Hairy Root Clones Research Articles

Development of an efficient selection procedure for high imperatorin-yielding hairy root clones of Urena lobata L. using morphological and molecular markers

Development of an efficient selection procedure for high imperatorin-yielding hairy root clones of Urena lobata L. using morphological and molecular markers

Metabolic engineering of hairy root cultures in Beta vulgaris for enhanced production of vanillin, 4-hydroxybenzoic acid, and vanillyl alcohol.

The flavor of vanilla is a complex blend of compounds, with vanillin as the most prominent, along with vanillyl alcohol and 4-hydroxybenzoic acid. Natural vanillin extracted from vanilla beans is expensive, so researchers use heterologous synthesis to produce nature-identical vanillin in plant hosts. Consequently, alternative traditional farming and gathering methods are required to bridge the significant disparity between supply and demand. The current research successfully developed a method to induce hairy root formation from leaves. It integrated the Vanillin synthase (VpVAN) gene into transgenic hairy root lines of Beta vulgaris, synthesizing vanillin-related compounds. The presence of the VpVAN gene in transgenic roots was confirmed using PCR analysis. Additionally, RT-qPCR analysis demonstrated the expression of the VpVAN gene in the transgenic root lines. The transgenic hairy root clones H1, H2, and H5 showed enhanced vanillin production, vanillyl alcohol, and 4-hydroxybenzoic acid. Elicitation with methyl jasmonate (MJ) and salicylic acid (SA) further improved the production of these compounds in B. vulgaris hairy roots. The maximum hairy root biomass was observed after 60 days, with the maximum synthesis of vanillin and 4-hydroxybenzoic acid obtained from hairy root clones H5 and HR2, respectively. Vanillyl alcohol HR2 was obtained on the 45th day of cultivation. Elicitation with wound-associated hormone methyl jasmonate and salicylic acid enhanced the yield of vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid, with a 215-fold increase in vanillin, a 13-fold increase in vanillyl alcohol, and a 21 fold increase in 4-hydroxybenzoic acid. The study results indicate that establishing transgenic hairy root cultures with the VpVAN gene is a promising alternative method for enhancing the production of vanilla flavor compounds such as vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid. A cost-effective protocol has been developed to mass-produce phenolic compounds using a hairy root culture of B. vulgaris. This approach addresses the increasing demand for these substances while reducing the cost of natural vanillin production, making it suitable for industrial-scale applications.

Efficient production and enhanced accumulation of Valerenic acid in Valeriana officinalis: Early identification of high-performing hairy root clones and Pioneering use of hydrogen peroxide as an elicitor

Valeriana officinalis (valerian) roots and rhizomes possess a long history of medicinal use due to their sedative, antiepileptic, and anticonvulsant properties. Valerenic acid, a bioactive sesquiterpene with therapeutic potential, is present in limited quantities within these tissues. This study explores the application of hairy root cultures for enhanced valerenic acid production. Hairy root induction was attempted on valerian leaves and petioles using three Rhizobium rhizogenes strains (ATCC15834, A4, and MSU440) across three culture media (Murashige and Skoog (MS), Gamborg's B5 (B5), and Schenk and Hildebrandt (SH)). A non-destructive imaging system and periodic analyses were employed to identify superior hairy root clones exhibiting increased branching frequency. Leaf explants co-cultured with R. rhizogenes strain ATCC15834 yielded the most promising clones, characterized by the highest dry weight (1.03 mg) and valerenic acid content (0.384 mg/g dry weight) when grown in a half-strength SH liquid medium. Following strain and media optimization, the impact of 50 mM hydrogen peroxide as an elicitor on valerenic acid production was investigated. This treatment resulted in a significant 1.76-fold increase in valerenic acid accumulation compared to the control group at the first day post-treatment. This approach presents a valuable strategy for the early identification of high-yielding hairy root lines. Moreover, the utilization of hydrogen peroxide, a safe and cost-effective elicitor, offers a rapid method for enhancing valerenic acid production in the selected superior clone. This study establishes a promising platform for the sustainable production of valuable plant compounds within both research and industrial settings.

Non-Enzymatic and Enzymatic Antioxidant Responses of Hypericum perforatum L. Hairy Roots upon Photooxidative Stress

The aim of this study was to evaluate the non-enzymatic and enzymatic antioxidant response of fifteen Hypericum perforatum L. dark-grown (HR1 A-HR1 O) and photoperiod-exposed (HR2 A-HR2 O) hairy root clones. Dark-grown HR1 clones were characterized with high biomass accumulation and up-regulated phenylpropanoid metabolism through phenylalanine ammonia lyase (PAL)-mediated production of flavonoids, flavan-3-ols and anthocyanins. These groups of phenolics showed superior hydrogen-donating capability and significantly contributed to the antioxidant capacity of dark-grown HR1 clones. Photoperiod-exposed HR2 clones showed green coloration with shoot regenerative potential and reduced biomass accumulation. Photoperiod exposition improved the production of hypericins, as well as ferrous chelating properties and lipid peroxidation inhibition activity in HR2 cultures. Furthermore, HR2 clones were represented with enhanced activity of antioxidant enzymes (guaiacol peroxidase, catalase and superoxide dismutase) that resulted in suppression of oxidative stress markers (hydrogen peroxide, superoxide anion and malondialdehyde). These observations revealed the involvement of an efficient antioxidant defense system in the adaptive response of HR to photooxidative stress. Altogether, photoperiod-exposed H. perforatum HR2 clones were considered as a promising alternative for further scale-up production of naphthodianthrones that could be used in the pharmaceutical industry.

Improving yield of a recombinant biologic in a Brassica hairy root manufacturing process.

Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha‐ l‐iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4‐ d + 20.5 g/l KNO3) resulting in 3.4 fold eGFP secretion when compared to the non‐induced control. These optimized conditions were applied to the IDUA‐expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A > 150‐fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non‐induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.

Remediation of endosulfan-contaminated water by hairy roots: removal and phytometabolization assessment

Although many countries banned the insecticide endosulfan, it is still an environmental pollutant. Plants metabolize the two diastereomers of the formulations known as technical grade endosulfan (TGE) by two phase I pathways: hydrolysis leading to less toxic derivatives and oxidation giving endosulfan sulfate which is as toxic as endosulfan itself. We assessed the removal, bioaccumulation and phase I metabolization of TGE from water matrices using hairy root clones (HRs) of three edible species, Brassica napus, Raphanus sativus and Capsicum annuum. B. napus and C. annuum HRs removed 86% of TGE from the bioreaction media in 2 and 96 h, respectively, whereas R. sativus HRs removed 91% of TGE within 6 h of biotreatment. In the experiments with B. napus, only endosulfan sulfate was detected in both biomass and medium, whereas R. sativus and C. annuum accumulated endosulfan sulfate and endosulfan alcohol. Besides, endosulfan lactone was detected in C. annuum reaction medium. Acute ichthyotoxicity assays toward Poecilia reticulata showed that media contaminated with TGE lethal levels did not produce mortality after the phytotreatments. This research highlights the feasibility of using HRs to evaluate plant enzymatic abilities toward xenobiotics and their potential for the design of ex situ decontamination processes.

Harnessing the versatility of diverse pentacyclic triterpenoid synthesis through hairy root cultures of various Ocimum species: An unprecedented account with molecular probing and up-scaling access

Biosynthesis of two pharmaceutically and nutraceutically active Pentacyclic-triterpenes (PCTs), namely Corosolic [CA] and Arjunic [AA] acids, had been first time achieved in the hairy root (HR) clones of four Ocimum species (O. basilicum, O. grattisimum, O. kilimandscharicum and O. sanctum), contrasting to their absence in the respective controls. Additionally, compared with each in-vivo controls, their respective HR clones revealed enhanced synthesis of four more PCTs, (i.e., Ursolic acid [UA], Oleanolic acid [OA], Maslinic acid [MA], and Betulinic acid [BA]), in growth dependent manner with notable inter-species yield variations. Real-time analysis revealed noticeable up-regulations of functionally-defined hydroxylase and oxidase genes of CYP716 family in the HR clones over that of their parents, which postulates their possible involvements in the synthesis of CA & AA plus increased levels of the other PCTs in the HR clones. Bioreactor up-scaling further revealed rewarding potentials of the HR technology in Ocimum for synthesis of such distinctive PCTs of immense industrial applications.

Transcriptome-wide identification and expression pattern analysis for Dof gene family in Panax ginseng from Jilin

Dof(DNA binding with one finger), a unique class of transcription factors in plants, play an important role in seed development, tissue differentiation, and metabolic regulation. To identify the number and function of Dof gene family members in Panax ginseng, this study identified the members of Dof gene family in P. ginseng and systematically analyzed their structures, evolution, functional differentiation, expression patterns, and interactions using bioinformatics methods at the transcriptome level. At the same time, the association analysis of Dof genes from P. ginseng with key enzyme genes for ginsenoside synthesis was carried out to screen the candidate PgDof genes involved in the regulation of ginsenoside biosynthesis. The results showed that there were 54 genes belonging to the Dof gene family in P. ginseng from Jilin. All PgDof genes had Zf-Dof conserved motifs, implying that they were evolutionarily conserved and could be divided into five groups. Expression pattern analysis confirmed that the expression of PgDof gene family members in different tissues, different year-old P. ginseng, and different farm varieties varied significantly. Simultaneously, as revealed by "gene-saponin content" and "gene-gene" linkage analysis, an important candidate PgDof14-1 gene involved in the regulation of ginsenoside biosynthesis was obtained. From the established genetic transformation system of this gene in the hairy roots of P. ginseng, a positive hairy root clone was determined. This study has laid a theoretical foundation for the study of Dof gene family in P. ginseng.

Establishment of hairy root culture of Rubia yunnanensis Diels: Production of Rubiaceae-type cyclopeptides and quinones

Rubia yunnanensis is an important medicinal plant with various bioactive secondary metabolites. In order to reduce the dependence on wild populations of the species, we aim to establish in vitro culture system that can produce Rubiaceae-type cyclopeptides (RAs) and quinones. Agrobacterium rhizogenes-mediated transformation of stem segments of in vitro grown R. yunnanensis plants using four A. rhizogenes strains was studied and transformation conditions were optimized. Hairy roots appeared with the highest frequency (68.89%) when stem segments (with leaves) without pre-culture were immersed in A. rhizogenes A4 strain bacterial suspension for 30 min, co-cultured on Murashige and Skoog (MS) solid medium in the dark for three days, and afterwards incubated in darkness. PCR analysis of rolB and rolC genes confirmed transformed nature of six hairy root clones. The hairy roots grew rapidly, especially showing the highest accumulation of biomass in MS liquid medium compared to in vitro grown plants and calli. Histological observation of hairy root revealed anatomical difference in vascular cylinder, where the cells exhibited high mitotic activity characterized by vigorous growth. The UPLC-MS/MS analysis revealed that the amount of RAs in the hairy roots grown in ½MS liquid medium (4.611 μg g−1 DW) was higher than that in in vitro grown plants (0.331 and 4.096 μg g−1 DW for shoots and roots respectively) and calli (1.082 μg g−1 DW), but still far lower than that in the roots of seed-borne plants (80.296 μg g−1 DW). However, the hairy roots accumulated high level of quinones (2320.923 and 5067.801 μg g−1 DW for MS and ½MS liquid media respectively), of the same order of magnitude as the roots of seed-borne plants (7409.973 μg g−1 DW). Hairy root culture of R. yunnanensis, with high accumulation of biomass and production of quinones, may offer an attractive perspective for the production of the RAs and quinones that could be further optimized for pharmaceutical use.

Xanthones Production in Gentiana dinarica Beck Hairy Root Cultures Grown in Simple Bioreactors.

The hairy root clones of Gentiana dinarica cl-B, cl-D, cl-3, and cl-14 were cultivated in parallel in diverse simple bioreactors, including temporary immersion systems RITA® (TIS RITA®), bubble column bioreactors (BCB), and Erlenmeyer flasks (EF), and evaluated for biomass production and xanthone content. The obtained results showed that TIS RITA® and BCB containing ½ MS medium with 4% sucrose provided equally good growth conditions in which the majority of the clones displayed the higher percentage of dry matter (DM%), and xanthones norswertianin-1-O-primeveroside (nor-1-O-prim) and norswertianin production than those cultivated in EF. Thin and well branched hairy root clone cl-B grown in BCB for 7 weeks was superior regarding all growth parameters tested, including growth index (19.97), dry weight (2.88 g), and DM% (25.70%) compared to all other clones. Cl-B cultured in TIS RITA® contained the highest amount of nor-1-O-prim (56.82 mg per vessel). In BCB with constant aeration, cl-B accumulated the highest norswertianin content reaching 18.08 mg/vessel. The optimized conditions for cultivation of selected G. dinarica hairy root clones in highly aerated TIS RITA® and BCB systems contribute to the development of bioreactor technology designed for the large scale commercial production of xanthones nor-1-O-prim and norswertianin.

Enhanced expression of recombinant miraculin, a tasting modifying protein, in Nicotiana tabacum hairy roots using 18.2 heat shock protein promoter and terminator

Miraculin, a taste modifier, is a protein that was first isolated from miracle fruit (Richadella dulcifica). It can change a sour taste into a sweet taste when sour acids are consumed, although it does not elicit a sweet response. Miraculin may have the potential in industry as a substitute for sugars and as artificial sweeteners. Since the miracle plant has low fruit productivity, mass production of miraculin is limited. Transgenic hairy root culture is a potential alternative system for the mass production of miraculin. In this study, we investigated the expression of recombinant miraculin in tobacco (Nicotiana tabacum) hairy roots. To increase miraculin expression, the heat shock protein 18.2 promoter and terminator were used to drive the expression of miraculin gene in a potential host system. Synthetic miraculin gene was transformed into Nicotiana tobacum leaf explants via Agrobacterium rhizogenes. The transgenic hairy root clones that contained synthetic miraculin gene showed rapid growth and reached maximum growth after 35-day culture. When the expression of miraculin gene was regulated by heat shock protein 18.2 promoter and heat shock protein terminator, the expression of recombinant miraculin increased than the control regulated by CaMV 35S promoter and nopaline synthase terminator. The recombinant miraculin was 19.97 ng per µg of the total soluble protein and equivalently with approximately 2% of the total soluble protein. For the first time, a taste modifying miraculin was successfully expressed in tobacco hairy root. The results in this study have given a promising approach for the application of the transgenic hairy root system to produce recombinant miraculin.

Comparative investigation of sesquiterpene components of essential oils originating from intact plants and hairy root chamomile cultures

The importance of chamomile (Chamomilla recutita) inflorescence is widely known in classical and folk medicine, with the largest group of its effective constituents forming the essential oil (chamazulene, a-bisabolol, α-farnesene, trans-β-farnesene, spathulenol, cis/trans-en-in-dicycloethers). Among cultivated species, the Hungarian BK-2 contains more chamazulene in its essential oil than the German Degumil type, which is mainly cultivated for its a-bisabolol. Both components have important antiinflammatory activities. Wild populations can be easily distinguished from cultivated ones by their high amount of bisaboloides, particularly the flower of Hungarian Szabadkígyós wild type, which contained on average 48 % of the biologically active (-)-a-bisabolol. The population of Szabadkígyós has good salt tolerance which is important owing to global warming, because the proportion of saline areas is increasing worldwide. To keep the genome of Szabadkígyós having high (-)-a-bisabolol content, Szőke and research team used biotechnological methods. Sterile plantlets, were infected by Agrobacterium rhizogenes strains #A-4, #15834, #R-1601. The hairy root clones possessing the best growing and biosynthetical potential were multiplied for phytochemical investigations. Pharmacologically important compounds of their essential oils were followed in great detail. The amount of in vitro cultured terpenoids and polyin compounds was compared with that of in vivo plants. GC-MS studies showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristics of the mother plant. Berkheyaradulene, geranyl-isovalerat and cedrol as new components were identified in these sterile cultures. The main component of hairy root cultures (D/400, D/1, D/100 and Sz/400) was tr-b-farnesene and in addition one new compound: a-selinene was identified. Hairy root culture originated from chamomile collected in Szabadkígyós was intensive increased the essential oil content and pharmacological active compounds: (-) -α-bisabolol and β-eudesmol was also synthetized in large quantity. Furthermore, in vitro organized cultures were made from this population to obtain propagation material containing numerous active substances.

A new synthetic biology approach for the production of curcumin and its glucoside in Atropa belladonna hairy roots

Curcumin has ignited global interest as an elite drugable molecule, owing to its time-honoured pharmacological activities against diverse human ailments. Limited natural accessibility and poor oral bioavailability caused major hurdles in the curcumin-based drug development process. We report the first successful testimony of curcumin and its glucoside synthesis in Atropa belladonna hairy roots (HR) through metabolic engineering. Re-routing the inherent biosynthetic precursors of the phenylpropanoid pathway of A. belladonna by heterologous expression of key curcumin biosynthetic pathway genes (i.e., Diketide-CoA synthase-DCS and Curcumin synthase-CURS3) and glucosyltransferase gene (CaUGT2) resulted in the production of curcumin and its glucoside in HR clones. Under shake-flask cultivation, the PGD2-HR1clone bearing DCS/ CURS3 genes showed the maximum curcumin yield (180.62 ± 4.7 μg/g DW), while the highest content of curcumin monoglucoside (32.63 ± 2.27 μg/g DW) along with curcumin (67.89 ± 2.56 μg/g DW) were noted in the PGD3-HR3 clone co-expressing DCS/CURS3 and CaUGT2 genes. Bioreactor up-scaling showed yield improvements in the PGD2-HR1 (2.3 fold curcumin) and the PGD3-HR3 clone (0.9 and 1.65 folds of curcumin-monoglucoside and curcumin respectively). These findings proved the advantageous use of HR cultures as the production source for curcumin and its glucoside, which remained unexplored so far.

The direct and indirect transformation methods on expressing a recombinant Dermaseptin peptide in tobacco transgenic hairy root clones

Despite the increasing demand for production of recombinant proteins, only plant cell cultures are considered as the efficient systems for producing eukaryotic valuable products. Although plant cells are grown in the straightforward and simple conditions, optimization of culture conditions is crucial for a higher yield. In the present study, the establishment of transgenic tobacco hairy roots (HRs) expressing a Dermaseptin B1 recombinant antimicrobial peptide (C2-B1) is examined. Direct and indirect transformation (DT and IT) methods were applied to compare the amount of the recombinant peptide production. The integration, transcription and expression of the transgene in the HRs were confirmed by PCR, semi-quantitative RT-PCR and western blot analysis, respectively. The C2-B1 level was quantified by ELISA analysis of HRs extracts. The results of this study showed that the longer the inoculation and co-culture period, the higher the transformation efficiency. Antimicrobial activity by of crude protein extract from transgenic HR clones showed a significant (P ≤ 0.01) difference between HRs derived from DT and IT methodes methodsmethodes . Transgenic clones derived from both DT and IT methodes and grown in liquid medium showed a difference in terms of total protein content significantly. HRs derived from IT methodmethod produced a significantly higher amount of C2-B1 recombinant peptide in liquid B5 medium supplemented with sucrose.

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.

Genetic transfection, hairy root induction and solasodine accumulation in elicited hairy root clone of Solanum erianthum D. Don

An efficient genetic transfection technique has been established using A4 strain of Agrobacterium rhizogenes for the first time in a medicinally valuable plant Solanum erianthum D. Don. The explants were randomly pricked with sterile needle, inoculated with bacterial suspension. The infected leaves were then washed and transferred to MS basal medium fortified with cefotaxime for hairy root induction. A maximum transformation efficiency of 72 % has been recorded after two days of co-cultivation period. The transfer of rolA and rolB genes from the bacterium to the plant genome has been confirmed in five transformed hairy rootlines by standard Polymerase Chain Reaction technique. On the basis of growth analysis and secondary metabolite study two potential rhizoclones (A4-HR-A and A4-HR-B) were selected. Rhizoclone A4-HR-A can produce highest amount of alkaloid, phenolic and flavonoid, whereas A4-HR-B was observed to be highest tannin producer. Alkaloid like solasodine, commercially important for steroidal drug synthesis, was quantified from leaf and A4-HR-A clone by an improved High Performance Liquid Chromatography method. This showed a sustainable increase (1.33 fold) in production of solasodine in hairy rootline.

First-generation genome editing in potato using hairy root transformation.

SummaryGenome editing and cis‐gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens‐based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T‐DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%–98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%–30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ‐line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium‐mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.

Antibody-Mediated Resistance to Rhizomania Disease in Sugar Beet Hairy Roots.

Agrobacterium rhizogenes–mediated transformation of sugar beet hairy roots expressing single-chain variable fragment (scFv) was exploited to evaluate the efficacy of four antibody-based constructs for interfering with the Beet necrotic yellow vein virus infection. The scFv specific to a major coat protein of virus, p21, was targeted to various cellular compartments including the cytosol (pIC and pICC constructs), apoplast (pIA), and mitochondrion (pIM). After mechanical virus inoculation, most of the hairy root clones expressing scFv in the cytosol displayed low virus titers while the majority of transgenic hairy root clones accumulated antibody in outer membrane of mitochondria or apoplast were infected. This hairy root system provided an efficient and rapid approach to initially investigating root disease resistance like rhizomania prior to transform whole recalcitrant plants such as sugar beet.

Optimization of conditions for the induction of Tagetes patula L. hairy roots

The hairy root cultures are promising sources of secondary metabolites of plants, including rare and endangered species. They possess genetic and biochemical stability, unlimited growth rate in free-hormone medium, short doubling times, high biosynthetic activity and ecological purity of plant raw materials. The hairy root cultures of Tagetes patula L. can be used to produce biologically active substances with biocidal activity. The study aimed to determine the virulent strain of Agrobacterium rhizogenes and the most effective period of co-cultivation of T. patula leaf explants with an agrobacterium to induce actively growing hairy root cultures. We used 3 strains (A-4b, 8196RT and 15834). The time of infection ranged from 3 to 33 hours in increments of 3 hours. We found that 24 h is the best time of infection to induce hairy roots with the highest transformation efficiency (92%). The wild strain A. rhizogenes 15834 turned out to be the most virulent when infected leaf explants of spreading marigold. This strain provided the maximum transformation effect, reaching 85.4%. We have identified 5 actively growing clones of hairy roots with intensive branching, the growth indices of which were 64-75. In the future, they will be transferred to a liquid medium for biomass accumulation and scaling.

Growth and physiological alterations related to root-specific gene function of LrERF061-OE in Glycyrrhiza uralensis Fisch. hairy root clones under cadmium stress

Abiotic stresses, particularly heavy metals, are a threat to living organisms, including plants. Heavy metals activate ethylene biosynthesis and ultimately stimulate reactive oxygen species (ROS)-related gene expression to enhance stress tolerance. The identification and functional analysis of such stress-related genes is very important to increase the stress tolerance of valuable plants to heavy metals. Ethylene responsive factor ERF061 belongs to the ERF transcription factor family, controls plant growth and ontogenesis and regulates abiotic stress in model plant species. Therefore, in this study, we examined the tolerance function of LrERF061 via an overexpression vector in the growth and physiology of G. uralensis hairy roots under cadmium (Cd) stress. The effect of different Cd concentrations (0, 25, 50, 75, 100 mg/L) on the growth and physiology of LrERF061-OE hairy roots (LrE-HR) and control hairy roots (LrC-HR) of G. uralensis was measured. Initially, at low cadmium concentrations, there was a stimulating effect of Cd on G. uralensis LrE-HR and LrC-HR. However, at high cadmium doses, the growth of LrC-HR was retarded compared to that of LrE-HR. Intriguingly, a significant increase was observed in the relative expression level of LrERF061-OE in LrE-HR at 100 mg/L Cd. According to the results, compared to LrC-HR (11 mg/g), LrE-HR showed maximum Cd uptake (81 mg/g) at high Cd concentrations. The antioxidant enzyme activities (SOD, CAT and POD) in LrE-HR were enhanced at high Cd concentrations compared to those in LrC-HR. This work first highlighted the effect of LrERF061-OE, which is upregulated under Cd stress in G. uralensis hairy root clones, on heavy metal tolerance. Current study for the first time aimed to evaluate effect of ethylene responsive transcription factor (LrERF061) under Cd stress on growth and physiological properties of Glycyrrhiza uralensis hairy root clones.