Plant growth promoters mediated quality and yield attributes of milk thistle (Silybum marianum L.) ecotypes under salinity stress.

Silybum marianum (L.) Gaertn (Astraceae) is a well-reputed medicinal plant mostly utilized for silymarin (Sily) content and oil production, however, the information about Sily contents in achene part is still fragmented under different climatic conditions. In this study four milk thistle ecotypes from Faisalabad (FSD), Gujranwala (GUJ), Quetta (QTA), and Kallar kahar (KK) having an altered achene color were analyzed under salt stress. Application of plant growth promoters (PGPs) is one of the solution for ameliorating the effect of salinity and increasing the quantity and quality traits of milk thistle, so ascorbic acid (AsA), thiourea (TU), and moringa leaf extract (MLE) were soil supplied after developing salinity stress (120 mM with irrigation) at germination stage. Predetermined levels were selected for PGPs such as AsA (500 µM), MLE (3%), and TU (250 µM). Results revealed that all yield related attributes were significantly decreased, while secondary metabolites, pericarp epidermis, pericarp parenchyma, and pericarp seed integument increased under salinity stress. Data suggested that PGPs treatment was helpful to alleviate the deleterious effects of salinity stress and enhance the milk thistle quality and quantity parameters. The ecotypic variations with altered achene color patterns represent an advantage for QTA ecotypes for higher Sily extraction under salt stressed conditions.

Novel energy crops for Mediterranean contaminated lands: Valorization of Dittrichia viscosa and Silybum marianum biomass by pyrolysis.

Establishing energy crops could be a cost-efficient alternative towards the valorization of the plant biomass produced in contaminated lands, where they would not compete with food production for land use. Dittrichia viscosa and Silybum marianum are two native Mediterranean species recently identified as potential energy crops for degraded lands. Here, we present the first characterization of the decomposition of the biomass of these species during thermo-chemical conversion (pyrolysis). Using a greenhouse study we evaluated whether the quality of D. viscosa and S. marianum biomass for energy production through pyrolysis could be substantially influenced by the presence of high concentrations of soluble trace element concentrations in the growing substrate. For each species, biomass produced in two different soil types (with contrasted trace element concentrations and pH) had similar elemental composition. Behavior during thermal decomposition, activation energies and concentrations of pyrolysis gases were also similar between both types of soils. Average activation energy values were 295 and 300 kJ mol-1 (for a conversion value of α = 0.5) for S. marianum and D. viscosa, respectively. Results suggest that there were no major effects of soil growing conditions on the properties of the biomass as raw material for pyrolysis, and confirm the interest of these species as energy crops for Mediterranean contaminated lands.

The potential of native species as bioenergy crops on trace-element contaminated Mediterranean lands.

The establishment of energy crops could be an option for the management of degraded and contaminated lands, where they would not compete with food production for land use. Here, we aimed to explore the potential of certain native Mediterranean species for the revegetation of contaminated lands for energy production purposes. A field survey was conducted in a trace-element (TE) contaminated area from SW Spain, where the patterns of biomass production, TE accumulation and the calorific value of some thistle species were analyzed along a soil contamination gradient. In a greenhouse experiment the response of two thistle species (Cynara cardunculus and Silybum marianum) and the shrub Dittrichia viscosa to soil contamination was assessed, as well as the effects of these species on some soil microbial parameters involved in nutrient cycling (enzyme activities and arbuscular mycorrhizal colonization in roots). Silybum marianum was able to colonize highly contaminated soils. Its aboveground biomass accumulated Cd and had a relatively high calorific value; this value was similar in biomass obtained from both heavily and moderately contaminated soils. Greenhouse experiment confirmed that S. marianum biomass production and calorific value is scarcely affected by soil contamination. In addition, some soil enzyme activities were clearly enhanced in the S. marianum rhizosphere. Dittrichia viscosa is another promising species, given its capacity to produce a high biomass with appreciable calorific value in acid contaminated soils. Germination of both species was hampered in the acid contaminated soil, and therefore soil pH correction would have to be accomplished before establishing these species on extremely acid soils. Further assessment of the risk of transfer of Cd and other TE to the food chain would be needed to confirm the suitability of these species for the revegetation of contaminated lands with energy production purposes.

[Establishment of culture system of Silybum marianum hairy roots and determination of silybin].

This research uses six Agrobacterium rhizogenes R1601, R15384, R1000, A4, R1025 and R1 to infect silymarin explants to induce hairy roots and silibin. All of the six A. rhizogenes can induce Silybum marianum to generate hairy roots and the A. rhizogene A4 shows comparatively high infection on the plant. This research determines the condition to induce silymarin hairy roots by the factors of infection time, pre-culturing, co-culturing and pH value. The fact that MS liquid medium fits the proliferation of silymarin hairy roots is determined. Through PCR molecular identification, it can be seen that the DNA plasmids in the A. rhizogenes are successfully integrated into the genome of transformed roots. Using liquid chromatography, it is determined that the silibin content in silymarin hairy roots is 2.5 times that in the plant In this research, the silymarin hairy roots culturing system is established, which lays a foundation for the study of culturing silymarin hairy roots and producing silibin.

Solid-state fermentation of industrial solid wastes from the fruits of milk thistle Silybum marianum for feed quality improvement.

The industrial solid wastes generated during the production of silymarin from the fruits of milk thistle Silybum marianum was used as the substrate. Preparation and evaluation of the feeds produced by solid-state fermentation (SSF) of the industrial solid wastes was carried out. The protein content of the fermented feed (FF) from a combination of Aspergillus niger and Candida tropicalis was the highest among the examined strains. The optimal process parameters for protein enrichment with SSF using A. niger and C. tropicalis included incubation temperature of 30.8 °C, fermentation time of 87.0 h, and initial moisture content of 59.7 %. Under these conditions, the value additions of FF occurred. The fiber of FF was decreased by 25.07 %, while the digestibility of protein, protein content, and the ratio of total essential amino acids to total amino acids were increased by 79.85, 16.22, and 8.21 %, respectively. The analysis indicated that FF contained 1.44 mg/kg flavonoids and 0.5 mg/kg silybin, which significantly increased by 2.42 and 1.63 times, respectively than those in unfermented substrates. FF recorded reduced molecular weight of proteins from 20.1 to 44.3 kDa to below 14.3 kDa. The results of feeding trial of FF replacement with soybean meal in broilers diets for 8 weeks showed that FF significantly improved carcass characteristics including abdominal fat rate, serum biochemical parameters including aspartate transaminase, blood urea nitrogen and high density lipoprotein cholesterol, and immune responses of broilers. A potential feed quality improvement was achieved through mixed strains SSF of industrial solid wastes of S. marianum fruits.

An integrated proteomic approach to decipher the effect of methyl jasmonate elicitation on the proteome of Silybum marianum L. hairy roots.

Jasmonate and its methyl derivative, methyl jasmonate (MeJA), are naturally occurring compounds that mediate several plant physiological processes in response to pathogen attack, wounding, and ozone. Exogenous application of jasmonates triggers defense responses that resemble those initiated by pathogen infection and also modulates the production of certain secondary metabolites in a variety of plant species. In this study, we treated the hairy root cultures of Silybum marianum L. with 100 µM MeJA and then measured the content of Silymarin (SLM). We observed that the SLM content increased significantly after 48 h of MeJA treatment and remained constant for 120 h. However, MeJA treatment caused a significant growth reduction after 96 h incubation. The activity of lipoxygenase as a key enzyme in the jasmonate biosynthesis pathway and anti-oxidative enzymes; peroxidase and ascorbate peroxidase was also significantly increased after MeJA treatment. To elucidate the global effect of jasmonate on gene expression of S. marianum, we employed high resolution two-dimensional gel electrophoresis coupled with tandem mass spectrometry. Out of 670 reproducibly detected protein spots which were analyzed on each given gel, 32 spots were up- or down regulated upon MeJA treatment. Of them, ten proteins such as ER binding protein, glutamine synthetase, pathogenesis-related protein, caffeoyl CoA O-methyltransferase, and profilin-1 could be identified by mass spectrometry analysis. The possible implications of the identified proteins on physiological outcome of MeJA application in S. marianum hairy root culture will be discussed.

Elicitation of silymarin in cell cultures of Silybum marianum: effect of subculture and repeated addition of methyl jasmonate.

Production of silymarin and the effect of the elicitor, methyl jasmonate (MeJA), was monitored in cell cultures of Silybum marianum over 4 years. Silymarin concentrations gradually declined after prolonged subculture, making the success of elicitor strategy limited in long-term cultures. The continuous presence of MeJA in cultures for an extended period was necessary for induction of silymarin accumulation. A repeated elicitor strategy was not a good option for improving silymarin productivity in batch cultures. Removal of medium from elicited cultures and addition of fresh medium avoided the toxic effects of elicitor accumulation, allowing the system to respond to a repeated MeJA treatment without loss of productivity.

Screening for soil streptomycetes from North Jordan that can produce herbicidal compounds.

A total of 231 different soil Streptomyces isolates were recovered from 16 different locations in North Jordan. They were assessed for their phytotoxic activity on seeds of cucumber (Cucumis sativus L.) and ryegrass (Lolium perenne L.) placed adjacent to a 2 cm wide Streptomyces culture strips grown at 28C degrees for 3 weeks on starch casein nitrate (SCN) agar. Phytotoxicity was ascertained on the basis of suppressed seed germination, discoloration of the root tip, reduced root and the shoot growth and eventual death of the root. Twenty one of the isolates exhibited adverse effect against growth of germinated cucumber seeds, germination and growth of ryegrass seeds. Using filter paper bioassay method, culture filtrate from the SCN broth of the isolate R9; identified as Streptomyces aburaviensis, significantly inhibited seed germination, radicle and shoot growth ofryegrass, reduced radicle and shoot growth of cucumber and suppressed the shoot growth of milk thistle (Silybum marianum L.). Also, culture filtrate from the glucose-peptone-molasses (GPM) broth diluted (1:1) with sterilized distilled water caused complete inhibition of seed germination of redroot pigweed (Amaranthus retroflexus L.). Dichloromethane extracted fraction of S. aburaviensis (strain R9) culture filtrate from GPM broth completely inhibited seed germination of ryegrass when applied at doses of 3 and 5 mg of dry weight, and the seedling growth of cucumber and milk thistle was severely reduced by the same doses.

[Formation of flavanolignane in the white-flowered variety (Szibilla) of Silybum marianum in relation to fruit development]. / Silybum marianum fehérviráigú fajtájainak (Szibilla) flavanolignan képzése a termésfejlodéssel osszefüggésben.

In our present work we studied the white-flowered lady's thistle variety 'Szibilla' improved by our Institute and having received state acknowledgement in 1997. During our investigations we established the following facts: --flavanolignanes having liver-protecting, cytoprotective effects can be found only in the developing and ripe achene fruits. The vegetative organs contain flavanoids of different types; --around the tissues of the fruit (Silybi mariani fructus) known as drug raw material for therapy and the pharmaceutical industry, effective agents are contained only in the fruit wall having undergone significant changes as well as in the woody (ligneous) seed-coat. The endosperm and the plant embryo contain effective agents only in traces; --during ontogenesis--following the pistil's organization into fruit and seed, respectively--we revealed that the biogenesis and accummulation of flavanolignanes correlate with the wood formation (lignification) of the seed-coat.