Effect of Light Quality and Media Components on Shoot Growth, Rutin, and Quercetin Production from Common Buckwheat.

Common buckwheat (Fagopyrum esculentum Moench) seeds are important nutritious grains that are widely spread in several human food products and livestock feed. Their health benefits are mainly due to their bioactive phenolic compounds, especially rutin and quercetin, which have a positive impact on heart health, weight loss, and diabetes management. In this study, we evaluated different media and light treatments for the in vitro cultures of common buckwheat (CB) in order to find the most optimum one producing the highest yield with the highest purity of these compounds. The subcultured treated samples included in this study were shoots, leaves, stems, hairy roots, and calli. From the several treated samples and under different light stress conditions, the best production was achieved by growing the shoots of common buckwheat in hormone-free media containing activated charcoal and exposing to blue light, attaining 4.3 mg and 7.0 mg/g of extracts of rutin and quercetin, respectively, compared to 3.7 mg of rutin/g of extract and traces of quercetin in the seeds of CB. Continuous multiplication of CB shoots in the media containing charcoal and different concentrations of kinetin produced an extract with 161 mg/g of rutin and 26 mg/g of quercetin with an almost 20-fold increase in rutin content. The rutin content under these conditions reached up to 16% w/w of the extract. The hairy root cultures of the leaves exposed to red light showed a significantly high yield of quercetin attaining 10 mg/g of extract. Large-scale production of CB shootlets under the best conditions were carried out, which enabled the isolation of pure quercetin and rutin using a simple chromatographic procedure. The identity and purity of the isolated compounds were confirmed through NMR and HPLC analyses.

Genetic Stability, Phenolic, Flavonoid, Ferulic Acid Contents, and Antioxidant Activity of Micropropagated Lycium schweinfurthii Plants.

Lycium schweinfurthii is a Mediterranean wild shrub rich in plant secondary metabolites. In vitro propagation of this plant may support the production of valuable dietary supplements for humanity, introduction of it to the world market, and opportunities for further studies. The presented study aimed to introduce an efficient and reproducible protocol for in vitro micropropagation of L. schweinfurthii and assess the genetic stability of micropropagated plants (MiPs) as well as to estimate phenolic, flavonoid, ferulic acid contents, and the antioxidant activity in leaves of micropropagated plants. Two DNA-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR), and one biochemical technique, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were used to assess the genetic stability in MiPs. Spectrophotometric analysis was performed to estimate total phenolic and flavonoid contents and antioxidant activity of MiPs leaves, while ferulic acid content was estimated using high-performance thin-layer chromatography (HPTLC). Sufficient shoot proliferation was achieved at MS (Murashige and Skoog) medium supplemented with 0.4 mg L-1 kinetin and rooted successfully on half-strength MS medium fortified with 0.4 mg L-1 Indole-3-butyric acid (IBA). The Jaccard's similarity coefficients detected in MiPs reached 52%, 55%, and 82% in the RAPD, ISSR, and SDS-PAGE analyses, respectively. In the dried leaves of MiPs, the phenolic, flavonoid, and ferulic acid contents of 11.53 mg gallic acid equivalent, 12.99 mg catechin equivalent, and 45.52 mg were estimated per gram, respectively. However, an IC50 of 0.43, and 1.99 mg mL-1 of MiP dried leaves' methanolic extract was required to scavenge half of the DPPH, and ABTS free radicals, respectively. The study presented a successful protocol for in vitro propagation of a valued promising plant source of phenolic compounds.

High-Density SNP-Based Association Mapping of Seed Traits in Fenugreek Reveals Homology with Clover.

Fenugreek as a self-pollinated plant is ideal for genome-wide association mapping where traits can be marked by their association with natural mutations. However, fenugreek is poorly investigated at the genomic level due to the lack of information regarding its genome. To fill this gap, we genotyped a collection of 112 genotypes with 153,881 SNPs using double digest restriction site-associated DNA sequencing. We used 38,142 polymorphic SNPs to prove the suitability of the population for association mapping. One significant SNP was associated with both seed length and seed width, and another SNP was associated with seed color. Due to the lack of a comprehensive genetic map, it is neither possible to align the newly developed markers to chromosomes nor to predict the underlying genes. Therefore, systematic targeting of those markers to homologous genomes of other legumes can overcome those problems. A BLAST search using the genomic fenugreek sequence flanking the identified SNPs showed high homology with several members of the Trifolieae tribe indicating the potential of translational approaches to improving our understanding of the fenugreek genome. Using such a comprehensively-genotyped fenugreek population is the first step towards identifying genes underlying complex traits and to underpin fenugreek marker-assisted breeding programs.

Accumulation of amino acids and flavonoids in hairy root cultures of common buckwheat (Fagopyrum esculentum).

Transformed hairy root culture in common buckwheat (Fagopyrum esculentum Moench Rubra cultivar) was investigated for accumulation of amino acids and specific flavonoids. Leaves and stems of F. esculentum were used a starting material for induction of hairy roots via the Agrobacterium rhizogenes A4 strain. The transformed lines were confirmed by PCR detection of rol B gene, and their capability to continuously form hairy roots. Three lines from each explant types depending upon growth kinetics were observed. The hairy root lines were used to measure the contents of 17 amino acids and 3 flavonoids. Overall, the hairy root lines exhibited elevated accumulation of semi-essential amino acids such as lysine, isoleucine, valine, histidine and phenylalanine. Content of proline was increased 3-5 times, likely due to the biotic stress reaction induced with A. rhizogenes. Determination of flavonoids by high-performance liquid chromatography, hesperidine and kaempferol-3-rutinoside, were accumulated in hairy root cultures and didn't detected in non-transformed root. The increase in flavonoids positively correlated with the antioxidant capacity of the hairy root cultures.

Determination of lignans, phenolic acids and antioxidant capacity in transformed hairy root culture of Linum usitatissimum.

Hairy root culture is a promising alternative method for the production of secondary metabolites. In this study, transformed root of Linum usitatissimum was established using Agrobacterium rhizogenes A4 strain from root cultures for lignans, phenolic acids and antioxidant capacity determination. Total lignin content (secoisolariciresinol diglucoside, secoisolariciresinol and matairesinol) was 55.5% higher in transformed root cultures than in the non-transformed root culture. Secoisolariciresinol was detected in higher concentration (2.107 µmol/g DM) in the transformed root culture than non-transformed culture (1.099 µmol/g DM). Secoisolariciresinol diglucoside and matairesinol were exclusively detected in the transformed root culture, but were not found in the non-transformed root culture. The overall production of phenolic acids in transformed roots was approximately 3.5 times higher than that of the corresponding non-transformed culture. Free radical scavenging DPPH˙ and ABTS˙+ assays showed 2.9-fold and 1.76-fold higher anti-oxidant activity in transformed root culture as compared to non-transformed.

Enhancement of silymarin and phenolic compound accumulation in tissue culture of Milk thistle using elicitor feeding and hairy root cultures.

In the present study, the effects of the metabolite elicitors chitosan, methyl jasmonate (MeJA) and salicylic acid (SA) as well as the hairy root transformation were tested for silymarin and phenolic compound accumulation in in vitro cultures of Milk thistle. For callus induction, leaf explants were cultured on MS medium supplemented with 5 mg/l NAA + 2 mg/l Kin + 0.1 mg/l GA3. Chitosan, SA and MeJA were added separately in three concentrations 200, 400 and 800 mg/l; 10, 20 and 40 mg/l; 20, 40 and 80 mg/l, respectively, to hormone free B5 medium. Alternatively, cotyledons of 12 day old seedlings were transformed with Agrobacterium rhizogenes A4 strain. Overall, increasing the concentrations of the three elicitors dramatically increased the total silymarin content. Remarkably, the elicitors mainly enhanced the accumulation of silybine A&B that were not detected in un-treated callus culture (control). In addition, the hairy root culture triggered the accumulation of silybine A&B, and silydianin, which was not detected in the non-transgenic roots. The hairy root culture was superior in production of the phenolic compounds in comparison to the control and elicitor treatments. The hairy root cultures showed also higher antioxidant capacities than non-transformed cultures and/or chemically elicited-callus cultures. Thus hairy root provide instrumental in enhancing the production of economically valuable metabolite.

Effects of different encapsulation agents and drying process on stability of betalains extract.

Red beet plants are rich in betalains that can be used as food natural colorants. Betalains were extracted from red beet and encapsulated with different carrier agents and freeze or spray dried. Effect of different encapsulating agents as maltodextrin, guar gum, gum Arabic, pectin and xanthan gum with different concentration (as encapsulating agents) were studied on the betalain stability. Encapsulated betalains with xanthan gum with maltodextrin showed about 65 % more recovery than the control. Encapsulation showed a higher recovery of betalains during freeze drying by 1.3 times than during spray drying. Spray dried samples has L* (lightness) higher than the freeze dried samples. The variations of maltodextrin with xanthan and guar gum freeze dried have highest chroma value of 21. The stabilization of pure betalain pigments may boost the use of these colouring molecules in the food industry and promote their application.