Influence of thidiazuron on callus induction and crocin production in corm and style explants of <Emphasis Type="Italic">Crocus sativus</Emphasis> L.

Crocus sativus L., mostly famous as saffron, has gained more attention due to its crocin (crocetin ester) pigment responsible for its extensive pharmaceutical properties. In this study, we established two different callus cultures from corm and style explants of saffron to find out the best explant as a suitable source for crocin production. Comparative analyses of total phenolic, flavonoid, carotenoid and anthocyanin contents were also performed in the two callus cultures. For callus induction, different combinations of MS medium with name thidiazuron (TDZ), benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination were tested. Of the used media, all the combinations containing TDZ and NAA gave 100% callus induction. HPLC-DAD and HPLC–ESI-MS analysis were used for identification of crocin esters in established callus cultures. The highest amount of 0.35 mg g^?1 DW crocin was detected in style originated calli grown on the medium containing 3 mg L^?1 NAA?+?1 mg L^?1 TDZ while the corm calli showed the most abundant total carotenoid (0.73 mg g^?1 DW), phenolic (15.04 mg gallic acid equivalent g^?1 DW) and flavonoid (0.76 mg rutin equivalent g^?1 DW) contents. In general, style-derived calli showed longer time survival with a fine texture and good quality compared to corm-derived calli.     

The production and antiprotozoal activity of abietane diterpenes in Salvia austriaca hairy roots grown in shake flasks and bioreactor

The biomass and concentration of bioactive quinone methide-type diterpenes in hairy roots of Salvia austriaca were determined and compared with levels of these metabolites in roots of field-grown plants. The cultures were maintained in shake flasks and a nutrient sprinkle bioreactor. Diterpene production was more efficient in the shake flask root culture than the bioreactor one. Biomass and diterpene production within the shake flask culture was evaluated using Schenk and Hildebrandt (SH), Gamborg (B5), and woody plant medium (WPM), with both full- and half-strength macro and micronutrient concentrations (1/2 SH, 1/2 B5, and 1/2 WPM). Among the tested media, SH medium proved to be most effective for biomass and diterpene production. In this medium, the transformed roots accumulated the levels of taxodone (3.89?mg?g^?1 DW; equivalent to 63.3?mg?L^?1), taxodione (1.15?mg?g^?1 DW; equivalent to 17.4?mg?L^?1), 15-deoxy-fuerstione (2.15?mg?g^?1 DW; equivalent to 32.5?mg?L^?1), and 7-(2′-oxohexyl)-taxodione (0.076?mg?g^?1 DW; equivalent to 1.1?mg?L^?1). Three diterpenes were also detected in the roots of S. austriaca intact plants, but their concentrations were lower than those in hairy root culture. No 7-(2′-oxohexyl)-taxodione was found in the roots of field-grown plants. The hairy roots were able to maintain high metabolite levels even for 6 years of cultivation. Taxodone, taxodione, 15-deoxy-fuerstione, and 7-(2′-oxohexyl)-taxodione were tested for in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum and their cytotoxicity was determined using L6 cells. Among these compounds, taxodione was the most active against T. brucei rhodesiense [IC₅₀?=?0.05?µM with high selectivity, selectivity index (SI)?=?38]. Taxodione was found to inhibit the growth of P. falciparum and T. cruzi by 50% at respective concentrations of 1.9 and 7.1?µM (SI values of 1.0 and 0.27). Other diterpenoids demonstrated weaker activity against tested parasites (IC₅₀ values ranging from 0.62 to 194.7?µM) and lower selectivity (SI value ranged from 0.4 to 5.0).     

Production of bioactive phenolic acids and furanocoumarins in in vitro cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams. under different light conditions

Extracts from the biomass of Ruta graveolens and Ruta graveolens ssp. divaricata cultured in vitro under different light conditions (far-red, red and blue light, UV-A irradiation, in darkness and white light) were tested for the amounts of free phenolic acids and cinnamic acid (twelve compounds) as well as furanocoumarins and umbelliferone (seven compounds) using HPLC methods. Total amounts of the investigated groups of compounds in the cultures of both plants increased from 2.6 to 6.7 times, depending on light quality, and the maximum values reached were 106.50 and 1,276.74?mg?100?g^?1 DW (in R. graveolens), and 106.97 and 262.54?mg?100?g^?1 DW (in the subspecies), respectively. Both white light and blue light were equally beneficial for the total production of phenolic acids in cultures of both plants, whereas the total production of furanocoumarins was clearly better stimulated by blue light in R. graveolens and by darkness in the subspecies (i.e. the amounts were respectively 1.44 and 1.7 times higher than in the biomass cultivated under white light). The amounts of individual compounds in both plant cultures increased from about 2.2 to 26.3 times depending on light quality. The following bioactive compounds were obtained in quantities which are of interest from a practical perspective: in R. graveolens culture??protocatechuic acid (45?mg?100?g^?1 DW), isopimpinellin (about 500?mg?100?g^?1 DW) and bergapten (about 270?mg?100?g^?1 DW), and in the subspecies culture: p-coumaric acid (70?mg?100?g^?1 DW) and isopimpinellin (about 210?mg?100?g^?1 DW).     

Production of biologically active phenolic acids in Aronia melanocarpa (Michx.) Elliott in vitro cultures cultivated on different variants of the Murashige and Skoog medium

Phenolic acids, both benzoic and cinnamic acid derivatives, are plant metabolites with high therapeutic and cosmetic values. Methanolic extracts from the biomass of shoot and callus cultures of Aronia melanocarpa growing on seven variants of the Murashige and Skoog (MS) medium with different concentrations of plant growth regulators, BA and NAA, ranging from 0.1 to 3.0 mg l^?1, were examined for the production of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. The extracts from the shoot and callus cultures were confirmed to contain five of the twelve compounds tested for: caffeic, p-coumaric, p-hydroxybenzoic, syringic and vanillic acids. The shoot extracts contained additionally salicylic acid. Both the total amounts and the amounts of individual compounds in either the shoot or callus extracts were dependent on the concentration of cytokinin and auxin in the MS medium variants. The total amounts in the shoot and callus cultures were in the range from 93.52 to 217.00 mg 100 g^?1 DW and from 47.11 to 83.83 mg 100 g^?1 DW, respectively. The amounts of individual compounds showed wide variation, from 1.31 to 91.86 mg 100 g^?1 DW in the shoot extracts, and from 2.58 to 40.16 mg 100 g^?1 DW in the callus extracts. Salicylic acid (max. 91.86 mg 100 g^?1 DW), p-coumaric acid (max. 62.39 mg 100 g^?1 DW) and p-hydroxybenzoic acid (max. 50.66 mg 100 g^?1 DW) dominated in the shoot extracts, while syringic acid (max. 40.16 mg 100 g^?1 DW) and p-hydroxybenzoic acid (max. 23.59 mg 100 g^?1 DW) were the main metabolites in the callus extracts. This is the first report on the quantitative analysis of benzoic and cinnamic acid derivatives in shoot and callus cultures of A. melanocarpa growing on MS-based media with different concentrations of selected plant growth regulators—BA and NAA. The obtained maximum amounts of some metabolites are of interest from a practical perspective.     

Accumulation of hydroxybenzoic acids and other biologically active phenolic acids in shoot and callus cultures of Aronia melanocarpa (Michx.) Elliott (black chokeberry)

Phenolic acids are plant metabolites important in phytotherapy and also in cosmetology. In this study, proliferating shoot and callus cultures of Aronia melanocarpa were established and maintained on Linsmaier and Skoog (L-S) medium containing different levels of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), ranging from 0.1 to 3.0 mg l^?1. Methanolic extracts from the biomass of these cultures and from the fruits of soil-grown plants were used to determine the amounts of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. Out of a total of twelve analyzed compounds, all of the extracts contained four of them: caffeic acid, p-hydroxybenzoic acid, syringic acid, and vanillic acid. Moreover, shoot extracts also contained salicylic acid (o-hydroxybenzoic acid), while callus extracts contained p-coumaric acid. On the other hand, fruit extracts also contained both salicylic acid and p-coumaric acid. The total amount of the analyzed compounds in extracts from both shoot and callus cultures depended on the L-S medium used, and varied between 103.05 and 150.95 mg 100 g^?1 dry weight (DW), and between 50.23 and 81.56 mg 100 g^?1 DW, respectively. Both types of culture contained higher levels of phenolic acids than the fruit extracts (32.43 mg 100 g^?1 DW). In shoot cultures, p-hydroxybenzoic acid and salicylic acid were the predominant metabolites (reaching 55.14 and 78.25 mg 100 g^?1 DW, respectively), while in callus cultures, p-hydroxybenzoic acid (25.60 mg 100 g^?1 DW) and syringic acid (41.20 mg 100 g^?1 DW) were the main compounds. In fruit extracts, salicylic acid (15.60 mg 100 g^?1 DW) and p-hydroxybenzoic acid (5.29 mg 100 g^?1 DW) were predominant.     

Scale-up of adventitious root cultures of Echinacea angustifolia in a pilot-scale bioreactor for the production of biomass and caffeic acid derivatives

In an attempt to scale-up of adventitious root cultures of Echinacea angustifolia for the production of biomass and caffeic acid derivatives, i.e. echinacoside, chlorogenic acid, cichoric acid, caftaric acid, and cynarin, the effects of Murashige and Skoog (MS) medium dilutions, and initial sucrose concentrations were investigated in a 5-L airlift bioreactor. In addition, the kinetics of adventitious root growth and accumulation of secondary metabolites were also studied. The greatest root dry weight (6.50 g L^?l) and accumulation of total phenolics [22.06 mg g^?1 DW (dry weight)], total flavonoids (5.77 mg g^?1 DW) and total caffeic acid derivatives (10.63 mg g^?1 DW) were obtained at quarter-strength MS medium. Of the various gradients of sucrose tested, 5 % sucrose supplementation was regarded as an optimal concentration for enhancing productivity of biomass and bioactive compounds. Neither higher salt strength (3/4–2 MS) nor sucrose concentrations (7 and 9 %) showed promotive effect on root growth and metabolite production. The kinetic studies revealed that 4 weeks of culture period is the optimal time to achieve highest productivity of metabolites. Based on these results, a large-scale (20 L) and a pilot-scale (500 L) adventitious root culture system was established. In the pilot-scale bioreactor, adventitious roots were elicitor-treated with 100 μM methyl jasmonate (MJ) on day 28. After 1 week of elicitation, 1.75 kg dry root biomass was harvested containing 60.41 mg g^?1 DW of total phenolics, 16.45 mg g^?1 DW of total flavonoids, and 33.44 mg g^?1 DW of total caffeic acid derivatives. Among the caffeic acid derivatives, the accumulation of echinacoside (the major bioactive compound) in MJ-treated adventitious roots grown in the 500-L bioreactor was the highest (12.3 mg g^?1 DW), which is approximately threefold more than the non-MJ-treated roots cultured in 5- and 20-L bioreactors.     

Hairy root cultures of Rehmannia glutinosa and production of iridoid and phenylethanoid glycosides

Hairy root lines through the infection of Agrobacterium rhizogenes strain (A4) were established from shoot tips and leaves of Rehmannia glutinosa Libosch. Ten lines of hairy roots were selected on the basis of biomass increase in half-strength Gamborg medium (¹/₂ B5). Transgenic status of the roots was confirmed by polymerase chain reaction using rolB and rolC specific primers. Iridoid glycosides (catalposide, loganin, aucubin and catalpol) and phenylethanoid glycosides (verbascoside and isoverbascoside) identified using HPLC?CESI?CMS, and their contents were compared with untransformed root culture and roots of 1-year-old field-grown plants of R. glutinosa by RP-HPLC. The growth and production of secondary metabolites in ten hairy root lines varied considerably as to the media. Woody plant (WP) medium displayed higher growth in terms of fresh (FW) and dry weights (DW) compared to ¹/₂ B5 medium. High-yielding hairy root lines produced higher amounts of loganin, catalposide, verbascoside and isoverbascoside in comparison to the untransformed root culture and roots of 1-year-old field-grown plants. The highest amounts of catalposide and loganin in transformed roots were 4.45?mg?g^?1 DW (RS-2 hairy root line) and 4.66?mg?g^?1 DW (RS-1 hairy root line), respectively. Aucubin and catalpol were detected in some lines in trace amounts. The highest amounts of verbascoside (16.9?mg?g^?1 DW) and isoverbascoside (3.46?mg?g^?1 DW) were achieved in RS-2 root line. The contents of catalposide, verbascoside and isoverbascoside in high-producing lines were several times higher than in untransformed root culture and roots of R. glutinosa plants grown in soil. Loganin and aucubin could not be detected in roots of field-grown plants. However, the levels of catalpol were much lower in the in vitro roots.     

Effect of elicitation and precursor feeding on accumulation of 20-hydroxyecdysone in <Emphasis Type="Italic">Achyranthes aspera</Emphasis> Linn. cell suspension cultures

20-Hydroxyecdysone is one of the most common ecdysteroids in plants with potential therapeutic applications. In this study, cell suspension cultures of Achyranthes aspera were raised in shake flasks to investigate the production of 20-hydroxyecdysone. The quantification and characterization of 20-hydroxyecdysone in the cultures were done by High performance liquid chromatography (HPLC) and Liquid Chromatography-quadrupole time-of- flight mass spectrometry (LC-Q-TOF) analyses. For raising the suspension, calli initiated from in vitro grown leaf explants were cultured in liquid Murashige and Skoog (MS) medium augmented with combinations of 2, 4-dichlorophenoxyacetic acid (1 mg L^?1) and α-naphthaleneacetic acid (1 mg L^?1). Maximum growth index of the cell suspension was 9.9, which was achieved during 20th day of culture (final phase of exponential growth). At this stage, the biomass accumulated was 1.09 ± 0.09 g dry weight (DW) and the 20-hydroxyecdysone concentration was 0.24 mg g^?1 DW. Eliciting the cultures with 0.6 mM Methyl jasmonate for 6 days; enhanced the production of 20-hydroxyecdysone production to 0.35 mg g^?1 DW. By augmenting the cultures with the precursors namely cholesterol (10 mg L^?1) and 7-dehydrocholesterol (10 mg L^?1), production of 20-hydroxyecdysone was boosted to 0.31 mg g^?1 DW and 0.28 mg g^?1 DW respectively.     

Characterization of phytohormonal and postharvest senescence responses of balsam fir (Abies balsamea (L.) Mill.) exposed to short-term low temperature

To fulfill the US Thanksgiving and Christmas tree markets, balsam fir (Abies balsamea (L.) Mill.) is generally harvested before the cold season, anecdotally leading to premature needle senescence. Accordingly, we tested the hypothesis that LT exposure before harvest induces specific hormonal changes and delays postharvest senescence and/or abscission in balsam fir. Two hundred and six seedlings exposed to two temperature treatments for 48?h, LT at 5?°C and controls at 22?°C were severed off roots and monitored for their postharvest needle senescence. Root and shoot (needles and buds) tissues were examined for major endogenous hormone metabolites. LT increased shoot ABA (2,007?ng?g^?1 DW) by 2.5× and decreased GA₄₄ (9.84?ng?g^?1 DW) by 3.5× over those in roots. LT did not alter cytokinins, auxins or any root hormonal concentration. With auxins, only IAA, IAA-Asp, IAA-Leu and IAA-Glu were detected and the concentrations of IAA and IAA-Asp in shoots were lower than those found in roots. Among cytokinins, shoot c-ZR (58.95?ng?g^?1 DW) and t-ZR (4.17?ng?g^?1 DW) were 3× higher than those in roots. Apart from GA₄₄, GA₉ (136.76?ng?g^?1 DW) was abundant in shoots. The PBL and PNL were 46 and 1.2?%, irrespective of treatments. LT seedlings held needles 11?days longer than the controls (122?days). In balsam fir, short-term LT exposure augmented ABA and decreased GA₄₄ levels in shoots and delayed postharvest needle senescence.     

Improved accumulation of betulin and betulinic acid in cell suspension culture of Betula pendula roth by abiotic and biotic elicitors

Abstract

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5?mg L^?1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200?mg L^?1 of chitosan was found to be 5.9?×?(6.5?mg g^?1 DW) higher over control cells. Treating the cells with 2?mg L^?1 of CCC, after 7?days, led to 149.3× enhancement of B content (19.4?mg g^?1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation.     

Effect of culture container and carbohydrate content on in vitro slow growth storage of the cherry rootstock ‘Gisela<Superscript>®</Superscript>5’

The present study investigated the effects of gas-tight and gas-permeable culture containers and different sucrose concentrations, as well as sucrose and mannitol combinations on the development of an effective in vitro slow growth storage protocol (at 4 °C, in darkness) for ‘Gisela^®5’ shoot cultures. ‘Gisela^®5’ is the most widely used cherry rootstock in Europe. This dwarf triploid hybrid has many advantages over the conventional cherry rootstocks. Optimizations for the cold storage of ‘Gisela^®5’ in vitro shoot cultures included use of storage medium supplemented with 10, 20, 30, 45, and 60 g L^?1 sucrose and sucrose (15, 30 g L^?1) and mannitol (15 g L^?1) combinations, contained in gas-tight glass jars and gas-permeable ‘Star Pac?’ bags. Cold storage was prolonged to 12 months, during which in every 3 months, cultures were evaluated. Possibility of 16 month-cold storage in gas-tight glass jars was also explored, during which gas chromatographic analysis was performed for the detection of CO₂ and ethylene accumulation for the first 5 months of cold storage. Our results showed that both the 12- and 16-month conservations were possible, especially when 45 or 60 g L^?1 sucrose was supplemented to storage medium, contained in glass jars. Mannitol inclusion to the storage medium was also effective to reduce the metabolic activity of the shoot cultures during storage; however, it did not have a significant positive influence on shoot quality in post-conservation.     

An efficient callus proliferation protocol and rhaponticin accumulation of Rheum franzenbachii Munt., a medicinal plant

An efficient callus proliferation system for Rheum franzenbachii Munt., a rare medicinal plant, has been developed. Callus induced from leaf explants incubated on Murashige and Skoog (MS) medium with appropriate supplements of plant growth regulators. In the 6-benzylaminopurine (6-BAP) in combination with α-naphthalene acetic acid (NAA) treatments, different concentrations of NAA showed different induction effects on explants. When concentration of 6-BAP was as high as 2.0 mgl^?1 in combination with 0.5 mgl^?1 NAA, the callus induction rate reached 58.3%. N-phenyl-N’-1,2,3-thiadiazol-5-ylure (TDZ) in combination with NAA was very suitable for callus proliferation compared to TDZ in combination with 2,4-dicholorophenoxy acetic acid (2,4-D) or TDZ in combination with indole-3-acetic acid (IAA). Fresh and dry weight of callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ in combination with 0.2 mgl^?1 NAA increased 26.3 and 15.0 times within 35 days culture, respectively. Quantitative analysis of rhaponticin by HPLC showed that the phytochemical profile of callus was similar to that of wild plants, and the content of rhaponticin in callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ and 0.2 mgl^?1 NAA was 16.6 mgg?¹DW compared to that of 4.0 mgg^?1 DW in wild plants.     

Light-induced production of antidepressant compounds in etiolated shoot cultures of Hypericum hookerianum Wight & Arn. (Hypericaceae)

Hypericum hookerianum is a lesser known ethnomedicinal plant having wound healing, antitumor and anti-HSV-1 properties. Isolated nodes of in vitro shoots sub-cultured in the dark for 4 weeks on half strength Murashige and Skoog medium solidified with Gelzan (1.5 g l^?1), and supplemented with 2.325 μM kinetin produced 8.0 ± 0.40 etiolated shoots of 5.0 ± 0.62 cm length at 74 % efficiency versus 9.2 ± 0.6 healthy shoots of 4.4 ± 0.5 cm obtained from nodes in light at 96 % efficiency. Low concentrations of hypericin were found in wild plant [0.35 ± 0.09 mg g^?1 dry weight (DW)] and control green shoot cultures (0.91 ± 0.03 mg g^?1 DW). Etiolated shoots exposed to a 12 h photoperiod (50 μmol m^?2 s^?1) through 1–25 days turned red incrementally due to synthesis and accumulation of 0.1–3.83 mg g^?1 DW hypericin in sub-epidermal cortical cells of the stem and varied shaped cells of the distorted mesophyll. Flavonoid and anthocyanin concentrations of the etiolated shoots subjected to the 12 h photoperiod were 3–5 fold higher than the control shoot cultures while total chlorophylls [1.97 ± 0.05 mg g^?1 fresh weight (FW)] of the light exposed shoots were significantly less compared to the control (2.86 ± 0.18 mg g^?1 FW) and natural plant (6.82 ± 0.29 mg g^?1 FW). HPLC analysis of shoot extracts revealed the presence of 0.14 ± 0.03, 0.16 ± 0.02 and 1.45 ± 0.16 mg g^?1 DW hyperforin in wild plant, control shoot cultures and etiolated shoot cultures illuminated for 25 days, respectively. Despite a reasonable presence in etiolated shoots (0.61 ± 0.15 g^?1 FW), total phenols did not increase significantly during illumination. The results indicate light induced synthesis of anti-depressant phenolic derivatives (hypericin, hyperforin and flavonoids) in etiolated shoot cultures of H. hookerianum.     

Osmotic stress induced-capsaicin production in suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili

The influence of osmotic stress on capsaicin production was investigated in cell suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili, a chili species native to Northeastern India. The sterilized seeds were germinated in Murashige and Skoog medium. Two-week-old hypocotyls were excised from in vitro germinated seedlings and implanted in MS medium containing 2, 4-dichlorophenoxyacetic acid (2?mg/l), and Kinetin (0.5?mg/l) for callus induction. Capsaicin production in the suspension cultures was significantly affected using sucrose, mannitol, and NaCl in the medium. Stoichiometric analysis with different combinations of sucrose and non-sugar osmotic agent (NaCl) showed that osmotic stress was an important factor for enhancing capsaicin production in cell suspension cultures of C. chinense. The capsaicin content of 1,644.1???g?g^?1 f.wt was recorded on day 15 in cultures grown in MS medium containing 87.64?mM sucrose in combination with 40?mM NaCl. However, osmotic stress treatment at 160?mM NaCl with sucrose resulted in lowering capsaicin accumulation and separation of cell wall from their cytoplasm, under microscopic observation.     

Use of Commercial Sugar, Isabgol and Ordinary Water in Culture Medium for Conservation of Curcuma longa L

Commercial sugar (market sugar or cube sugar) as carbon source, psyllium seed hask commonly known as isabgol (Plantago ovata) as gelling agent, and medium prepared in ordinary water [OW; pH: 7.48; electrical conductivity (EC): 299 μS cm^?1] instead of deionized distilled water (DW; pH: 5.67; EC: 5 μS cm^?1), did not show any significant effect on micropropagation and in vitro conservation of Curcuma longa cv Prathibha as compared to the control medium (CM) that was prepared with sucrose, gelled with agar in DW. The isabgol-gelled media not only supported better survival of cultures than those on agargelled media, but was also very cost-effective. After 12 months of conservation, significantly higher survival of 33–44% cultures was recorded in isabgol-gelled media, whereas only 16% of cultures survived on CM. By substituting inexpensive sources of three major components of the medium — market sugar, isabgol and OW, up to 84% decrease in cost of medium for plant regeneration and in vitro conservation could be achieved.     

Exogenous application of salicylic acid enhanced the rutin accumulation and influenced the expression patterns of rutin biosynthesis related genes in Fagopyrum tartaricum Gaertn leaves

Except for buckwheat, no cereal or pseudocereal contains any detectable rutin. This study was carried out to compare the rutin content in leaves treated with various concentrations of salicylic acid (SA) (50?mg?l^?1, 100?mg?l^?1, 150?mg?l^?1) for various lengths of time (2?C120?h) to those of untreated F. tartaricum. Rutin content in leaves during the early stages of SA treatment (100?mg?l^?1, 150?mg?l^?1; 24?C48?h) were higher than at other times. The highest rutin content (40.3?mg?gFW^?1) was found in leaves treated with 150?mg?l^?1 SA for 48?h. We cloned partial cDNAs of five genes known to be related to rutin biosynthesis from the leaves of F. tataricum using RACE. These genes were chalcone synthase (FtCHS), flavonol synthase (FtFLS-like), flavone 3-hydroxylase (FtF3H), 4-coumaroyl CoA ligase (Ft4CL), and 4-coumaroyl CoA ligase 2 (Ft4CL2). The protein sequences of these genes were analyzed by similarity searching and phylogenetic trees. Apart from the FtFLS-like gene, which had poor identity with F. esculentum, the other genes showed high similarity to those in F. esculentum. During the early stages of SA treatment (24?C48?h), expression levels of four genes (FtCHS, FtFLS-like, FtF3H, and Ft4CL) were higher than controls but that of Ft4CL2 was not. Most notably, the expression level of FtFLS-like 24?h after application of 150?mg?l^?1 SA was 14.79 times of the control. These results suggest that rutin content can be enhanced to great extent by SA treatment and the gene expression patterns of rutin-biosynthesis-related genes are regulated by SA.     

Influence of culture age on the phytochemical content and pharmacological activities of five Scenedesmus strains

Five axenic Scenedesmus strains (MACC-411, MACC-422, MACC-493, MACC-720, and MACC-727) were cultured and harvested after 5 and 10 days in culture. Using colorimetric methods, the concentrations of total phenolic, condensed tannin, and iridoids in 50 % methanol extracts from both 5- and 10-day-old cultures were quantified. Different solvent extracts from the strains were also tested for antioxidant, acetylcholinesterase inhibitory (AChEI), and antimicrobial activities using various in vitro test systems. Phenolic content was highest (3.6?±?0.42 mg GAE g^?1 DW) in 10-day-old MACC-727. This was approximately fourfold and significantly higher than in the 5-day-old cultures of MACC-727. Among the tested Scenedesmus strains, 5-day-old MACC-411 had the highest iridoid content (3.4?±?0.3 mg HE g^?1 DW), and this was significantly higher than the level detected in the 10-day-old MACC-411. Scenedesmus strains showed better antioxidant potential in the β-carotene–linoleic acid model compared to the DPPH free radical scavenging assay. The AChEI activity (IC₅₀?μg mL^?1) in all strains (besides MACC-422) was higher in 10-day-old cultures compared to the 5-day-old cultures. Although a broad-spectrum of antibacterial activity was observed, the tested microalgae strains demonstrated varying degrees of antimicrobial potential depending on the harvest time, strain-type, and extracting solvent. Thus, the Scenedesmus strain and time of harvest played a significant role in determining their phytochemical content and resultant pharmacological activity. The promising bioactivity in the tested Scenedesmus strains indicates their potential as possible sources of novel/alternative antioxidants and AChE inhibitors.     

Enhanced production of the pharmaceutically important polyphenolic compounds in Vitex agnus castus L. shoot cultures by precursor feeding strategy

Agitated Vitex agnus castus L. shoot cultures were established to analyse the content of selected pharmaceutically important flavonoids and phenolic acids. Two variants (selected from nine ones) of MS medium were prepared: A (BAP 1 mg/L; NAA 0.5 mg/L; GA₃ 0.25 mg/L) and B (BAP 2 mg/L; NAA 0.5 mg/L). The biomass was harvested after 1, 2, 3,4, 5 and 6 weeks. Four‐week cultures (variant A) were selected to perform the precursor feeding experiment. The L‐phenylalanine dose of 1.6 g/L appears to be the most advantageous. Compared to the control cultures, the content of the individual compounds increased in a range from 1.4 to 17.3‐fold (e.g. p‐coumaric acid – 17.3 fold; casticin – 4.8‐fold). The biomass from in vitro cultures is richer in neochlorogenic acid (16‐fold), p‐coumaric acid (5.3‐fold), rutin (2.8‐fold), caffeic acid (1.5‐fold) and cinaroside (1.5‐fold) than the leaves of its parent greenhouse‐cultivated plants. Extracts contained 30 mg/100 g DW of casticin, but after the hydrolysis its amount increased up to 200 mg/100 g DW and twice exceeded the content in the greenhouse leaves. The results indicate that V. agnus castus agitated shoot cultures might be considered as a potential biotechnological source of some pharmaceutically important compounds, especially casticin, rutin, neochlorogenic and p‐coumaric acids.     

An efficient micropropagation protocol for Citrus jambhiri Lush. and assessment of clonal fidelity employing anatomical studies and RAPD markers

Citrus jambhiri (rough lemon) is considered a major rootstock source for a number of Citrus species. A simple method for micropropagation from nodal segments is reported. Nodal segments of C. jambhiri were inoculated on MS medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin, and N6-(2-isopentenyl) adenosine (2iP). Maximum multiple shoot regeneration response (75?%) was observed with BAP at 3?mg?l^?1. Shoots were multiplied for 30?d on fresh medium with similar composition. A total of 67?% of the cultures showed multiplication with the optimum number of shoots (4.02) and height of shoots (1.81?cm) with BAP (3?mg?l^?1) alone. Maximum rooting response (87?%) was observed with naphthaleneacetic acid at 0.5?mg?l^?1. Transverse sections of shoot stems obtained in vivo (sampled from seedlings) and in vitro (regenerated from nodal segments), showed similar anatomies. Randomly amplified polymorphic DNA analysis confirmed that all the regenerated plants were genetically identical to their donor plant, suggesting absence of detectable genetic variation in the regenerated plantlets.