Growth and isoflavonoid accumulation of Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures

We established cell suspension cultures derived from leaf, stem, and root calli of Pueraria candollei var. candollei and P. candollei var. mirifica using liquid Murashige and Skoog (MS) medium supplemented with 0.56 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Growth of the cell suspension cultures progressed to the stationary phase within 15–24 days. Methanolic extracts of cell suspension cultures of both varieties of P. candollei were analyzed using a validated HPLC protocol. All cell lines derived from leaf, stem, and root explants produced four major isoflavonoids: daidzein, daidzin, genistein, and genistin; these isoflavonoids were detected only in the roots of intact plants. Furthermore, the isoflavonoid contents of the cell suspension cultures were higher than those of intact plants. Thus, cell suspension culture of both varieties of P. candollei may be an effective tool for isoflavonoid production.     

Increased miroestrol, deoxymiroestrol and isoflavonoid accumulation in callus and cell suspension cultures of Pueraria candollei var. mirifica

Miroestrol and deoxymiroestrol are highly active phytoestrogens derived from the tuberous roots of Pueraria candollei var. mirifica. To date, there have been no reports regarding the production of miroestrol and deoxymiroestrol in in vitro cell culture. In this study, callus and cell suspension cultures were established for the purpose of investigating miroestrol and deoxymiroestrol content in P. candollei var. mirifica cells. Stem-derived callus cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg l⁻¹ thidiazuron (TDZ), 0.5 mg l⁻¹ naphthaleneacetic acid (NAA), and 1.0 mg l⁻¹ benzyladenine (BA) provided optimal conditions for the accumulation of deoxymiroestrol and total isoflavonoids. The calli produced 184.83 ± 20.09 μg g⁻¹ dry weight of total chromene and 20.72 ± 2.38 mg g⁻¹ dry weight of total isoflavonoid. This is the first report to suggest that callus culture is a suitable alternative method for producing miroestrol and deoxymiroestrol. Carbon sources were evaluated for the cell suspension cultures of P. candollei var. mirifica. Sucrose provided optimal conditions for biomass production, whereas fructose was the most suitable carbon source for deoxymiroestrol and isoflavonoid production. The information from our study can be employed for enhancing the production of miroestrol, deoxymiroestrol, and total isoflavonoids using in vitro cell culture of P. candollei var. mirifica.     

Effects of abiotic and biotic elicitors on growth and isoflavonoid accumulation in Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures

This study demonstrates the effects of various concentrations of abiotic and biotic elicitors on the cell growth and isoflavonoid accumulation of P. candollei var. mirifica (PM) and P. candollei var. candollei (PC) cell suspension cultures. The two plant varieties exhibited different growth responses and varied isoflavonoid accumulation after the addition of elicitors. Copper sulfate, methyl jasmonate (MeJA), and yeast extract did not significantly affect the growth of either plant variety, whereas oligosaccharide and the biotic elicitors used in this study [i.e., 50 mg l⁻¹ chitosan and all concentrations of laminarin (LAM)] suppressed the growth of PM. The addition of MeJA to the medium principally induced an effect on the isoflavonoid content in both PM and PC, with 2.0 μM MeJA inducing the highest isoflavonoid content, as indicated by the induction index—4.41 in PM and 9.62 in PC cells on the 12th and ninth day of culture, respectively. A maximum total isoflavonoid content of 40.49 mg g⁻¹ dry weight was achieved in PM 21 days after elicitation with 2.0 μM MeJA. LAM elicited the PM cell suspension culture to produce puerarin, which was not found in the unelicited culture. The results of this study provide information that will be useful for enhancing the accumulation of isoflavonoids in P. candollei cell suspension cultures.     

Enhanced production of isoflavones by elicitation in hairy root cultures of Soybean

A combined treatment of sonication (2 min) and vacuum infiltration (2 min) stimulated isoflavones production of 75.26 mg g^?1 DW which was 15.11-fold higher than control hairy root line at optimal harvest time of 40 days. Addition of MeJ at 100 μM concentration with 72 h exposure time on 30 day-old hairy root culture further enhanced total isoflavones production of 53.16 mg g^?1 DW (10.67-fold) and SA at 200 μM concentration with 96 h exposure period enhanced the production of isoflavones (28.79 mg g^?1 DW; 5.78-fold). MeJ-treated hairy roots reduced biomass accumulation whereas sonication, vacuum infiltration and SA did not exhibit a negative effect on biomass growth.     

Improved cardenolide production in Calotropis gigantea hairy roots using mechanical wounding and elicitation

A hairy root culture system of Calotropis gigantea was established and effects of mechanical wounding (MW) and elicitors [methyl jasmonate (MJ), yeast extract (YE) and chitosan (CS)] on cardenolide production were investigated. All treatments stimulated the production of cardenolide in hairy root cultures of C. gigantea. CS was the most effective elicitor, followed by MJ. YE and MW also improved cardenolide yield in individual treatments. The highest cardenolide yield (1,050 ± 55 mg/l) was obtained after adding 50 mg CS/l for 20 days, which was 2.7-fold higher than the control.     

Statistical analysis of elicitation strategies for thiarubrine A production in hairy root cultures of Ambrosia artemisiifolia

Elicitation strategies were studied for yield enhancement of thiarubrine A, a secondary metabolite and a potential pharmaceutical, produced by hairy root cultures of Ambrosia artemisiifolia. Abiotic elicitation was performed using vanadyl sulfate solution and biotic elicitation using autoclaved cell wall filtrates of the fungi Protomyces gravidus, a pathogen of A. artemisiifolia and Botrytis cinereae. The factors considered were age of the culture, concentration of the elicitor used and the time period of exposure or contact. Statistical methods were used to determine the strength of the interaction between the various factors and their response on the yield of the secondary metabolite. The maximum increase in the yield relative to the control, 8-fold corresponding to 569 microg g(-1) of biomass, was observed when 16-day-old cultures were elicited with 50 mg l(-1) of vanadyl sulfate for a time period of 72 h. The maximum yield of 647 microg g(-1) was achieved when the cultures were exposed to 5 microM autoclaved cell wall filtrates of P. gravidus for a time period of 48 h. The yield increase was 3-fold in the case of elicitation with autoclaved cell wall filtrates of B. cinereae. The methodology used in this report can be extended to determine the optimum conditions of other elicitors.     

Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding

We have studied the effects of precursor amino acids (phenylalanine and cystein), elicitors (salicylic and acetylsalicylic acids, methyl jasmonate, β-aminobutyric acid, yeast extract), PAL-inhibitor (1-amino-2-phenylethylphosphonic acid) applied alone or in combination on glucotropaeolin production and myrosinase activity in hairy root cultures of Tropaeolum majus. Short, 24-h treatment, and subsequent transfer of hairy roots to the fresh medium enabled avoiding detrimental effect of studied stimulators on biomass growth. In control cultures the highest glucotropaeolin content, 58.1 ± 6.7 mg g⁻¹ DW, was detected on the 3rd day after transfer of the roots to the fresh medium but glucotropaeolin yield (mg 100 ml^–1 culture volume) had been increasing until the 9th day after transfer as a result of continuous biomass growth. Glucotropaeolin content and yield were 2-fold enhanced after treatment with precursor amino acids or PAL-inhibitor alone, but their combination additively led to 4-fold increase in glucotropaeolin production. Among the studied elicitors acetylsalicylic acid induced the highest, 3-fold increase in glucotropaeolin production, it also enhanced myrosinase activity, but to a smaller extend (by about 50%). Acetylsalicylic acid also potentiated induced by precursors, PAL-inhibitor, methyl jasmonate and yeast extract production of glucotropaeolin. The highest, 4.8-fold increase in glucotropaeolin production was found after combined acetylsalicylic acid and precursors treatment. Additive effect of acetylsalicylic acid-combined treatment on myrosinase activity was not detected. The obtained results indicate that amino acid precursors, phenylalanine and cystein, availability may be a limiting factor in the process of stimulation of glucotropaeolin production in T. majus hairy root cultures.     

Methyl jasmonate elicitation enhances glycyrrhizin production in Glycyrrhiza inflata hairy roots cultures

Hairy roots were induced by infecting stems and leaves of Glycyrrhiza inflata with Agrobacterium rhizogenes ATCC 15834. The optimization of growth and glycyrrhizin accumulation of G. inflata hairy roots was studied. Sucrose (6%, w/v) was optimal for growth and glycyrrhizin accumulation in G. inflata hairy roots. Effects of elicitors like chitosan, methyl jasmonate, and yeast extract on glycyrrhizin production were studied. Methyl jasmonate (100 microM) was most efficient in enhancing glycyrrhizin production up to almost 109 microg/g dry weight on day 5 of elicitation. These results indicate that application of elicitors can enhance the capacity of G. inflata hairy roots to produce glycyrrhizin.     

Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora

In an attempt to increase productivity, the effect of elicitation on tropane alkaloids (TA) biosynthesis was studied in adventitious hairy root cultures of Scopolia parviflora. Two Gram-positive strains and one Gram-negative strain of bacteria were used as biotic elicitors. The raw bacterial elicitors affected the tropane alkaloid profile by increasing the scopolamine concentration, while the autoclaved bacterial elicitors produced similar effects on the control. The conversion ratio of hyoscyamine to scopolamine was increased following elicitation using raw bacterial elicitors. The bacterial elicitor inhibited the expression of H6H (hyoscyamine 6β-hydoxylase) whereas the expression of PMT (putrescine N-methyltransferase) was raised by elicitation. These results have important implications for the large-scale production of tropane alkaloids.     

Alkaloid production by hairy root cultures in Atropa belladonna

Hairy roots were induced by inoculation of stems of sterile plants of Atropa belladonna with Agrobacterium rhizogenes. The axenic culture of the hairy roots isolated from the stems proliferated 60 fold as based on the initial fresh weight after one month of culture. The presence of atropine and scopolamine in hairy roots were examined by TLC and HPLC. Their amounts were analyzed by GLC. The results show that the amount of the two alkaloids in the axenic cultures was the same as or even higher than those of normal plants grown in the field.     

Enhanced accumulation of secondary metabolites in hairy root cultures of Scutellaria lateriflora following elicitation

Hairy root cultures of Scutellaria lateriflora were established using Agrobacterium rhizogenes A4 and produced acteoside (18.5?mg?g(-1) dry wt), baicalin (14.5?mg?g(-1) dry wt) and wogonoside (12?mg?g(-1) dry wt). Yeast extract (50?μg?ml(-1)) increased acteoside production 1.4-fold and flavone production 1.7-fold after 7 and 14?days of elicitation. Addition of Pectobacterium carotovorum lysate in the stationary phase of the hairy root culture stimulated only the accumulation of wogonin to 30?mg?g(-1) dry wt. The production of wogonin in hairy roots could be associated with its role as a phytolaexin.     

Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization

Azadirachtin is one of the most potent biopesticides so far developed from a plant sources. Influence of different culture media and elicitation on growth and production of azadirachtin by hairy root cultures of Azadirachta indica was studied. Out of the three media tested, namely Ohyama and Nitsch, Gamborg's and Murashige and Skoog's basal media, hairy roots cultured on Ohyama and Nitsch's basal medium produced maximum yield of azadirachtin (0.0166% dry weight, DW). Addition of biotic elicitor enhanced the production of azadirachtin by approximately 5-fold (0.074% DW), while signal compounds such as jasmonic acid and salicylic acid showed a approximately 6 (0.095% DW) and approximately 9-fold (0.14% DW) enhancement, respectively, in the production of azadirachtin as compared to control cultures on Ohyama and Nitsch medium. Extracts from hairy roots were found to be superior to those from the leaves for antifeedant activity against the larvae of Spodoptera litura.     

Hairy roots induced by Agrobacterium rhizogenes and production of regenerative plants in hairy root cultures in maize

Hairy roots of maize were induced by infecting 15-d calli with Agrobacterium rhizogenes. The hairy roots cultured in hormone-free media showed the vigorous growth and typical hairy root features. The regenerated plants were produced from hairy roots in MS media supplemented with 1.6 mg/L ZT and 0.4 mg/L NAA. The PCR-Southern hybridization demonstrated that T-DNA had been integrated into the chromosome of regenerated plants.     

Enhanced production of phytoestrogenic isoflavones from hairy root cultures of Psoralea corylifolia L. Using elicitation and precursor feeding

The effect of biotic elicitors (yeast extract, chitosan), signaling molecule (salicylic acid), and polyamines (putrescine and spermidine) was studied with respect to isoflavones accumulation in hairy root cultures of Psoralea corylifolia L. Untreated hairy roots (control) accumulated 1.55% dry wt of daidzein and 0.19% dry wt of genistein. In precursor feeding experiment, phenylalanine at 2 mM concentration led to 1.3 fold higher production of daidzein (1.91% dry wt) and genistein (0.27% dry wt). In biotic elicitors, chitosan (2 mg/L) was found to be the most efficient elicitor to induce daidzein (2.78% dry wt) and genistein (0.279% dry wt) levels in hairy roots. Salicylic acid at 1 mM concentration stimulated the maximum accumulation of daidzein (2.2% dry wt) and genistein (0.228% dry wt) 2 days after elicitation. In case of polyamines, putrescine (50 mM) resulted in highest accumulation of daidzein (3.01% dry wt) and genistein (0.227% dry wt) after 5 days of addition. Present results indicated the effectiveness of elicitation and precursor feeding on isoflavones accumulation in hairy roots of P. corylifolia. This is the first report of elicitation on isoflavones production by hairy roots of P. corylifolia.     

Biosynthetic studies of lactucin derivatives in hairy root cultures of Lactuca floridana

Biosynthetic studies of the guaianolide-type sesquiterpene lactones 11βH,13-dihydrolactucin-8-O-acetate and 8-desoxylactucin were performed in Agrobacterium rhizogenes—transformed hairy root cultures of blue-flowered lettuce, Lactuca floridana. The ¹³C NMR spectra of the two guaianolides labelled by incorporation of [1-¹³C], [2-¹³C], [1,2-¹³C₂]acetate and [2-¹³C]mevalolactone showed patterns of enrichment consistent with a previously proposed biogenetic pathway for guaianolide-type sesquiterpene lactones via the acetate-mevalonate-germacradiene route.     

Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation

Valerenic acid (VA) is a pharmacologically-active sesquiterpene found in valerian (Valeriana officinalis L., Valerianaceae) roots and rhizomes. The plant produces only small amounts of this metabolite naturally. So, induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, Wild-type strain ‘A13’ of Agrobacterium rhizogenes was used to induce hairy roots in valerian. The influence of three different elicitors including Fusarium graminearum extract (FE), methyl jasmonate (MJ) and salicylic acid (SA) on VA production in the selected hairy root line ‘LeVa-C4’ was also investigated. The 23-day-old cultures were treated with different concentrations of the elicitors at exposure time of 3 and 7 days. FE (1%) and MJ (100 µM L^?1) highly promoted VA production at 7 days after elicitation, to a level of 12.31- and 6-fold higher than that of non-elicited controls, respectively, and FE did not exert any negative effects on biomass yield of hairy root. SA did not significantly increase the production of VA. This is the first time study to assess the elicitation of hairy root cultures to promote VA biosynthesis in valerian and the resulting experiments demonstrated that F. graminearum extract and MJ were indeed a potent inducer of VA biosynthesis.     

Overexpression of the Arabidopsis thaliana squalene synthase gene in Withania coagulans hairy root cultures

Squalene synthase (SS) dimerizes two molecules of farnesyl diphosphate to synthesize squalene, a shared precursor in steroid and triterpenoid biosynthesis in plants. The SS1 gene encoding SS from Arabidopsis thaliana was introduced in Withania coagulans under the control of the CaMV35S promoter together with the T-DNA of Agrobacterium rhizogenes A4. The engineered hairy roots were studied for withanolide production and phytosterol accumulation and the results were compared with those obtained from control roots harbouring only the T-DNA from pRiA4. The increased capacity of the engineered roots for biosynthesizing phytosterols and withanolides was strongly related with the expression level of the transgene, showing the effectiveness of overexpressing 35SS1 to increase triterpenoid biosynthesis.     

Root specific elicitation and antimicrobial activity of rosmarinic acid in hairy root cultures of Ocimum basilicum

Rosmarinic acid (RA) is a multifunctional caffeic acid ester present in sweet basil (Ocimum basilicum L.). Untransformed normal roots of O. basilicum harbored the maximum titers (0.98% g fresh weight basis) of RA compared to leaves and shoots. Hairy root cultures of O. basilicum transformed with Agrobacterium rhizogenes (ATCC-15834) showed three-fold increases in growth and RA production compared to the untransformed normal roots. Upon elicitation with fungal cell wall elicitors (CWE) from Phytophthora cinnamoni, the production of RA was enhanced 2.67-fold compared with the untreated control. Roots were induced to exude RA by fungal in situ challenge with Pythium ultimum, to our knowledge an undocumented observation. Absence of RA in the root exudates of unchallenged root cultures proves that RA under normal circumstances is not exuded. RA showed antimicrobial activity against a range of soil-borne microorganisms, with its most deleterious effects against Pseudomonas aeruginosa, an opportunistic soil bacterium and human pathogen. Confocal and scanning imaging of Aspergillus niger hyphae treated with RA (250 μM) exhibited damaged cytoskeletons with broken interseptas and convoluted cell surfaces resulting in a multinucleated stage compared to the untreated control. Both strains of P. aeruginosa tested, PAO1 and PA14, showed increased spatial division and condensation of DNA upon RA treatment compared to the untreated control. Our findings suggest that in nature RA is a constitutive antimicrobial compound that may be released into the surrounding rhizosphere upon microbe challenge.     

Genista tinctoria hairy root cultures for selective production of isoliquiritigenin

Hairy root cultures were established after inoculation of Genista tinctoria in vitro shoots with Agrobacterium rhizogenes, strain ATCC 15834. In transformed roots of G. tinctoria grown in Schenk-Hildebrandt medium without growth regulators the biosynthesis of isoflavones, derivatives of genistein and daidzein, and flavones, derivatives of luteolin and apigenin, characteristic for the intact plant, was completely inhibited. The only compound synthesized in G. tinctoria hairy roots was isoliquiritigenin (2.3 g/100 g DW), a daidzein precursor absent in the intact plant. This compound was stored entirely within cells and it was not until abscisic acid was added (37.8 microM supplement on day 42) that approx. 80% of it was released into the experimental medium. The paper discusses the effect of abscisic acid on the growth of G. tinctoria hairy root cultures, the biosynthesis of isoliquiritigenin and the way it is stored. A prototype basket-bubble bioreactor was designed and built to upgrade the scale of the G. tinctoria hairy root cultures. With immobilized roots and a new aeration system, large amounts of biomass were obtained (FWmax 914.5 g l(-1)) which produced high contents of isoliquiritigenin (2.9 g/100 g DW). The abscisic acid-induced release of the metabolite from the tissue into the growth medium greatly facilitated subsequent extraction and purification of isoliquiritigenin.