeadamente a produção de culturas de raízes vegetais. Neste trabalho, foi estudado o potencial geroprotetor de culturas de raízes de Hedysarum neglectum Ledeb e Panax ginseng C. A. Mey. Eles foram cultivados em condições in vitro pela transformação do rizoma (H. neglectum) e mudas de sementes (P. ginseng) com Agrobacterium rhizogenes 15834 Swiss. Para identificar o potencial geroprotetor, o método antimicrobiano de difusão em disco e a atividade antioxidante foram analisados por titulação de extratos de KMnO4 de raízes de plantas. A composição qualitativa e quantitativa foi analisada por cromatografia líquida de alta eficiência, cromatografia em camada delgada e cromatografia gasosa com espectrometria de massa. No decorrer do trabalho, foi constatada a presença de atividade antimicrobiana e antioxidante dos extratos de raízes de plantas. Substâncias biologicamente ativas contidas em extratos de raízes de H. neglectum Ledeb e P. ginseng C. A. Mey são caracterizadas pelo potencial geroprotetor, podendo atuar como fonte de antioxidantes naturais na nutrição funcional da orientação geroprotetora.

Adventitious root culture of Lessertia frutescens for the production of triterpenoid saponins and polysaccharides.

Lessertia frutescens is a perennial shrub of commercial importance in South Africa, but the scarcity of plant resources has limited current product production. In this study, to provide an alternative approach for obtaining L. frutescens material, adventitious roots (ARs) were induced from sterilized seedlings and cultured in a suspension culture system. During this process, selection tests were conducted to find a suitable auxin and its concentration for AR induction and a suitable basal medium for AR growth and metabolite accumulation; a kinetic study was then performed to constructure kinetic models. The results showed that compared to other auxins and concentrations, indole-3-butyric acid at 3 mg/L was suitable for increasing the number and length of ARs during AR induction. In AR suspension culture, Schenk and Hildebrandt (SH) was better than other basal media, and the maximum AR fresh (86.9 g/L) or dry weight (5.5 g/L), total triterpenoid saponin (92.6 mg/g DW), and polysaccharide (114.7 mg/g DW) contents were determined in the 1.5×SH medium. In addition, AR biomass and metabolite contents reached the maximum on day 42. The kinetic models for AR growth and triterpenoid and polysaccharide production were constructed, providing the basis for further optimization of culture conditions and large-scale culture.

Production of Phenolic Compounds and Antioxidant Activity in Hairy Root Cultures of Salvia plebeia.

Salvia plebeia (Lamiaceae) is a medicinal plant containing diverse bioactive constituents that have biological properties. In this study, we determined the optimal conditions (media and auxin) for the hairy root culture of S. plebeia for the growth and accumulation of phenolic compounds and evaluated its antioxidant activities. Rosmarinic acid and five phenylpropanoids were detected using high-performance liquid chromatography. The hairy roots grown in 1/2 SH medium with 1 mg/L NAA had a high level of rosmarinic acid content. Hairy roots cultured in 1 mg/L NAA had the highest total content of five phenylpropanoids. Compared to wild-type roots grown in the field, hairy roots (NAA 1) expressed similar levels of rosmarinic acid but significantly enhanced phenylpropanoid accumulation. Furthermore, the total phenolic content and total flavonoid content of hairy roots (NAA 1) were 2.22 and 1.73 times higher than those of wild-type roots. In the results of DPPH, ABTS, and reducing power assays, the hairy roots (NAA 1) showed higher free radical scavenging effects and reduction potential than the wild-type roots. These results suggest that S. plebeia hairy roots cultured under optimal conditions, which exhibit enhanced phenolic compound accumulation and antioxidant activity, can potentially be used as sources of antioxidants.

Critical Role of Regrowth Conditions in Post-Cryopreservation of In Vitro Plant Germplasm.

Cryopreservation is an effective option for the long-term conservation of plant genetic resources, including vegetatively propagated crops and ornamental plants, elite tree genotypes, threatened plant species with non-orthodox seeds or limited seed availability, as well as cell and root cultures useful for biotechnology. With increasing success, an arsenal of cryopreservation methods has been developed and applied to many species and material types. However, severe damage to plant material accumulating during the multi-step cryopreservation procedure often causes reduced survival and low regrowth, even when the optimized protocol is applied. The conditions at the recovery stage play a vital role in supporting material regrowth after cryopreservation and, when optimized, may shift the life-and-death balance toward a positive outcome. In this contribution, we provide an overview of the five main strategies available at the recovery stage to improve post-cryopreservation survival of in vitro plant materials and their further proliferation and development. In particular, we discuss the modification of the recovery medium composition (iron- and ammonium-free), exogenous additives to cope with oxidative stress and absorb toxic chemicals, and the modulation of medium osmotic potential. Special attention is paid to plant growth regulators used at various steps of the recovery process to induce the desired morphological response in cryopreserved tissues. Given studies on electron transport and energy provision in rewarmed materials, we discuss the effects of light-and-dark conditions and light quality. We hope that this summary provides a helpful guideline and a set of references for choosing the recovery conditions for plant species that have not been cryopreserved. We also propose that step-wise recovery may be most effective for materials sensitive to cryopreservation-induced osmotic and chemical stresses.

Current status and future prospects in cannabinoid production through in vitro culture and synthetic biology.

For centuries, cannabis has been a rich source of fibrous, pharmaceutical, and recreational ingredients. Phytocannabinoids are the most important and well-known class of cannabis-derived secondary metabolites and display a broad range of health-promoting and psychoactive effects. The unique characteristics of phytocannabinoids (e.g., metabolite likeness, multi-target spectrum, and safety profile) have resulted in the development and approval of several cannabis-derived drugs. While most work has focused on the two main cannabinoids produced in the plant, over 150 unique cannabinoids have been identified. To meet the rapidly growing phytocannabinoid demand, particularly many of the minor cannabinoids found in low amounts in planta, biotechnology offers promising alternatives for biosynthesis through in vitro culture and heterologous systems. In recent years, the engineered production of phytocannabinoids has been obtained through synthetic biology both in vitro (cell suspension culture and hairy root culture) and heterologous systems. However, there are still several bottlenecks (e.g., the complexity of the cannabinoid biosynthetic pathway and optimizing the bioprocess), hampering biosynthesis and scaling up the biotechnological process. The current study reviews recent advances related to in vitro culture-mediated cannabinoid production. Additionally, an integrated overview of promising conventional approaches to cannabinoid production is presented. Progress toward cannabinoid production in heterologous systems and possible avenues for avoiding autotoxicity are also reviewed and highlighted. Machine learning is then introduced as a powerful tool to model, and optimize bioprocesses related to cannabinoid production. Finally, regulation and manipulation of the cannabinoid biosynthetic pathway using CRISPR- mediated metabolic engineering is discussed.

Biotechnological Intervention and Secondary Metabolite Production in Centella asiatica L.

Centella asiatica L., commonly known as Gotu kola, Indian pennywort, and Asiatic pennyworts, is an herbaceous perennial plant that belongs to the family Apiaceae and has long been used in the traditional medicine system. The plant is known to produce a wide range of active metabolites such as triterpenoids including asiatic acid, asiaticoside, brahmoside, and madecassic acid along with other constituents including centellose, centelloside, and madecassoside, etc., which show immense pharmacological activity. Due to its beneficial role in neuroprotection activity, the plant has been considered as a brain tonic. However, limited cultivation, poor seed viability with low germination rate, and overexploitation for decades have led to severe depletion and threatened its wild stocks. The present review aimed to provide up-to-date information on biotechnological tools applied to this endangered medicinal plant for its in vitro propagation, direct or indirect regeneration, synthetic seed production, strategies for secondary metabolite productions including different elicitors. In addition, a proposed mechanism for the biosynthesis of triterpenoids is also discussed.

Green root cultures for enhanced production of camptothecin in Pyrenacantha volubilis Hook.

The roots of Pyrenacantha volubilis contain camptothecin (CPT), a high-value bioactive compound possessing anticancer and anti-HIV properties. Isolated root cultures of P. volubilis established in half MS media fortified with 0.3 mgL-1 indole-3-acetic acid and 0.2 mgL-1 indole-3-butyric acid and transferred to light conditions resulted in induction of green roots which obtained a maximum biomass content of 1.09 ± 0.03 g fresh weight with a growth index of 2.07 ± 0.02 in 60 days. The chlorophyll and camptothecin content of the roots was found to increase throughout the culture period attaining a maximum total chlorophyll content of 2.97 ± 0.004 mgg-1 fresh weight in 60 days and CPT content of 2.59 ± 0.02 mgg-1dry weight in 50 days. In the elicitation studies, the green roots treated with 200 mgL-1 yeast extract yielded a maximum camptothecin content of 5.3 ± 0.40 mgg-1 dry weight in 7 days of incubation, which was a 1.94 fold increase in camptothecin content than the control cultures. The thin layer chromatography, high performance liquid chromatography, and liquid chromatography-mass spectroscopy analysis further confirmed the presence of camptothecin (2.17 ± 0.04 mgg-1DW) in the 30 day old in vitro root samples. The overall results suggest the feasibility of green root cultures of P. volubilis as an efficient system for sustainable in vitro production of camptothecin provided further scaling up experiments in bio reactors are imperative.

In vitro production of atractylon and ß-eudesmol from Atractylodes chinensis by adventitious root culture.

Atractylodes chinensis is a medicinal plant widely used for the treatment of gastric disorders, and its main bioactive compounds are atractylon and ß-eudesmol. This study was purposed to establish the adventitious root culture system of A. chinensis for in vitro production of atractylon and ß-eudesmol. The main parameters in the adventitious root induction and suspension cultures were optimized to maximize the culture efficiency. Adventitious roots were induced most efficiently from leaf explants on Murashige and Skoog (MS) solid medium containing 1.5 mg/L naphthaleneacetic acid (NAA) and 30 g/L sucrose with the highest root induction rate of approximately 92% and 12.9 roots per explant. During the adventitious root suspension culture, the root biomass and the accumulated content of the target compounds simultaneously increased to reach the maximum values after 8 weeks of culture. The maximum yield of the target compounds (total concentration 3.38 mg/g DW, total yield 2.66 mg) was achieved in the roots cultured in ½ MS liquid medium supplemented with 2.0 mg/L IBA, 3.2 mg/L NAA, and 40 g/L sucrose with the inoculum density of 8 g/L. Through the central composite design experiment, it was found that the combined use of different types of auxins in the suspension culture could further improve root growth and metabolite accumulation than the application of only one type of auxin. This work provides a new possibility to have a promising candidate for the industrial production of A. chinensis pharmaceuticals without relying on wild resources or field cultivation. KEY POINTS: • The induction culture was optimized for efficient root induction. • Suspension culture was optimized for the atractylon and ß-eudesmol production. • Combined use of different auxins improves root growth and metabolite accumulation.

Biotechnological interventions and indole alkaloid production in Rauvolfia serpentina.

Rauvolfia serpentina (L). Benth. ex Kurz. (Apocynaceae), commonly known as Sarpagandha or Indian snakeroot, has long been used in the traditional treatment of snakebites, hypertension, and mental illness. The plant is known to produce an array of indole alkaloids such as reserpine, ajmaline, amalicine, etc. which show immense pharmacological and biomedical significance. However, owing to its poor seed viability, lesser germination rate and overexploitation for several decades for its commercially important bioactive constituents, the plant has become endangered in its natural habitat. The present review comprehensively encompasses the various biotechnological tools employed in this endangered Ayurvedic plant for its in vitro propagation, role of plant growth regulators and additives in direct and indirect regeneration, somatic embryogenesis and synthetic seed production, secondary metabolite production in vitro, and assessment of clonal fidelity using molecular markers and genetic transformation. In addition, elicitation and other methods of optimization of its indole-alkaloids are also described herewith. KEY POINTS: • Latest literature on in vitro propagation of Rauvolfia serpentina • Biotechnological production and optimization of indole alkaloids • Clonal fidelity and transgenic studies in R. serpentina.

Phosphodiesterase-5 Inhibitory Activity of Canthin-6-One Alkaloids and the Roots of Eurycoma longifolia and Eurycoma harmandiana.

Eurycoma longifolia (EL) and Eurycoma harmandiana (EH) are natural medicinal plants belonging to the Simaroubaceae family, and are well-known for their ability to enhance male sexual performance. The present study investigated the phosphodiesterase-5 (PDE-5) inhibitory activity of intact roots of EL and EH. Additionally, canthin-6-one alkaloids, ß-carboline alkaloids, and quassinoids were also screened for PDE-5 inhibitory activity. We developed in vitro root and callus cultures of EL and EH to determine their PDE-5 inhibitory activity. Our results indicated that canthin-6-one alkaloids, which include canthin-6-one-9-O-ß-D-glucopyranoside, 9-methoxycanthin-6-one, canthin-6-one, and 9-hydroxycanthin-6-one, exhibited PDE-5 enzymatic inhibitory activity, with IC50 values of 2.86±0.23, 3.30±1.03, 4.31±0.52, and 4.66±1.13 µM, respectively. The ethanolic extract of the intact roots of EL and EH, and the in vitro root culture of EH had large amounts of canthin-6-one alkaloids (1.50±0.04, 2.12±0.03, and 3.48±0.08 mg/g dry weight, respectively), and showed potent PDE-5 inhibition. Our findings indicate that in vitro root cultures of EH may be used to replace intact plants, and canthin-6-one-9-O-ß-D-glucopyranoside should be further investigated for development as a health supplement.

Biotechnology for micropropagation and camptothecin production in Ophiorrhiza sp.

Camptothecin (CPT) is a monoterpenoid-alkaloid, an anticancer compound from plant. Ever since its discovery in 1996 from the bark of Camptotheca acuminata, various researches have been conducted for enhancing its production. CPT has also been reported in several other species belonging to the plant families Icacinaceae, Rubiaceae, Apocynaceae, Nyssaceae, Betulaceae, Violaceae, Meliaceae, and Gelseminaceae. Out of these, Ophiorrhiza sp. (Rubiaceae) is the next possible candidate for sustainable CPT production after C. acuminata and Nothapodytes nimoonia. Various biotechnological-studies have been conducted on Ophiorrhiza sp. for searching the elite species and the most optimal strategies for CPT production. The genus Ophiorrhiza has been used as medicines for antiviral, antifungal, antimalarial, and anticancer activities. Phytochemical analysis has revealed the presence of alkaloids, flavonoids, triterpenes, and CPT from the plant. Because of the presence of CPT and its herbaceous habit, Ophiorrhiza sp. has now become a hot topic in research area. Currently, for mass production of the elite spp., tissue culture techniques have been implemented. In the past decades, several researchers have contributed on the diversity assessment, phytochemical analysis, mass production, and in vitro production of CPT in Ophiorrhiza sp. In this paper, we review the on the biotechnological strategies, optimal culture medium, micropropagation of Ophiorrhiza sp., effect of PGR on shoot formation, rhizogenesis, callus formation, and enhanced production of CPT for commercial use. KEY POINTS: • Latest literature on in vitro propagation of Ophiorrhiza sp. • Biotechnological production of camptothecin and related compounds • Optimization, elicitation, and transgenic studies in Ophiorrhiza sp.

Systemic Regulation of Iron Acquisition by Arabidopsis in Environments with Heterogeneous Iron Distributions.

Nutrient distribution within the soil is generally heterogeneous. Plants, therefore, have evolved sophisticated systemic processes enabling them to optimize their nutrient acquisition efficiency. By organ-to-organ communication in Arabidopsis thaliana, for instance, iron (Fe) starvation in one part of a root drives the upregulation of a high-affinity Fe-uptake system in other root regions surrounded by sufficient levels of Fe. This compensatory response through Fe-starvation-triggered organ-to-organ communication includes the upregulation of Iron-regulated transporter 1 (IRT1) gene expression on the Fe-sufficient side of the root; however, the molecular basis underlying this long-distance signaling remains unclear. Here, we analyzed gene expression by RNA-seq analysis of Fe-starved split-root cultures. Genome-wide expression analysis showed that localized Fe depletion in roots upregulated several genes involved in Fe uptake and signaling, such as IRT1, in a distant part of the root exposed to Fe-sufficient conditions. This result indicates that long-distance signaling for Fe demand alters the expression of a subset of genes responsible for Fe uptake and coumarin biosynthesis to maintain a level of Fe acquisition sufficient for the entire plant. Loss of IRON MAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) leads to the disruption of compensatory upregulation of IRT1 in the root surrounded by sufficient Fe. In addition, our split-root culture-based analysis provides evidence that the IMA3/FEP1-MYB10/72 pathway mediates long-distance signaling in Fe homeostasis through the regulation of coumarin biosynthesis. These data suggest that the signaling of IMA/FEP, a ubiquitous family of metal-binding peptides, is critical for organ-to-organ communication in response to Fe starvation under heterogeneous Fe conditions in the surrounding environment.

Root Cultures, a Boon for the Production of Valuable Compounds: A Comparative Review.

Medicinal plants are an inevitable source of pharmaceutical drugs and most of the world population depends on these plants for health benefits. The increasing global demand for bioactive compounds from medicinal plants has posed a great threat to their existence due to overexploitation. Adventitious root and hairy root culture systems are an alternative approach to the conventional method for mass production of valuable compounds from medicinal plants owing to their rapid growth, biosynthetic and genetic stability. The main purpose of this review is to investigate the recent scientific research published worldwide on the application of adventitious and hairy root cultures to produce valuable compounds from medicinal plants. Furthermore, a comparison of adventitious root vs. hairy root cultures to produce valuable compounds has also been discussed. Various aspects such as medium composition, carbon source, pH, amount of macronutrients, optimization strategy, scale-up cultures, and use of biotic abiotic and nano-elicitors at various concentrations are the topic of discussion in this review. Several studies on adventitious and hairy root cultures of Polygonum multiflorum¸ Withania somnifera¸ Echinacea purpurea and Ajuga bracteosa have been discussed in detail which highlights the importance of elicitation strategies and bioreactor system, presenting commercial applications.

Production of recombinant ß-glucocerebrosidase in wild-type and glycoengineered transgenic Nicotiana benthamiana root cultures with different N-glycan profiles.

Gaucher disease is an inherited lysosomal storage disorder caused by an insufficiency of active ß-glucocerebrosidase (GCase). Exogenous recombinant GCase via enzyme replacement therapy is considered the most practical treatment for Gaucher disease. Mannose receptors mediate the efficient uptake of exogenous GCase into macrophages. Thus, terminal mannose residues on N-glycans are essential for the delivery of exogenous GCase. In this study, recombinant GCase was produced in root cultures of wild-type (WT) and glycoengineered transgenic Nicotiana benthamiana with downregulated N-acetylglucosaminyltransferase I expression. Root cultures of WT and glycoengineered transgenic N. benthamiana plants were successfully generated by the induction of plant hormones. Recombinant GCases produced in both root cultures possessed GCase enzyme activity. Purified GCases derived from both root cultures revealed different N-glycan profiles. The WT-derived GCase possessed the predominant plant-type N-glycans, which contain plant-specific sugars-linkages, specifically ß1,2-xylose and α1,3-fucose residues. Notably, the mannosidic-type N-glycans with terminal mannose residues were abundant in the purified GCase derived from glycoengineered N. benthamiana root culture. This research provides a promising plant-based system for the production of recombinant GCase with terminal mannose residues on N-glycans.

Development of Hairy Root Cultures for Biomass and Triterpenoid Production in Centella asiatica.

Centella asiatica (Apiaceae) is a tropical/subtropical medicinal plant, which contains a variety of triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. In this study, we tested the efficiency of hairy root (HR) induction in C. asiatica from leaf and petiole explants. Leaves and petioles collected from C. asiatica plants were suspended in agro-stock for 30 min and co-cultured with Agrobacterium rhizogenes for 3 days to induce HR formation. The transformation efficiency of leaf and petiole explants was approximately 27% and 12%, respectively. A total of 36 HR lines were identified by PCR-based amplification of rol genes, and eight of these lines were selected for further analysis. Among all eight HR lines, the petiole-derived lines HP4 and HP2 displayed the highest growth index (37.8) and the highest triterpenoids concentration (46.57 mg∙g-1), respectively. Although triterpenoid concentration was >2-fold higher in leaves than in petioles of C. asiatica plants, the accumulation of triterpenoids in petiole-derived HR cultures was 1.4-fold higher than that in leaf-derived HR cultures. Additionally, in both leaf- and petiole-derived HR cultures, terpenoid production was higher in HRs than in adventitious roots. These results demonstrate that the triterpenoid content in the explant does not affect the triterpenoid content in the resultant HRs. The HR culture of C. asiatica could be scaled up to enable the mass production of triterpenoids in bioreactors for the pharmaceutical and cosmetic industries.

Impact of copper treatment on phenylpropanoid biosynthesis in adventitious root culture of Althaea officinalis L.

Althaea officinalis has been widely used in various pharmaceutical applications. The biological effects and significance of phenylpropanoids in numerous industries are well studied. However, fulfilling consumer demand for these commercially important compounds is difficult. The effect of heavy-metal toxic influence on plants is primarily due to a strong and rapid suppression of growth processes, as well as the decline in activity of the photosynthetic apparatus, also associated with progressing senescence processes. Some of the secondary metabolite production was triggered by the application of heavy metals, but there was not a stress response. In the adventitious root culture of A. officinalis, copper-mediated phenylpropanoid biosynthesis has been investigated in both concentration-and duration-dependent manners. High-performance liquid chromatography (HPLC) analysis revealed a total of nine different phenolic compounds in response to different concentrations of copper chloride. In this study, high productivity of phenolic compounds was observed in the copper chloride treated-adventitious root culture of A. officianalis. In particular, a low concentration of copper chloride led to a significant accumulation of phenolic compounds under optimal conditions. Moreover, all genes responsible for phenylpropanoid biosynthesis may be sensitive to phenolic compound production following copper treatment. Especially, the highest change in transcript level was observed from AoANS at 6 h. According to our findings, treatment with copper chloride (0.5 mM) for 48 or 96 h can be an appropriate method to maximize phenylpropanoid levels in A. officinalis adventitious root culture.