Arbuscular mycorrhizal responsiveness of in vitro tomato root lines is not related to growth and nutrient uptake rates

2003 ◽  
Vol 81 (7) ◽  
pp. 645-656 ◽  
Author(s):  
Karine Labour ◽  
Mario Jolicoeur ◽  
Marc St-Arnaud
Keyword(s):  
Nutrient Uptake ◽  
Hairy Root ◽  
Hairy Roots ◽  
Inorganic Phosphate ◽  
Minimal Medium ◽  
Glomus Intraradices ◽  
Phosphate Concentration ◽  
Tomato Cultivar ◽  
Uptake Rates

Variability in growth and nutritional dynamics of in vitro tomato hairy root lines and their relationship with responsiveness to mycorrhizal colonization were studied. Four tomato cultivars were transformed with three Agrobacterium rhizogenes strains to obtain several hairy root lines, which were compared for growth and receptivity to Glomus intraradices. Four transformed hairy root lines were further characterized and compared with excised roots of the nontransformed tomato cultivar 'Cobra' and with Ri-T-DNA carrot hairy roots. Lines were compared during 4 months on minimal medium in terms of growth, nutrient uptake, and mycorrhizal colonization. In a subexperiment, the cultures were grown on a modified minimal medium to assess the contribution of initial inorganic phosphate concentration in mycorrhizal susceptibility of the three initially nonreceptive lines. On minimal medium, growth and nutrient uptake rates were highly correlated, but both were unrelated to mycorrhizal receptiveness. All the lines successfully established the symbiosis when the initial phosphate concentration was significantly reduced. No association was found between the origin of lines from the different tomato cultivar – bacterial strain combinations and the absence of symbiosis establishment on minimal medium. Decrease of inorganic phosphate concentration at the beginning of the culture was a key factor involved in precolonization steps of mycorrhizal symbiosis.Key words: Glomus intraradices, hairy roots, Lycopersicon esculentum, mycorrhizal responsiveness, root nutrition, inorganic phosphate.

Agrobacterium rhizogenes-Transformed Roots of Medicago truncatula for the Study of Nitrogen-Fixing and Endomycorrhizal Symbiotic Associations

2001 ◽  
Vol 14 (6) ◽  
pp. 695-700 ◽  
Author(s):  
Aurélien Boisson-Dernier ◽  
Mireille Chabaud ◽  
Fernand Garcia ◽  
Guillaume Bécard ◽  
Charles Rosenberg ◽  
...  
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Medicago Truncatula ◽  
Hairy Root ◽  
Hairy Roots ◽  
Glomus Intraradices ◽  
Subsequent Development ◽  
Nitrogen Fixing ◽  
Root Explants ◽  
Symbiotic Associations

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous “composite plants.” In addition, we found that kanamycin can be used to select for the co-transformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter p MtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbus-cules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.

scholarly journals Resveratrol Biosynthesis in Hairy Root Cultures of Tan and Purple Seed Coat Peanuts

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 975
Author(s):  
Ye-Eun Park ◽  
Chang-Ha Park ◽  
Hyeon-Ji Yeo ◽  
Yong-Suk Chung ◽  
Sang-Un Park
Keyword(s):  
Arachis Hypogaea ◽  
Hairy Root ◽  
Hairy Roots ◽  
Seed Coat ◽  
Biological Properties ◽  
Fresh Weight ◽  
Good Source ◽  
Plant Parts ◽  
Seedling Roots

Peanut (Arachis hypogaea) is a crop that can produce resveratrol, a compound with various biological properties, such as those that exert antioxidant, anticancer, and anti-inflammatory effects. In this study, trans-resveratrol was detected in the roots, leaves, and stems of tan and purple seed coat peanuts (Arachis hypogaea) cultivated in a growth chamber. Both cultivars showed higher levels of resveratrol in the roots than the other plant parts. Thus, both cultivars were inoculated with Agrobacterium rhizogenes, in vitro, to promote hairy root development, thereby producing enhanced levels of t-resveratrol. After 1 month of culture, hairy roots from the two cultivars showed higher levels of fresh weight than those of seedling roots. Furthermore, both cultivars contained higher t-resveratrol levels than those of their seedling roots (6.88 ± 0.21 mg/g and 28.07 ± 0.46 mg/g, respectively); however, purple seed coat peanut hairy roots contained higher t-resveratrol levels than those of tan seed coat peanut hairy roots, ranging from 70.16 to 166.76 mg/g and from 46.61 to 54.31 mg/g, respectively. The findings of this study indicate that peanut hairy roots could be a good source for t-resveratrol production due to their rapid growth, high biomass, and substantial amount of resveratrol.

scholarly journals Improving hairy root induction of Urena lobata L. by Agrobacterium rhizogenes ATCC 15834 by some factors

2018 ◽  
Vol 21 (3) ◽  
pp. 90-97 ◽  
Author(s):  
Vu Thi Bach Phuong ◽  
Pham Thi Anh Hong ◽  
Quach Ngo Diem Phuong
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Culture Medium ◽  
Transformation Frequency ◽  
Root Induction ◽  
Infection Time ◽  
Fresh Biomass ◽  
In Vitro Plants ◽  
Original Procedure

Introduction: Our previous study showed that Urena lobata L. hairy root is a potential pharmaceutical source for type 2 diabetes treatment. In order to improve the transformation efficacy and the quality of hairy roots, this study examined the effects of several factors including age, parts of plants, infection time and culture medium in inducing hairy roots in Urena lobata L. Methods: In this study, we investigated four factors to improve the hairy root induction in Urena lobata L. These factors include: age of plant (15-day-old in vitro plants, 45-day-old in vitro plants and after two subculture generations plants), different parts of plant (roots, stems, and leaves), infection time (10, 20 and 30 minutes), and culture medium (Murashige and Skoog (MS), Gamborg B5 medium (GB5) and Woody plant medium (WPM)). All experiments were repeated three times, with uninfected leaf explants of 15-day-old in vitro as the negative control. The transformation frequency and the fresh biomass of hairy roots were recorded at four weeks after infection. Results: The results showed that the optimized procedure which used 15-day-old in vitro plants, the leafy part, the infection time of 10 minutes and culture in the WPM medium was better than the original procedure. The optimized procedure achieved a transformation frequency of 100%. In addition, the fresh biomass of hairy roots formed on an explant in the optimized procedure was 3.2 times higher than the ones induced by the original procedure. Conclusion: The results showed that the optimized procedure was more effective than the original procedure in inducing Urena lobata hairy roots.  

scholarly journals An Efficient Root Transformation System for Recalcitrant Vicia sativa

2022 ◽  
Vol 12 ◽  
Author(s):  
Vy Nguyen ◽  
Iain R. Searle
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Functional Gene ◽  
Gene Analysis ◽  
Vicia Sativa ◽  
Transformation System ◽  
Common Vetch ◽  
Root Transformation ◽  
Functional Gene Analysis

Common vetch (Vicia sativa) is a multi-purpose legume widely used in pasture and crop rotation systems. Vetch seeds have desirable nutritional characteristics and are often used to feed ruminant animals. Although transcriptomes are available for vetch, problems with genetic transformation and plant regeneration hinder functional gene studies in this legume species. Therefore, the aim of this study was to develop a simple, efficient and rapid hairy root transformation system for common vetch to facilitate functional gene analysis. At first, we infected the hypocotyls of 5-day-old in vitro or in vivo, soil-grown seedlings with Rhizobium rhizogenes K599 using a stabbing method and produced transgenic hairy roots after 24 days at 19 and 50% efficiency, respectively. We later improved the hairy root transformation in vitro by infecting different explants (seedling, hypocotyl-epicotyl, and shoot) with R. rhizogenes. We observed hairy root formation at the highest efficiency in shoot and hypocotyl-epicotyl explants with 100 and 93% efficiency, respectively. In both cases, an average of four hairy roots per explant were obtained, and about 73 and 91% of hairy roots from shoot and hypocotyl-epicotyl, respectively, showed stable expression of a co-transformed marker β-glucuronidase (GUS). In summary, we developed a rapid, highly efficient, hairy root transformation method by using R. rhizogenes on vetch explants, which could facilitate functional gene analysis in common vetch.

scholarly journals Alterations in oleanolic acid and sterol content in marigold (Calendula officinalis) hairy root cultures in response to stimulation by selected phytohormones

2021 ◽  
Vol 43 (3) ◽  
Author(s):  
Abdulwadood S. M. Alsoufi ◽  
Klaudia Staśkiewicz ◽  
Michał Markowski
Keyword(s):  
Oleanolic Acid ◽  
Hairy Root ◽  
Hairy Roots ◽  
Naphthaleneacetic Acid ◽  
Hairy Root Cultures ◽  
Root Cultures ◽  
Calendula Officinalis ◽  
Plant Metabolites ◽  
Triterpenoid Saponins

AbstractHairy root cultures are an efficient tool for the biotechnological production of plant metabolites and a convenient experimental model for analyzing the effect of various compounds on plant metabolism. In contrast to many other types of in vitro plant cultures, hairy roots do not require an external supply of phytohormones to the medium. Consequently, plant growth regulators such as auxins and cytokinins are rarely used as elicitors in hairy root in vitro cultures; however, they can strongly influence plant defense responses. The aim of this study was to investigate the influence of two auxins: natural indole-3-acetic acid (IAA) and synthetic 1-naphthaleneacetic acid (NAA), as well as two cytokinins: natural kinetin and synthetic 6-benzylaminopurine (BAP) at a concentration of 0.75 mg/L on the metabolism of sterols and triterpenoids in Calendula officinalis hairy roots. Auxins prevented the accumulation of triterpenoid saponins (oleanolic acid glycosides), while cytokinin BAP increased their accumulation by 17% and their release into the culture medium by a factor of 10. Other cytokinins and kinetins increased the sterol levels by 17%, the level of stigmasterol by 15%, and the level of isofucosterol by 7 times.

scholarly journals Kultur akar rambut Cinchona ledgeriana dan C. succirubra dalam kultur in vitro Hairy root culture of Cinchona ledgeriana and C. succirubra by in vitro culture

2016 ◽  
Vol 72 (2) ◽  
Author(s):  
Nurita TORUAN-MATHIUS ◽  
. REFLINI ◽  
. NURHAIMI-HARIS ◽  
. JOKO-SANTOSO ◽  
A PRIANGANI-ROSWIEM
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Basal Medium ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Chain Reaction ◽  
Ri Plasmid ◽  
Polymerase Chain ◽  
Cinchona Ledgeriana

Summary Problems encountered in hairy root culture  of  C. ledgeriana and C. succirubra are low percentage of transformation of explants by Agrobacterium rhizogenes and slow growth of hairy root. The objective of this research was to evaluate the potential of several A. rhizogenes strains for initiation  hairy roots of  C. succirubra and C. ledgeriana, and to obtain the best medium for hairy root culture of Cinchona spesies. Axenic shoot and leaves explants of eight-month-old of C. ledgeriana and  C. succirubra seedlings were inoculated with A. rhizogenes strain ATCC-15834, ATCC-8196,    R-20001, 07-20001, A4, R-MAFFA, TISTR509, TISTR510 and LBA9457. Inoculated explants were cultured in solid MS medium with the addition of 100 mg/L amphicylin. Subculture of the hairy root was performed by transferred of root pieces into fresh liquid basal medium MS, B5, White and Heller. Hairy roots from the best of basal medium were subcultured on the same medium with the addition of 50  and 100 mg/L   L-tryptophane, three or five times concentration of MS vitamins. The integration of T-DNA of   A. rhizogenes in hairy root was confirmed with specific primer for TL and TR-DNA of plasmid by Polymerase Chain Reaction analysis. The results showed that only A. rhizogenes strain  LBA 9457 were effective for  transformation of explants from both Cinchona species. The fastest hairy roots growth were found  in MS medium, while growth in others medium was poor. Hairy roots of  C. ledgeriana has vigor and growth better than hairy roots of C. succirubra. MS with the addition of 50 mg/L  L-tryptophane and  three times the concen-trations of vitamin  is the best medium for hairy root growth and vigor. Hairy roots of  C. succirubra and C. ledgeriana used in this studies were confirmed that hairy roots  contained TL and TR-DNA region of Ri plasmid with molecular weight 780 and 1600 bp.  The results showed that strain of A. rhizogenes, plant species, source of explant and composition of medium affect the initiation, growth, development  and vigor of hairy roots.Ringkasan Masalah dalam kultur akar rambut  C. ledgeriana dan C. succirubra adalah rendahnya tingkat keberhasilan transformasi eksplan dengan Agrobacterium rhizogenesdan pertumbuhannya yang lambat. Penelitian ini bertujuan untuk mengevaluasi  potensi dari beberapa galur A. Rhizogenes untuk inisiasi, mendapatkan komposisi medium terbaik untuk pertumbuhan akar rambut C. ledgeriana dan C. succirubra, serta konfirmasi terintegrasinya TR dan TL-DNA Ri plasmid ke dalam jaringan eksplan.  Eksplan batang  dan  daun  berasal  dari kecambah aksenik C. ledgeriana dan C. succirubra berumur delapan bulan diinokulasi dengan A. rhizogenes galur 15834, 8196, R-20001, 07-20001, A4, R.MAFFA,TISTR 509, TISTR 510 dan LBA 9457. Eksplan yang sudah diinokulasi dikulturkan dalam medium MS padat. Subkultur dilakukan dengan cara mentransfer potongan ujung akar rambut ke dalam medium cair MS, B5, White dan Heller. Akar rambut dari medium kultur yang terbaik kemudian disubkultur ke dalam medium yang sama dengan penambahan 50 dan 100 mg/L L-triptofan dengan konsentrasi vitamin sebanyak tiga kali dan lima kali dari konsentrasi normal MS. Integrasi T-DNA dalam akar rambut dikonfirmasi meng-gunakan Polymerase Chain Reaction  dengan primer spesifik untuk TL dan TR-DNA plasmid. Hasil yang diperoleh menunjukkan bahwa hanya A.rhizogenes galur LB9457 yang efektif menginfeksi eksplan baik batang maupun daun dari kedua spesies kina. Induksi, pertumbuhan dan vigor akar rambut yang terbaik diperoleh dari medium MS dengan penambahan 50 mg/L L-triptofan dan tiga kali konsentrasi vitamin. Hasil konfirmasi akar rambut baik dari batang maupun daun menggunakan PCR, menunjukkan bahwa TL dan TR-DNA dari Ri plasmid  A. rhizogenes mampu menghasilkan pita-pita DNA dengan BM780 dan 1600 pb. Hasil yang diperoleh menunjukkan bahwa galur  A. rhizogenes, spesies tanaman, sumber eksplan dan komposisi medium berpengaruh terhadap inisiasi, pertumbuhan,  perkembangan dan vigor akar rambut.

Extracellular ribonuclease secretion by Ustilago hordei

1971 ◽  
Vol 49 (11) ◽  
pp. 2035-2039
Author(s):  
N. T. Bech-Hansen ◽  
C. O. Person
Keyword(s):  
Stationary Phase ◽  
Gel Electrophoresis ◽  
Inorganic Phosphate ◽  
Culture Media ◽  
Phosphate Concentration ◽  
Glucose Content ◽  
Ustilago Hordei ◽  
Parasitic Phase

RNase assays and gel electrophoresis of culture media have provided evidence that U. hordei, grown in vitro during the haploid (non-parasitic) phase, produced several extracellular RNases. The secretion of the RNases occurred during the end of log phase and at the beginning of stationary phase. Secretion was not affected by the addition of RNA, or by varying the inorganic phosphate concentration; it was enhanced in media of increased glucose content.

scholarly journals Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes‑mediated transformation

2011 ◽  
Vol 46 (9) ◽  
pp. 1070-1075 ◽  
Author(s):  
Ricardo Luís Mayer Weber ◽  
Maria Helena Bodanese‑Zanettini
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Fluorescent Protein ◽  
Root Culture ◽  
Transformation Frequency ◽  
Plant Production ◽  
Uniform Size ◽  
Soybean Genotypes

The objective of this work was to perform the screening of soybean genotypes as to their ability to respond to the induction of hairy roots by Agrobacterium rhizogenes‑mediated transformation. Four Brazilian soybean cultivars (BRSMG 68 Vencedora, BRS 137, Embrapa 48, and MG/BR 46 Conquista) and two North American ones adapted to Brazilian cropping conditions (Bragg and IAS‑5) were screened for their capacity to respond to A. rhizogenes in protocols for in vitro hairy root culture and ex vitro composite plant production. Four‑day‑old seedlings with uniform size were injected with A. rhizogenes harboring the plasmid p35S‑GFP. Seedlings expressing green fluorescent protein (GFP) in at least one hairy root were used to determine the transformation frequency. Using an axenic in vitro protocol, excised cotyledons from four‑day‑old seedlings were infected with A. rhizogenes harboring the pCAMBIA1301 plasmid, containing the gusA reporter gene. The transformation frequency and the number of days for hairy root emergence after bacterial infection (DAI) were evaluated. The transformation frequency and DAI varied according to the genotype. Cultivars MG/BR 46 Conquista and BRSMG 68 Vencedora are more susceptible to A. rhizogenes and can be recommended for transformation experiments.

scholarly journals Kultur akar rambut Cinchona ledgeriana dan C. succirubra dalam kultur in vitro Hairy root culture of Cinchona ledgeriana and C. succirubra by in vitro culture

2016 ◽  
Vol 72 (2) ◽  
Author(s):  
Nurita TORUAN-MATHIUS ◽  
. REFLINI ◽  
. NURHAIMI-HARIS ◽  
. JOKO-SANTOSO ◽  
A PRIANGANI-ROSWIEM
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Basal Medium ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Chain Reaction ◽  
Ri Plasmid ◽  
Polymerase Chain ◽  
Cinchona Ledgeriana

Summary Problems encountered in hairy root culture  of  C. ledgeriana and C. succirubra are low percentage of transformation of explants by Agrobacterium rhizogenes and slow growth of hairy root. The objective of this research was to evaluate the potential of several A. rhizogenes strains for initiation  hairy roots of  C. succirubra and C. ledgeriana, and to obtain the best medium for hairy root culture of Cinchona spesies. Axenic shoot and leaves explants of eight-month-old of C. ledgeriana and  C. succirubra seedlings were inoculated with A. rhizogenes strain ATCC-15834, ATCC-8196,    R-20001, 07-20001, A4, R-MAFFA, TISTR509, TISTR510 and LBA9457. Inoculated explants were cultured in solid MS medium with the addition of 100 mg/L amphicylin. Subculture of the hairy root was performed by transferred of root pieces into fresh liquid basal medium MS, B5, White and Heller. Hairy roots from the best of basal medium were subcultured on the same medium with the addition of 50  and 100 mg/L   L-tryptophane, three or five times concentration of MS vitamins. The integration of T-DNA of   A. rhizogenes in hairy root was confirmed with specific primer for TL and TR-DNA of plasmid by Polymerase Chain Reaction analysis. The results showed that only A. rhizogenes strain  LBA 9457 were effective for  transformation of explants from both Cinchona species. The fastest hairy roots growth were found  in MS medium, while growth in others medium was poor. Hairy roots of  C. ledgeriana has vigor and growth better than hairy roots of C. succirubra. MS with the addition of 50 mg/L  L-tryptophane and  three times the concen-trations of vitamin  is the best medium for hairy root growth and vigor. Hairy roots of  C. succirubra and C. ledgeriana used in this studies were confirmed that hairy roots  contained TL and TR-DNA region of Ri plasmid with molecular weight 780 and 1600 bp.  The results showed that strain of A. rhizogenes, plant species, source of explant and composition of medium affect the initiation, growth, development  and vigor of hairy roots.Ringkasan Masalah dalam kultur akar rambut  C. ledgeriana dan C. succirubra adalah rendahnya tingkat keberhasilan transformasi eksplan dengan Agrobacterium rhizogenesdan pertumbuhannya yang lambat. Penelitian ini bertujuan untuk mengevaluasi  potensi dari beberapa galur A. Rhizogenes untuk inisiasi, mendapatkan komposisi medium terbaik untuk pertumbuhan akar rambut C. ledgeriana dan C. succirubra, serta konfirmasi terintegrasinya TR dan TL-DNA Ri plasmid ke dalam jaringan eksplan.  Eksplan batang  dan  daun  berasal  dari kecambah aksenik C. ledgeriana dan C. succirubra berumur delapan bulan diinokulasi dengan A. rhizogenes galur 15834, 8196, R-20001, 07-20001, A4, R.MAFFA,TISTR 509, TISTR 510 dan LBA 9457. Eksplan yang sudah diinokulasi dikulturkan dalam medium MS padat. Subkultur dilakukan dengan cara mentransfer potongan ujung akar rambut ke dalam medium cair MS, B5, White dan Heller. Akar rambut dari medium kultur yang terbaik kemudian disubkultur ke dalam medium yang sama dengan penambahan 50 dan 100 mg/L L-triptofan dengan konsentrasi vitamin sebanyak tiga kali dan lima kali dari konsentrasi normal MS. Integrasi T-DNA dalam akar rambut dikonfirmasi meng-gunakan Polymerase Chain Reaction  dengan primer spesifik untuk TL dan TR-DNA plasmid. Hasil yang diperoleh menunjukkan bahwa hanya A.rhizogenes galur LB9457 yang efektif menginfeksi eksplan baik batang maupun daun dari kedua spesies kina. Induksi, pertumbuhan dan vigor akar rambut yang terbaik diperoleh dari medium MS dengan penambahan 50 mg/L L-triptofan dan tiga kali konsentrasi vitamin. Hasil konfirmasi akar rambut baik dari batang maupun daun menggunakan PCR, menunjukkan bahwa TL dan TR-DNA dari Ri plasmid  A. rhizogenes mampu menghasilkan pita-pita DNA dengan BM780 dan 1600 pb. Hasil yang diperoleh menunjukkan bahwa galur  A. rhizogenes, spesies tanaman, sumber eksplan dan komposisi medium berpengaruh terhadap inisiasi, pertumbuhan,  perkembangan dan vigor akar rambut.

scholarly journals AGROBACTERIUM-MEDIATED TRANSFORMATION OF PANAX VIETNAMENSIS HA ET GRUSHV.

2018 ◽  
Vol 15 (4) ◽  
pp. 641-650
Author(s):  
Duong Tan Nhut ◽  
Nguyen Phuc Huy ◽  
Trinh Thi Huong ◽  
Vu Quoc Luan ◽  
Vu Thi Hien ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Genetic Transformation ◽  
Hairy Root ◽  
Hairy Roots ◽  
Large Scale ◽  
Continuous Production ◽  
Pcr Amplification ◽  
Limited Area ◽  
Root Induction

In recent years, the Agrobacterium-mediated genetic transformation system has become the most useful method widely used for the introduction of foreign genes into plant cells followed by regeneration of genetically improved plants. Panax vietnamensis Ha et Grushv. is a highly valued medicinal plant native to Vietnam with limited area of distribution. This report illustrates the possibilities of biotechnology for genetic transformation aimed at establishing an effective production of secondary metabolites in P. vietnamensis. In the present investigation, 0.5 cm2 leaf blades, 1 cm long leaf petioles and 0.5 cm3 callus clusters were used for the hairy root induction. Results indicated that hairy roots were induced on P. vietnamensis callus clusters co-cultivated with Agrobacterium rhizogenes strain ATCC15834 at OD600 of 0.5 with an infection time of 20 min and a supplementation of 100 mM acetosyringone. PCR amplification of the DNA isolated from the resulting hairy roots was used to confirm the presence of rol genes. Compared to in vitro rhizome cultures, hairy root cultures appear to be potential for continuous production of valuable secondary metabolites with similar saponin profiles. The protocol described in this study is simple and rapid and therefore, can be used for large-scale experiments for the rapid production of valuable compounds.

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