Growth Of Hairy Roots Research Articles

The effect of silver nanoparticles on the growth and antioxidants of transgenic hairy roots in hyssop (Hyssopus officinalis, H. angustifolius)

The effect of silver nanoparticles on the growth and antioxidants of transgenic hairy roots in hyssop (Hyssopus officinalis, H. angustifolius)

Smi-miR396b targeted SmGRFs, SmHDT1, and SmMYB37/4 synergistically regulates cell growth and active ingredient accumulation in Salvia miltiorrhiza hairy roots.

MIR396b had been cloned and overexpressed in Salvia miltiorrhiza hairy roots. MiR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to regulate cell growth and secondary metabolism in S. miltiorrhiza hairy roots. Danshen (Salvia miltiorrhiza Bunge) is a valuable medicinal herb with two kinds of clinically used natural products, salvianolic acids and tanshinones. miR396 is a conserved microRNA and plays extensive roles in plants. However, it is still unclear how miR396 works in S. miltiorrhiza. In this study, an smi-MIR396b has been cloned from S. miltiorrhiza. Overexpression of miR396b in danshen hairy roots inhibited hairy root growth, reduced salvianolic acid concentration, but enhanced tanshinone accumulation, resulting in the biomass and total salvianolic acids respectively reducedto 55.5 and 72.1% of the control and total tanshinones increased up to 1.91-fold of the control. Applied degradome sequencing, 5'RLM-RACE, and qRT-PCR, 13 targets for miR396b were identified including seven conserved SmGRF1-7 and six novel ones. Comparative transcriptomics and microRNomics analysis together with qRT-PCR results confirmed that miR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to mediate the phytohormone, especially gibberellin signaling pathways and consequentially resulted in the phenotype variation of miR396b-OE hairy roots. Furthermore, miR396b could be activated by methyl jasmonate, abscisic acid, gibberellin, salt, and drought stresses. The findings in this study indicated that smi-miR396b acts as an upstream and central regulator in cell growth and the biosynthesis of tanshinones and salvianolic acids, shedding light on the coordinated regulation of plant growth and biosynthesis of active ingredients in S. miltiorrhiza.

Overexpression of Ath-MIR160b increased the biomass while reduced the content of tanshinones in Salvia miltiorrhiza hairy roots by targeting ARFs genes

MicroRNAs (miRNAs) have attracted increasing attention in the field of medicinal plant research. Our previous analyses indicated that the mature miR160a level was related to the accumulation of tanshinones in Salvia miltiorrhiza (S. miltiorrhiza). Therefore, the aim of this research was to reveal how the miR160a regulates the tanshinones biosynthesis. The overexpression of miR160a in hairy roots of S. miltiorrhiza down-regulated the levels of the target genes ARF10, 16, and 17, and inhibited tanshinones biosynthesis but increased hairy root biomass. In overexpression line 1, the content of four major tanshinones dropped sharply to 9.06% (dihydrotanshinone I), 12.56% (cryptotanshinone), 12.02% (tanshinone I), and 8.52% (tanshinone IIA) compared with that of the wildtype (WT). The fresh weight (FW) of hairy roots increased to 1.72-fold of the WT, and in overexpression lines 4, tanshinones were not detected. However, the FW of hairy roots in overexpression line 4 increased to 1.90-fold of the WT. We also observed the down regulated genes in the tanshinones biosynthesis pathway. In particular, the transcription level of KSL2 dropped to 3% as compared to the WT, which was consistent with the decrease in tanshinones content. We further analyzed hormone changes and found an increase in indole acetic acid accumulation, while the accumulation of salicylic acid (SA) and jasmonic acid (JA) decreased. These findings indicate that miR160a may negatively regulate the biosynthesis of tanshinones, while promote the growth of hairy roots by targeting ARF10, 16, and 17. Our findings demonstrate that miRNAs could be used as an alternative strategy to dissect the complex network of secondary metabolism in medicinal plants. MiR160a overexpression promoted the IAA level and the growth of hairy roots of Salvia miltiorrhiza, but reduced the GH3-like level and production of tanshinones by targeting ARF10, ARF16, and ARF17.

Production of phenolic acids in hairy root cultures of medicinal plant Mentha spicata L. in response to elicitors.

In this study, hairy root induction in leaf and stem explants of Mentha spicata using various Agrobacterium rhizogenes strains was established for the first time. Although inoculation of explants by immersion method resulted in tissue necrosis, direct injection of explants by all examined strains (A13,R318,A4,GMI 9534 and ATCC15834) was effective. All different parts of the stem were susceptible to A. rhizogenes infection. However, the middle and lower internodes showed a higher rate of transformation. Among the different strains, the strain A13 exhibited the highest infection efficiency (almost 75% of the explants). A13 and R318-infected hairy roots showed the highest biomass production (close to 60 mg/flask), while infection with GMI 9534 produced the highest content of phenolic acids. Finally, the effect of phytohormone elicitation on hairy root growth and phenolic acid biosynthesis was investigated. A substantial increase in root growth and phenolic acids accumulation was obtained followed by 0.3 mg L-1 IBA and 100 µM MeJA treatment, respectively.

SmGRAS3 negatively responds to GA signaling while promotes tanshinones biosynthesis in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge is the origin plant of traditional Chinese medicine, Danshen. Demand of high-quality S. miltiorrhiza is increasing because of its excellent pharmaceutical value. Tanshinones and salvianolic acids (SAs) are the main active ingredients of S. miltiorrhiza. GA was reported to induce tanshinones and SAs accumulation, however, its mechanism was unclear. Here, we got a GA responsive protein, SmGRAS3. SmGRAS3 expressed highest in the periderm where tanshinones produced and accumulated. Results of SmGRAS3 over and antisense expression indicated that SmGRAS3 is a positive regulator of tanshinones biosynthesis in S. miltiorrhiza hairy roots. But overexpression of SmGRAS3 depressed SAs and GA biosynthesis as well as hairy roots growth, and application of GA could partially recover these depressions. Pathway enzyme genes expressions of tanshinones, SAs and GA biosynthesis were comprehensively changed in SmGRAS3 transgenic hairy roots. SmGRAS3 acted as a transcription factor that located in nucleus and has the transcription activation activity. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that SmGRAS3 directly bound to GARE-motif in promoter of KSL1 and activated KSL1 transcription. Our study suggested that the SmGRAS3 may be a good potential target for further metabolic engineering of bioactive components biosynthesis in S. miltiorrhiza.

Enhanced biosynthesis of triterpenoids in Centella asiatica hairy root culture by precursor feeding and elicitation

Centella asiatica contains various triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. Among them, they are mainly contained in leaf, and among them asiaticoside is known as tissue-specific compound that are mainly accumulated in the leaves. Therefore, in the present study, C. asiatica hairy root cultures were established from petiole and leaf explants using Agrobacterium rhizogenes-mediated transformation. To promote the biosynthesis of these triterpenoids, hairy root cultures were treated with precursors (squalene and pyruvic acid) and elicitors [methyl jasmonate (MeJA) and salicylic acid (SA)] at various concentrations. No significant difference was detected in the growth index of hairy root cultures between precursor feeding and elicitation, except pyruvic acid feeding, suggesting that elicitors do not affect the growth of hairy roots. Treatment with 2.5 mM squalene and 5 mM pyruvic acid enhanced the biosynthesis of target triterpenoids (57.53 and 29.13 mg g−1 DW, respectively) compared with other treatments. However, compared to squalene, MeJA showed a greater effect on the triterpene saponin content in C. asiatica. The highest quantity of triterpenoids (60.25 mg g−1 DW) was produced in hairy root cultures treated with 400 μM MeJA. These results indicate that precursor- and elicitor-treated C. asiatica hairy root cultures rapidly accumulate biomass and could be used to enhance the production of triterpenoids for industrial purposes.

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.

Effect of Media Strength and pH on the Growth of Hairy Roots and Production of Gymnemic Acid from Gymnema Sylvestre

Gymnema sylvestre (Madhunashini) is one of the most important medicinal plants used as a crude drug for its preventive and therapeutic properties. Among other constituents of Gymnema, gymnemic acid is a major component responsible for biological and pharmacological actions. The present study deals with the influence of different media strength and initial medium pH on the growth of hairy roots and gymnemic acid production from Gymnema sylvestre. Higher strength of the media (1.5X) favoured the biomass production (114.64 g/L FW and 12.63 g/L DW) and gymnemic acid content (11.7 mg/g DW) in the tested range of 0.25, 0.50, 0.75, 1.0, 1.5 and 2.0 X strength. Among the different hydrogen ion concentration (pH) of 4.0, 4.5, 5.0, 5.5, 5.8, 6.0 and 6.5, initial medium pH of 6.0 favoured the biomass production (102.41 g/L FW and 11.52 g/L DW) and medium pH of 5.8 favoured the gymnemic acid production (11.30 mg/g DW).

UHPLC-HRMSn Analysis Reveals the Dynamic Metabonomic Responses of Salvia miltiorrhiza Hairy Roots to Polysaccharide Fraction from Trichoderma atroviride

We have previously reported that Trichoderma atroviride, an endophytic fungus isolated from S. miltiorrhiza, promotes S. miltiorrhiza hairy root growth and significantly stimulates the biosynthesis of tanshinones specifically the polysaccharide fraction (PSF). However, this study only focused exclusively on six metabolites whilst ignoring changes to the whole metabolite composition of the S. miltiorrhiza hairy roots. In the present study, the dynamic metabonomic responses of S. miltiorrhiza hairy roots were investigated using ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMSn). UHPLC-HRMS typical total ions chromatograms (TICs) of PSF-treated hairy root samples were different from the control. Moreover, the results of principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and hierarchical clustering analysis (HCA) indicated that PSF-treated samples were significantly different from the control. Through the analysis of PLS-DA, a total of 114 and 99 differential metabolites were found from the positive and negative models respectively and a total of 33 differential metabolites were identified. Thus, S. miltiorrhiza hairy roots had been induced to regulate the metabolic profiling in response to PSF and the changes of the metabolic profiling contributed to promoting the biosynthesis of tanshinones notably whilst the biosynthesis of phenolic acids were slightly inhibited.

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.

Structure of a polysaccharide from Trichoderma atroviride and its promotion on tanshinones production in Salvia miltiorrhiza hairy roots

This study evaluates the chemical structure of a heteropolysaccharide (PSF-W-1) from the endophytic fungus Trichoderma atroviride and its effects on the production of tanshinones in Salvia miltiorrhiza hairy roots. The total carbohydrate content of isolated PSF-W-1 was determined to be 97.72%. PSF-W-1 has a relative molecular weight of 36.13 kDa and contains mannose, glucose and galactose in molar ratios of 1.00:4.86:2.25. Through methylation analysis, IR and NMR, PSF-W-1 was determined to possess a backbone of →4)-β-D-Glcp-(1→6)-α-D-Galp-(1→4)-β-D-Manp-(1→6)-α-D-Galp-(1→ with two side chains β-D-Glcp-(1→4)-β-D-Glcp-(1→ attached to O3 of 1,6-α-D-Galp. Bioactivity tests suggested that PSF-W-1 was responsible for boosting the S. miltiorrhiza hairy root growth and the biosynthesis of dihydrotanshinone I, tanshinone I, tanshinone IIA and cryptotanshinone in hairy roots. According to this study, PSF-W-1 might be utilized as a potent stimulator of tanshinones synthesis.

The Roles of GmERF135 in Improving Salt Tolerance and Decreasing ABA Sensitivity in Soybean.

Abscisic acid (ABA) mediates various abiotic stress responses, and ethylene responsive factors (ERFs) play vital role in resisting stresses, but the interaction of these molecular mechanisms remains elusive. In this study, we identified an ABA-induced soybean ERF gene GmERF135 that was highly up-regulated by ethylene (ET), drought, salt, and low temperature treatments. Subcellular localization assay showed that the GmERF135 protein was targeted to the nucleus. Promoter cis-acting elements analysis suggested that numerous potential stress responsive cis-elements were distributed in the promoter region of GmERF135, including ABA-, light-, ET-, gibberellin (GA)-, and methyl jasmonate (MeJA)-responsive elements. Overexpression of GmERF135 in Arabidopsis enhanced tolerance to drought and salt conditions. In addition, GmERF135 promoted the growth of transgenic hairy roots under salt and exogenous ABA conditions. These results suggest that soybean GmERF135 may participate in both ABA and ET signaling pathways to regulate the responses to multiple stresses.

High Yield of Bioactive Abietane Diterpenes in Salvia sclarea Hairy Roots by Overexpressing Cyanobacterial DXS or DXR Genes.

Abietane diterpenoids, containing a quinone moiety, are synthesized in the roots of several Salvia species. Promising cytotoxicity and antiproliferative activities have been reported for these compounds in various cell and animal models. We have recently shown that aethiopinone, an o-naphto-quinone diterpene, produced in the roots of different Salvia species, is selectively cytotoxic against the A375 melanoma cell line. To enhance the synthesis of this abietane diterpenoid, we have engineered the plastidial 2-C-methyl-D-erythritol 4-phosphate-derived isoprenoid pathway in Salvia sclarea hairy roots by ectopic expression and plastid targeting of cyanobacterial genes encoding the 1-deoxy-D-xylulose 5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene, the first two enzymatic steps of the plastidial MEP pathway, from which plant diterpenes primarily derive. Plastid-targeted expression of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase proteins significantly enhanced the yield of aethiopinone by a 3-fold and about 6-fold increase, respectively. The accumulation of other abietane-type diterpenes (ferruginol, salvipisone, and carnosic acid), with interesting antiproliferative activity, was also increased. Compared to our previous data obtained by overexpressing the plant orthologous 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase genes in S. sclarea hairy roots, the results presented here confirm that the bacterial 1-deoxy-D-xylulose-5-phosphate reductoisomerase enzyme plays a major role than the DXS enzyme in the biosynthetic pathway of this class of compounds and that its ectopic expression does not conflict with active hairy root growth, resulting in a balanced trade-off between the transgenic hairy root final biomass and the increased content of o-naphto-quinone diterpenes, with interesting biological activities.

Study on the growth of Agrobacterium rhizogenes-induced Catharanthus roseus hairy roots in liquid media

Catharanthus roseus hairy roots induced by Agrobacterium rhizogenes get potential to apply to many fields. The suitable conditions for culturing depend on each hairy root line. In shaken liquid media in dark at 25 oC, VIN002-12 and VIN005-07 hairy root lines had the best growth in hemi-concentrated Gamborg’B5 media while VIN072-15 and VIN077-09 lines showed the best growth in in hemi-concentrated White media. The appropriate initial pH of medium for the lines was in range of 5.7–6.5. The hairy root lines used 2–5% sucrose as an optimal source of carbon for their growth. In selected conditions, the growth kinetics curves showed the end of exponential phase at the 28th day of culture with VIN005-07 and VIN072- 15 lines whereas at the 35th day with VIN002-12 and VIN077-09 lines. The initial results are quite possible to produce biomass for researches on four hairy root lines in the future.

Особенности роста волосовидных корней табака с конститутивной экспрессией гена экспансина NtEXPA5

Особенности роста волосовидных корней табака с конститутивной экспрессией гена экспансина NtEXPA5

Preliminary study on the preservation of Catharanthus roseus (L.) G. Don hairy root lines induced by Agrobacterium rhizogenes isolated in Vietnam

Agrobacterium rhizogenes-transformed hairy roots from Catharanthus roseus have been widely used in research and in life. Consequently, methods for preservation are essential to maintain valuable hairy root lines. Our results showed that Gamborg’B5 was the most suitable medium for hairy roots from VIN002 and VIN005 while solid White media was more comfortable for the hairy lines from VIN022 and VIN077. All hairy root lines must be preserved in the dark at 25–27 oC. Under suitable conditions, the rate of lines growing normally reached 69.3, 67.0, 57.3 and 60.7 % for VIN002, VIN005, VIN072, and VIN077, respectively. There were 90.0, 93.3, 80.0 and 93.3 % of lines could preserve rolB, a gene which has important effect on the growth of hairy roots.

Morphological and Molecular Analysis of Isolated Cultures of Tobacco Adventitious Roots Obtained by the Methods of Biolistic Bombardment and Agrobacterium-Mediated Transformation

Plant infection with Agrobacterium rhizogenes leads to the development of a hairy root disease notable for the rapid agravitropic growth of roots on hormone-free nutrient media. In order to look into the interaction of A. rhizogenes with plants and assess opportunities of practical application of hairy root culture, new approaches to their production are elaborated. A method of bacterium-free and plasmid-free production of genetically modified roots (hairy roots) by means of biolistic transformation of leaf explants with a DNA fragment (size of 5461 bp) consisting of genes rolA, rolB, rolC, and rolD are proposed. In most cases, such transformation resulted in the emergence of only adventitious roots with transient expression of rol-genes, and the growth of such roots on hormone-free media ceased in 2–3 months in contrast to genuine hairy roots capable of unrestricted growth. Molecular analysis of different systems of target genes’ expression showed an important role of transgene rolC and host gene of cyclin-dependent protein kinase CDKB1-1 in the maintenance of rapid growth of hairy roots in vitro (in isolated cultures).

Optimization of Hairy Root Induction in Solanum tuberosum

An efficient protocol for hairy root induction in Solanum tuberosum was established using Agrobacterium rhizogenes. Transformation of Desiree was more efficient than with Shepody or Purple Majesty. Transformation efficiency of Desiree tuber discs (97% transformation efficiency) was higher in comparison to root (67% transformation efficiency), stem (61% transformation efficiency) and leaf (14% transformation efficiency) explants. Bacterial density, culture parameters, explant type, and genotype influenced the transformation efficiency, as well as the growth of hairy roots. Hairy root transformation efficiency in explants treated with acetosyringone was enhanced from 72 to 89% in Purple Majesty tubers and 56 to 80% in roots during co-cultivation. The growth rate of hairy roots from tuber discs was about 5 times greater than that of hairy roots from leaves. Tuber hairy roots were highly resistant to kanamycin in the absence of NPT II. This study describes the efficient generation of hairy roots in different potato tissues and cultivars that can provide a fast method to generate transformed tissue or be used for production of secondary metabolites.

Enhancing Astragaloside Production in Astragalus membranaceus through Growth Media and Auxins

The secondary metabolites of triterpenoid saponins, isoflavonoids and polysaccharides are present in Astragalus membranaceus those are biologically active. Astragalosides are a major group of identified compounds that have various pharmacological properties. The present study was considered to enhance astragaloside biosynthesis in hairy root cultures of A. membranaceus by using different growth media and concentrations of different auxins. Our findings revealed that Schenk and Hildebrandt (SH) medium responded greatly for the production of the highest dry weight. However, the highest astragaloside production was detected while using SH medium of its half strength. The growth of hairy roots and accumulation of astragaloside in response to different auxinlike Indole-3-Acetic Acid (IAA), Indole-3-Butyric Acid (IBA) and 1-naphthaleneacetic acid (NAA) were different. Hairy root growth rate was increased by the treatment of IBA and NAA, but was unaffected by IAA. Accumulation of Astragaloside was enhanced in response to the treatment of IBA and NAA. In most cases, treatment with the 0.5 mg L-1 NAA accumulated the highest level of stragaloside in the hairy root of A. membranaceus, which might, therefore, be utilized as an ideal mass-scale production system for astragalosides.

Agrobacterium rhizogenes-mediated genetic transformation in Cichorium spp.: hairy root production, inulin and total phenolic compounds analysis

ABSTRACTChicory (Cichorium spp.) is a valuable vegetable crop worldwide for its edible leaves and for the production of coffee substitutes from roots. Agrobacterium rhizogenes-mediated genetic transformation of two species of chicory (C. intybus and C. endivia) was investigated using Agrobacterium strain K599 harbouring p35SGFPGUS+ plasmid and two types of explants: leave and leaf stalks. This Agrobacterium strain proved to be competent in the transformation with transformation rate about (23.1%) in leaf explants of C. intybus. However, the transformation rate with C. endivia was much lower (3.6%). Moreover, the hairy roots appeared from different infection sites of the leaf explants. Several hairy roots of the two species were acquired, out of them 11 clones (C. intybus) and two clones (C. endivia) were selected due to their fast-growing character. Growth of hairy roots was determined on the basis of total root biomass accumulation. It was found that the liquid MS-basal medium seems to be the most suitable for biomass production. PCR analysis revealed foreign DNA integration in the selected transgenic hairy root clones. Notable, the transgenic hairy roots exhibited substantially higher growth rates and accumulated higher amount of inulin than non transgenic roots (WT). Also, the total phenolic compounds were determined.