Are tissue cultures of Peganum harmala a useful model system for studying how to manipulate the formation of secondary metabolites?

This article reviews our present knowledge on the formation of tryptophan derived secondary metabolites in tissue cultures of Peganum harmala. With the presence of -carboline alkaloids and serotonin, P. harmala contains two rather simple, interrelated biosynthetic pathways. The long term disadvantage of low and unstable productivity of P. harmala suspension culture has recently been overcome by establishing highly productive hairy root cultures. The first -carboline alkaloid biosynthetic enzymes, specific for the O-methylation of harmalol and harmol as well as for the oxidation of harmaline to harmine, have been detected in these cultures, and they should thus provide a suitable source for studying the yet unknown initial two enzymatic steps of -carboline alkaloid biosynthesis. Seedlings of P. harmala have also been successfully transformed with constructed strains of Agrobacterium, as demonstrated by the overexpression of a tryptophan decarboxylase gene from Catharanthus roseus in cultures of P. harmala. In such transgenic cultures a large overproduction of serotonin was observed. The relative simplicity of these pathways and the rather easy handling of the cultures could make P. harmala a useful and attractive model system for studying the interaction, regulation and manipulation of secondary pathways in cultured cells.Abbreviations TDC tryptophan decarboxylase - tdc gene of tryptophan decarboxylase     

Flavonoid Production in Transformed Scutellaria baicalensis Roots and Ways of Its Regulation

A root culture of skullcap (Scutellaria baicalensisGeorgi) transformed with pRi T-DNA was initiated by the inoculation of sterile seedlings with Agrobacterium rhizogenes(wild-type strain A-4). The flavonoid concentration in cultured roots comprised 5% of the root dry weight and was maintained essentially constant during a subculture. For four weeks of culturing, the weight of the roots increased by 20–30 times; when the roots were cultured for a longer time and with periodic enrichment of the nutrient medium, their weight increased 50-fold. Skullcap roots were shown to synthesize flavones characteristic of intact roots (wogonin, baicalein, and baicalin). The addition of 0.01–1 mM L-phenylalanine (a precursor of flavonoids) to the nutrient medium affected neither root growth, nor their flavonoid concentration. Root elicitation with 100 M methyl jasmonate for 72 h increased the flavonoid content per flask and per root dry weight by 1.8 and 2.3 times, respectively.     

Artificial seed preparation as the efficient method for storage and production of healthy cultured roots of medicinal plants

The technique for the refinement of pRi T-DNA-transformed root cultivation by the root fragment encapsulation in the gel coat, i.e., so-called “artificial seed” (AS) production, was studied. AS were produced from genetically transformed roots of Baikal skullcap (Scutellaria baicalensis Georgi) and common rue (Ruta graveolens L.). The effects of duration of AS storage at 4°C on their subsequent growth activity and a capability for resumption of actively growing root cultures were analyzed. Encapsulation of contaminated Baikal skullcap root culture with the addition of antibiotic and storage during 2–5 weeks at low above-zero temperature resulted in a complete elimination of infection, i.e., obtaining the healthy root culture. Growth activity and total flavone concentration were markedly increased in this culture, so that total productivity of this renewed root culture increased substantially. Using AS produced from the root fragments of common rue, it was shown that, after long-term storage at low above-zero temperature, they are capable of not only root growth resumption but also active shoot formation, which is of interest for plant micropropagation. Long-term retaining growth activity of AS produced from root cultures of valuable medicinal plants permits their usage as a reserve and also, in the case of necessity, for long-distance transport as compact axenic root inocula. The storage of viable root fragments within AS also helps to optimize intervals between numerous subculturings of root cultures required for the maintenance of IPPRAS collection in the active state.     

Artificial seeds as a way to produce ecologically clean herbal remedies and to preserve endangered plant species

Influence of entrapment of the segments of isolated pRi T-DNA transformed roots of skullcap (Scutellaria baicalensis Georgi) in alginate gel capsules on the morphological, anatomical, physiological, and biochemical parameters of root cultures obtained from so-called artificial seeds (ASs) has been studied. The obtained ASs remain viable even after long-term storage at 4°C. Using the encapsulation technique, we have renewed a skullcap root culture and made it healthier. During anatomical study of the renewed root culture, the formation of chloroplasts has been observed in the cells of roots cultivated under lighting. Total chlorophyll content, chlorophyll a: chlorophyll b ratio, and the flavone content in the green roots have been determined.     

Poplar and shrub willow energy crops in the United States: field trial results from the multiyear regional feedstock partnership and yield potential maps based on the PRISM‐ELM model

To increase the understanding of poplar and willow perennial woody crops and facilitate their deployment for the production of biofuels, bioproducts, and bioenergy, there is a need for broadscale yield maps. For national analysis of woody and herbaceous crops production potential, biomass feedstock yield maps should be developed using a common framework. This study developed willow and poplar potential yield maps by combining data from a network of willow and poplar field trials and the modeling power of PRISM‐ELM. Yields of the top three willow cultivars across 17 sites ranged from 3.60 to 14.6 Mg ha^?1 yr^?1 dry weight, while the yields from 17 poplar trials ranged from 7.5 to 15.2 Mg ha^?1 yr^?1. Relationships between the environmental suitability estimates from the PRISM‐ELM model and results from field trials had an R² of 0.60 for poplar and 0.81 for willow. The resulting potential yield maps reflected the range of poplar and willow yields that have been reported in the literature. Poplar covered a larger geographic range than willow, which likely reflects the poplar breeding efforts that have occurred for many more decades using genotypes from a broader range of environments than willow. While the field trial data sets used to develop these models represent the most complete information at the time, there is a need to expand and improve the model by monitoring trials over multiple cutting cycles and across a broader range of environmental gradients. Despite some limitations, the results of these models represent a dramatic improvement in projections of potential yield of poplar and willow crops across the United States.     

Biosynthesis of rutacridone in tissue cultures of Ruta graveolens L.

The dihydrofuroacridone, rutacridone, is the main alkaloid in tissue cultures of Ruta graveolens L. strain R-19. The biosynthesis of this particular acridone alkaloid was investigated by using calluses and suspension cultures of strain R-19. Anthranilic acid is specifically incorporated into ring A of rutacridone. Some further evidence was provided that acetate via a polyketide is involved in acridone biosynthesis. Mevalonic acid gave a poor incorporation into rutacridone. Thus the origin of the isopropyldihydrofuran moiety of the investigated alkaloid is still obscure.     

Lead uptake and translocation by willows in pot and field experiments

Plant growth and lead (Pb) uptake by seven willow varieties were investigated in pot and field experiments to assess the suitability of willows for phytoremediation of Pb at heavily contaminated sites such as skeet ranges. Differences in uptake and translocation of Pb in Salix were observed between pot and field experiments. In the pot experiment, willows grown in Pb-contaminated field soil for 6 months showed tolerance to very high soil Pb concentration (21,360 mg kg(-1)), and with the addition of EDTA were able to take up and translocate more than 1000 mg kg(-1) Pb into above-ground tissues. In the field experiment, all willow varieties showed tolerance to heterogeneously high soil Pb concentrations. Plants were also able to take up and translocate Pb into above-ground tissues. However, after 4.5 months, the lead concentration in the above-ground tissues of willows grown in soil amended with EDTA was less than 200 mg kg(-1). The results from the pot experiment suggest that Salix varieties have the potential to take up and translocate significant amounts of Pb into above-ground tissues using EDTA. However, to verify the phytoextraction abilities of Salix in the field, additional research is needed.     

Bioremediation and phytoremediation of total petroleum hydrocarbons (TPH) under various conditions

Remediation of contaminated soils is often studied using fine-textured soils rather than low-fertility sandy soils, and few studies focus on recontamination events. This study compared aerobic and anaerobic treatments for remediation of freshly introduced used motor oil on a sandy soil previously phytoremediated and bioacclimated (microorganisms already adapted in the soil environment) with some residual total petroleum hydrocarbon (TPH) contamination. Vegetated and unvegetated conditions to remediate anthropogenic fill containing residual TPH that was spiked with nonaqueous phase liquids (NAPLs) were evaluated in a 90-day greenhouse pot study. Vegetated treatments used switchgrass (Panicum virgatum). The concentration of aerobic bacteria were orders of magnitude higher in vegetated treatments compared to unvegetated. Nevertheless, final TPH concentrations were low in all saturated soil treatments, and high in the presence of switchgrass. Concentrations were also low in unvegetated pots with fertilizer. Acclimated indigenous microbial communities were shown to be more effective in breaking down hydrocarbons than introducing microbes from the addition of plant treatments in sandy soils. Remediation of fresh introduced NAPLs on pre-phytoremediated and bioacclimated soil was most efficient in saturated, anaerobic environments, probably due to the already pre-established microbial associations, easily bioavailable contaminants, and optimized soil conditions for microbial establishment and survival.     

Cadmium and copper uptake and translocation in five willow (Salix L.) species

The efficacy for phytoremediation of five willow species was tested by experimental copper and cadmium uptake in a greenhouse hydroponic system. Five treatments included two concentrations (5 and 25 microM for each metal) and a control. Metal concentrations in solution as well as solution uptake were monitored. Metal resistance was assessed through effects on the dry weight of roots and shoots. The willow species tested were generally resistant of increased Cu and Cd content. Metal accumulation was found in all plant organs of all species. Growth and transpiration were not decreased by 5 microM of copper and 25 microM of cadmium in the solution for most species. 25 microM copper caused injury and reduced the dry weight for all species after 21 d. Salix nigra was highly resistant of both Cu and Cd and accumulated more metals than other species. Future field study should be conducted to confirm the findings and feasibility of the phytoremediation technology using those species.