A comparative study of bioactive secondary metabolite production in diploid and tetraploid Echinacea purpurea (L.) Moench

The impact of the ploidy level on biomass accumulation and the production of high-value secondary metabolites was studied in Echinacea purpurea (L.) Moench. Tetraploid E. purpurea was obtained by treating diploid explants with colchicine. The morphology, biomass yield, the contents of caffeic acid derivatives and alkamides, and the activities of phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) were compared between diploid plants and tetraploid plants of E. purpurea. The total fresh root weight and total dry root weight of the tetraploid plants were 39.32 and 40.48 % higher than those of the diploid plants, respectively. The chemical profiles of the diploid and tetraploid E. purpurea plants were similar, as determined through a comparison of their FTIR spectra and second derivative spectra. The caffeic acid derivatives and alkamides in the diploid and tetraploid plants were determined by HPLC. The tetraploid plants had higher contents of both of these types of molecules. In addition, the tetraploid plants had higher PAL and C4H activities compared with the diploid plants. The enhancement in the PAL and C4H activities was accompanied with an increase in the cichoric acid content, which indicates that the induction of polyploidy in E. purpurea resulted in higher PAL and C4H expression and promoted the biosynthesis of cichoric acid. Therefore, the induction of polyploidy may be a valid strategy to achieve a higher yield of biomass and bioactive compounds in E. purpurea.     

Active polyphenolic compounds,nutrient contents and antioxidant capacity of extruded fish feed containing purple coneflower (Echinacea purpurea (L.) Moench.)

The growth of fish is directly dependent on feed composition and quality. Medicinal plants can be added to fish feed as adjuvant therapy for the prevention of fish diseases. The purple coneflower (Echinacea purpurea (L.) Moench.) has been reported to have multiple biological effects, including immunomodulatory and antioxidant activity. The most active compounds of E. purpurea are polyphenols - caffeic acid derivatives: caftaric acid, chlorogenic acid, cynarin, echinacoside and cichoric acid.Due to a relatively limited number of studies on the use of the purple coneflower as a nutritional supplement for fish feeding, extruded fish feed with addition of Echinacea roots was produced. In the feed total phenolic content, selected polyphenol contents, the energetic value, nutrient contents and antioxidant capacity were examined.The results indicate that fish feed with addition of the Echinacea has a great potential to be a good source of natural radical scavengers, for example polyphenols, and nutritive ingredients. Antioxidant properties of feed were well correlated with the coneflower content. The study findings confirmed that high-temperature extrusion-cooking process does not deactivate phenolic antioxidant compounds, which are present both in the Echinacea roots and in the final product. Fish feed with addition of E. purpurea can be used as a nutritional supplement in the prevention of fish diseases caused by oxidative stress.     

Flavonoids from Echinacea purpurea

Two compounds, nicotiflorin and rutin, are isolated and idenitified from flowers of Echinacea purpurea (L.) Moench., of the family Asteraceae, cultivated in Samara oblast. It is established that nicotiflorin (3-O-rutinoside campherol) is the dominant flavonoid of Echinacea purpurea; it is described along with rutin for the first time for this plant, cultivated in the Russian Federation and CIS countries.     

Anther culture and haploid plant regeneration in purple coneflower (Echinacea purpurea L.)

Anthers containing microspores at the uninucleate stage were excised from capitula of field grown plants of purple coneflower, Echinacea purpurea, and cultured on medium conducive to callus growth. In callus induction cultures, N6 basal medium was more effective than Murashige and Skoog (MS), and a combination of benzyladenine (BA) at 2.22 μM with naphthaleneacetic acid (NAA) at 0.054 μM was more effective than 2,4-dichlorophenoxyacetic acid (2,4-D) alone at 4.52, 9.05 and 13.57 μM. Callus induction rate as high as 85.8% was achieved, and no statistically significant differences were found among cultures with various densities of 20, 40, 60, 80 and 100 anthers per bottle each filled with 40-ml medium. Shoots were regenerated from calluses on MS medium containing 2.22 μM BA and various concentrations of NAA, with the highest regeneration rate of 95.24% obtained when 0.27 μM NAA was applied. Although a large portion of the␣regenerated shoots had prominent symptom of vitrification, some normal shoots could be easily rooted on MS medium containing 0.054 μM NAA. Thirty regenerated plants were randomly selected and 19 of them were confirmed to be haploid by observation of chromosome number of root-tip cells.     

Alkamides and a neolignan from Echinacea purpurea roots and the interaction of alkamides with G-protein-coupled cannabinoid receptors

Multiple chromatographic separations of the CHCl₃-soluble extract of the roots of Echinacea purpurea led to the isolation of 19 compounds. Four natural products, three alkamides and nitidanin diisovalerianate, were identified, and five further compounds were detected for the first time in this species. Additionally, 10 known E. purpurea metabolites were isolated. The structures were determined by mass spectrometry and advanced 1D and 2D NMR techniques. The bioactivity of the isolated compounds was studied in [³⁵S]GTPγS-binding experiments performed on rat brain membrane preparations. Both partial and inverse agonist compounds for cannabinoid (CB1) receptors were identified among the metabolites, characterized by weak to moderate interactions with the G-protein signaling mechanisms. The G-protein-modulating activities of the Echinacea compounds are rather far from the full agonist effects seen with the CB1 receptor agonist reference compound arachidonyl-2′-chloroethylamide (ACEA). However, upon coadministration with ACEA, a number of them proved capable of inhibiting the stimulation of the pure agonist, thereby demonstrating cannabinoid receptor antagonist properties.     

Indirect somatic embryogenesis of purple coneflower (Echinacea purpurea (L.) Moench): a medicinal-ornamental plant: evaluation of antioxidant enzymes activity and histological study

Molecular Biology Reports - Purple coneflower (Echinacea purpurea (L.) Moench) is a widely used medicinal and ornamental plant. In the present study, the callus embryogenesis was examined using...     

Further amides from Echinacea purpurea

The aerial parts of Echinacea purpurea afforded, in addition to known compounds, five further highly unsaturated amides and a derivative of linolen     

The Distribution of Silicon Deposits in the Roots of Molinia caerulea (L.) Moench. and Sorghum bicolor (L.) Moench.

The occurrence of silica in relation to meristematic zones andthe thickening of the endodermis in the roots of Molinia caerulea(L.) Moench. and Sorghum bicolor (L.) Moench. has been investigatedby means of the electron-probe microanalyser and the scanningelectron microscope. In proximal regions of mature roots ofM. caerulea, the central strengthening tissue of the stele,the vessel walls, the endodermis and the sub-epidermal sclerenchymaare areas of heavy accumulation. The distal regions of suchroots are relatively free of silicon and show little thickeningof the inner tangential walls of the endodermis or of the cellsof the strengthening tissues. The thickening of these elementsis shown to be associated with their location and the age ofthe root. In the proximal regions of S. bicolor, silicon is detected andlargely confined to the inner tangential walls of the endodermiswhich display some thickening. In addition, discrete and evenly-distributeddeposits varying in size partly fill the lumen of this layer.Some cells exhibit a number of smaller protrusions. High magnificationsof these lumen deposits show a distinct granular structure incontrast to the very uniform pattern of the wall deposits. The results are compared with deposits in grass leaves and inflorescencebracts and in woody perennials. The presence of silicon in additionto suberin, lignin and polyphenols in the thickened endodermalwall is also discussed in relation to the recognized functionof the endodermis.     

Somaclonal variation from Sorghum bicolor (L.) Moench cell culture

Plant Cell, Tissue and Organ Culture (PCTOC) - Sorghum bicolor (L.) Moench, plants were regenerated from 4 to 5 month old callus cultures originally derived from seedling explants. Somaclonal...     

Binding studies of an arabinogalactan-protein from Echinacea purpurea to leucocytes.

Flow cytometric investigations show binding of an isolated arabinogalactan-protein (AGP) from pressed juice of the aerial parts of Echinacea purpurea to the cell surface of human leucocytes. AGP demonstrates binding to lymphocytes, monocytes and granulocytes of different donors (n=8). Competition assays with two antibodies, directed against CD4 and CD8, revealed no interaction of AGP with these receptors, leading to the conclusion that binding of AGP to leucocytes is mediated via other structures.     

Anther culture of Sorghum bicolor (L.) Moench

The sequence of pollen development from the tetrad stage to the mature tricellular grain was studied in freshly harvested anthers of Sorghum bicolor. This pattern of development was then compared with that occurring during panicle pretreatment and subsequent anther incubation in vitro. It was found that during pretreatment at 7° C mitoses of the vegetative cell were induced in up to 30% of the pollen. During anther incubation procallus development was highly polarised with contributions from both the generative and vegetative cells. After pretreatment at 14 or 20° C the generative cell became detached from the pollen wall and it was not possible to determine whether subsequent development involved only the vegetative cell or both the vegetative and generative cells.Although retarded pollen grains were observed both in vivo and in vitro, and were occasionally seen to divide in culture, they did not appear to be the source of the procalluses produced.     

Elicitation of secondary metabolism in Echinacea purpurea L. by gibberellic acid and triazoles

In vitro bioreactor production of Echinacea purpurea L. was used to facilitate the screening of compounds capable of eliciting increased secondary metabolite production. Based on previous experience with various bioreactors, the Southern Sun Liquid Lab Rocker was selected for this study as it produces healthy, vigorous, whole plants. The focus of the present study was to quantify the concentration of the medicinally important secondary metabolites caftaric acid and cichoric acid and to study the effect of growth regulators on the production of these compounds in this system. Both marker compounds were produced at levels that compared favorably to field grown plants. Application of gibberellic acid (GA₃), paclobutrazol, uniconazole and a combined treatment of GA₃ with paclobutrazol to the in vitro plants generally increased the concentration of caftaric acid in the roots with little effect on the concentration in the shoots. The concentration of cichoric acid was higher in the roots of treated plants than in the roots of control plants, but lower in corresponding shoots. The present study highlights the use of in vitro production of whole plants as a model system for studying the regulation of plant secondary metabolism in a controlled environment.     

In vitro regeneration of Echinacea purpurea from leaf explants

Efficient plant regeneration was achieved via organogenesis from callus cultures derived from leaf tissue of Echinacea purpurea. Proliferating shoot cultures were obtained by placing leaf explants on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) combinations. MS medium supplemented with BAP (4.44 M) and NAA (0.054 M) was the most effective, providing high shoot regeneration frequencies (100%) associated with a high number of shoots per explant (7.7 shoots/explant). Plantlets were rooted on MS medium alone or in combination with different concentrations of indole-3-butyric acid (IBA), and high rooting and survival was achieved using MS media without plant growth regulators (PGR). All plantlets survived acclimatization producing healthy plants in the greenhouse. This study demonstrated that adventitious shoot regeneration of E. purpurea from leaf explants can be a useful method for the multiplication of this important medicinal plant.     

Plant regeneration from cultured immature embryos of Sorghum bicolor (L.) Moench

Summary Immature embryos of 20 sorghum genotypes were cultured on MS 5 medium containing MS mineral salts supplemented with 2,4-D, zeatin, glycine, niacinamide, Ca-pantothenate, L-asparagine, and vitamins. For regeneration, calli were transferred onto the same medium with the exception that IAA was substituted for 2,4-D. In general, immature embryos obtained 9–12 days after pollination resulted in the best redifferentiation. Ability of calli to regenerate varied among genotypes; cultivars C401-1 and C625 had the highest redifferentiation frequencies. Ability to redifferentiate was heritable and acted as a dominant trait. At least two gene pairs were involved. Regenerated R₀ plants were planted in a greenhouse and their selfed (R₁ and R₂) progenies were planted in the field and examined for morphological and cytological variations. The majority of the phenotypic variations noted in R₀ were not transmitted to later generations. However, variants for plant height, degree of fertility, and midrib color persisted in R₁ and R₂ generations. A variation in tallness was attributable to one dominant mutant gene. Short stature and male sterility variants appeared to be consequences of recessive mutant genes controlling those traits. Minor variations in peroxidase banding patterns were found among R₀ plants.This study was supported by a research grant from Kansas Sorghum Commission and by a Research Fellowship to the senior author from the Ministry of Agriculture, Animal Husbandry, and Fisheries, China. Contribution 86-456-J from the Kansas Agricultural Experiment Station     

Agrobacterium rhizogenes-mediated transformation of Echinacea purpurea

Summary Echinacea purpurea seedlings were inoculated with several Agrobacterium rhizogenes strains in order to obtain hairy roots. Infection with A. rhizogenes strains LMG63 and LMG150 resulted in callus formation. Upon infection with strains ATCC 15834 and R1601 hairy roots were obtained. Opine detection confirmed transformation of E. purpurea. Comparative HPLC fingerprint analysis of the alkamides from natural plant source, control tissues, and transformed callus and roots indicated that transformed callus and hairy roots might be a promising source for continuous and standardized production of the dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide and related amides.Abbreviations HPLC high-pressure liquid chromatography - MS Murashige and Skoog culture medium     

Competition for nutrients between Calluna vulgaris (L.) Hull and Molinia caerulea (L.) Moench

Summary A combination of a removal and fertilization experiment in the field and a replacement experiment in containers in a cold frame was carried out to investigate the role of nutrient supply in the interactions between Calluna vulgaris and Molinia caerulea. It is concluded that the growth of Calluna as well as that of Molinia increased with increasing nutrient availability. However, the increase in biomass of Molinia was much larger than that of Calluna. It is also concluded that increased nutrient availability favours the competitive vigour of Molinia in interaction with Calluna.     

Wide hybridization: pollination of Zea mays L. by Sorghum bicolor (L.) Moench

Summary Foreign pollen tubes in the stigma of Zea mays can be prevented from reaching the ovary cavity by the unusual length of the pollen tube pathway. A simple and rapid procedure is described for overcoming this difficulty by pollinating the basal parts of the stigmas without removing the ensheathing bracts (husks). The method maintains high humidity in the vicinity of the ovaries, and by conserving photosynthetic tissues probably also ensures a more normal O₂ /CO₂ balance in the neighbourhood of the stigmas than do bagging procedures. It is shown that Sorghum pollen tubes readily reach the ovary after pollination by the method. Their presence induces some of the characteristic post-pollination effects caused by Zea pollen tubes, but they frequently also stimulate premature enlargement of the nucellus and lysis of nucellar cells. Although Sorghum tubes have been traced across the inner ovary wall, they have not been seen to enter the micropyle, and hybrid embryos have not yet been obtained.     

In vitro propagation of mature trees of Alnus incana (L.) Moench.

Mature trees of European grey alder (Alnus incana) were micropropagated on a modified MS medium containing 2.5 M BA, 6.2 mM (500 mg l^-1) NH₄NO₃ and 1.5% glucose. Prior to in vitro culture, mature scions were multiplied through grafting and cutting techniques. Shoot tips from cuttings were established in vitro. After six months of culture, shoots were rooted either in vitro or in vivo and plantlets were transferred to greenhouse conditions.