Production Of Podophyllotoxin Research Articles

Enhanced podophyllotoxin production of endophyte Fusarium proliferatum TQN5T by host extract and phenylalanine.

Fermentation technology using endophytes is considered a potential alternative approach for producing pharmaceutical compounds like podophyllotoxin (PTOX). In this study, fungus TQN5T (VCCM 44284) was selected from endophytic fungi isolated from Dysosma versipellis in Vietnam for PTOX production through TLC. The presence of PTOX in TQN5T was further confirmed by HPLC. Molecular identification indicated TQN5T as Fusarium proliferatum with 99.43% identity. This result was asserted by morphological characteristics such as white cottony, filamentous colony, layer and branched mycelium, and clear hyphae septa. Cytotoxic assay indicated both biomass extract and culture filtrate of TQN5T presented strong cytotoxicity on LU-1 and HepG2 with IC50 of 0.11, 0.20, 0.041, and 0071, respectively, implying anti-cancer compounds were accumulated in the mycelium and secreted into the medium. Further, the production of PTOX in TQN5T was investigated in the fermentation condition supplemented with 10µg/ml of host plant extract or phenylalanine as elicitors. The results revealed a significantly higher amount of PTOX in the PDB + PE and PDB + PA at all studied time points in comparison with PDB (control). Especially, after 168h of culture, PTOX content in the PDB with plant extract reached the peak with 314µg/g DW which is 10% higher than the best yield of PTOX in previous studies, denoting F. proliferatum TQN5T as a promising PTOX producer. This is the first study on enhancing the PTOX production in endophytic fungi by supplementing phenylalanine-a precursor for PTOX biosynthesis in plants into fermented media, suggesting a common PTOX biosynthetic pathway between host plant and endophytes. KEY POINTS: • Fusarium proliferatum TQN5T was proven for PTOX production. • Both mycelia extract and spent broth extract of Fusarium proliferatum TQN5T presented strong cytotoxicity on cancer cell lines LU-1 and HepG2. • The supplementation of 10µg/ml host plant extract and phenylalanine into fermentation media of F. proliferatum TQN5T improved the yield of PTOX.

Transcriptome analysis reveals differentially expressed genes involved in somatic embryogenesis and podophyllotoxin biosynthesis of Sinopodophyllum hexandrum (Royle) T. S. Ying.

Sinopodophyllum hexandrum (Royle) T. S. Ying, an important source of podophyllotoxin (PTOX), has become a rare and endangered plant because of over-harvesting. Somatic embryogenesis (SE) is the main way of seedling rapid propagation and germplasm enhancement, but the regeneration of S. hexandrum has not been well established, and the PTOX biosynthesis abilities at different SE stages remain unclear. Therefore, it is extremely important to elucidate the SE mechanism of S. hexandrum and clarify the biosynthesis variation of PTOX. In this study, the transcriptomes of S. hexandrum at different SE stages were sequenced, the contents of PTOX and 4'-demethylepipodophyllotoxin were assayed, and the transcript expression patterns were validated by qRT-PCR. The results revealed that plant hormone (such as auxins, abscisic acid, zeatin, and gibberellins) related pathways were significantly enriched among different SE stages, indicating these plant hormones play important roles in SE of S. hexandrum; the expression levels of a series of PTOX biosynthesis related genes as well as PTOX and 4'-demethylepipodophyllotoxin contents were much higher in embryogenic callus stage than in the other stages, suggesting embryogenic callus stage has the best PTOX biosynthesis ability among different SE stages. This study will contribute to germplasm conservation and fast propagation of S. hexandrum, and facilitate the production of PTOX.

Production of podophyllotoxin, kaempferol and quercetin in callogenesis of Dysosma versipellis — an endangered medicinal plant

• Dysosma versipellis is an endangered medicinal herb found in southwest regions of China. • Callus initiation was achieved in B5 salts with 2 mg/L 2,4-D, and 0.2 mg/L kinetin under complete darkness. • The maximum callus biomass proliferation was obtained on B5 basal salts supplemented with 2 mg/L 2,4-D. • Leaf callus showed maximum production of podophyllotoxin, kaempferol and quercetin than that of control. • UPLC analysis revealed that roots and rhizomes were the primary source of secondary compounds accumulation. Dysosma versipellis is an endangered medicinal herb found in southwest regions of China. It has long been used in Traditional Chinese Medicine for general remedies in China. This herb is commonly used as an alternative to Podophyllum species. The large-scale removal of D. versipellis rhizomes from natural habitat continues at higher rates than natural regeneration. This study reports the callus induction from D. versipellis explants, callus proliferation and production of tumor inhibitory compounds such as podophyllotoxin, kaempferol and quercetin. The results revealed that tender leaves of D. versipellis exhibited the highest callus induction efficiency of 86% than other explants tested. The best callus initiation was obtained in Gamborg's medium (B5) augmented with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 mg/L kinetin, 3% sucrose, 0.1% activated charcoal and 0.7% agar and incubated under complete darkness. The maximum callus biomass proliferation was obtained on B5 basal salts supplemented with 2 mg/L 2, 4-D. The UPLC analysis of leaf callus showed maximum production of podophyllotoxin, kaempferol and quercetin than that of control callus. Furthermore, analysis of whole plant parts for their secondary compounds found roots and rhizomes were the primary source of secondary compounds accumulation. Therefore, this bioactive-enriched callus biomass protocol may be used as a possible technique for sustainable production of podophyllotoxin, kaempferol and quercetin at commercial scale.

Influence of light quality and some growth regulators in inducing the production of Podophyllotoxin, a bioactive compound against cancer, in adventitious roots formed in the leaves of Hyptis suaveolens

Podophyllotoxin (PTOX) is an aryltetraline lignan used to treat skin lesions, such as warts, human papilloma virus, and genital infections. Hyptis suaveolens has PTOX in its roots and the culture of its plant tissues in vitro has been increasingly used to produce rare and high-quality medicinal compounds. Adventitious roots are an attractive source of phytochemicals due to their genetic and biosynthetic stability. Thus, the aim of the present study is to investigate adventitious roots’ induction in leaves and nodal segments under different light qualities and auxins in H. suaveolens. Adventitious roots have only emerged in H. suaveolens leaves grown in semi-solid MS medium, in the absence of light and under red light-emitting diode (LED). Root dry weight values recorded for these treatments were statistically equal to each other, i.e., 21.84 and 29.81 mg, respectively. PTOX quantification was performed in the dark (10.72 µg g−1) and under red LED (9.93 µg g−1). Indole-3-butyric acid (IBA) and naphthaleneacetic acid (NAA) (0.5, 1.0 and 2.0 mg L−1) concentrations induced adventitious roots’ formation in leaves of plants belonging to this species. Explant supplemented with 2.0 mg L−1 NAA led to higher root dry weight (45.53 mg) in leaves. However, the 1.0 mg L−1 IBA treatment produced more PTOX (9.96 µg g−1 root). Nodal segments of H. suaveolens, IBA and NAA concentrations (0.5, 1.0 and 2.0 mg L−1) also induced adventitious roots’ formation. The treatment with 0.5 mg L−1 IBA resulted in higher root dry weight (37.62 mg) in the nodal segment. However, 1 and 2 mg L−1 IBA resulted in lower root dry weight and higher PTOX (9.97 µg g−1 and 9.95 µg g−1). In conclusion, adventitious root formation and PTOX induction in H. suaveolens leaves grown in semi-solid MS medium only happen in plants grown in the dark and under monochromatic red LED light. Growth regulator NAA was the best element to induce adventitious roots’ emergence in leaves, whereas IBA was the best one for such a purpose in nodal segment. Growth regulator NAA can inhibit the synthesis of PTOX, but IBA induces this secondary compound.

In vitro propagation from seeds and enhanced synthesis of podophyllotoxin from root callus of SinoPodophyllum hexandrum Royle T.S. Ying (Himalayan Mayapple) − An endangered medicinal plant

In vitro propagation and secondary metabolite production are the vital biotechnological approaches for the multiplication, conservation, and phytochemicals production from rare, endangered, and industrially important medicinal plants . Himalayan Mayapple ( SinoPodophyllum hexandrum Royle T.S. Ying) is a perennial venerate herb of the alpine Himalayas, rhizomes of which yield podophyllotoxin (PTOX), widely used in the pharmaceutical industry for the synthesis of anti-cancer drugs. The low regeneration rate and high demand for this plant emphasized the need for the development of a non-conventional method for its propagation and PTOX production using biotechnological interventions. In the present study, a single-step protocol for in vitro propagation of S. hexandrum through seeds and PTOX production from seedlings-induced leaf and root calli of three geographical locations in Himachal Pradesh (India) is reported. Maximum in vitro seed germination (56.23%) was obtained after 77 days followed by the highest shoot multiplication on MS medium containing BA (2.0 mg/L), NAA (0.2 mg/L), and calcium chloride (0.7 mg/L) after the fourth sub-culture. Rooting was induced at the base of shoots during in vitro shoot multiplication hence, the shoots were directly transferred for hardening without adopting additional rooting passage. For PTOX production, callus was induced from in vitro leaves and roots on B5 medium supplemented with BA (0.1 mg/L) + NAA (1.0 mg/L) and BA (0.5 mg/L) + 2,4-D (0.5 mg/L) under dark incubation. High-performance liquid chromatography (HPLC) analysis revealed maximum PTOX content (0.27 mg/g) from in vitro root callus of district Kinnaur followed by Lahaul-Spiti (0.26 mg/g), and Kullu (0.25 mg/g), thus signifying the role of high altitudes in enhanced PTOX accumulation. The current study offers an insight into the high-frequency regeneration and PTOX production of S. hexandrum , which may serve as significant baseline information for the production of high-quality plant material in the pharmaceutical industry. • Developed single-step in vitro propagation protocol from seeds of Himalayan Mayapple. • Effect of altitude on enhanced podophyllotoxin production was studied.

In vitro propagation from rhizomes, molecular evaluation and podophyllotoxin production in Himalayan May Apple (Sinopodophyllum hexandrum Royle T.S. Ying): an endangered medicinal plant

In vitro propagation from rhizomes, molecular evaluation and podophyllotoxin production in Himalayan May Apple (Sinopodophyllum hexandrum Royle T.S. Ying): an endangered medicinal plant

Optimum Conditions for Enhanced Production of Podophyllotoxin from Penicillium sp. Isolated from Khanspur, Pakistan

Optimum Conditions for Enhanced Production of Podophyllotoxin from Penicillium sp. Isolated from Khanspur, Pakistan

Research progress in biosynthesis of podophyllotoxin and its derivatives

Podophyllotoxin (PTOX) is an aryl-tetralin lignan of plant origin found in some species of Podophyllum such as Dysosma versipellis, Diphylleia sinensis, and Sinopodophyllum hexandrum. Etoposide and teniposide are produced semisynthetically from PTOX and used clinically to treat several forms of cancer. As a typical representative of new drug discovery from natural products, the production of PTOX solely depends on extraction from plants, resulting in severe contradiction between supply and demand. With the advantages of unconstrained resources and eco-friendly reaction conditions, biosynthesis method has become a trend in the production of PTOX and its derivatives. In this review, we summarize the research progress of PTOX biosynthesis in plants and expound the functions of the key enzymes as well as their subcellular location. The synthetic biology for production of PTOX intermediates in a tobacco chassis is also introduced. Finally, the heterologous expression and biotransformation of PTOX in microorganisms is summarized, which sets the foundation for the efficient microbial production of PTOX using cell factories.

Essential Oil Composition and Bioactivity of Two Juniper Species from Bulgaria and Slovakia.

Juniperus excelsa M. Bieb and J. sabina L. contain essential oil (EO), while J. sabina also contains podophyllotoxin, which is used as a precursor for anti-cancer drugs. Two studies were conducted. The first assessed the variability in the EO profile and podophyllotoxin concentration of the two junipers, depending on the location and tree gender. The main EO constituents of J. excelsa were α-cedrol, α-limonene and α-pinene, while the constituents in J. sabina were sabinene, terpinen-4-ol, myrtenyl acetate and α-cadinol. The podophyllotoxin yield of 18 J. sabina accessions was 0.07–0.32% (w/w), but this was not found in any of the J. excelsa accessions. The second study assessed the effect of hydrodistillation (Clevenger apparatus) and steam distillation (in a semi-commercial apparatus) on the EO profile and bioactivity. The extraction type did not significantly alter the EO composition. The EO profiles of the two junipers and their accessions were different and may be of interest to the industry utilizing juniper leaf EO. Breeding and selection programs could be developed with the two junipers (protected species) in order to identify chemotypes with (1) a high EO content and desirable composition, and (2) a high concentration of podophyllotoxin in J. sabina. Such chemotypes could be established as agricultural crops for the commercial production of podophyllotoxin and EO.

BRIDELIA SCANDENS WILD. A NEW SOURCE FOR PODOPHYLLOTOXIN PRODUCTION IN VITRO BY FEEDING CONIFERYL ALCOHOL

Objective: In the present study, a new method for the production of anticancerous compound podophyllotoxin (PTOX) was developed for Bridelia scandens Wild. by feeding coniferyl alcohol.
 Methods: The production of anticancerous compound PTOX through leaf explant derived calli of B. scandens. Murashige and Skoog (MS) medium fortified with 0.5 mg/l 6-Benzylaminopurine (BAP) and 0.5 mg/l 2,4-D (2,4-Dichlorophenoxyacetic acid) induced luxuriant mass of callus growth. Suspension culture was initiated by sterile MS media fortified with 0.1–1.0 mg/l BAP and 0.1–1.0 mg/l 2,4-D. and growth product was analyzed by the high-pressure liquid chromatography method.
 Results: Phytochemical analysis of the B. scandens leaf and leaf calli showed the presence of PTOX at the concentrations of 0.69 and 1.81, respectively. The callus cell suspension was established with the same callogenic media also it is augmented with 10–70 mg/l of coniferyl alcohol to elicit the biosynthesis of PTOX. Successive cultures of the calli suspension yielded stable production of PTOX of 3.91 mg/g dry cell weight at 50 mg/l coniferyl alcohol in the media. The biosynthesis of PTOX was ideal when plant cells were cultivated in the dark with an agitation speed of 100 rpm.
 Conclusion: The growth and production of PTOX were found to be better with glucose than with sucrose as the medium carbon source. The harvesting of the secondary metabolite from the in vitro grown leaf calli of B. scandens is a better way to stop the exploitation of medicinal plants.

<p><strong>Role of polyamines on <em>in vitro </em>regeneration and podophyllotoxin production in <em>Podophyllum hexandrum</em> Royle</strong></p>

Podophyllum hexandrum is a popular medicinal plant endemic to Himalayas. In the present investigation, we report an efficient mass propagation protocol and Podophyllotoxin (PTOX) production in in vitro-derived plants of P. hexandrum, cultured on MS medium containing various plant growth regulators and polyamines. A combination of BA (1.5 mg/L), IAA (0.2 mg/L) and spermidine (20 mg/L) produced the highest number of multiple shoots per explant (23 shoots) after 6 weeks of culture; the regenerated shoots were elongated on the same medium. The elongated-shoots were rooted on root induction medium (half strength MS medium) supplemented with IBA (1.5 mg/L) and putrescine (15 mg/L). The rooted plantlets were successfully hardened and acclimatized with a survival rate of 68% in the greenhouse. The highest content of PTOX (4.23 mg/g DW) was recorded in in vitro derived roots followed by leaves when compared to field-grown parent plants. The present system offers the possibility to use in vitro culture techniques for mass propagation and PTOX production for commercial utilization.

Spatial transcriptional dynamics of geographically separated genotypes revealed key regulators of podophyllotoxin biosynthesis in Podophyllum hexandrum

Podophyllum hexandrum is the major source of podophyllotoxin (PTOX), a highly bioactive lignan of great pharmacological importance with anti-cancerous activities. The industrial demand of PTOX relies on the highly endangered natural resources only. It is therefore, desirable to elucidate global molecular processes and identify key genes for enhancing PTOX biosynthesis by overexpressing the targeted candidates. Transcriptome of leaf, rhizome, and stalk was generated to analyse the spatial regulation of PTOX biosynthesis in genetically diverse genotypes. Overall, 198 million high-quality paired-end reads were assembled into 85,531 transcripts. In addition, 32,341 transcripts were assigned gene ontologies with 6570 hits in distinct pathways and 15,886 transcription factors representing 70 families. Interestingly, comparative expression analyses revealed that 12 of 31 genes of PTOX biosynthesis were upregulated in rhizome. However, shikimate and phenylalanine pathways that generate PTOX precursors were abundantly upregulated in leaves. Thus, a further insight on the inducers of these genes can be extended to enrich the aerial tissues for downstream pathway through genetic manipulations. Additionally, higher expression of transcription factors WRKY, MYB, bZIP, bHLH, and AP2, transporters ABCB and ABCC, UGTs, CYP450s, and jasmonate pathway in rhizome supported the secondary metabolism. The comprehensive genomic resource created during this study will provide deeper understanding of lignan biosynthesis and its regulation. This will further enable selection of the elite genotypes and potential genes that can be directed to enhance PTOX production and yield at an industrial scale.

Enhanced production of podophyllotoxin, kaempferol, and quercetin from callus culture of Dysosma pleiantha (Hance) Woodson: An endangered medicinal plant.

Dysosma pleiantha (Hance) Woodson is one of the endangered traditional Chinese medicinal herbs, highly valued for its medicinal properties by Taiwan's mountain tribes. The present study aims to develop an efficient protocol for callus biomass by optimizing suitable culture medium, carbon source culture condition, and enhanced production of pharmaceutically important podophyllotoxin, kaempferol, and quercetin from callus culture of D. pleiantha under the influence of different additives. Best callus induction was achieved in Gamborg's medium (B5) with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) along with 0.2 mg/L kinetin under dark condition. Tender leaves of D. pleiantha showed the maximum of 86% callus induction among the different explants tested. Highest leaf callus proliferation was noted in B5 medium with 1 mg/L 2,4-D incubated under complete darkness. In addition, it was found that B5 medium with 1 mg/L 2,4-D along with 2 g/L peptone produced more leaf callus biomass and enhanced production of podophyllotoxin (16.3-fold), kaempferol (12.39-fold), and quercetin (5.03-fold) compared to control. Therefore, D. pleiantha callogenesis can provide an alternative source for enhanced production of secondary compounds regardless of the exploitation of its natural plant population.

Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets.

Ten Hyptis suaveolens hairy root lines were established by infecting nodal explants with K599+pGus-GFP+ and ATCC15834+pTDT strains from Agrobacterium rhizogenes. Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth in absence of growth regulators. Cytotoxicity was determined using the sulforhodamine B method, and the production of podophyllotoxin (PTOX) was measured by high performance thin layer chromatography scanning. Through these methodologies, HsTD10 was identified as the hairy root line with the highest cytotoxicity and PTOX production, which was corroborated by liquid chromatography-mass spectrometry and micrOTOF-Q II. A suspension culture of HsTD10 was established in which PTOX and carbohydrate consumption during growth kinetics were quantified by high-performance liquid chromatography. Procedures to increase the production and retrieval of PTOX in the HsTD10 line included selection of culture medium, addition of thiamine, and modification of the PTOX extraction method. The best combination of these variables was MS medium at 75% of its components with the addition of 2 mg L-1 of thiamine, extraction with methanol-dichloromethane, and sonication at 40 ± 5°C. During kinetics, growth-associated PTOX accumulation was observed. The specific growth rate (μ) was 0.11 d-1. The highest concentration of PTOX obtained with HsTD10 (5.6 mg g-1 DW) was 100 times higher than that reported for roots of wild plants and 56 times higher than that for in vitro nontransformed roots of H. suaveolens.

Effects of exogenous IBA and fungal elicitor on growth of in vitro roots culture of Dysosma versipellis and production of podophyllotoxin

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 μg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

Biotechnological Approaches for Production of Anti-Cancerous Compounds Resveratrol, Podophyllotoxin and Zerumbone.

Secondary metabolites from numerous plant sources have been developed as anti- cancer reagents and compounds such as resveratrol, podophyllotoxin and zerumbone are of particular importance in this regard. Since their de novo chemical synthesis is both arduous and commercially expensive, there has been an impetus to develop viable, biotechnological methods of production. Accordingly, this review focuses on the recent developments in the field, highlighting the use of micropropagation, cell suspension cultures, callus cultures, hairy root cultures, recombinant microbes and genetically modified higher plants. Optimization of media and culture conditions, precursor feeding, immobilization and the use of chemical or physical elicitation in various protocols has led to an increase in resveratrol and podophyllotoxin production. Heterologous gene transformation of higher plants with stilbene synthase derived from Arachis hypogaea or Vitis vinifera lead to resveratrol production with the concomitant increase in resistance to plant pathogens. Interestingly, genetic transformation of Podophyllum hexandrum and Linum flavum with Agrobacterium rhizogenes resulted in Ri-T-DNA gene(s)-mediated enhancement of podophyllotoxin production. Zerumbone yields from tissue cultured plantlets or from suspension cultures are generally low and these methods require further optimization. In microbes lacking the native resveratrol or zerumbone biosynthesis pathway, metabolic engineering required not only the introduction of several genes of the pathway, but also precursor feeding and optimization of gene expression to increase their production. Data pertaining to safety and toxicity testing are needed prior to use of these sources of anti-cancer compounds in therapy.

Effect of Precursor and Phytohormones on Podophyllotoxin Production in Juniperus virginiana Suspension Cultures

Our results showed that cinnamic acid can increase podophyllotoxin production in Juniperus virginiana suspension cultures. The best effect was manifested after a 24-hours application of a 10 mmol/L concentration. The highest podophyllotoxin content was determined at 1.47 mg/g DW and the production was statistically significantly stimulated by about 444% in comparison with the control. Comparison of podophyllotoxin production in the cinnamic acid- and salicylic acid-elicited J. virginiana suspension cultures confirms that the maximum increase in both cases was induced by the 24-hours application of the 10 mmol/L concentration. In contrast, the best effect of jasmonic acid was manifested after the longest 168-hours application of a 5 mmol/L concentration.

Abiotic factors influencing podophyllotoxin and yatein overproduction in Leptohyptis macrostachys cultivated in vitro

This work describes an in vitro propagation protocol for the large-scale cultivation of Leptohyptis macrostachys (Benth.) Harley & JFB Pastore and the influence of abiotic factors on podophyllotoxin and yatein production. The plant was established from seeds collected in Chapada Diamantina, BA, Brazil and submitted to different growth mediums and physical conditions. The podophyllotoxin and yatein contents were quantified by HPLC/DAD and with pure standards and these two lignans were present in all experiments. The lignan quantities were evaluated using the Sisvar Program, compared by Tukey’s test and hierarchical cluster analysis and principal component analysis. In all experiments, podophyllotoxin and yatein were detected at different concentrations. The best protocol cultivar of L. macrostachys was established from seeds in MS ½ medium supplemented with 1.5% sucrose and 11.55μM of gibberellic acid A3 (GA3) at 30°C, which yielded the highest concentration of podophyllotoxin (5.831mgg−1). These results are important findings for the production of podophyllotoxin from the tissue culture.

Methyl jasmonate treatment increases podophyllotoxin production in Podophyllum hexandrum roots under glasshouse conditions

Background and aimThe endangered Podophyllum hexandrum is an important industrial source of podophyllotoxin, which is a precursor for the anticancer drugs etoposide and teniposide. Attempts to obtain podophyllotoxin through cell cultures or chemical synthesis have still a long way to go before being economical feasible. The objective of this study was to increase the root formation and podophyllotoxin production of P. hexandrum cultivated in a glasshouse.MethodsRoot formation and podophyllotoxin production of P. hexandrum in sand or peat-perlite soil at 15 °C or 25 °C was determined. Furthermore, the influence of methyl jasmonate on the podophyllotoxin production was determined.ResultsMore root formation was observed in peat-perlite soil than in sand soil. Furthermore, root formation was higher at 15 °C than at 25 °C. This resulted in the highest podophyllotoxin production per plant in peat-perlite at 15 °C (160 ± 22 mg/plant d.w.). Furthermore, methyl jasmonate treatment of the leaves increased the podophyllotoxin production in the roots by 21%.ConclusionWe were able to cultivate P. hexandrum in a glasshouse in the Netherlands and improve the root formation and podophyllotoxin production. This paves the way for large-scale cultivation of P. hexandrum in the temperate latitudes for the production of the pharmaceutical interesting podophyllotoxin.

In vitro polyploidy induction: changes in morphology, podophyllotoxin biosynthesis, and expression of the related genes in Linum album (Linaceae).

Induction of tetraploidy was performed and podophyllotoxin production increased by upregulating the expression level and enzyme activity of genes related to its biosynthesis in tetraploid compared to diploid Linum album. Linum album is a valuable medicinal plant that produces antiviral and anticancer compounds including podophyllotoxin (PTOX). To achieve homogeneous materials, in vitro diploid clones were established, and their nodal segments were exposed to different concentrations and durations of colchicine. This resulted in successful tetraploidy induction, confirmed by flow cytometry, and is being reported for the first time. The highest efficiency of tetraploid induction (22%) was achieved after 72h exposure to 2.5-mM colchicine treatment. The stable tetraploids were produced after being subcultured three times, and their ploidy stability was confirmed after each subculture. The effects of autopolyploidy were measured on the morphological and phytochemical characteristics, as well as enzyme activity and the expression levels of some key genes involved in the PTOX biosynthetic pathway, including phenylalanine ammonia-lyase (PAL), cinnamoyl-Coa reductase (CCR), cinnamyl-alcohol dehydrogenase (CAD), and pinoresinol-lariciresinol reductase (PLR). The tetraploid plants had larger leaves and stomata (length and width) and lower density stomata. Increasing the ploidy level from diploid to tetraploid resulted in 1.39- and 1.23-fold enhancement of PTOX production, respectively, in the leaves and stem. The increase in PTOX content was associated with upregulated activities of some enzymes studied related to its biosynthetic pathway and the expression of the corresponding genes. The expression of the PAL gene and PLR enzymatic activity had the most positive correlation with the ploidy level in both leaf and stem tissues. Our results verified that autotetraploid induction is a useful breeding method, remarkably increasing the PTOX content in the leaves and stem of L. album.