Rhizogenes Infection Research Articles

Optimization of factors affecting Agrobacterium-mediated hairy root induction in Vitex negundo L. (Lamiaceae)

Vitex negundo L. is an aromatic, woody, blooming shrub in the Verbenaceae family which can grow into a small tree. Traditionally, V. negundo root has been used to cure diabetes, colic, boils, leprosy, and rheumatism. Keeping the importance of its roots in mind, an attempt has been taken for development of a protocol for efficient hairy root proliferation system. The Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364) were used for hairy root induction. For A. rhizogenes infection, both in vitro and in vivo leaves as well as internodes were used as explant. In vitro leaves and internodal explants were obtained by the inoculation of matured nodal segments on the optimum medium [MS + 2.0 mg/L N6-Benzylaminopurine (BAP)] with c.a. 91.6% shoot regeneration and an average of 8.1 shoots per explants. In vitro leaf showed best hairy root induction followed by in vitro internode on ½ MS medium augmented with acetosyringone. Highest transformation efficiency was achieved using MTCC 2364 strain, while no transformation was observed in MTCC 532 strain. Different factors affecting transformation including co-cultivation period, infection time and optical density (O.D.) value were standardized. The highest efficacy, 88.8% hairy root induction was observed in in vitro leaves infected by MTCC 2364 for 60 minutes infection time with an O.D. value of 0.29 maintained over a 44-48 hours of co-cultivation period. The prescribed protocol may be used as a reference for development of industrial scale hairy root production for bioactive compound located in root of V. negundo.

Upscaling biomass production of rosmarinic acid-rich hairy root cultures of Agastache rugosa (Fisch. & C.A.Mey.) Kuntze

Agastache rugosa (Fisch. & C. A. Mey.) Kuntze known as Korean Mint is a medicinal and aromatic plant containing rosmarinic acid as a major bioactive polyphenol but its content in this herbal drug is variable. Plant in vitro culture is one of the approaches used to overcome the complexity of environmental factors influencing phytochemical profiles in medicinal plants. In this study, hairy root cultures of A. rugosa were established through the infection of Rhizobium rhizogenes. Four hairy roots lines were selected on the basis of biomass production in liquid media. Transformation was confirmed by PCR using rol C specific primers. The hairy roots were successfully cultured in 300 mL conical flasks and scaled-up using three bioreactor types (nutrient sprinkle bioreactor—NSB and two modular temporary immersion systems—RITA® and Plantform®). The UPLC analysis of A. rugosa transformed roots methanol extracts showed the presence of 24 polyphenolic compounds with the predominance of rosmarinic acid (RA), which level ranged between 3.82 and 9.16 mg/g of dry weight, depending on the culture system. Nineteen compounds were identified in hairy roots growing in NSB system, 9 of them were identified in roots cultured in RITA® or Plantform® and 7 compounds were identified in roots from Erlenmeyer flasks. The R. rhizogenes infection (strain A4) was found to be an effective method of hairy root culture establishment of A. rugosa.

The Highly Embryogenic Brassica napus DH4079 Line Is Recalcitrant to Agrobacterium-Mediated Genetic Transformation.

Brassica napus is a species of high agronomic interest, used as a model to study different processes, including microspore embryogenesis. The DH4079 and DH12075 lines show high and low embryogenic response, respectively, which makes them ideal to study the basic mechanisms controlling embryogenesis induction. Therefore, the availability of protocols for genetic transformation of these two backgrounds would help to generate tools to better understand this process. There are some reports in the literature showing the stable transformation of DH12075. However, no equivalent studies in DH4079 have been reported to date. We explored the ability of DH4079 plants to be genetically transformed. As a reference to compare with, we used the same protocols to transform DH12075. We used three different protocols previously reported as successful for B. napus stable transformation with Agrobacterium tumefaciens and analyzed the response of plants. Whereas DH12075 plants responded to genetic transformation, DH4079 plants were completely recalcitrant, not producing any single regenerant out of the 1784 explants transformed and cultured. Additionally, an Agrobacterium rhizogenes transient transformation assay was performed on both lines, and only DH12075, but no DH4079 seedlings, responded to A. rhizogenes infection. Therefore, we propose that the DH4079 line is recalcitrant to Agrobacterium-mediated transformation.

Agrobacterium rhizogenes-Mediated Transformation of Crotalaria ochroleuca: Production of Flavonoids from Hairy Roots

Crotalaria ochroleuca (Fabaceae) is rich in bioactive compounds used for nematode control. Transformed root culture allows the production of a large amount of hairy root biomass, conditioned by auxin biosynthesis-related genes integrated via the process of Agrobacterium rhizogenes infection, which is known to cause a hairy root phenotype. We transformed A. rhizogenes to increase the biomass of hairy roots and optimized the process of production of bioactive compounds. The transformed nature of hairy roots was confirmed by polymerase chain reaction, which confers kanamycin resistance. The transformed roots were cultured in full-strength in liquid media to realize the growth of hairy roots and the production of bioactive compounds. The transformed roots were grown in a culture medium supplemented with elicitors to produce total phenols, and flavonoids. Chromatographic analysis of transformed roots revealed the presence of flavonoids apigenin-6,8-C-diglucoside and luteolin-6-C-glucoside. The results were obtained by conducting physiological and biochemical studies with the flavonoids and studying the pathways that led to the production of large amounts of bioactive compounds from the hairy roots of C. ochroleuca. It was observed that the extraction of the compounds significantly affected nematodes and insect larvae, resulting in significantly high levels of economic damage to crops.

Altered carbon status in Glycine max hairy roots induced by Agrobacterium rhizogenes

ABSTRACT Plants fix CO2 into carbohydrates through photosynthesis, and various organisms interact with plants to obtain carbohydrates. Agrobacterium rhizogenes is a soil bacterium known as a plant pathogen that induces hairy root disease. Through A. rhizogenes-plant interactions, transfer-DNA (T-DNA) of the Ri plasmid is inserted into the host plant genome, leading to excessive formation of hairy roots and the synthesis of opines that are carbon and nitrogen sources for A. rhizogenes. In this study, we analyzed the carbohydrate contents in soybean (Glycine max) hairy roots. We found that the starch content was strongly increased in hairy roots, whereas the glucose was significantly decreased. On the other hand, no significant differences were observed in sucrose levels between the main roots and hairy roots of A. rhizogenes-inoculated plants. This result suggests that A. rhizogenes infection caused a change in primary carbon metabolism in the host plant cells.

Metabolic gene expression and centelloside production in elicited Centella asiatica hairy root cultures

The most economically important centellosides, madecassoside and asiaticoside, are produced in very low amounts in Centella asiatica (L.) Urban roots. With the aim of overcoming this limitation, in this work we compared the effect of different elicitors on centelloside production in hairy root cultures established by Agrobacterium rhizogenes infection. Additionally, elicitor-induced changes in the expression of key biosynthetic genes were studied to shed light on the regulation of the triterpene metabolic pathway. The elicitors tested were coronatine and methyl jasmonate, added separately or together, and salicylic acid. The content of the four main centellosides (asiaticoside, madecassoside, asiatic acid and madecassic acid) was determined by HPLC/DAD, and the expression level of genes after elicitation was analyzed by real-time quantitative polymerase chain reaction. The greatest increase in the production of centellosides, especially madecassoside, was achieved with coronatine, applied alone or with methyl jasmonate. This treatment also enhanced the expression of the target genes, particularly at the start of elicitation. By far the most highly expressed were those involved in oxidations, indicating that the tested elicitors did not act specifically on key genes in the centelloside biosynthetic pathway. These results support that hairy roots are a promising biotechnological platform for improved centelloside production and that this approach warrants further research.

Rhizobium rhizogenes infection in threatened Indian orchid Dendrobium ovatum mobilises ‘Moscatilin’ to enhance plant defensins

The present study illustrates the transformation ability of two wild-type bacterial strains of Rhizobium rhizogenes (MTCC 532 and MTCC 2364) on the embryogenic callus and callus-derived plantlets of a threatened Indian orchid, Dendrobium ovatum. Co-culture of the bacterium with the explants gave marginal hairy root phenotype that failed to multiply in the culture medium. Some primary and secondary metabolites were subdued in infected explants. Moscatilin, the stilbenoid active principle in D. ovatum, was found below the detection limit. The presence of two metabolites viz., Laudanosine, a benzyltetrahydroisoquinoline alkaloid and Lyciumin B, a cyclic peptide, were detected exclusively in the infected explants. The subjugated amino acids and phenolics in the infected plantlets were routed to produce phytoanticipins, and phenanthrenes, strengthening the defence mechanism in infected tissues. This research implies that the plant's defence mechanism activation could have prevented the extensive hairy root formation in the explants, even though nodulations and phenotype transitions were witnessed. Moscatilin has a structural resemblance with Resveratrol, a phytoalexin that combats bacterial and fungal pathogens. The study favours the possibility of Moscatlin being a precursor for phenanthrene compounds, thereby serving as a ‘phytoanticipin’ during the infection phase.

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.

Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean

Small guide RNA (sgRNA) is an important component of the CRISPR/Cas9 system. The gene editing efficiency of the CRISPR/Cas9 system could be enhanced by using highly active U6 promoters to drive the expression of sgRNA. Therefore, we constructed various expression vectors based on the 11 GmU6 promoters predicted and cloned in the whole soybean genome. The expression of truncated GUS driven by 11 GmU6 promoters was tested in hairy roots and by Arabidopsis thaliana transformation. The results indicated that higher transcriptional levels were driven by 5 GmU6 promoters (GmU6-4, GmU6-7, GmU6-8, GmU6-10 and GmU6-11) in both soybean hairy roots and Arabidopsis thaliana. In addition, three genes, Glyma03g36470, Glyma14g04180 and Glyma06g136900, were selected as targets to detect the transcriptional levels of multiple GmU6 promoters. Mutations in these three genes were detected in soybean hairy roots after Agrobacterium rhizogenes infection, indicating efficient target gene editing, including nucleotide insertion, deletion, and substitution. Mutation efficiencies differed among the 11 GmU6 promoters, ranging from 2.8% to 20.6%, and markedly higher efficiencies were obtained with all three genes using the GmU6-8 (20.3%) and GmU6-10 (20.6%) promoters. These two GmU6 promoters also showed higher ability to drive truncated GUS transcription in both soybean hairy roots and transformed Arabidopsis thaliana. These results will help to construct an efficient CRISPR-Cas9 gene editing system and promote the application of the CRISPR-Cas9 genome editing system in soybean molecular breeding.

Variability of Bioactive Glucosinolates, Isothiocyanates and Enzyme Patterns in Horseradish Hairy Root Cultures Initiated from Different Organs.

Horseradish hairy root cultures are suitable plant tissue organs to study the glucosinolate–myrosinase–isothiocyanate system and also to produce the biologically active isothiocyanates and horseradish peroxidase, widely used in molecular biology. Fifty hairy root clones were isolated after Agrobacterium rhizogenes infection of surface sterilized Armoracia rusticana petioles and leaf blades, from which 21 were viable after antibiotic treatment. Biomass properties (e.g., dry weight %, daily growth index), glucosinolate content (analyzed by liquid chromatography-electronspray ionization-mass spectrometry (LC-ESI-MS/MS)), isothiocyanate and nitrile content (analyzed by gas chromatography-mass spectrometry (GC-MS)), myrosinase (on-gel detection) and horseradish peroxidase enzyme patterns (on-gel detection and spectrophotometry), and morphological features were examined with multi-variable statistical analysis. In addition to the several positive and negative correlations, the most outstanding phenomenon was many parameters of the hairy root clones showed dependence on the organ of origin. Among others, the daily growth index, sinigrin, glucobrassicin, 3-phenylpropionitrile, indole-3-acetonitrile and horseradish peroxidase values showed significantly higher levels in horseradish hairy root cultures initiated from leaf blades.

Coupling Nicotiana tabaccum Transgenic Plants with Rhizophagus irregularis for Phytoremediation of Heavy Metal Polluted Areas

Several studies have shown that hairy roots (HRs) can increase the phytoextraction of organic and inorganic pollutants. In addition, microorganisms colonizing the rhizosphere of hairy roots have demonstrated the efficacy of HRs either in removing or in stabilizing of pollutants. This growth chamber study aimed to determine the effect of colonization by the arbuscular mycorrhizal (AM) fungus (Rhizophagus irregularis) on the HRs of tobacco (Nicotiana tabaccum L. cv. Virginia gold) under heavy metals (Cu, Pb and Zn) conditions. Seedlings of N. tabacum were grown in contaminated substrate without infection caused by Agrobacterium rhizogenes expressed in the form of HRs disease, a second one with infection, and the third treatment contained AM fungus beside the A. rhizogenes infection. In the order they have been described, these treatments were coded with C, CT and CTM. The experiment demonstrated that HRs mutants resisted better in terms of higher biomass content in the contaminated soil than the normal plants. Even more, the transgenic plants also had a very strong interaction with mycorrhizal fungi. Thus, soil respiration, biomass and some biochemical variables such as assimilating pigments, protein content and lipid peroxidation indicate a decrease in stress due to the presence of heavy metals in the CTM treatment. Furthermore, CTM treatment significantly alleviated the concentration of toxic elements in plants, compared with the CT treatment, in which a significant increase was registered when compared to treatment C.

Enhancement of anthraquinone production and release by combination of culture medium selection and methyl jasmonate elicitation in hairy root cultures of Rubia tinctorum

Anthraquinones (AQs) are secondary metabolites widely distributed in nature. Interesting applications of plant extracts containing AQs include the treatment of Hepatitis C and cancer. Plant in vitro culture is an alternative for producing plant-derived pharmaceuticals in controlled conditions and with low environmental impact. Moreover, it allows the application of different strategies for enhancing secondary metabolite production. Hairy root cultures, obtained after Agrobacterium rhizogenes infection, are able to produce high amounts of secondary metabolites at high growth rates. In this work, growth kinetics of hairy root cultures of Rubia tinctorum and AQ production were evaluated in two different culture media, Gamborg B5 with half of the saline strength (B51/2) and Lloyd & Mc Cown’s Woody Plant Medium (WPM). Although WPM allowed higher biomass production (58.6% higher) than B51/2, specific AQ production was higher in B51/2 (between 1.2 and 2.1 fold increases from day 21 to the end of the experiment). Moreover, AQ release to the culture medium was observed in B51/2 (∼10% of total AQs). The different performance of hairy roots in these culture media may be due to a limiting nutrient (other than carbon source) in B51/2. Elicitation in B51/2 with methyl jasmonate (100μM) resulted in a massive accumulation of intracellular (between 1.5 and 2.4-fold increases) and also extracellular AQs (up to 8.1 fold-increase compared with control at 4days post-elicitation), which could ease AQ purification. These results prove the usefulness of combining different approaches to enhance secondary metabolite accumulation in plant in vitro cultures, in order to develop an optimized productive process.

Establishment, Culture, and Scale-up of Brugmansia candida Hairy Roots for the Production of Tropane Alkaloids.

Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6β-hydroxyhyoscyamine (anisodamine), and scopolamine are the most important ones due to their anticholinergic activity. These bioactive compounds have been historically and widely applied in medicine and their demand is continuous. Their chemical synthesis is costly and complex, and thereby, these alkaloids are industrially produced from natural producer plants. The production of these secondary metabolites by plant in vitro cultures such as hairy roots presents certain advantages over the natural source and chemical synthesis. It is well known that hairy roots produced by Agrobacterium rhizogenes infection are fast-growing cultures, genetically stable and able to grow in hormone-free media. Additionally, recent progress achieved in the scaling up of hairy root cultures makes this technology an attractive tool for industrial processes. This chapter is focused on the methods for the induction and establishment of B. candida hairy roots. In addition, the scaling up of hairy root cultures in bioreactors and tropane alkaloid analysis is discussed.

Hairy root induction and phytoremediation of textile dye, Reactive green 19A-HE4BD, in a halophyte, Sesuvium portulacastrum (L.) L.

In this study, we report phytoremediation of textile dyes using hairy roots derived through Agrobacterium rhizogenes (NCIM 5140) infection of in vitro leaf and stem explants of a halophyte Sesuvium portulacastrum (L.) L. Leaf explants showed higher frequency of hairy root induction (70%) than stem explants (30%), and maximum number of roots (leaf 42.3±2.4 and stem 50.3±1.7). Transformed nature of hairy roots was ascertained by amplifying 970bp region of T-DNA of Ri plasmid. Hairy roots were screened for phytoremediation of various textile dyes and results showed that HRs were able to degrade Reactive green 19A HE4BD upto 98% within 5 days of incubation. Spectrophotometric analysis showed decrease in dye concentration while HPLC and FTIR analysis confirmed its degradation. Seed germination assay demonstrated non-toxic nature of the extracted metabolites. This is the first report on induction of hairy root culture in Sesuvium portulacastrum and phytoremediation of textile dyes.

Targeted mutagenesis in soybean using the CRISPR-Cas9 system

Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2–9.7% using the pCas9-AtU6-sgRNA vector and 14.7–20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules.

Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants.

BackgroundCapsicum annuum and Capsicum frutescens, also known as “chilies”, belong to the Solanaceae family and have tremendous beneficial properties. The application of hairy root culture may become an alternative method for future development of these species by adding value, such as by increasing secondary metabolites and improving genetic and biochemical stability compared with normal Capsicum plants. Therefore, in this research, different types of explants of both species were infected with various Agrobacterium rhizogenes strains to provide more information about the morphology and induction efficiency of hairy roots. After 2 weeks of in vitro seed germination, young seedling explants were cut into three segments; the cotyledon, hypocotyl, and radical. Then, the explants were co-cultured with four isolated A. rhizogenes strains in Murashige & Skoog culture media (MS) containing decreasing carbenicillin disodium concentrations for one month.ResultsIn this experiment, thick and short hairy roots were induced at all induction sites of C. annuum while thin, elongated hairy roots appeared mostly at wound sites of C. frutescens. Overall, the hairy root induction percentages of C. frutescens were higher compared with C. annuum. Hairy root initiation was observed earliest using radicles (1st week), followed by cotyledons (2nd week), and hypocotyls (3rd week). Cotyledon explants of both species had the highest induction frequency with all strains compared with the other explants types. Strains ATCC 13333 and ATCC 15834 were the most favourable for C. frutescens while ATCC 43056 and ATCC 43057 were the most favourable for C. annuum. The interactions between the different explants and strains showed significant differences with p-values < 0.0001 in both Capsicum species.ConclusionsBoth Capsicum species were amenable to A. rhizogenes infection and hairy root induction is recommended for use as an alternative explants in future plant-based studies.

Hairy Root Induction in Linum mucronatum ssp. mucronatum, an Anti-Tumor Lignans Producing Plant

Transgenic hairy root system is a promising source of secondary metabolites in medicinal plants with high pharmaceutical value.For the first time, hairy roots were established in different explants of Linum mucronatum, an anti-cancer agent producing plant, via amikimopine type strain of Agrobacterium rhizogenes, ‘A13’. The percentage of hairy root induction varied from 0 to 60% depended onthe explants and hypocotyl (including cotyledonary node) explants were found to be highly susceptible to A. rhizogenes infection withthe highest (60%) rate of hairy root induction. four different Murashige and Skoog (MS)-based liquid culture media were used for wellestablishment of hairy roots. Hairy root growth medium D (HRGM-D) containing hormone-free MS basal medium with an extra oneday pre-incubation period at 35°C was found to be more efficient for profuse growth (fresh weight; 8500 mg per 25 ml culture medium)of hairy roots. Hairy root system presented in this study may offer a suitable platform for optimization and production of satisfactorylevel of aryltetralin lignans like podophyllotoxin and its derivatives from L. mucronatum.

Hairy roots induction in Nepeta crispa (Lamiaceae) from Iran by using Agrobacreium rhizogenes

Nepeta crispa Willd. (Lamiaceae) is an aromatic endemic plant of Iran. This plant with the common local name Mofarrah has been of great interest to Iranian traditional medicine. Infusion and beverage obtained from the aerial parts of N. crispa were used traditionally as sedative, relaxant, carminative, restorative tonic for nervous and respiratory disorders. N. crispa has antimicrobial activity against bacteria and fungi. The main constituents were 1,8-cineole (47.9%), 4-aα,7α,7aβ-nepetalactone (20.3%), α-pinene (5%) and β-terpineol (4.1%). Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. Hairy roots were induced from cotyledon explants excised from seven day old aseptically grown seedlings of N.crispa using Agrobacterium rhizogenes 15834. The cefotaxime concentration of 250 mgL-1 was found to be most suitable for hairy root induction in N.crispa.To confirm transformation, PCR analysis was performed by using of specific primers for rol B gene amplification. Results of PCR analysis showed the presence of diagnostic 780 bp rol B product amplification and thus confirmed the transformation of hairy roots. This is the first report on the induction of hairy roots in N.crispa.

Engineering secondary metabolite production in hairy roots

Plants are a rich source of valuable secondary metabolites. Plant cell culture technologies developed in the past as possible tools for secondary metabolites production. In most cases, for the large scale production of these compounds, production is too low for commercialization. The cultured cell suspensions being undifferentiated have not gained momentum because of their instability and non-uniformity of the product formation. Secondary metabolite production is under strict metabolic regulation and tissue specific localization. Hence the differentiated cultures such as hairy root cultures are widely studied. Agrobacterium rhizogenes causes hairy root disease in plants. Transformed roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce amounts of secondary metabolites comparable to that of intact plants. Elicitation of hairy roots leads to increased production of secondary metabolites and also helps in designing of metabolic traps to allow adsorption of product, preventing feedback inhibition and protection of metabolites from degradation in the culture media. Permeabilization and in situ product adsorption result in many fold increase in product yield. T- DNA activation tagging allows overexpressing the respective gene and increasing the product formation in transformed hairy roots. Recent progress in transgenic research has opened up the possibility of the metabolic engineering of biosynthetic pathways to produce high-value secondary metabolites. Metabolic engineering offers promising perspectives to improve yields; however it requires the understanding of the regulation of the secondary metabolite pathways involved in the regulation of levels of product, enzymes and genes, including aspects as transport and compartmentation. This article reviews the recent advances in secondary metabolites production in transformed hairy roots.

Increased furanocoumarin production by Glehnia littoralis roots induced via Agrobacterium rhizogenes infection

The objective of this study was to determine a possible application of hairy roots as biotechnological producers of furanocoumarins, by establishing root clones of Glehnia littoralis after infection from Agrobacterium rhizogenes. None of the root clones grew under hormone-free conditions, but when cultured in the presence of indole-3-butyric acid, growth varied substantially across clones. Analysis of the insertion and expression of pathogenic rol (A, B TL , B TR , and C ) genes for 10 clones showed that the stable expression of rolC and irregular expression of rolB were detected in 2 clones, which exhibited active growth and higher furanocoumarin induction. Following ascorbic acid treatment, the productivity of xanthotoxin and bergapten in a root clone harbouring a pathogenic gene was 2.8 and 3.7 times greater than that recorded in wild-type roots, respectively. These results indicate that root cultures possessing pathogenic rol genes offer a potential means to produce furanocoumarins.