Optimized production of isoflavones in cell 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 cell suspension cultures of corylifolia L. Untreated cell suspension (control) accumulated 1.66% dry wt of daidzein and 0.165% dry wt of genistein. In precursor feeding experiment, phenylalanine at 0.5 mM concentration led to 1.3 fold higher production of daidzein (1.99% dry wt) and genistein (0.22% dry wt). In biotic elicitors, yeast extract (100 mg/L) was found to be the most efficient elicitor to induce higher production levels of daidzein (2.21% dry wt) and genistein (0.293% dry wt) in suspension cultures. Salicylic acid (signaling molecule) at 1 mM concentration stimulated the maximum accumulation of daidzein (3.4% dry wt) and genistein (0.41% dry wt) 2 days after elicitation. In case of polyamines, spermidine (100 mM) resulted in highest accumulation of daidzein (3.2% dry wt) and genistein (0.475% dry wt) after 7 days of addition, which was 2.4 fold of that in control. This is the first report on kinetics of isoflavone production in response to elicitation in cell suspension of P. corylifolia.     

Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation

The effect of abiotic and biotic elicitors (methyl jasmonate, chitosan, salicylic acid, Agrobacterium, and yeast extract) at various concentrations on total isoflavonoid accumulation was studied in the hairy root cultures of Pueraria candollei. All elicitors stimulated isoflavonoid production. Yeast extract (0.5 mg/ml) was the most efficient giving total isoflavonoids at 60 ± 1 mg/g dry wt, which was 4.5-fold higher than control hairy roots on day 3 of elicitation.     

Influence of polyamines on growth and formation of secondary metabolites in hairy root cultures of Beta vulgaris and Tagetes patula

Growth of hairy roots of Beta vulgaris, which produces betalaines, and of Tagetes patula, which produces thiophenes, was studied under the influence of externally treated polyamines. Of the three polyamines, viz. putrescine, spermidine and spermine, administered singly at 1.5 mM concentration, putrescine and spermidine at 0.75 mM concentration influenced increase in the accumulation of biomass of B. vulgaris and T. patula hairy roots by 1.42 and 1.30 fold over the control. Whereas, the treatment of spermine (1.5 mM) alone resulted in decrease in the biomass in both the systems. Combined administration of putrescine (0.75 mM) and spermidine (0.75 mM) enhanced growth in both B. vulgaris and T. patula than that observed in individual treatments. Polyamines administered alone or in combination did alter production of betalaine and thiophene content. Dose response experiments showed that, when putrescine and spermidine was administered at 0.75 mM concentration, it resulted in maximum biomass and production of beta-laine and thiophene in B. vulgaris and T. patula respectively as compared to the control and the media treated with double and triple strength of nitrates and in combination with putrescine and spermidine at equimolar concentration. In B. vulgaris and T. patula hairy root cultures, endogenous spermine titers were maximum in putrescine and spermidine 0.75 mM each treated, cultures, which was 1.63 and 2.0 fold higher than in control on 28^th and 35^th days respectively.     

Improvement of artemisinin production by chitosan in hairy root cultures of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg⁻¹ dry wt over 6 days by adding 150 mg chitosan l⁻¹. The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml⁻¹) increased artemisinin production to 1.5 and 0.9 μg mg⁻¹ dry wt, respectively.     

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.     

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g⁻¹ dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.     

Saponin production by hairy root cultures ofPanax ginseng CA meyer: Influence of PGR and polyamines

A culture of hairy roots ofPanax ginseng C.A. Meyer was set up in order to investigate the possibility of producing ginseng saponin. Roots cultured in 1/2 MS medium in the presence of 2 mg/L IAA and 0.1 mM spermidine showed the maximal growth rate, whereas other polyamines increased the growth of hairy roots only slightly or not at all. High saponin root contents were obtained in culture media supplemented with 0.5 mg/L GA and 1 mM putrescine.     

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.Abbreviations Cht chitosan - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog(1962)     

Lactofen induces isoflavone accumulation and glyceollin elicitation competency in soybean

Lactofen, the active ingredient of the soybean disease resistance-inducing herbicide, Cobra, induces large accumulations of isoflavone conjugates and aglycones in soybean tissues. The predominant isoflavones induced in cotyledon tissues are daidzein (and its conjugates) and formononetin and glycitein aglycones. The latter two isoflavones are usually present only at very low levels in soybean seedling tissues. In leaves, the predominant lactofen-induced isoflavones are daidzein and formononetin aglycones and the malonyl-glucosyl conjugate of genistein. Isoflavone induction also occurs in cells distal to the point of treatment, but is only weakly systemic. Lactofen also induces elicitation competency, the capacity of soybean cells to accumulate the pterocarpan phytoalexin glyceollin in response to glucan elicitors from the cell wall of the pathogen Phytophthora sojae. Comparison of the activity of a series of diphenyl ether herbicides demonstrated that while all diphenyl ethers tested induced some degree of elicitation competency, only certain ones induced isoflavone accumulation in the absence of glucan elicitor. As a group the diphenyl ethers are thought to inhibit protoporhyrinogen oxidase, eventually leading to singlet oxygen generation. Another singlet oxygen generator, rose bengal, also induced elicitation competency, but little isoflavone accumulation. It is hypothesized that diphenyl ether-induced activated oxygen species mimic some aspects of hypersensitive cell death, which leads to elicitation competency in infected tissues.     

Putrescine influences growth and production of coumarins in transformed and untransformed root cultures of witloof chicory (Cichorium intybus L. cv. Lucknow local)

The effect of putrescine on growth and production of two coumarins, esculin, and esculetin in the transformed and untransformed roots of chicory (Cichorium intybus L. cv. Lucknow local) was examined. To study the role of putrescine (Put) on growth and production of coumarins, polyamine inhibitors namely α-DL-difluromethylornithine (DFMO) and α-L-difluromethylarginine (DFMA) were used at 1 mM levels. Treatment with 1.5 mM of putrescine (Put) produced 1.96 - fold and 4.0 - fold increase in the growth of transformed and untransformed roots of chicory, respectively. The treatment with polyamine inhibitors showed much lower growth, as well as production compared with both 1.5 mM putrescine treatment and control in both transformed and untransformed chicory roots. The endogenous polyamines, both free and conjugated, were studied over the whole culture period, and it was seen that conjugated titers of all three polyamines viz., putrescine, spermidine and spermine were higher than level of free polyamines, throughout the culture period in both transformed and untransformed roots of chicory. Treatment in which polyamine inhibitors were used showed lower level of endogenous polyamines as compared with the 1.5 mM putrescine treated sample in both the systems. The treatment wherein putrescine was added at 1.5 mM level showed maximum accumulation of endogenous conjugated putrescine (2098.86±157.6 nmoles g⁻¹ FW; 896.8±67.2 nmoles·g⁻¹ FW), on the 14^th day in both transformed and untransformed roots respectively. The production of esculin and esculetin was strictly correlated with growth in every treatment in both systems. Putrescine at 1.5 mM resulted in greater length of primary root in transformed (18.3±1.4 cm) and untransformed (6.86±0.51 cm) as compared with their respective controls (11±0.9 cm; 2.9±0.1cm) and greater number of secondary and tertiary roots. This study suggests that putrescine influences plant root development and differentiation, and it also provides insight into the morphological changes that occur in roots in response to the external supply of polyamines.     

Synergistic effect of auxins and polyamines in hairy roots of <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. during growth,coumarin production and morphogenesis

Hairy roots of Cichorium intybus obtained by infecting with different Agrobacterium rhizogenes strains (LMG-150 and A20/83) were studied for total endogenous indole-3-acetic acid (IAA) levels and indole-3-acetic acid oxidase (IAAO) activity. The roots initiated by LMG- 150 showed higher endogenous IAA levels as well as IAAO activity as compared to the roots from A20/83. Coumarin production in roots obtained by both of these strains strictly correlated with growth, with higher content in the roots obtained by LMG- 150. Moreover roots from LMG-150 showed increased growth index, length of primary roots and number of secondary and tertiary roots. The roots derived from LMG-150 were studied for total endogenous IAA and IAAO activity under the exogenous administration of polyamines and fungal elicitors. The treatment with putrescine (Put) at 1.5 mM level showed maximum endogenous IAA levels and IAAO activity as compared to the control and other polyamine administration, it also supported faster growth in terms of biomass accumulation, and total coumarin production. Of the various treatments, mycelial extract (ME) and culture media filtrate (CMF) of Pythium aphanidermatum and Phytopthora parasitica var. nicotiana, the treatment with 1 % CMF of P. parasitica var. nicotiana, resulted in maximum IAA levels and IAAO activity, which was supported by maximum biomass, coumarin production as compared to the control and other elicitor treatments. Two different regenerants of chicory obtained through A. rhizogenes LMG-150 designated as T-I and T-II, were studied for total endogenous IAA levels and IAAO activity. T-II showed higher titers of IAA with higher activity of IAAO as compared to T-I. Endogenous titer of IAA and IAAO activity was found to be maximum in transformed roots as compared to T-I, T-II, normal roots and normal plants. Our work showed a variation in endogenous auxin levels in these transformed plants. There exists a synergistic effect of endogenous IAA titers and polyamines in regulating root morphogenesis. Fungal elicitors influenced growth and coumarin production and an elicitor preparation of 1 % CMF of P. parasitica var. nicotiana gave spontaneous regeneration of shoots. The implications of these results are discussed.     

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.     

Isoflavones Daidzein and Genistein: Preparation by Acid Hydrolysis of Their Glycosides and the Effect on Phospholipid Peroxidation

A mixture of isoflavones was obtained by acid hydrolysis of isoflavone glycosides isolated from the products of soybean processing by successive extraction with aqueous acetone and methanol. The homogeneous isoflavones daidzein and genistein were isolated from the aglycone mixture by adsorption chromatography and identified by spectral and chromatographic methods. The effect of both isoflavones on lipid peroxidation of soy phospholipids in multilamellar vesicles was studied at various concentrations. These aglycones were found to inhibit the formation of lipid hydroperoxides and malonic dialdehyde at concentrations as low as 1 mM.     

Eugenol from normal and transformed root cultures of Coluria geoides

Transformed root cultures of Coluria geoides Ledeb. were established with the use of Agrobacterium rhizogenes LBA 9402. Both normal and transformed root cultures were investigated for their growth and yield of eugenol. Normal roots were grown in B5 medium-supplemented with 0.2 mg l^-1 of kinetin and 0.2 mg l^-1 of 1-naphthaleneacetic acid (NAA). Hairy roots grew well in hormone-free B5 medium. Both hairy roots and normal roots produced glycosidic bound eugenol. as with the roots of intact plants, eugenol was the main component of the total essential oils obtained from hairy root and normal root cultures. The yield of eugenol from normal roots was 0.1–0.25% of the dry wt. and depended on the development stage of the culture. Yield of eugenol from hairy roots was 0.08–0.1% of the dry wt. NAA modified the hairy root morphology and influenced the yield of eugenol.Abbreviations NAA 1-naphthaleneacetic acid     

Characterization of the elicitor-induced biosynthesis and secretion of genistein from roots of Lupinus luteus L

Genistein is a multi-functional isoflavonoid naturally secreted from roots of hydroponically grown legume plants. Roots of hydroponically cultivated yellow lupin (Lupinus luteus L.) plants, transferred into water secreted minor amounts of genistein (about 5 g g^-1 fr. wt.). Secretion of genistein from L. luteus roots (rhizosecretion) was stimulated dramatically to over 100 g g^-1 root fresh weight by soluble chitosan, salicylic acid (SA) and potassium cyanide (KCN) supplied at 0.12% (w/v), 800 M and 400 M, respectively. Other identified elicitors caused a smaller induction of genistein rhizosecretion. Increased levels of genistein in root exudates corresponded to greater amounts of genistein in root tissue. Elicitor-induced rhizosecretion of genistein was based on de novo synthesis and was inhibited by glyphosate and other less specific metabolic inhibitors. Except for NaF:AICI3, all tested elicitors of genistein rhizosecretion produced a distinct bell-shaped dose-response curve. Most of the elicitor-induced rhizosecretion of genistein occurred during the first day, followed by a gradual decline. Further addition of elicitor treatments had little effect of genistein rhizosecretion, indicating that the induction of genistein rhizosecretion by the identified elicitors is a once only event.Keywords: Genistein, isoflavonoids, Lupinus luteus, elicitation, exudation.      

Development of Linum flavum hairy root cultures for production of coniferin

Linum flavum hairy roots were initiated from leaf discs using Agrobacterium rhizogenes strains LBA9402 and TR105 though two other strains, 15834 and A4, were relatively ineffective for induction. Significant variation in coniferin accumulation was observed between hairy root lines originating from different L. flavum seedlings and/or A. rhizogenes strains. Coniferin reached 58 mg g^–1 dry wt by culturing the roots in Linsmaier and Skoog (LS) medium with 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid as growth regulators.     

Biotic elicitor enhanced production of psoralen in suspension cultures of Psoralea corylifolia L.

Cell cultures of Psoralea corylifolia L. were established from the leaf disk derived callus. The effect of different biotic elicitors prepared from the fungal extract (Aspergillus niger and Penicillium notatum), yeast extract and chitosan with different concentrations was studied. The increased synthesis of psoralen in 16-day old cell cultures under 16 h of light and 8 h of dark period was studied. Elicitation of psoralen in A. niger elicitor treated cells was found 9-fold higher over control cells. Treating the cells with P. notatum, yeast extract and chitosan elicitors lead to four to seven-fold higher psoralen accumulation over control cells. The extract of A. niger at 1.0% v/v increased the significant accumulation of psoralen (9850 μg/g DCW) in the cultured cells. Our study clearly shows that all the elicitors had the potential to increase the accumulation of psoralen but the A. niger elicitor at 1.0% v/v induced maximum accumulation.     

Elicitation of thiophene production by cultured hairy roots of Tagetes patula

Hairy roots of Tagetes patula were initiated by infecting the seedlings with Agrobacterium rhizogenes. The hairy roots were grown in liquid medium MS (Murashige and Skoog 1962) supplemented with sucrose. The roots were treated with three different elicitors obtained from mycelial culture of Aspergillus niger, Rhizopus oligosporus and Pencillium notatum. Accumulation of biomass and the production of thiophenes were studied over a period of six weeks in culture. The HPLC separation profile of the thiophenes indicated the presence of several structurally different thiophenes. α-terthienyl being predominent. Maximum production of thiophene was recorded at the end of the fourth week in culture with a content of 0.138 % (w/w on dry weight basis). Treatment of hairy roots with mycelial extract of Aspergillus niger (1.5 % v/v) elicited an increase in thiophene content by 1.6 folds over the control.     

粉葛主要营养物质、异黄酮成分的积累规律及其相关性分析

为确定粉葛的最佳采收时期及各部位的应用价值，探索粉葛主要营养物质与异黄酮成分的积累规律及相关关系，该研究以广西藤县粉葛为材料，采用测试盒法、酶重量法、索氏抽提法、高效液相色谱法等生理生化技术，测定了成熟期不同部位异黄酮成分的含量及不同生长时期粉葛块根主要营养物质、异黄酮成分的含量，并分析了各营养物质与异黄酮成分积累的相互关系。结果表明：(1)在成熟期，粉葛不同部位的染料木素含量差异均不显著，但叶片的大豆苷元含量显著小于中藤，根头和藤的总异黄酮、葛根素、大豆苷的含量显著大于叶片和块根，并且根头和藤的葛根素含量均超过1.00%。(2)不同生长时期，粉葛的淀粉、多糖、粗蛋白、可溶性蛋白的积累在11月和12月达到最大，不溶性膳食纤维的积累在12月最小，可溶性膳食纤维在8月和12月的积累量最大，总异黄酮和葛根素在8月和9月的积累量最大。(3)总异黄酮、葛根素的积累与淀粉、多糖的积累呈显著负相关，总异黄酮、葛根素的积累与不溶性膳食纤维的积累呈显著正相关。综上结果认为：粉葛的根头、藤蔓含有丰富的总异黄酮、葛根素、大豆苷，具有很好的药用开发价值；粉葛药用的最佳采收期为8月和9月，粉葛食用的最佳采收期为...