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Abstract Background Rehmannia glutinosa is a rich source of terpenoids with a high medicinal reputation. The present study compared dedifferentiated cells (DDCs) and cambial meristematic cells (CMCs) cell cultures of R. glutinosa for terpenoid (catalpol) and indole alkaloid (IA) biosynthesis. In this regard, we used widely targeted metabolomics and transcriptome sequencing approaches together with the comparison of cell morphology, cell death (%), and catalpol production at different time points. Results We were able to identify CMCs based on their morphology and hypersensitivity to zeocin. CMCs showed higher dry weight content and better catalpol production compared to DDCs. The metabolome analysis revealed higher concentrations of IA, terpenoids, and catalpol in CMCs compared to DDCs. The transcriptome sequencing analysis showed that a total of 27,201 genes enriched in 139 pathways were differentially expressed. The higher catalpol concentration in CMCs is related to the expression changes in genes involved in acetyl-CoA and geranyl-PP biosynthesis, which are precursors for monoterpenoid biosynthesis. Moreover, the expressions of the four primary genes involved in monoterpenoid biosynthesis (NMD, CYP76A26, UGT6, and CYP76F14), along with a squalene monooxygenase, exhibit a strong association with the distinct catalpol biosynthesis. Contrarily, expression changes in AADC, STR, and RBG genes were consistent with the IA biosynthesis. Finally, we discussed the phytohormone signaling and transcription factors in relation to observed changes in metabolome. Conclusions Overall, our study provides novel data for improving the catalpol and IA biosynthesis in R. glutinosa.

Cambial meristematic cells (CMCs) culture has received a fair share of scientific and industrial attention among the trending topics of plant cell culture, especially their potential toward secondary metabolites production. However, the conventional plant cell culture is often not commercially feasible because of difficulties associated with culture dedifferentiated cells. Several reports have been published to culture CMCs and bypass the dedifferentiation process in plant cell culture. Numerous mitochondria, multiple vacuoles, genetic stability, self-renewal, higher biomass, and stable metabolites accumulation are the characteristics features of CMCs compared with dedifferentiated cells (DDCs) culture. The CMCs culture has a broader application to produce large-scale natural compounds for: pharmaceuticals, food, and cosmetic industries. Cutting-edge progress in plant cellular and molecular biology has allowed unprecedented insights into cambial stem cell culture and its fundamental processes. Therefore, regarding sustainability and natural compound production, cambial cell culture ranks among the most vital biotechnological interventions for industrial and economic perspectives. This review highlights the recent advances in plant stem cell culture and understands the cambial cells induction and culture mechanisms that affect the growth and natural compounds production. [ABSTRACT FROM AUTHOR]

Background: Rehmannia glutinosa is a rich source of terpenoids with a high medicinal reputation. The present study compared dedifferentiated cells (DDCs) and cambial meristematic cells (CMCs) cell cultures of R. glutinosa for terpenoid (catalpol) and indole alkaloid (IA) biosynthesis. In this regard, we used widely targeted metabolomics and transcriptome sequencing approaches together with the comparison of cell morphology, cell death (%), and catalpol production at different time points. Results: We were able to identify CMCs based on their morphology and hypersensitivity to zeocin. CMCs showed higher dry weight content and better catalpol production compared to DDCs. The metabolome analysis revealed higher concentrations of IA, terpenoids, and catalpol in CMCs compared to DDCs. The transcriptome sequencing analysis showed that a total of 27,201 genes enriched in 139 pathways were differentially expressed. The higher catalpol concentration in CMCs is related to the expression changes in genes involved in acetyl-CoA and geranyl-PP biosynthesis, which are precursors for monoterpenoid biosynthesis. Moreover, the expressions of the four primary genes involved in monoterpenoid biosynthesis (NMD, CYP76A26, UGT6, and CYP76F14), along with a squalene monooxygenase, exhibit a strong association with the distinct catalpol biosynthesis. Contrarily, expression changes in AADC, STR, and RBG genes were consistent with the IA biosynthesis. Finally, we discussed the phytohormone signaling and transcription factors in relation to observed changes in metabolome. Conclusions: Overall, our study provides novel data for improving the catalpol and IA biosynthesis in R. glutinosa. [ABSTRACT FROM AUTHOR]

Catharanthus roseus is a perennial herb of the Apocynaceae family, from which about 200 kinds of alkaloids have been characterized. Most alkaloids from C. roseus are terpenoid indole alkaloids (TIAs), such as vinblastine and vincristine, which are widely used in the clinic for their good antitumor activity. However, they were only biosynthesized in C. roseus, and their content in C. roseus is extremely low. The access to these valuable compounds is by plant extraction or chemical semisynthesis from their precursors catharanthine and vindoline. Since catharanthine and vindoline are also obtained from C. roseus, the supply of vinblastine and vincristine makes it difficult to meet market demands. Therefore, how to improve the yield of TIAs is an attractive issue. In this study, we compared the regulatory effect of two critical transcription factors, octadecanoid-derivative responsive Catharanthus AP2-domain protein 3 (ORCA3) and octadecanoid-derivative responsive Catharanthus AP2-domain protein 4 (ORCA4), on the biosynthesis of TIAs in C. roseus. The results showed that overexpressing both two transcription factors could increase the accumulation of TIAs. The effect was more significant when ORCA4 was overexpressed. To acquire C. roseus TIAs on a continuous and consistent basis, we then created and acquired C. roseus stem cells stably overexpressing ORCA4. This is the first time a recombinant C. roseus stem cell system with stable ORCA4 overexpression has been developed, which not only provides new ideas for future research in this area but also breaches new life into the industrial application of using plant cell culture to obtain natural products. [Display omitted] • Transcription factor ORCA3 regulated the expression of ORCA4 in Catharanthus roseus. • ORCA4 overexpression resulted in accumulation of catharanthine and vindoline. • C. roseus stem cells overexpressing ORCA4 were built for the first time. • Overexpressing ORCA4 is beneficial for terpenoid indole alkaloids biosynthesis. [ABSTRACT FROM AUTHOR]

Squalene synthase (SQS), squalene epoxidase (SE), and oxidosqualene cyclase (OSC) are encoding enzymes in downstream biosynthetic pathway of triterpenoid in plants, but the relationship between three genes and celastrol accumulation in Tripterygium wilfordii still remains unknown. Gene transformation system in plant can be used for studying gene function rapidly. However, there is no report on the application of cambial meristematic cells (CMCs) and dedifferentiated cells (DDCs) in genetic transformation systems. Our aim was to study the effects of individual overexpression of TwSQS, TwSE, and TwOSC on terpenoid accumulation and biosynthetic pathway related gene expression through CMCs and DDCs systems. Overexpression vectors of TwSQS, TwSE, and TwOSC were constructed by Gateway technology and transferred into CMCs and DDCs by gene gun. After overexpression, the content of celastrol was significantly increased in CMCs compared with the control group. However, there was no significant increment of celastrol in DDCs. Meanwhile, the relative expression levels of TwSQS, TwSE, TwOSC, and terpenoid biosynthetic pathway related genes were detected. The relative expression levels of TwSQS, TwSE, and TwOSC were increased compared with the control group in both CMCs and DDCs, while the pathway-related genes displayed different expression trends. Therefore, it was verified in T. wilfordii CMCs that overexpression of TwSQS, TwSE, and TwOSC increased celastrol accumulation and had different effects on the expression of related genes in terpenoid biosynthetic pathway, laying a foundation for further elucidating the downstream biosynthetic pathway of celastrol through T. wilfordii CMCs system. [ABSTRACT FROM AUTHOR]

Abstract Background Tripterygium wilfordii Hook. f. (T. wilfordii) is an important medicinal plant with anti-inflammatory, immunosuppressive and anti-tumor activities. The main bioactive ingredients are diterpenoids and triterpenoids, such as triptolide, triptophenolide and celastrol. However, the production of terpenoids from original plants, hairy roots and dedifferentiated cells (DDCs) are not satisfactory for clinical applications. To find a new way to further improve the production of terpenoids, we established a new culture system of cambial meristematic cells (CMCs) with stem cell-like properties, which had strong vigor and high efficiency to produce large amounts of terpenoids of T. wilfordii. Results CMCs of T. wilfordii were isolated and cultured for the first time. CMCs were characterized consistent with stem cell identities based on their physiological and molecular analysis, including morphology of CMCs, hypersensitivity to zeocin, thin cell wall and orthogonal partial least square-discriminant analysis, combination of transcriptional data analysis. After induction with methyl jasmonate (MJ), the maximal production of triptolide, celastrol and triptophenolide in CMCs was 312%, 400% and 327% higher than that of control group, respectively. As for medium, MJ-induced CMCs secreted 231% triptolide and 130% triptophenolide at the maximum level into medium higher than that of control group. Maximal celastrol production of induced CMCs medium was 48% lower than that of control group. Long-term induction significantly enhanced the production of terpenoids both in cells and medium. The reason for increasing the yield of terpenoids was that expression levels of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA synthase (HMGS) were upregulated in CMCs after induction. Conclusions For the first time, CMCs of T. wilfordii were isolated, cultured, characterized and applied. Considering the significant enrichment of terpenoids in CMCs of T. wilfordii, CMCs could provide an efficient and controllable platform for sustainable production of terpenoids, which can be a better choice than DDCs.

In Fraxinus mandshurica , we successfully isolated and identified the loose, uniform and creamy-white cambial meristematic cells (CMCs) from newborn shoots, and established a culture technology for induction, proliferation and differentiation of CMCs. In this technology, higher induction rate (83.0%, 0.57-fold to the control) was obtained by an effective pretreatment after 28-day induction culture, CMCs can be better proliferation cultured than common calli and maintain same growth states after several times of cultures and 3.3% CMCs primarily realized differentiation. Gene expressions in the differentiated CMCs revealed that, low expression of FmWOX5 (regulator in establishment of competence for shoot formation, 0.09-fold to the control) and high expressions of FmWOX4 (cambium stem cell regulator, 16.7-fold to the control) and 9 key genes in shoot regeneration (2.4-fold–72.1-fold to the control) function in CMCs differentiation. In addition to the function of high expression of PHAVOLUTA (FmPHV) in CMCs differentiation (5.4-fold–157.3-fold to undifferentiated CMCs), functions of high expression of FmPHV in CMCs identification (22.4-fold to common calli) and generating more shoots (2.3-fold to the control) by significantly changing expressions of key regulators in HD-Zip Class III related shoot regeneration networks in positive transgenic plants through the hypocotyl transforming system in F. mandshurica , were further revealed. These works were of profound significance in providing the culture technology of CMCs from newborn shoots in F. mandshurica for the first time and revealing the positive functions of FmPHV in CMCs identification and differentiation in F. mandshurica and promoting the shoot regeneration by hypocotyls • Cambial meristematic cells (CMCs) from newborn shoots in Fraxinus mandshurica were successfully isolated and identified. • Cultures for higher induction, fast proliferation and primary differentiation of CMCs in F. mandshurica were established. • High expression of FmPHV functions in identification and differentiation of CMCs and shoot regeneration in F. mandshurica. [ABSTRACT FROM AUTHOR]

The application of plant suspension culture to produce valuable compounds, such as the triterpenoids oleanolic acid and ursolic acid, is a well-established alternative to the cultivation of whole plants. Cambial meristematic cells (CMCs) are a growing field of research, often showing superior cultivation properties compared to their dedifferentiated cell (DDC) counterparts. In this work, the first-time establishment of O. basilicum CMCs is demonstrated. DDCs and CMCs were cultivated in shake flasks and wave-mixed disposable bioreactors (wDBRs) and evaluated regarding triterpenoid productivity and biomass accumulation. CMCs showed characteristic small vacuoles and were found to be significantly smaller than DDCs. Productivities of oleanolic and ursolic acid of CMCs were determined at 3.02 ± 0.76 mg/(l*d) and 4.79 ± 0.48 mg/(l*d) after 19 days wDBR cultivation, respectively. These values were consistently higher than any productivities determined for DDCs over the observed cultivation period of 37 days. Elicitation with methyl jasmonate of DDCs and CMCs in shake flasks resulted in increased product contents up to 48 h after elicitor addition, with the highest increase found in CMCs at 232.30 ± 19.33% (oleanolic acid) and 192.44 ± 18.23% (ursolic acid) after 48 h. Key message: For the first time, cambial meristematic cells of Ocimum basilicum were established and cultivated in a disposable bioreactor system. These cells outperform dedifferentiated cells of the same organism regarding productivity. [ABSTRACT FROM AUTHOR]

Ethnopharmacological importance Catharanthus roseus (L.) G. Don. is an important medicinal plant with rich sources of remarkable health benefits consisting more than 100 alkaloids and significant amounts of bioactive compounds, which have been widely used as a folk medicine for treatment of several pathologies. The aim of the study In the present study, we isolated and cultured innately undifferentiated cambium meristematic cells (CMCs), which were observed stable cell growth, enhancement of bioactive compounds from C.roseus . Materials and methods We attempted to determine the effect of association between time-course growth rates, bioactive compounds and terpenoids indole alkaloid (TIA) contents as well as antioxidant and anticancer efficacies of C. roseus CMC suspension culture treated by UV-C. Results The bioactive compounds, vincristine contents, and antioxidant power were noticed significantly higher in 60 min exposure at 5 cm distances and with the directly collected sample (T7). A similar trend has also been noticed from the anticancer activity. Demonstration of TIA accumulation was found higher at 5 min exposure, at 20 cm distances and 48 h of incubation (T21) and the result of TIA contents had the highest correlation effects of anticancer activities. Conclusion In the current study, we demonstrated that UV-C light could enhance the production of the essential compounds and bioactivities in the CMCs of C. roseus , and thus, C. roseus CMCs have the potential to serve as an industrial platform for the production of bioactive alkaloids and antioxidant, anticancer activity. Moreover, additional efforts should be made to irradiate CMC suspension cultures from C. roseus with UV-C to achieve better pharmacological profiles. [ABSTRACT FROM AUTHOR]

The undifferentiated cambial meristematic cell (CMC) has been recognized as a value-added production platform for plant natural products in comparison to the dedifferentiated plant cell line (DDC). In a time-based approach at 0, 24, 48, and 72 h, the present study aimed at investigating the phytochemical metabolome of methyl jasmonate (MeJA)-elicited CMC cultures derived from sweet basil (Ocimum basilicum L.), including primary and secondary metabolites analyzed using GC/TOF-MS post-silylation and RP-UPLC-C 18 -FT-MS/MS, respectively, as well as the analysis of aroma composition using headspace SPME-GC-MS. The results revealed a stress response in primary metabolism manifested by an increase in amino and organic acids reaching their maximum levels after 48 (1.3-fold) and 72 (1.7-fold) h, respectively. In addition, phenolic acids (e.g., sagerinic acid, rosmarinic acid, and 3- O -methylrosmarinic acid) followed by flavonoid aglycones (e.g., salvigenin and 5,6,4′-trihydroxy-7,3′-dimethoxyflavone) were the most abundant with prominent increases at 48 (1.2-fold) and 72 (2.1-fold) h, respectively. The aroma was intensified by the elicitation along the time, especially after 48 and 72 h. Furthermore, multivariate data analyses, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) confirmed elicitation effect, especially post 48 and 72 h. The study further assessed the effect of MeJA elicitation on the antioxidant and polyphenolic content. The cultures at 48 h demonstrated a significant (p < 0.05) antioxidant activity concurrently with correlation with total polyphenolic content using Pearson's correlation. Our study provides new insights to the elicitation impact on primary and secondary metabolism, in addition to aroma profile, to orchestrate the stress response and in relation to antioxidant effect. [Display omitted] • Cambial meristematic cells (CMC) are a value-added production platform for natural products. • Methyl jasmonate-elicited CMC derived from sweet basil revealed an increase of phenolic metabolites and C6-volatiles. • Multivariate data analyses confirmed elicitation effect especially post 48 and 72 h. • The 48 h cultures demonstrated a significant (p < 0.05) antioxidant activity correlated with total polyphenolic content. [ABSTRACT FROM AUTHOR]

This study reported the inducing effect of Aspergillus flavus fungal elicitor on biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus cambial meristematic cells (CMCs) and its inducing mechanism. According to the results determined by HPLC and HPLC-MS/MS, the optimal condition of the A. flavus elicitor was as follows: after suspension culture of C. roseus CMCs for 6 day, 25 mg/L A. flavus mycelium elicitor were added, and the CMC suspensions were further cultured for another 48 h. In this condition, the contents of vindoline, catharanthine, and ajmaline were 1.45-, 3.29-, and 2.14-times as high as those of the control group, respectively. Transcriptome analysis showed that D4H, G10H, GES, IRS, LAMT, SGD, STR, TDC, and ORCA3 were involved in the regulation of this induction process. The results of qRT-PCR indicated that the increasing accumulations of vindoline, catharanthine, and ajmaline in C. roseus CMCs were correlated with the increasing expression of the above genes. Therefore, A. flavus fungal elicitor could enhance the TIA production of C. roseus CMCs, which might be used as an alternative biotechnological resource for obtaining bioactive alkaloids.

Plant cell culture technology has been introduced for the mass production of the many useful components. A variety of plant-derived compounds is being used in various fields, such as pharmaceuticals, foods, and cosmetics. Plant cell cultures are believed to be derived from the dedifferentiation process. In the present study, an undifferentiated cambial meristematic cell (CMCs) of Catharanthus is isolated using histological and genetic methods, and compared with dedifferentiation-derived callus (DDCs) cultures. Furthermore, differential culture conditions for both DDCs- and CMCs-derived cell lines were established. A suitable media for the increased accumulation of terpenoid indole alkaloids (TIAs) was also standardized. Compared with DDCs, CMCs showed marked accumulation of TIAs in cell lines grown on media with 1.5 mg·mL −1 of NAA and 0.5 mg·mL −1 of kinetin. CMCs-derived cultures of Catharanthus , as a source of key anticancer drugs (viblastine and vincristine), would overcome the obstacles usually associated with the production of natural metabolites through the use of DDCs. Cell culture systems that are derived from CMCs may also provide a cost-effective and eco-friendly basis for the sustainable production of a number of important plant natural products. [ABSTRACT FROM AUTHOR]

Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM β-CD and 150 μM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that β-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that β-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that β-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus, and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids. [ABSTRACT FROM AUTHOR]

Background: Tripterygium wilfordii Hook. f. (T. wilfordii) is an important medicinal plant with anti-inflammatory, immunosuppressive and anti-tumor activities. The main bioactive ingredients are diterpenoids and triterpenoids, such as triptolide, triptophenolide and celastrol. However, the production of terpenoids from original plants, hairy roots and dedifferentiated cells (DDCs) are not satisfactory for clinical applications. To find a new way to further improve the production of terpenoids, we established a new culture system of cambial meristematic cells (CMCs) with stem cell-like properties, which had strong vigor and high efficiency to produce large amounts of terpenoids of T. wilfordii. Results: CMCs of T. wilfordii were isolated and cultured for the first time. CMCs were characterized consistent with stem cell identities based on their physiological and molecular analysis, including morphology of CMCs, hypersensitivity to zeocin, thin cell wall and orthogonal partial least square-discriminant analysis, combination of transcriptional data analysis. After induction with methyl jasmonate (MJ), the maximal production of triptolide, celastrol and triptophenolide in CMCs was 312%, 400% and 327% higher than that of control group, respectively. As for medium, MJ-induced CMCs secreted 231% triptolide and 130% triptophenolide at the maximum level into medium higher than that of control group. Maximal celastrol production of induced CMCs medium was 48% lower than that of control group. Long-term induction significantly enhanced the production of terpenoids both in cells and medium. The reason for increasing the yield of terpenoids was that expression levels of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA synthase (HMGS) were upregulated in CMCs after induction. Conclusions: For the first time, CMCs of T. wilfordii were isolated, cultured, characterized and applied. Considering the significant enrichment of terpenoids in CMCs of T. wilfordii, CMCs could provide an efficient and controllable platform for sustainable production of terpenoids, which can be a better choice than DDCs. [ABSTRACT FROM AUTHOR]

This study reported the inducing effect of Aspergillus flavus fungal elicitor on biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus cambial meristematic cells (CMCs) and its inducing mechanism. According to the results determined by HPLC and HPLC-MS/MS, the optimal condition of the A. flavus elicitor was as follows: after suspension culture of C. roseus CMCs for 6 day, 25 mg/L A. flavus mycelium elicitor were added, and the CMC suspensions were further cultured for another 48 h. In this condition, the contents of vindoline, catharanthine, and ajmaline were 1.45-, 3.29-, and 2.14-times as high as those of the control group, respectively. Transcriptome analysis showed that D4H, G10H, GES, IRS, LAMT, SGD, STR, TDC, and ORCA3 were involved in the regulation of this induction process. The results of qRT-PCR indicated that the increasing accumulations of vindoline, catharanthine, and ajmaline in C. roseus CMCs were correlated with the increasing expression of the above genes. Therefore, A. flavus fungal elicitor could enhance the TIA production of C. roseus CMCs, which might be used as an alternative biotechnological resource for obtaining bioactive alkaloids. [ABSTRACT FROM AUTHOR]