https://cyberleninka.org/article/n/132565.pdf

Discovery and resupply of pharmacologically active plant-derived natural products: A review.

Plant tissue culture as an important tool for the continuous production of active compounds including secondary metabolites and engineered molecules. Novel methods (gene editing, abiotic stress) can improve the technique. Humans have a long history of reliance on plants for a supply of food, shelter and, most importantly, medicine. Current-day pharmaceuticals are typically based on plant-derived metabolites, with new products being discovered constantly. Nevertheless, the consistent and uniform supply of plant pharmaceuticals has often been compromised. One alternative for the production of important plant active compounds is in vitro plant tissue culture, as it assures independence from geographical conditions by eliminating the need to rely on wild plants. Plant transformation also allows the further use of plants for the production of engineered compounds, such as vaccines and multiple pharmaceuticals. This review summarizes the important bioactive compounds currently produced by plant tissue culture and the fundamental methods and plants employed for their production.

/pdf/in-vitro-plant-tissue-culture-means-for-production-of-5cvlep1j9n.pdf

In vitro plant tissue culture: means for production of biological active compounds

The present study aims to investigate the effects of supplemental ultraviolet B (3.6 kJ m(-2)day(-1) above ambient) radiation on secondary metabolites and phenylpropanoid pathway enzymes of Withania somnifera under field conditions at 40, 70, and 100 days after transplantation. Secondary metabolites' (alkaloids, anthocyanins, carotenoids, flavonoids, lignin, phytosterols, saponins, and tannins) concentrations were analysed at the end of the treatments. Activities of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4-coumarate-CoA ligase (4CL), chalcone-flavanone isomerase (CHI), and dihydroflavonol reductase (DFR) were also determined. In treated plants, secondary metabolite-concentrations generally increased (higher concentrations being recorded in roots compared to leaves). Anomalies were recorded for lycopene in roots and phytosterols in leaves (all sampling ages); β-carotene declined in leaves at third sampling age. s-UV-B-treated plants depicted decrease in withanolide A content with concomitant increase in withaferin A (two major alkaloids analysed by HPLC) compared to their respective controls. Phenylpropanoid pathway enzyme-activities increased in leaves and roots under s-UV-B treatment, the latter showing greater increase. The study concludes that s-UV-B is a potent factor in increasing the concentrations of secondary metabolites and their biosynthetic pathway enzymes in W. somnifera.

Secondary metabolites and phenylpropanoid pathway enzymes as influenced under supplemental ultraviolet-B radiation in Withania somnifera Dunal, an indigenous medicinal plant.

Anisodus luridus hairy root cultures were established to test biological effects of acetylsalicylic acid (ASA) and ultraviolet ray-B (UV-B) on gene expression, tropane alkaloid (TA) biosynthesis and efflux. The TAs-pathway gene expression was ASA dosage dependant. The expression of PMT, TRI and CYP80F1 showed no significant difference in hairy root cultures in treatment of 0.01 and 0.1 mM ASA, compared with those without ASA treatment; while 0.01 or 0.1 mM ASA slightly upregulated H6H expression. All the four genes including PMT, TRI, CYP80F1 and H6H had a dramatic increase in 1 mM ASA-treated hairy root cultures compared with control. The expressing levels of all the four genes were much significantly higher in 1 mM ASA-treated hairy root cultures than those in 0.01 and 0.1 mM ASA-treated ones. As expected, hairy root cultures treated with 1 mM ASA had the highest capacity of TAs biosynthesis, in which the content of scopolamine and hyoscyamine reached respectively 57.2 and 14.7 μg g−1 DW. Surprisingly, it was found that 1 mM ASA dramatically induced the efflux of scopolamine. In the liquid medium with 1 mM ASA, the content of scopolamine was 153.4 μg flask−1, about 6.2 folds compared with that of control. At the same time, hyoscyamine was detected at trace levels in liquid medium. In the UV-B stressed hairy root cultures, all the four genes had a very strong increase of gene expression that led to more accumulation of scopolamine and lower accumulation of hyoscyamine. Only trace amounts of hyoscyamine and scopolamine were detected in the liquid medium when hairy root cultures were stressed under UV-B, and this suggested that UV-B did not affect TAs efflux.

Effects of acetylsalicylic acid and UV-B on gene expression and tropane alkaloid biosynthesis in hairy root cultures of Anisodus luridus

An efficient protocol for hairy root induction in Berberis aristata DC. was established using two different strains of Agrobacterium rhizogenes, MTCC 532 and 2364 from IMTECH (Institute of Microbial Technology), Chandigarh, India. The strain 532 was more effective than strain 2364 in hairy root induction and in vitro grown callus (61.11 ± 1.60 % transformation frequency) was found to be suitable explant in comparison to leaves (42.59 ± 0.92 % transformation frequency) and nodal segments (34.25 ± 0.92 % transformation frequency) of in vitro grown microshoots for hairy root induction. The presence of rol A and rol B genes during amplification confirmed the transgenic nature of hairy roots and transformed callus. Transformation frequency of callus was further enhanced (from 61.11 ± 1.60 % to 72.22 ± 1.60 %; when infection time was 1 h) by using acetosyringone (100 µM) during co-cultivation period (48 h) on semisolid MS (Murashige and Skoog) medium. In conclusion, this study describes the protocol for hairy root induction which could further be useful for the production of berberin and may reduce the overharvesting of this endangered species from its natural habitat.

/pdf/agrobacterium-rhizogenes-mediated-hairy-root-induction-in-4z5hs7yvsi.pdf

Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC.

Leaves of Withania somnifera contained more withaferin A and withanolide A than roots indicating that these compounds mainly accumulate in leaves. With an increase in age of the plant, withaferin A was enhanced with a corresponding decrease in withanolide A. Hairy root cultures were induced from leaf explants using Agrobacterium rhizogenes and the transgenic nature of hairy roots was confirmed by partial isolation and sequencing of rolB gene, which could not be amplified in untransformed plant parts. In hairy roots, withaferin A accumulated at 2, 3 and 4% but not at 6% sucrose, the highest amount being 1733 microg/g dry weight at 4% level. High and equal amounts of withaferin A and withanolide A accumulated (890 and 886 microg/g dry tissue respectively) only at 3% sucrose. Increasing concentrations of glucose enhanced withaferin A and it peaked at 5% level (3866 microg/g dry tissue). This amount is 2842 and 34% higher compared to untransformed roots and leaves (collected from 210-day-old plants) respectively. Withanolide A was detected at 5% glucose but not at other concentrations. While chitosan and nitric oxide increased withaferin A, jasmonic acid decreased it. Acetyl salicylic acid stimulated accumulation of both withaferin A and withanolide A at higher concentrations. Triadimefon, a fungicide, enhanced withaferin A by 1626 and 3061% (not detected earlier) compared to hairy and intact roots respectively.

http://nopr.niscair.res.in/bitstream/123456789/14330/1/IJEB%2050%287%29%20484-490.pdf

Gauri Abhayankar

Papers

Carbohydrate and elicitor enhanced withanolide (withaferin A and withanolide A) accumulation in hairy root cultures of Withania somnifera (L.).