Non-transformed Roots Research Articles

Application of AtMYB75 as a reporter gene in the study of symbiosis between tomato and Funneliformis mosseae.

Composite plants containing transgenic hairy roots produced with Agrobacterium rhizogenes-mediated transformation have become an important method to study the interaction between plants and arbuscular mycorrhizal fungi (AMF). Not all hairy roots induced by A. rhizogenes are transgenic, however, which leads to requirement of a binary vector to carry a reporter gene to distinguish transgenic roots from non-transformed hairy roots. The beta-glucuronidase gene (GUS) and fluorescent protein gene often are used as reporter markers in the process of hairy root transformation, but they require expensive chemical reagents or imaging equipment. Alternatively, AtMYB75, an R2R3 MYB transcription factor from Arabidopsis thaliana, recently has been used as a reporter gene in hairy root transformation in some leguminous plants and can cause anthocyanin accumulation in transgenic hairy roots. Whether AtMYB75 can be used as a reporter gene in the hairy roots of tomato and if the anthocyanins accumulating in the roots will affect AMF colonization, however, are still unknown. In this study, the one-step cutting method was used for tomato hairy root transformation by A.rhizogenes. It is faster and has a higher transformation efficiency than the conventional method. AtMYB75 was used as a reporter gene in tomato hairy root transformation. The results showed that the overexpression of AtMYB75 caused anthocyanin accumulation in the transformed hairy roots. Anthocyanin accumulation in the transgenic hairy roots did not affect their colonization by the arbuscular mycorrhizal fungus, Funneliformis mosseae strain BGC NM04A, and there was no difference in the expression of the AMF colonization marker gene SlPT4 in AtMYB75 transgenic roots and wild-type roots. Hence, AtMYB75 can be used as a reporter gene in tomato hairy root transformation and in the study of symbiosis between tomato and AMF.

In Vitro and in Silico Insights on the Biological Activities, Phenolic Compounds Composition of Hypericum perforatum L. Hairy Root Cultures

Three Hypericum perforatum hairy root lines (HR B, HR F and HR H) along with non-transformed roots were analyzed for phenolic compounds composition and in vitro enzyme inhibitory properties. In silico molecular modeling was performed to predict the interactions of the most representative phenolic compounds in HR clones with enzymes related to depression, neurodegeneration and diabetes. Chromatographic analyses revealed that HR clones represent an efficient source of quinic acid and hydroxybenzoic acids, epicatechin and procyanidin derivatives, quercetin and kaempferol glycosides, as well numerous xanthones. In vitro antidepressant activity of HR extracts through monoamine oxidase A (MAO-A) inhibition was attributed to the production of oxygenated and prenylated xanthones. The neuroprotective potential of HR extracts was related to the accumulation of quercetin 6-C-glucoside, epicatechin, procyanidins and γ-mangostin isomers as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Vanillic acid and prenylated xanthones in HR clones as promising inhibitors of tyrosinase additionally contributed to the neuroprotective activity. Five preeminent xanthones in HR (γ-mangostin, mangiferin, garcinone C, garcinone E and 1,3,7-trihydroxy-6-metoxy-8-prenyl xanthone) along with the flavonol quercetin 6-C-glucoside effectively inhibited α-amylase and α-glucosidase indicating the antidiabetic properties of HR extracts. Transgenic roots of H. perforatum can be exploited for the preparation of novel phytoproducts with multi-biological activities.

Establishment of Gypsophila paniculata root culture for biomass, saponin, and flavonoid production

Baby’s breath (Gypsophila paniculata L.) roots are valuable as pharmaceuticals due to the content of triterpenoid saponins and other bioactive phytochemicals. However, the long root harvest period and fluctuation of these constituent’s content are among the constraints to traditional agricultural production. The present study shows an efficient and reliable liquid root culture of G. paniculata cv. ‘Perfecta’ established in a shake flask system using different auxins, media, and sugars. Hairy root (HR) induction in G. paniculata through Rhizobium rhizogenes-mediated transformation was also investigated. α-Naphthaleneacetic acid (NAA) was optimized for adventitious roots (AR) biomass, saponin, and flavonoid production at 1 or 2 mg/L compared to IBA. Full strength Gamborg’s medium (B5) recorded higher saponin content; however, the highest yield of total saponin and total flavonoids was achieved by full strength Murashige and Skoog's (MS) medium. Sucrose was more essential for root growth and accumulated total saponins and flavonoids rather than fructose and glucose. Polymerase chain reaction (PCR) analysis showed that G. paniculata HR carried rolC gene of R. rhizogenes A4 strain in its genome but not virD2 gene. Compared to non-transformed root, saponin content of leaf and stem-derived HR was 2.7 and 2.3-fold, while total flavonoid content was 2.1 and 2.0-fold, respectively. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was also higher in HR extracts than in non-transgenic roots. This study established an efficient protocol for G. paniculata root cultures for sustainable production of important natural saponins and flavonoids.

Auxin sensitivity improves production of rosmarinic acid in transformed hairy roots of <em>Lavandula angustifolia</em>

Hairy root culture is a promising approach to improve production of plant secondary metabolites. The genes, which are located in T-DNA of a root-inducing plasmid, regulate auxin sensitivity of hairy roots. Therefore, this study was aimed to improve the growth and rosmarinic acid production of Lavandula angustifolia hairy roots. Lateral branches of hairy roots were transferred to ½ MS and ½ B5 liquid media. To assess auxin sensitivity, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with four different concentrations (0, 0.1, 0.5 and 1 mg/l) were also applied. The growth of hairy roots in ½ MS medium was two-fold higher than in ½ B5 medium. In addition, both auxins were found to significantly improve the growth of hairy roots whereas non-transformed roots stopped growing in the presence of the auxins. The highest dry weight and rosmarinic acid production of hairy roots were obtained from ½ MS medium supplemented with IBA irrespective of its concentration. As a result, the hairy roots grown in ½ MS medium supplemented with IBA produced the maximum amount of rosmarinic acid (7.98 mg/g dry weight of hairy roots). This first report of rosmarinic acid production in L. angustifolia hairy roots provides new insights into the auxin sensitivity of L. angustifolia hairy roots.

Phytohormone profiles in non-transformed and AtCKX transgenic centaury (Centaurium erythraea Rafn) shoots and roots in response to salinity stress in vitro

Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations (0, 50, 100, 150, 200 mM). The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content.

A Comparative Study of Apigenin Content and Antioxidant Potential of CosmosBipinnatus Transgenic Root Culture

Background: Flavonoid-derived components have been studied for their therapeutic properties. Objectives: Apigenin has shown remarkable antioxidant and anti-inflammatory features, so we should have a reliable source of apigenin. Methods: In this study, we used high-performance liquid chromatography method to compare the amount of apigenin in flower, root, leaf, and stem of three varieties of osmos bipinnatus, i.e., ‘Dazzler,’ ‘Xanthos,’ ‘Sensation Pinkie’, and in transgenic root culture of C. bipinnatus ‘Dazzler’. Besides, the antioxidant activity of C. bipinnatus ‘Dazzler’ transgenic root culture was evaluated using Ferric Reducing Antioxidant Power (FRAP) assay. Results: Dazzler variety flowers showed the highest recovery of apigenin with 0.799 mg/100 mg Dry Weight (DW). However, the Sensation pinkie variety leafs had the lowest recovery with 0.089 mg/100mg. Apigenin content in transformed roots (0.797 mg/100 mg DW) of C. bipinnatus ‘Dazzler’ was significantly higher than non-transformed roots (0.42 mg/100 mg DW). The ethanolic extract of hairy root showed the FRAP value of 668.1 µM Fe2+/mg that was comparatively more than the wild root FRAP value (426.2 µM Fe2+/mg). Conclusion: In conclusion, the presence of apigenin in high amounts in hairy root cultures of C. bipinnatus ‘Dazzler’ indicates its great potential for the future pharmaceutical industry.

Preliminary Phytochemical Analysis and Evaluation of the Biological Activity of Leonotis nepetifolia (L.) R. Br Transformed Roots Extracts Obtained through Rhizobium rhizogenes-Mediated Transformation.

According to the present knowledge, this is the first report on establishing transformed root cultures of Leonotis nepetifolia after Rhizobium rhizogenes-mediated transformation. The preliminary phytochemical analysis showed differences in the content of phenols and flavonoids in transformed and nontransformed roots. The dominant compounds in the analyzed extracts were (+)-catechin (5464 and 6808 µg/g DW), p-coumaric acid (2549 and 4907 µg/g DW), m-coumaric acid (1508 and 2048 µg/g DW) and rosmarinic acid (1844 and 2643 µg/g DW) for nontransformed (LNNR) and transformed (LNTR4) roots, respectively. Initial biological studies carried out on LNNR, and LNTR4 extracts showed a cytotoxic effect on the A549 lung, HCC1937 breast and leukemia NALM-6 cell lines, antioxidants, as well as repair and protection against DNA damage induced by H2O2 in HUVEC cells. Due to the stronger effect of the LNTR4 root extract, which can be a relatively efficient and cheap source of bioactive secondary metabolites, further biological analyses are needed to discover in detail their potentially valuable biological properties.

Effects associated with insertion of rol genes on morphogenic potential in explants derived from transgenic Bacopa monnieri (L.) Wettst

The present study deals with the establishment of rolA-transgenic and rolB-transgenic plants for the first time through Agrobacterium tumefaciens mediated transformation, exploiting the inherent morphogenic potential of an important medicinal plant, Bacopa monnieri (L.) Wettst. The rolA-transgenic and rolB-transgenic plants showed integration and expression of rolA and rolB genes respectively, whereas Ri-transformed plants showed integration and expression of rolA, rolB, rolC and rolD genes. Morphogenic potential of different types of explants derived from rolA-transgenic, rolB-transgenic and Ri-transformed plants on basal medium was evaluated. Shoot organogenesis was enhanced significantly in leaf (1.6-fold) and internode (1.4-fold) explants derived from rolA-transgenic plants, rolB-transgenic leaf (2.4-fold) and internode (1.6-fold) explants as well as leaf (5.2-fold) and internode (3.3-fold) explants derived from Ri-transformed plants compared to explants from non-transformed plants. Substantial increase in root organogenesis was also noticed in rolA-transgenic leaf (1.7-fold) explants, rolB-transgenic leaf (3.6-fold) and internode (1.4-fold) explants as well as leaf (4.1-fold) and internode (1.9-fold) explants derived from Ri-transformed plants compared to non-transformed ones. In addition to this, growth of root tip and shoot regeneration was also noticed from Ri-transformed root explants, but not in rolA-transgenic, rolB-transgenic and non-transformed roots. Clones of each transgenic plant line harboring rol genes depicted notable phenotypic changes including reduced shoot and internode length, increased number of nodes/plant, leaves/plant and roots/plant. The leaf morphology was altered in rolB-transgenic and Ri-transformed plants but not in rolA-transgenic plants.

Induction of Hairy Roots and Phenolics Biosynthesis of Genetically-Transformed Brassica campestris L

Brassica campestris could be used for medicinal purposes. The plant is rich in flavonoids and phenolics that have potential for pharmacological purposes. As such, enhancement of secondary metabolites in Brassica campestris plants is highly recommended. Therefore, the aim of this study is to obtain cultures of hairy roots of Brassica campestris, which has undergone a bacterial transformation using Agrobacterium rhizogenes in an attempt to enhance the production of secondary metabolites. The level of expression of root inducing gene in transformed hairy roots was estimated using quantitative real-time PCR analysis. Results revealed higher root inducing gene expression levels in the transgenic plants as compared to the non–transformed roots. Transformed hairy roots showed higher increases in the total phenolic content, as compared to non-transformed roots. This study indicated the enhanced secondary metabolites biosynthesis by hairy roots induction in Brassica campestris plants.

Effect of different Agrobacterium rhizogenes strains for in-vitro hairy root induction, total phenolic, flavonoids contents, antibacterial and antioxidant activity of (Cucumis anguria L.)

The present study aimed to investigate the effect of different Agrobacterium rhizogenes on the induction of hairy root of Cucumis anguria and determine its total phenolic, flavonoids contents, antibacterial and antioxidant activity. In this investigation A. rhizogenes strains such as, 15834, 13333, A4, R1200, R1000, LBA9402, R1301 and R1601 are all investigated, were developing hairy root conception in cotyledon and leaf tissue explants. Polymerase chain response (PCR) and the converse transcription-PCR are transgenic clones of hairy roots has been utilized rolA and rolB particular primers. In the middle of the different attention of better regulators the extreme transformation frequency was achieved in (IBA + NAA) cotyledon explant. Transgenic hairy roots increase in MS liquid medium added to with IBA + NAA (2.46 + 1.07) displayed the maximum accumulation of biomass 0.68 g/l dry weight (DW) and 6.52 ± 0.49 g/l fresh weight (FW) were obtained at the 21 days of cotyledon explant. The flavonoid and total phenolic contents were estimated using aluminium chloride method and Folin-Ciocalteu method. The amount of phenolic compounds in Cucumis anguria L non transformed root (124.46 ± 6.13 mg GA/g) was lower than that in the methanol extracts of Cucumis anguria L. hairy roots (160.38 ± 5.0 mg GA/g), being was Cucumis anguria L non transformed root lower (42.93 ± 1.58 mg rutin/g) than that in the concentration of total flavonoids in Cucumis anguria L. hairy root (16.26 ± 1.84 mg rutin/g). Additionally, transgenic hairy roots professionally produced various phenolic and flavonoid composites. The total antimicrobial activity, phenolics, flavonoids content and antioxidant were more in the hairy roots related to non-transformed roots. In our discovery, the A. rhizogenes R1000 is promising candidate for hairy root initiation of C. anguria from cotyledone explants were realized large number of hairy roots compared with leaf explants. The antioxidant potential of methanol extracts of flavonoid and phenolic compounds from the hairy roots have great potential to treat various diseases.

Evidences for Preferential Localization of Cytokinin Receptors of Potato in the Endoplasmic Reticulum

Cytokinins, classical plant hormones, are recognized by the cell through receptors—hybrid sensor histidine kinases, multidomain transmembrane proteins. The character of the hormonal effect on the cell and the mode of the hormonal signal transduction to the primary targets depends on the subcellular localization of the receptors. Recently, we cloned and investigated a family of cytokinin receptors from a new model plant, potato Solanum tuberosum L. Two typical representatives of this family, StHK2a and StHK4b, were selected to study their subcellular localization. In the current study, total membranes isolated from tobacco leaves, in which the selected recombinant potato receptors fused to GFP-reporter were transiently expressed, were fractionated by aqueous two-phase partitioning. Two membrane fractions were obtained, one tentatively enriched with endoplasmic reticulum (ER) membranes and the other, with the plasma membrane (PM). The content of StHK2a and StHK4b in each fraction was determined by the radioligand method. Both receptors were detected in the two fractions, with a slight predominance of StHK4b in the ER-enriched fraction. Given that the content of ER membranes in the cell is markedly higher than the content of the PM, the bulk of the receptors should be attributed to the ER. Specific cytokinin-binding sites were detected in the analogous membrane fractions isolated from non-transformed potato roots. Although the specific content of the cytokinin-binding sites was 1.8-fold higher in the PM, here also, the main part of these sites, i.e., cellular receptors, still resided in the ER. In order to find out whether the receptors in the ER are functional, a recently proposed pH criterion was applied. According to this criterion, potato cytokinin receptors, like their orthologs from other species, seem to be tuned for functioning in the ER but not in the PM. The results of molecular modeling of the dimerization interface of the receptors at different pH values also evidenced in favor of the ER. Thus, the main site of subcellular localization of the functionally active potato cytokinin receptors is the ER, although a certain amount of the receptors resides within the PM, too. The question of the functional role of PM-located receptors remains open.

Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets.

Ten Hyptis suaveolens hairy root lines were established by infecting nodal explants with K599+pGus-GFP+ and ATCC15834+pTDT strains from Agrobacterium rhizogenes. Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth in absence of growth regulators. Cytotoxicity was determined using the sulforhodamine B method, and the production of podophyllotoxin (PTOX) was measured by high performance thin layer chromatography scanning. Through these methodologies, HsTD10 was identified as the hairy root line with the highest cytotoxicity and PTOX production, which was corroborated by liquid chromatography-mass spectrometry and micrOTOF-Q II. A suspension culture of HsTD10 was established in which PTOX and carbohydrate consumption during growth kinetics were quantified by high-performance liquid chromatography. Procedures to increase the production and retrieval of PTOX in the HsTD10 line included selection of culture medium, addition of thiamine, and modification of the PTOX extraction method. The best combination of these variables was MS medium at 75% of its components with the addition of 2 mg L-1 of thiamine, extraction with methanol-dichloromethane, and sonication at 40 ± 5°C. During kinetics, growth-associated PTOX accumulation was observed. The specific growth rate (μ) was 0.11 d-1. The highest concentration of PTOX obtained with HsTD10 (5.6 mg g-1 DW) was 100 times higher than that reported for roots of wild plants and 56 times higher than that for in vitro nontransformed roots of H. suaveolens.

State of antioxidant systems and phenolic compounds’ production in Hypericum perforatum L. hairy roots

Hypericum perforatum hairy root clones (HR A–HR O) transformed with Agrobacterium rhizogenes A4 were evaluated for growth characteristics, phenolic compounds production, antioxidant/radical scavenging activity, antioxidant enzymes, and oxidative stress markers. The screening of growth characteristics revealed that HR clones were characterized with higher biomass accumulation, root elongation, and lateral branching in comparison to non-transformed roots. Significant increase of phenolics production in HR clones was related to phenylalanine ammonia lyase activity indicating an up-regulation of phenylpropanoid/flavonoid metabolism. Positive correlation of phenolics, flavonoids, and tannins with antioxidant assays indicated that these secondary metabolites significantly contributed to the antioxidant/radical scavenging properties of HR cultures. Regarding the enzymatic antioxidant state, an enhancement of superoxide dismutase activity in HR lines coincided with decrease in O 2 •− production rate, while ascorbate peroxidase and catalase greatly contributed to the reduction of H2O2 levels. The substantial accumulation of malondialdehyde in HR clones indicated an efficiency of antioxidant enzymes to reduce O 2 •− and H2O2 at levels that are not deleterious for membrane lipids. These results confirmed the involvement of an efficient antioxidant defense system in the response adjustment of H. perforatum HR cultures to transformation process. Two superior clones denoted as HR B and HR F were selected as fast-growing and high biomass yielding lines with up-regulated phenolic compounds’ production, antioxidant, and radical scavenging activity. Altogether, the best performing H. perforatum HR clones could be proposed as a prospective biotechnological system for scale-up production of antioxidant phenolic compounds.

Evaluation of Polyphenolic Compounds and Pharmacological Activities in Hairy Root Cultures of Ligularia fischeri Turcz. f. spiciformis (Nakai).

A considerable amount of bioactive compounds have been used for the biopharmaceutical engineering to help human health and nutrition. Hairy root culture (HRC) or transgenic root is a favourable alternative technique for phytochemical production. Ligularia fischeri is a significant source of pharmaceutically important active compounds with an enormous range of health care applications. HRC of L. fischeri was developed using Agrobacterium rhizogenes for the production of polyphenolic compounds with antioxidant, antimicrobial, antidiabetic, anticancer and anti-inflammatory pharmaceutical activities. Hairy roots (HRs) were selected by morphological assessment, genetic and molecular analyses. The maximum accumulation of fresh mass (94.15 g/L) and dry mass (9.45 g/L) was recorded in MS liquid medium supplemented with 30 g/L sucrose at 28 days. Furthermore, HRs successfully produced numerous polyphenolic compounds, including six hydroxycinnamic acids, seven flavonols, seven hydroxybenzoic acids, vanillin, resveratrol, pyrogallol, homogentisic, and veratric acids, which were identified by UHPLC analysis. HRs produced higher total phenolic (185.65 mg/g), and flavonoid (5.25 mg/g) contents than non-transformed roots (125.55 mg/g and 3.75 mg/g). As a result of these metabolic changes, pharmaceutical activities were found higher in HRs than non-transformed roots (NTRs). The present study indicates that HRC has the potential to increase the content of beneficial polyphenolic compounds with higher potential pharmaceutical activities. To the best of our knowledge, the present study is the first report on enhancing the production of polyphenolic compounds with pharmaceutical activities from the HRCs of L. fischeri.

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.

Hairy root cultures of Salvia viridis L. for production of polyphenolic compounds

Salvia viridis is a containing bioactive phenols plant, which has been used in Turkish medicine. The aim of the study was to establish hairy root culture of the species in order to increase the production of the compounds with pharmacological activity. Transformed roots are promising biotechnological systems that produce great amounts of secondary metabolites; this is attributed to their fast growth and their genetic and biosynthetic stability. Stable hairy root lines of S. viridis were established from shoot explants using Agrobacterium rhizogenes strain A4. The highest frequency of transformation (45%) was achieved when the shoot was inoculated at the node or internode by bacteria cultivated on Yeast/Mannitol/Broth (YMB) medium supplemented with acetosyringone. The transformation of the root clones was confirmed by polymerase chain reaction using aux1, aux2, rolB and rolC primers. Of the five obtained transformed root clones, the highest biomass was achieved for clone K3 (13.63 ± 0.5 g/L after 5 weeks) grown in Woody Plant (WP) medium in darkness. The UPLC-PDA-ESI–MS analysis of hydromethanolic extracts of transformed roots of S. viridis revealed the presence of 10 compounds identified as caffeic acid derivatives. Quantitative analysis showed that rosmarinic acid (RA) was the predominant compound in all clones. The highest RA (35.8 mg/g dry weight) and total polyphenol (41.24 mg/g dry weight) content were evaluated in clone K3 cultured in WP medium. These values were 8-fold higher than those of the non-transformed roots, indicating that hairy root cultivation could be a promising technique for the production of valuable compounds from S. viridis.

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.

Growth kinetics and withanolide production in novel transformed roots of Withania somnifera and measurement of their antioxidant potential using chemiluminescence

Markedly increased withanolide content was found in transformed roots (TR) of Withania somnifera germplasm grown in low mineral minimal media and withanolides showed high antioxidant potential when analysed using acidic potassium permanganate chemiluminescence. Transformation frequency of explants infected with Agrobacterium rhizogenes strain A4 varied between the three germplasms tested with the highest observed as 75 ± 0.9. Transformed root production was explant specific with leaves being the most productive among the different explants used. Withanolides, namely withaferin A, withanolide A, withanolide B and 12-deoxywithastramonolide were detected in TR cultures and differences in their content were found between germplasms. The highest concentrations of secondary metabolites were found in 4-week-old cultures and concentrations declined by the 8th and 12th week of culture. In 4-week-old cultures, the biomass of TR cultures was 4.5 fold higher than their respective non-transformed roots (NTR). Withaferin A was found in TR at levels that were 28–34 times higher than that found in NTR. A rapid method for the determination of the antioxidant potential of W. somnifera TR extracts was developed using post-column acidic potassium permanganate chemiluminescence (APPC) detection. The APPC chromatographic peaks for extract constituents showed strong alignment with those found for ultraviolet absorbance detection. The methods developed in this study for TR culture establishment and the use of a fast and sensitive way for the qualitative and quantitative determination of the antioxidant activity of their metabolites provides a new platform that will have use for similar studies in other species.

Study of artemisinin and sugar accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures

ABSTRACTWe studied the effect of genetic transformation on biologically active compound (artemisinin and its co-products (ART) as well as sugars) accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures. Glucose, fructose, sucrose, and mannitol were accumulated in A. vulgaris and A. dracunculus “hairy” root lines. Genetic transformation has led in some cases to the sugar content increasing or appearing of nonrelevant for the control plant carbohydrates. Sucrose content was 1.6 times higher in A. vulgaris “hairy” root lines. Fructose content was found to be 3.4 times higher in A. dracunculus “hairy” root cultures than in the control roots. The accumulation of mannitol was a special feature of the leaves of A. vulgaris and A. dracunculus control roots. A. vulgaris “hairy” root lines differed also in ART accumulation level. The increase of ART content up to 1.02 mg/g DW in comparison with the nontransformed roots (up to 0.687 mg/g DW) was observed. Thus, Agrobacterium rhizogenes-mediated genetic transformation can be used for obtaining of A. vulgaris and A. dracunculus “hairy” root culture produced ART and sugars in a higher amount than mother plants.

Genetic Transformation and Hairy Root Induction Enhance the Antioxidant Potential of Lactuca serriola L.

Lactuca serriola L. is a herbaceous species, used for human nutrition and medicinal purposes. The high antioxidant capacity of L. serriola indicates the possibility of enhancing its edible and health potential by increasing the flavonoid and phenolic contents. The present study aimed at enhancing the production of phenolics and flavonoids by hairy root cultures in Lactuca serriola transformed with Agrobacterium rhizogenes strain AR15834 harbouring the rolB gene. The genetic transformation of rolB in transformed roots was validated, and rolB expression level was evaluated using real-time qPCR analysis. Expression levels of flavonoid biosynthesis genes (CHI, PAL, FLS, and CHS) were assessed in the hairy and nontransformed roots. Results showed higher expression levels in the transgenic roots than in the nontransformed ones (p < 0.01). Transgenic hairy roots exhibited a 54.8–96.7% increase in the total phenolic content, 38.1–76.2% increase in the total flavonoid content, and 56.7–96.7% increase in the total reducing power when compared with the nontransgenic roots (p < 0.01). DPPH results also revealed that the transgenic hairy roots exhibited a 31.6–50% increase in antioxidant potential, when compared to normal roots. This study addressed the enhancement of secondary metabolite biosynthesis by hairy root induction in L. serriola.