Hairy root induction in Catharanthus roseus by various strains of Agrobacterium rhizogenes isolated in Vietnam

Catharanthus roseus is a well known medicinal plant. It produces several phytocompounds and many of which show anticancerous properties. However the yields of these compounds are very low. Recently, induction of C. roseus hairy roots by Agrobacterium rhizogenes, is interested as a promising tool for the enhanced production of these metabolites. In this research, wounded leaves from four strains of C. roseus were infected with various strains of A. rhizogenes isolated in Vietnam to provide more information about the induction efficiency of hairy roots. In this experiment, after 3 weeks of infection, the presence of rolB gene in hairy roots were analysed by polymerase chain reaction. All of 13 A. rhizogenes strains could induce the formation of hairy root in C. roseus. The A. rhizogenes C18 strain had the highest induction percentage in C. roseus VIN002, VIN005, and VIN072 with 59.4 %, 50.3 %, and 40.0 % respectively. And the same result was ontained at 26.7% by A. Rhizogenes C26 for C. roseus VIN077 rhizogenes strains. This result identified two A. rhizogenes strains C18 and C26 as potential transformation tools for hairy root production from C. roseus.

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Effect of Different Strains of Agrobacterium rhizogenes and Nature of Explants on Plumbago indica Hairy Root Culture with Special Emphasis on Root Biomass and Plumbagin Production

Natural Product Communications ◽

10.1177/1934578x1000501215 ◽

2010 ◽

Vol 5 (12) ◽

pp. 1934578X1000501

Author(s):

Moumita Gangopadhyay ◽

Saikat Dewanjee ◽

Somnath Bhattacharyya ◽

Sabita Bhattacharya

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Root Biomass ◽

Leaf Explants ◽

Pcr Analysis ◽

Stem Explants ◽

Root Induction ◽

Rolb Gene ◽

Plumbago Indica

The aim of the present study was to determine the effect of three strains of Agrobacterium rhizogenes (ATCC 15834, A4 and LBA 9402) and the nature of explants (leaf and stem) on hairy root induction, growth and plumbagin production in Plumbago indica. The first appearance of hairy roots, the transformation frequency, dry root biomass and plumbagin accumulation were found to be maximum in hairy roots induced in leaf explants infected with A. rhizogenes ATCC 15834 as compared with the other two bacterial strains. The hairy roots generated from stem explants infected with all three strains were not found to be productive in terms of the selected parameters. Finally, the insertion of the rolB gene of A. rhizogenes ATCC 15834 in hairy roots of P. indica derived from leaf explants was confirmed by PCR analysis.

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Technology and Application of Hairy Root Culture in Monocotyledons

Current Biochemical Engineering ◽

10.2174/2212711907666210525115018 ◽

2021 ◽

Vol 07 ◽

Author(s):

Chang-Qi Hao ◽

Shuai-Run Wang ◽

Yi Wang ◽

Xin-Yi Hou ◽

Ya-Xuan Jiang ◽

...

Keyword(s):

Medicinal Plants ◽

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Root Culture ◽

Hairy Root Culture ◽

Food Crops ◽

Root Induction ◽

Hairy Root Induction ◽

Selection Of

Background: Hairy root culture has been widely used in the production of metabolites in dicotyledons, and a large number of food crops and medicinal plants in monocotyledons need to be developed, but there are many difficulties in the induction of hairy roots in monocotyledons. The purpose of this paper is to introduce the inducing methods, influencing factors and application of hairy roots in monocotyledons, and to promote the development of hairy root system in monocotyledons. Methods: The mechanism of action of Agrobacterium rhizogenes and the current situation of hairy root induction, induction methods and influencing factors of monocotyledons were summarized so as to provide convenience for efficient acquisition of hairy root of monocotyledons. Results: Monocotyledons are not easy to produce phenols, cells are prone to lignification, adverse differentiation and selective response to Agrobacterium rhizogenes strains. It is proposed that before induction, plant varieties and explants should be selected, and different infection strains should be screened. In the process of hairy root induction, exogenous inducers such as acetosyringone can be added. Although these factors can provide some help for the induction of hairy roots in monocotyledons, we still need to pay attention to the disadvantages of monocotyledons from dicotyledons at the cellular level. Conclusion: A large number of food crops and medicinal plants are monocotyledons. Hairy root culture can be used to help the breeding and production of medicinal substances. Therefore, it is necessary to pay attention to the selection of varieties and explants, the selection of Agrobacterium rhizogenes and the addition of acetosyringone in the process of hairy root induction so as to improve the production efficiency and facilitate the development and utilization of monocotyledons.

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Evaluating α-glucosidase inhibitory activity of hairy roots cultured from some plants in malvaceae family induced by Agrobacterium rhizogenes ATCC 15834

Science and Technology Development Journal - Natural Sciences ◽

10.32508/stdjns.v2i6.877 ◽

2020 ◽

Vol 2 (6) ◽

pp. 165-174

Author(s):

Vu Thi Bach Phuong ◽

Pham Thi Anh Hong ◽

Quach Ngo Diem Phuong

Keyword(s):

Type 2 Diabetes ◽

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Inhibitory Activity ◽

Root Culture ◽

Root Induction ◽

Hibiscus Sabdariffa ◽

Sida Acuta

One of the most effective methods for type 2 diabetes treatments is inhibition of enzyme α-glucosidase in the intestines to slow down the release of glucose from carbohydrates in the diet, reduce plasma glucose levels and prevent hyperglycemia after meals. Therefore, seeking α-glucosidase inhibitors used in the treatment of diabetes from plant is the attention of many scientists. Based on the potential of the hairy root culture technology in increasing valuable chemical compounds accumulating, this study aimed to induce hairy roots from six plants of the Malvaceae family including Urena lobata, Abutilon indicum, Hibiscus Sabdariffa, Hibiscus rosa-sinensis, Sida acuta, Sida rhombifolia, and screening which materials has the highest in α-glucosidase inhibitory activity. We have successfully induced hairy roots from six plant species by using the Agrobacterium rhizogenes ATCC 15834 strain. The highest rates of hairy root induction were observed in Hibiscus Sabdariffa and Urena lobata. The stable introduction of rolB and rolC genes to plant genomes was confirmed by PCR. Under liquid-shake culture conditions on MS medium, hairy roots of Hibiscus sabdariffa, Urena lobata and Sida acuta showed better development than other species, and therefore, they are selected for the study of α-glucosidase inhibitory activity. This study proved that Urena lobata was stronger in inhibiting α-glucosidase activity than other studied plants, with the IC50 value of 7.65 μg/ml. The results of this study demonstrated Urena lobata hairy root might be considered as a potential supply of medicinal plants for the treatment of type 2 diabetes.

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Hairy root culture of white mulberry (Morus alba L.) for a source of tyrosinase inhibitors

Journal of Biological Research - Bollettino della Società Italiana di Biologia Sperimentale ◽

10.4081/jbr.2020.8611 ◽

2020 ◽

Vol 93 (1) ◽

Author(s):

Hanh Thi My Nguyen ◽

Tan Quoc Tran ◽

Anh Lan Bui ◽

Phuong Ngo Diem Quach

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Root Culture ◽

Hairy Root Culture ◽

Morus Alba ◽

Root Induction ◽

Skin Whitening ◽

Tyrosinase Inhibitors ◽

White Mulberry

White mulberry (Morus alba L.) root was reported to contain potential tyrosinase inhibitors such as norartocarpetin, streppogenin, oxyresveratrol (5, 7, 2′, 4′-tetrahydroxy-flavavone) and 2-arylbenzofuran. This study aims at investigating Morus alba L. hairy root culture to create a source of tyrosinase inhibitors for skin-whitening application. Morus alba L. leaf (including mesophyll and petiole) and stem explants were randomly wounded and inoculated with Agrobacterium rhizogenes ATCC 15834 in 10-40 minutes, and coculturing for 4 days. After inoculation, Agrobacterium rhizogenes were removed by using MS medium adding 200mg/L cefotaxime as antibiotics. Then, fresh inducted hairy roots were grown in nonphytohormone liquid medium (MS, White, Gamborg B5 or WPM) on a shaker (80rpm) at 25 ±1°C. Inducted hairy roots were tested for the present of rolB by PCR analysis and its effect on tyrosinase activity using a 96-well plate reader. The suitable explants for hairy root induction are leaves, especially petioles in 10 minutes (root induction frequency: 83%). After induction, hairy roots on MS was grown more efficiently (increased 3.933 times in FW) than on WPM, B5 and White medium. Inducted hairy roots were successfully transgenes by the present of rolB in their genome and had the same anti-tyrosinase activity as the natural roots without significance differences (IC50: 3.860 and 3.597 μg/mL, respectively). Cultured Morus alba L. hairy roots are potential source of tyrosinase inhibitors for future skin-whitening ingredient.

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Comparison of Different Strains of Agrobacterium rhizogenes for Hairy Root Induction and Betulin and Betulinic Acid Production in Morus alba

Natural Product Communications ◽

10.1177/1934578x1701200403 ◽

2017 ◽

Vol 12 (4) ◽

pp. 1934578X1701200

Author(s):

Chang Ha Park ◽

Shicheng Zhao ◽

Hyeon Ji Yeo ◽

Ye Eun Park ◽

Thanislas Bastin Baska ◽

...

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Betulinic Acid ◽

Acid Production ◽

Leaf Tissue ◽

Dry Weight ◽

Morus Alba ◽

Root Induction ◽

Different Strains

Three Agrobacterium rhizogenes strains were tested for their ability to transform the plant Morus alba L. and to induce production of the secondary metabolites betulin and betulinic acid. All the tested strains of A. rhizogenes (R1601, LBA9402 and R1000) were able to induce hairy root formation in leaf tissue explants. Strain LBA9402 had the highest rate of infection (92.7% ± 8.8%), whereas strain R1601 had the lowest rate (87.4% ± 9.3%). The highest number of hairy roots per explant (5.6 ± 0.5) and the greatest root length (2.4 ± 0.2 mm) were obtained with strain LBA9402. We also evaluated dry weight (a measure of growth) and betulin and betulinic acid production in hairy roots and found that the highest growth (167.8 ± 14.5 mg/flask) occurred after infection with strain LBA9402. Furthermore the highest production of betulin (5.4 ± 0.4 mg/g dry weight) and betulinic acid (2.3 ± 0.2 mg/g dry weight) was noted using strain LBA9402. Among three elicitors, yeast extract showed the highest induction of betulin production (8.7 ± 0.4 mg/g) and silver nitrate induced the highest yield of betulinic acid (4.1 ± 0.2 mg/g). Our study showed that A. rhizogenes strain LBA9402 was the most effective of the three tested strains for production of transformed root cultures and betulin and betulinic acid.

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

Science and Technology Development Journal - Natural Sciences ◽

10.32508/stdjns.v2i1.667 ◽

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.

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High-Efficiency Agrobacterium rhizogenes-Mediated Transgenic Hairy Root Induction of Lens culinaris

Agronomy ◽

10.3390/agronomy10081170 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1170

Author(s):

Chrysanthi Foti ◽

Ourania Pavli

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

High Efficiency ◽

Culture Media ◽

Abiotic Stress Tolerance ◽

Root Induction ◽

Media Composition ◽

Transgenic Hairy Root ◽

Transgenic Hairy Roots

Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant nature, giving rise to particularly low transformation and regeneration frequencies. This study aimed at optimizing an Agrobacterium rhizogenes-mediated transformation protocol for the generation of composite lentil plantlets, comprised of transgenic hairy roots and wild-type shoots. Transformation was performed by inoculating the cut hypocotyl of young lentil seedlings, while optimization involved the use of different bacterial strains, namely R1000, K599 and Arqua, and protocols differing in media composition with respect to the presence of acetosyringone and MES. Composite plantlets had a transgenic hairy root system characterized by an increased number of hairy roots at the hypocotyl proximal region, occasionally showing plagiotropic growth. Overall findings underline that transformation frequencies are subject to the bacterial strain, media composition as well as their combined effect. Among strains tested, R1000 proved to be the most capable of hairy root formation, while the presence of both acetosyringone and MES in inoculation and culture media yielded considerably higher transformation rates. The transgenic nature of hairy roots was demonstrated by the Ri T-DNA-mediated transfer of the rolB2 gene and the simultaneous absence of the virCD sequence of A. rhizogenes. Our findings provide strong evidence that A. rhizogenes-mediated transformation may be employed as a suitable approach for generating composite seedlings in lentil, a species whose recalcitrance severely hampers all efforts addressed to transformation and whole plant regeneration procedures. To the best of our knowledge, this is the first report on the development of a non-laborious and time-efficient protocol for the generation of transgenic hairy roots in lentil, thus providing an amenable platform for root biology and gene expression studies in the context of improving traits related to biotic and abiotic stress tolerance.

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Isolation and identification of Agrobacterium rhizogenes inducing hairy roots of Catharanthus roseus in vivo

Science and Technology Development Journal - Natural Sciences ◽

10.32508/stdjns.v1it1.434 ◽

2017 ◽

Vol 1 (T1) ◽

pp. 48-59

Author(s):

Nhut Nhu Nguyen ◽

Le Van Bui

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Catharanthus Roseus ◽

Root Formation ◽

Isolation And Identification ◽

16S Rrna Sequence ◽

Pathogenicity Tests ◽

Foreign Genes

Agrobacterium rhizogenes is a common tool for transforming foreign genes into plant cell and inducing hairy root formation. A total of 274 colonies of Agrobacterium species was isolated on selective MG-Te medium from 235 samples of rhizophere soils. There were only thirteen colonies belonged to A. rhizogenes being able to efficiently induce the hairy root formation of Catharanthus roseus in vivo based on the morphological, biochemical, and pathogenicity tests. These thirteen colonies were identified as A. rhizogenes based on 16S rRNA sequence and possessing rolABC genes. The results indicated A. rhizogenes was universal in rhizophere soil in Vietnam and riched transgenic tools for researching C. roseus hairy roots in the future.

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Induction of Platycodon grandiflorum hairy roots through the mediation of four Agrobacterium rhizogenes strains

Science and Technology Development Journal ◽

10.32508/stdj.v19i4.624 ◽

2016 ◽

Vol 19 (4) ◽

pp. 64-75

Author(s):

Phuong Dong Tra ◽

Phuong Thi Bach Vu ◽

Phuong Ngo Diem Quach

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Biological Activities ◽

Root Induction ◽

Infection Frequency ◽

Platycodon Grandiflorum ◽

Metabolites Production ◽

Valuable Compounds ◽

Agrobacterium Rhizogenes Strains

Balloon flower (Platycodon grandiflorum (Jacq.) A. DC.), the only species in Platycodon genus (Campanulaceae), is mainly distributed in East Asia. The rhizomes of P. grandiflorum, a traditional herbal medicine, have been widely used for the treatment of cough, sore throat, asthma, tuberculosis and other diseases. Recently, pharmacological researches identified important biological activities compounds in the rhizomes. Thus, to study and extract valuable compounds, a hairy root induced technique was achieved on P. grandiflorum for stable material with fast growth rates (in hormone-free media) and metabolites production. To achieve this, the “natural genetic tool” Agrobacterium rhizogenes, which can transfer DNA segments into genome of plant, was exploited. The results suggested two (A. rhizogenes ATCC 15834 and C34) of four A. rhizogenes strains could induce hairy roots. RolB and rolC genes, which are responsible for the induction of hairy roots, were inserted into the genome of hairy roots. Leaves had the highest infection frequency of hairy root induction 100 %. The optimization of protocol, including time of immersion and co-culture, had the best results with 10 and 15 mins (10 mins for A. rhizogenes ATCC 15834 and 15 mins for A. rhizogenes C34) and 72 hours, respectively. In the future, this protocol, which was described in this paper, should be useful for studying and isolating valuable compounds from P. grandiflorum hairy root cultures.

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Hairy Root Induction on Justicia gendarussa by Various Density of Agrobacterium rhizogenes strain LB 510

Biosaintifika Journal of Biology & Biology Education ◽

10.15294/biosaintifika.v9i1.8223 ◽

2017 ◽

Vol 9 (1) ◽

pp. 26

Author(s):

Dwi Kusuma Wahyuni ◽

Siti Nur Hafida ◽

Tri Muji Ermayanti ◽

Bambang Prajoga Eko Wardoyo ◽

Hery Purnobasuki ◽

...

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Solid Medium ◽

Leaf Explants ◽

Root Culture ◽

Hairy Root Culture ◽

Root Induction ◽

Hairy Root Induction ◽

Industrial Needs

Gandarusa (Justicia gendarussa Burm.f.) is an Indonesian medicinal plant that has many benefits as drug and male contracetive. For industrial needs, Gandarusa must be available in large quantity. Hairy root culture is one of methode to produce phytochemistry compound. The objective of the study was to examine the effect of various density of Agrobacterium rhizogenes strain LB510 on hairy roots induction of gandarusa (Justicia gendarussa Burm.f.) leaf plant. Leaf explants were inoculated in MS liquid medium with various density of OD600 = 0.1; 0.2; 0.3; 0.4; and 0.5. Explants were co-cultivated for 2 days on MS solid medium without any hormone then sub-cultured on MS solid medium containing antibiotic cefotaxim 300 ppm, in dark condition. The data were analyzed descriptively and statistically. The results showed that various density of Agrobacterium rhizogenes strain LB510 was affected the lenght of hairy roots induction of J. gendarussa Burm.f., but these was not effected toward lenght formation time and number of hairy root. The treatment of OD600 0.2 was the best treatment for hairy root induction on Justicia gendarussa Burm. f. This data could be used for optimized the quality of methode of hairy root induction.

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