scholarly journals Establishment of Hairy Root Cultures ofRhaponticum carthamoides(Willd.) Iljin for the Production of Biomass and Caffeic Acid Derivatives

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Ewa Skała ◽  
Agnieszka Kicel ◽  
Monika A. Olszewska ◽  
Anna K. Kiss ◽  
Halina Wysokińska
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Leaf Explants ◽  
Biomass Accumulation ◽  
Flavonoid Glycosides ◽  
Transformed Roots ◽  
Caffeoylquinic Acids ◽  
Best Response ◽  
Hairy Root Line ◽  
Half Strength

The aim of the study was to obtain transformed roots ofRhaponticum carthamoidesand evaluate their phytochemical profile. Hairy roots were induced from leaf explants by the transformation ofAgrobacterium rhizogenesstrains A4 and ATCC 15834. The best response (43%) was achieved by infection with A4 strain. The effects of different liquid media (WPM, B5, SH) with full and half-strength concentrations of macro- and micronutrients on biomass accumulation of the best grown hairy root line (RC3) at two different lighting conditions (light or dark) were investigated. The highest biomass (93 g L−1of the fresh weight after 35 days) was obtained in WPM medium under periodic light. UPLC-PDA-ESI-MS3and HPLC-PDA analyses of 80% aqueous methanol extracts from the obtained hairy roots revealed the presence of eleven caffeoylquinic acids and their derivatives and five flavonoid glycosides. The production of caffeoylquinic acids and their derivatives was elevated in hairy roots grown in the light. Only light-grown hairy roots demonstrated the capability for the biosynthesis of such flavonoid glycosides as quercetagetin, quercetin, luteolin, and patuletin hexosides. Chlorogenic acid, 3,5-di-O-caffeoylquinic acid and a tentatively identified tricaffeoylquinic acid derivative were detected as the major compounds present in the transformed roots.

scholarly journals Methyl jasmonate enhances ursolic, oleanolic and rosmarinic acid production and sucrose induced biomass accumulation, in hairy roots of Lepechinia caulescens

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11279
Author(s):  
Victor M. Vergara-Martínez ◽  
Samuel E. Estrada-Soto ◽  
Susana Valencia-Díaz ◽  
Karlina Garcia-Sosa ◽  
Luis Manuel Peña-Rodríguez ◽  
...  
Keyword(s):  
Methyl Jasmonate ◽  
Bioactive Compounds ◽  
Hairy Root ◽  
Hairy Roots ◽  
Leaf Explants ◽  
Biomass Accumulation ◽  
Wild Plants ◽  
Bioactive Metabolites ◽  
Hairy Root Cultures ◽  
Root Cultures

Background Ursolic (UA), oleanolic (OA) and rosmarinic (RA) acids are bioactive metabolites found in Lepechinia caulescens that have generated interest for their health benefits, which include antimicrobial, antioxidant, antimutagenic, gastroprotective, antidiabetic, antihypertensive and anti-inflammatory properties, among others. To date, very few attempts have been made to evaluate the potential for simultaneous production of these bioactive compounds, using a biotechnological approach. Hairy root cultures offer a biotechnology approach that can be used to study the factors affecting the biosynthesis and the production of UA, OA and RA. In the current study, we established hairy root cultures of L. caulescens and evaluated the effect of sucrose on biomass accumulation, and the effect of different concentrations and times of exposure of methyl jasmonate (MeJA), on the accumulation of UA, OA and RA. Methods Leaves from plants of L. caulescens were inoculated with Agrobacterium rhizogenes strain ATCC 15834. PCR of rolB gene confirmed the transgenic nature of hairy roots. Hairy roots were subcultured in semisolid MSB5 medium, supplemented with 15, 30, 45 or 60 g/L sucrose and after 4 weeks, dry weight was determined. The accumulation of UA, OA and RA of wild plants and hairy roots were determined by HPLC. Finally, the hairy roots were treated with 0, 100, 200 and 300 µM of MeJA and the content of bioactive compounds was analyzed, after 24, 48 and 72 h. Results High frequency transformation (75%) was achieved, using leaf explants from axenic seedlings, infected with A. rhizogenes. The hairy roots showed an enhanced linear biomass accumulation, in response to the increase in sucrose concentration. The hairy root cultures in MSB5 medium, supplemented with 45 g/L sucrose, were capable to synthesizing UA (0.29 ± 0.00 mg/g DW), OA (0.57 ± 0.00 mg/g DW) and RA (41.66 ± 0.31 mg/g DW), about two, seven and three times more, respectively, than in roots from wild plants. Elicitation time and concentration of MeJA resulted in significant enhancement in the production of UA, OA and RA, with treatments elicited for 24 h, with a concentration of 300 µM of MeJA, exhibiting greatest accumulation. Conclusion This is the first report on development of hairy root cultures of L. caulescens. Future studies should aim towards further improving triterpenes and polyphenolic compound production in hairy roots of L. caulescens, for use in the pharmaceutical and biotechnological industry.

A reliable and efficient protocol for induction of hairy roots in Agastache foeniculum

Biologia ◽  
2014 ◽  
Vol 69 (7) ◽  
Author(s):  
Elnaz Nourozi ◽  
Bahman Hosseini ◽  
Abbas Hassani
Keyword(s):  
Salicylic Acid ◽  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
High Performance ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Biochemical Properties ◽  
Separate Experiment ◽  
Transformed Roots

AbstractHairy root culture system is a valuable tool to study the characteristics of gene expression, gene function, root biology, biochemical properties and biosynthesis pathways of secondary metabolites. In the present study, hairy roots were established in Anise hyssop (Agastache foeniculum) via Agrobacterium rhizogenes. Three strains of Agrobacterium rhizogenes (A4, A7 and 9435), were used for induction of hairy roots in four various explants (hypocotyl, cotyledon, one-month-old leaf and five-month-old leaf) of Anise hyssop. The highest frequency of transformation was achieved using A4 strain in one-month-old leaves (51.1%). The transgenic states of hairy root lines were confirmed by PCR (Polymerase chain reaction) method. High performance liquid chromatography analysis revealed that the production of rosmarinic acid (RA) in transformed roots of A. foeniculum was almost 4-fold higher than that of the non-transformed roots. In a separate experiment, hairy roots obtained from one-month-old leaves inoculated with A4 strain, were grown in liquid medium and the effects of different concentrations of salicylic acid (0.0, 0.01, 0.1 and 1 mM) and chitosan (0, 50, 100 and 150 mg L−1) (as elicitor) and sucrose (20, 30, 40 and 50 g L−1) on the growth of hairy roots were evaluated. The results showed that, 30 g L−1 sucrose and 100 mg L−1 chitosan increased the biomass of hairy root cultures and application of salicylic acid reduced the growth of hairy roots compared with control roots.

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

2019 ◽  
Vol 9 (3) ◽  
pp. 415-420
Author(s):  
L. P. Khlebova ◽  
E. S. Brovko ◽  
O. V. Bychkova ◽  
N. V. Pavlova
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Wild Strain ◽  
Leaf Explants ◽  
Biologically Active ◽  
Infected Leaf ◽  
Hairy Root Cultures ◽  
Root Cultures ◽  
Tagetes Patula ◽  
Time Of Infection

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.

scholarly journals Influences of various factors on hairy root induction in Agastache foeniculum (Pursh) Kuntze

2016 ◽  
Vol 107 (1) ◽  
pp. 45 ◽  
Author(s):  
Elnaz NOUROZI ◽  
Bahman HOSSEINI ◽  
Abbas HASSANI
Keyword(s):  
Growth Rate ◽  
Hairy Root ◽  
Hairy Roots ◽  
Acid Content ◽  
Rosmarinic Acid ◽  
Culture Media ◽  
Root Induction ◽  
Transformed Roots ◽  
Inoculation Methods ◽  
Hairy Root Induction

<em>Agrobacterium rhizogenes</em> is known as a natural tool of genetic engineering in many plant species. For the first time, hairy root induction in <em>Agastache foeniculum</em> using <em>A. rhizogenes</em>,<em> </em>rosmarinic acid content<em> </em>and the effect of different culture media and inoculation methods on hairy root growth rate were investigated. Hairy root culture of <em>A. foeniculum</em> was established by inoculation of the 1-month-old leaf explant with A4 strain of <em>A. rhizogenes</em> and the effectiveness of light – dark conditions and two inoculation methods (immersion and injection) were tested. Furthermore, in immersion method, the effects of inoculation time (3, 5 and 7 min) on root induction were investigated. In the second part of the study, the hairy root culture of <em>A. foeniculum</em> was studied using different<em> </em>basal culture media (MS, 1/2 MS and B5). Rosmarinic acid content in hairy roots and non- transformed roots was analyzed using high-performance liquid chromatography (HPLC). There was no significant difference between various inoculation methods in the ability of hairy roots induction. Observations showed that percentage of hairy root induction was higher when the explants were immersed for 5 min in bacterial suspension. Light conditions displayed the highest hairy root induction rates compared with dark condition. Various culture media are different in terms of types and amounts of nutrients and have influence on growth rate. The maximum growth rate (1.61 g fr wt/50 ml) of hairy roots were obtained in 1/2 MS medium. Rosmarinic acid content in transformed roots (213.42 µg/g dry wt) was significantly higher than non-transformed roots (52.28 µg/ g dry wt).

scholarly journals High-efficient transgenic hairy roots induction in chicory: re-dawn of a traditional herb

2016 ◽  
Vol 107 (2) ◽  
pp. 321 ◽  
Author(s):  
Sara Kabirnataj ◽  
Ghorbanali Nematzadeh ◽  
Jafar Zolala ◽  
Ahmad Farhad Talebi
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Biomass Accumulation ◽  
Ms Medium ◽  
Heterologous Proteins ◽  
Bacterial Strains ◽  
Root Cells ◽  
Whole Process ◽  
Cell Factories ◽  
High Efficient

<p>Plant roots can be manipulated by <em>Agrobacterium rhizogenes</em> to stimulate the production of heterologous proteins for pharmaceutical applications as green cell-factories. During the present study, four bacterial strains (A4, ATCC15834, ATCC11325 and A13) in combination with three co-cultivation media (MS, B5, LS) were examined to establish an efficient and reliable transformation system for chicory (<em>Cichorium intybus</em> L.) using <em>A. rhizogenes</em>. The maximum chicory hairy roots induction was achieved using A13 strain. The observation confirmed that MS medium was more effective on hairy root growth. Dried biomass accumulation of hairy roots infected by A13 strain was 1.10 g l<sup>-1</sup> in MS medium which was significantly higher than those grown in LS and B5 medium (0.88 and 0.72 g l<sup>-1</sup>, respectively). Beta-glucuronidase (GUS) gene was introduced by A13 strain carrying the pCAMBIA1304 binary vector. The results showed that the highest frequency of transformation (63.15 %) was achieved using A13 strain and MS cultivation medium. Detection of GUS and <em>hpt</em>II genes by PCR and GUS histochemical localization confirmed the integrative transformation in hairy roots. In conclusion, the whole process was successfully optimized as a pre-step to manipulate the chicory hairy root cells to improve the unique potential of secondary metabolite production.</p>

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

2019 ◽  
Vol 2 (1) ◽  
pp. 5-12
Author(s):  
Nguyen Nhu Nhut ◽  
Bui Van Le
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Catharanthus Roseus ◽  
Growth Kinetics ◽  
Exponential Phase ◽  
Liquid Media ◽  
Hairy Root Line ◽  
Initial Ph ◽  
Initial Results

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.  

Establishment of Salvia officinalis L. Hairy Root Cultures for the Production of Rosmarinic Acid

2006 ◽  
Vol 61 (5-6) ◽  
pp. 351-356 ◽  
Author(s):  
Izabela Grzegorczyk ◽  
Aleksandra Królicka ◽  
Halina Wysokińska
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Rosmarinic Acid ◽  
Root Culture ◽  
Dry Weight ◽  
Salvia Officinalis ◽  
Root Cultures ◽  
Hairy Root Line ◽  
Salvia Officinalis L ◽  
Agrobacterium Rhizogenes Strains

Shoots of Salvia officinalis, a medicinally important plant, were infected with Agrobacterium rhizogenes strains ATCC 15834 and A4 which led to the induction of hairy roots in 57% and 37% of the explants, respectively. Seven lines of hairy roots were established in WP liquid medium under light and dark conditions. The transformed nature of the root lines was confirmed by polymerase chain reaction using rolB and rolC specific primers. Transformed root cultures of Salvia officinalis showed variations in biomass and rosmarinic acid production depending on the bacterial strain used for transformation and the root line analyzed. Both parameters (growth and rosmarinic acid content) of ATCC 15834-induced lines were significantly higher than the A4-induced lines. The maximum accumulation of rosmarinic acid (about 45 mg g-1 of dry weight) was achieved by hairy root line 1 (HR-1) at the end of the culture period (45D50 days). The level was significantly higher than that found in untransformed root culture (19 mg g-1 of dry wt).

Diterpenoid Production in Hairy Root Culture of Salvia sclarea L.

2008 ◽  
Vol 63 (7-8) ◽  
pp. 621-624 ◽  
Author(s):  
Łukasz Kuzma ◽  
Elzbieta Bruchajze ◽  
Halina Wysokińska
Keyword(s):  
Liquid Medium ◽  
Hairy Root ◽  
Total Content ◽  
Root Culture ◽  
Dry Weight ◽  
Hairy Root Culture ◽  
Growth Cycle ◽  
Transformed Roots ◽  
Salvia Sclarea ◽  
Half Strength

Growth and diterpenoid accumulation (salvipisone, ferruginol, aethiopinone and 1-oxoaethiopinone) during the growth cycle of a Salvia sclarea hairy root culture are described. The roots transformed by Agrobacterium rhizogenes (LBA 9402) were cultured in half-strength B5 liquid medium supplemented with 30 g L-1 sucrose under light (16 h/8 h light/dark). A culture period of 30 days was optimal for both biomass and diterpenoid production. The total content of four diterpenoids in the hairy roots [(27.3 ± 0.6) mg g-1 dry weight] was higher than that of roots of field-grown S. sclarea plants [(3.15 ± 0.15) mg g-1 dry weight]. In transformed roots, aethiopinone was the main diterpenoid, whereas the principal diterpenoid of natural roots was salvipisone.

scholarly journals Enhanced Production of β-Caryophyllene by Farnesyl Diphosphate Precursor-Treated Callus and Hairy Root Cultures of Artemisia vulgaris L.

2021 ◽  
Vol 12 ◽  
Author(s):  
Sundararajan Balasubramani ◽  
B. D. Ranjitha Kumari ◽  
Anil Kumar Moola ◽  
D. Sathish ◽  
G. Prem Kumar ◽  
...  
Keyword(s):  
Hairy Root ◽  
Leaf Explants ◽  
Farnesyl Diphosphate ◽  
Gas Chromatography Mass Spectrometry ◽  
Naphthaleneacetic Acid ◽  
Ms Medium ◽  
Artemisia Vulgaris ◽  
Enhanced Production ◽  
Wide Range ◽  
Half Strength

Artemisia vulgaris L. produces a wide range of valuable secondary metabolites. The aim of the present study is to determine the effects of various concentrations of farnesyl diphosphate (FDP) on β-caryophyllene content in both callus and hairy root (HR) cultures regeneration from leaf explants of A. vulgaris L. Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4D; 4–13 μM), α-naphthaleneacetic acid (NAA; 5–16 μM), and FDP (1 and 3 μM) was used for callus induction and HR regeneration from leaf explants of A. vulgaris L. In this study, precursor-treated (2,4D 13.5 μM + FDP 3 μM) callus displayed the highest biomass fresh weight (FW)/dry weight (DW): 46/25 g, followed by NAA 10.7 μM + FDP 3 μM with FW/DW: 50/28 g. Two different Agrobacterium rhizogenes strains (A4 and R1000) were evaluated for HR induction. The biomass of HRs induced using half-strength MS + B5 vitamins with 3 μM FDP was FW/DW: 40/20 g and FW/DW: 41/19 g, respectively. To determine β-caryophyllene accumulation, we have isolated the essential oil from FDP-treated calli and HRs and quantified β-caryophyllene using gas chromatography–mass spectrometry (GC–MS). The highest production of β-caryophyllene was noticed in HR cultures induced using A4 and R1000 strains on half-strength MS medium containing 3 μM FDP, which produced 2.92 and 2.80 mg/ml β-caryophyllene, respectively. The optimized protocol can be used commercially by scaling up the production of a β-caryophyllene compound in a short span of time.

scholarly journals TÉCNICAS DE TRANSFORMAÇÃO E LINHAGENS De Agrobacterium rhizogenes NA PRODUÇÃO DE HAIRY ROOTS DE Ocimum basilicum L.

Nativa ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 266
Author(s):  
José Nicomedes Júnior ◽  
Roberta Cristiane Ribeiro ◽  
Luis Louro Berbara ◽  
Elvia Mariam Lis Martinez Stark ◽  
Wagner Campos Otoni ◽  
...  
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Roots ◽  
Leaf Explants ◽  
Laboratory Strain ◽  
Root Culture ◽  
Ocimum Basilicum ◽  
Best Response ◽  
Agrobacterium Rhizogenes Strains ◽  
Whole Plants

O manjericão é uma planta medicinal e seus metabólitos especiais são utilizados com fins terapêuticos e industriais. Este trabalho teve por objetivo avaliar técnicas e linhagens de Agrobacterium rhizogenes na produção de raízes transformadas de duas variedades de Ocimum basilicum L., Dark Opal (roxo) e Minete Anão (verde). Verificou-se o efeito da inoculação com linhagens de A. rhizogenes selvagens (A4, 8196, 9402, 2659, 2659 G, 17242, LBA, 15834) ou transformadas (R1601) em plantas inteiras ou explantes (folhas e segmentos de caule) de manjericão, cultivados em casa de vegetação ou in vitro. A inoculação com as linhagens LBA e 8196 possibilitou redução no número de explantes de folha de manjericão-verde oxidados, quando comparado ao controle. Dentre as linhagens selvagens, as que proporcionaram melhor indução na produção de raízes transformadas foram A4, LBA e 8196, respectivamente. Entretanto, a cepa R1601 apresentou a melhor resposta. Raízes foram eficientemente induzidas mediante a inoculação de explantes de manjericão-roxo e manjericão-verde e expressaram o fenótipo típico de raízes em cabeleira (hairy roots). As culturas clonais de manjericão-verde apresentaram rápido crescimento em meio de cultura livre de reguladores de crescimento.Palavras-chave: agrobactéria, cultura de raízes, manjericão, plantas medicinais. TRANSFORMATION TECHNIQUES AND Agrobacterium rhizogenes Strains In The Production Of Hairy Roots Of Ocimum basilicum L. ABSTRACT:Basil is a medicinal plant and its special metabolites are used for therapeutic or industrial purposes. The aim of this work was to evaluate Agrobacterium rhizogenes techniques and strains in the production of hairy roots of two varieties of Ocimum basilicum L., Dark Opal (purple) and Dwarf Racer (green). The effects of inoculation of A. rhizogene (A4, 8196, 9402, 2659, 2659, 2659 G, 17242, LBA, 15834) or (R1601) lines on whole plants or explants (leaves and stem segments) of basil, grown in a greenhouse or in vitro, in the production of hairy roots and calluses made by subcultures. Inoculation with the LBA and 8196 lines allowed a reduction in the number of oxidized basil-green leaf explants when compared to the control. Among the wild strains, those that had best ability to induce hairy root of hairy roots were A4, LBA and 8196, respectively. However, the R1601 laboratory strain had the best response. Roots were efficiently induced by the inoculation of explants basil-purple and basil-green and expressed the typical phenotype of hairy roots. Green-basal clonal cultures showed rapid growth in culture medium free of growth regulators.Keywords: agrobacterim, basil, medicinal plants, root culture. DOI:

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