In the last years, hairy root HR cultures are gaining attention in the biotechnology industry. This particular plant cell culture derives from explants infected with Agrobacterium rhizogenes. They constitute a relatively new approach to in vitro plant biotechnology and modern HR cultures are far away from the valuables findings performed by Philip R. White in the 's, who obtained indefinite growth of excised root tips. HR cultures are characterized by genetic and biochemical stability and high growth rate without expensive exogenous hormones source.
These previous findings suggest that the hairy roots can be induced on a wide range of legumes with Gayt men having office sex A. Content of rutin in selected plant sources. Flavonoids in Health and Disease. Plant Biotech. The various explants of tea such as shoots, hypocotyl, cotyledon and cotyledonary nodes have been used to produce genetically modified plants either by using particle bombardment or Agrobacterium -mediated transformation. Metabolic engineering of morphinan alkaloids by over-expression of codeinone reductase in transgenic hairy roots of Papaver Hairy rootsthe Iranian poppy. Abstract Background Camellia sinensis var. Antioxidant and antimicrobial activities of tea infusions. Because rutin, which is present in tartary Hqiry, has various biological applications, its content can Nude pussy increased by the hairy root culture. Previously, we Hairy roots a hairy root culture of F.
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The Ri plasmids can be engineered to also contain T-DNAused aHiry genetic transformation biotransformation of the plant cells. Here's how Hairy roots works: Anybody can ask a question Anybody can answer The best answers are voted up and rise to the top. Plant Gen Res. J Biol Chem. Production of podophyllotoxin from roots and plantlets of Hyptis Results in teen curfews cultivated in Hairy roots. Int J Chem Pharm Sci. The production of PTOX was growth associated, Hairy roots a maximum Haiy of 5. A naturally occurring soil bacterium Rhizobium rhizogenes that contains root-inducing plasmids also called Ri plasmids can infect plant roots and cause them to produce a food source for the bacterium, opinesand to grow abnormally. Hairy root cultures can be used for phytoremediationand are particularly valuable for studies of the metabolic processes involved in phytoremediation. The opines are effective biochemical marker for identification of transformed roots has been done through paper electrophoresis. By Yao-Hua Law. Measurements of conductivity during growth kinetics showed that as anticipated, this parameter declined in inverse proportion to biomass increase. Depending on the extract, the HR-lines showed differential cytotoxic activity Table 3.
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- By Yao-Hua Law.
- Additionally, pathogenic Agrobacterium s pecies have recently been assigned to the genus Rhizobium.
- Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth in absence of growth regulators.
Bioactive Molecules and Medicinal Plants pp Cite as. Hairy roots in plants are the manifestation of infection caused by Agrobacterium rhizogenes , a gram negative soil bacterium.
This phytopathogen transfers its large root-inducing Ri plasmid carrying a set of genes into plant genome and thereby encoding enzymes capable of modifying the plant hormonal metabolism. Such new hormonal balances induce the formation of proliferating roots, called hairy roots that emerge at the wounding site. Hairy root cultures, owing to their stable and high productivity, have been investigated from several decades to produce the valuable metabolites present in wild-type roots.
The emergence of key molecules for overcoming the limiting culture parameters for the regulation of the metabolic pathways has made possible improvements in the production of secondary metabolites by hairy roots. Secretion and harvesting of these metabolites with the aid of trapping systems enhance the interest in such cultures.
The use of hairy roots to produce recombinant animal proteins represents an attractive system that may be extrapolated for industrial exploitation.
Equally, a good understanding of the underlying molecular mechanism, based on the transfer of the plasmid T-DNA of A. Indeed, hairy root systems allow gene gain- or loss-of-function techniques and transcriptome analyses for the discovery of new metabolic genes. Because of the prolific proliferation of the roots, hairy roots could be promising tools for phytoremediation. The hairy root system must be scaled up if they are to be used in industry for the mass production of secondary metabolites.
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World J Microbiol Biotechnol. Several decades have passed since the hairy root culture was established, however this system has not been utilized globally in commercial scale. Answer Now. Regeneration of transformed plants through hairy roots have been reported in medicinal plant species is limited. Arab J Chem.
Hairy roots. Book Details
Microscopy scans suggest that the roots carve their own way into rocks, rather than growing along cracks. These roots, found only in these two species so far, are the first known to dissolve rocks, the team says.
But the work has inspired plant physiologist Alex Valentine of the University of Stellenbosch in South Africa, who was not involved in the study. He now plans to search for such roots in Velloziaceae plants in South African mountain regions. Barbacenia tomentosa left and B. The plants have hardy roots that carve tunnels in the rock shown in this microscope image at right, arrows to access nutrients needed to survive. Plants living in other phosphorus-poor environments around the world have evolved either cluster roots or dauciform roots , with dense root hairs and acid secretions to harvest phosphorus from poor soils and sand, but not actual rock.
On average, each gram of rock contains only 0. By comparison, the lowest level found in a survey of 69 rock types worldwide was an average of 0. Further research into vellozioid roots might one day help develop more efficient crops.
The search for new natural sources that produce PTOX is mandatory, as is research for biotechnological alternatives, to achieve stable and controllable production of this compound [ 4 ]. Today, the most important PTOX producer species are Podophyllum hexandrum , Juniperus bermudiana , and Podophyllum peltatum , which produce 43, The highest concentration of PTOX obtained from in vitro plant cultures 8.
Hyptis suaveolens is a wild shrub native to Mexico that was employed throughout history, mainly for the treatment of gastric and bile problems, cancer, fever, spasms, cough, malaria, yellow fever and other diseases [ 14 ].
The aim of this research was to establish and select hairy root culture lines of Hyptis suaveolens , producing PTOX, as well as optimizing culture conditions to increase PTOX accumulation. A specimen of the plant was deposited voucher number CICY The sterilized seeds were immediately rinsed three times with sterile distilled water for 5 min. Light sources were cool white lamps of 40 watts. The in vitro multiplication of plantlets was achieved by placing nodal segment explants measuring 3 cm in length under the same conditions described previously.
Plantlet height and root length were registered. One hundred forty-four putative hairy roots were placed in Petri dishes with TQS MSB5 medium MSigma , which contains the same amount of inorganic salts and myo-inositol as MS medium, but it is supplemented with vitamins, as described by Gamborg B5 medium : nicotinic acid 1 mg L -1 , piridoxine 1 mg L -1 , and thiamine 10 mg L Putative hairy roots were maintained under these conditions for 45 days.
From these cultures, ten hairy root lines were selected based on their fast autonomous growth, hairy high branched appearance and plagiotropic growth to corroborate their genetic transformation, cytotoxic activity, and PTOX production. These selected hairy root lines HR-lines were transferred to TQS MSB5 liquid culture medium and kept at constant agitation of rpm under the same light, temperature and humidity conditions described for plantlet growth.
To observe the fluorescence, 1 cm of the roots was cut and directly placed on microscope slides. The retention factor from standard PTOX Sigma spot was compared with those spots from the extracts obtained through the MD and chloroformic methods.
HPTLC scanning of these spots complemented the results. To evaluate cytotoxicity, two types of extracts obtained through the MD and chloroformic methods from ten HR lines were tested.
To quantify PTOX and to include information obtained from previous experiments, extracts were obtained through the chloroformic and modified MD methods. Dry gas N2 : 10 L min -1 was employed. A Zorbax- Eclipse Plus 4. An isocratic elution of the mobile phase consisting of methanol-acetonitrile-water-acetic acid The effect of six different doses of thiamine 0.
Millipore Corp. The mobile phase consisted of an isocratic elution with methanol-acetonitrile-water and a flow rate of 0. To evaluate the consumption of carbohydrates during kinetics, three carbohydrate standards sucrose, fructose, and glucose from Sigma were utilized. Two milliliters of the culture medium were collected every four days for 48 days and frozen until use. The mobile phase consisted of an isocratic elution with acetonitrile-water at a flow rate of 0.
In total, putative hairy roots were induced; 15 days after infection, the roots were excised and placed in Petri dishes five to six per Petri dish containing hormone-free MSB5 culture medium M Sigma.
Ten induced roots that grew faster were individually transferred to Petri dishes. The roots showed the characteristic hairy root phenotype of the Agrobacterium rhizogenes -transformed roots and high branched, rapid and plagiotropic growth, which was observed in both semisolid Fig 1 HR-lines 1a — 10a and liquid culture medium Fig 1 HR-lines 1b — 10b. Individualized in solid MSB5 culture medium 1a — 10a and in liquid MSB5 liquid culture medium 1b — 10b without hormones.
Green or red epifluorescence was utilized to corroborate the genetic transformation of hairy roots. Observed in darkness and illuminated with blue a , green c , and white lights b , d , and f. When nontransformed roots were illuminated with blue or green light, epifluorescence was not observed e. All microphotographs are 20X. Developed-plate visualizations at nm a, and e , cerium sulfate revealed-plate at nm b, and f , and white light c, and g are shown. Absorbance spectra from the spots of PTOX in both extracts d, and h are shown.
Both extracts from the ten HR lines were evaluated for cytotoxicity using the sulforhodamine SRB method. Cytotoxic evaluation showed that all of the extracts were cytotoxic for at least one carcinoma cell line. Depending on the extract, the HR-lines showed differential cytotoxic activity Table 3. When the extraction of PTOX was performed using chloroform, a higher accumulation of this lignan was obtained from roots grown on MS medium followed by those grown on MSB5 and B5 media.
The results regarding the effect on PTOX yield of extra addition of thiamine at various concentrations to the MS culture medium 0. Measurements of conductivity during growth kinetics showed that as anticipated, this parameter declined in inverse proportion to biomass increase. The highest dry weight biomass was achieved at day 40 7. The production of PTOX was growth associated, observing a maximum accumulation of 5.
Concerning carbohydrate consumption, sucrose concentration on the medium decreased at a constant rate until day 20, when it was completely hydrolyzed; in contrast, fructose and glucose initially increased up to day 20, and the concentration of glucose began to diminish thereafter, while fructose concentration remained unchanged Fig 8C.
The best result for regenerated plantlets evaluated by height and root length was obtained with TQS MS medium. This result is similar to that reported in by Borges et al.
There is a direct relationship between plantlet height and root length. The corroboration of transformation has traditionally been reported by Southern blotting or using PCR.
The most important advantage of this method is that it reduces the carbon footprint and does not damage the obtained transformed tissues. Moreover, this approach has been successfully utilized for the structural elucidation of biological molecules and their interactions, as well as for in vitro assays or in vivo monitoring cellular research [ 29 ].
In the present work, we used the methodology described by Kamal et al. It was possible to confirm the presence of PTOX in the extracts by the development of spots with similar Rfs: 0. The slight difference in Rfs can be explained because the extract is a complex matrix composed of many metabolites which, in turn, influence chromatographic development. All HR-lines were cytotoxic for at least one of the carcinoma cell lines utilized.
Seven of the eight predictive structures proposed as resulting from ionization of the PTOX molecule in this work can also be observed, following links 4. It has been reported that the best culture medium for improving PTOX production depends on the plant species and the type of in vitro cultivation employed.
Considering the results described in this study, it was necessary to ascertain how much the culture medium or the method of extraction affected the yield of PTOX in the HsTD10 HR-line. When the HsTD10 HR-line was established in three culture media and extraction was performed using chloroform, the best result was obtained in roots grown in MS. However, when the extraction was performed using the modified MD method, the best result was obtained from roots grown in MSB5 medium 7 times more PTOX than that obtained in chloroform extracts from the same line, growing in the same culture medium.
The differences between MS and MSB5 culture medium pertain to the concentration of vitamins: times more thiamine and twice the amount of pyridoxine and nicotinic acid in the MSB5 medium. Then, the question was whether thiamine could modulate PTOX production.
Our results showed that there was no significant difference in biomass production, but there was a clear difference in PTOX accumulation. The increase in PTOX production was dose dependent on the thiamine concentration up to a maximum of 2 mg L -1 ; at this point, production began to decrease with each succeeding increased dose of thiamine.
To date, the relationship between the addition of thiamine to the culture medium and the increase in PTOX production has not been reported. Lignans are phenylpropanoid compounds that have been identified as plant-defenders [ 32 — 34 ]. Regarding the phenylpropanoid pathway, Boubakri et al. This vitamin increased plant defense activity [ 36 ], induced systemic acquired resistance SAR in Arabidopsis , rice, tobacco, and cucumber [ 37 ], and increased the concentration of callose and lignin in Arabidopsis [ 38 ].
The increase of thiamine in the HsTD10 HR-line of Hyptis suaveolens may activate a defense mechanism, possibly increasing the concentration of some phenylpropanoids but definitely that of PTOX. Several reports have analyzed the effect of temperature on PTOX extraction. In , Bedir et al. However, in , Renouard et al.
It is important to note that the modified MD method was performed as described in by Koulman et al. This change significantly increased PTOX extraction. In our work, it is clear that the culture medium and the extraction method were factors that determined the level of PTOX production. The highest dry weight biomass obtained under our culture conditions at day 40 was 9. Moreover, the production of PTOX associated with growth observed in this work is similar to that observed in hairy roots and in cell suspension cultures of L.
In this work, the consumption of carbohydrates was selective for sucrose and glucose because fructose was not consumed. This result is similar to that reported in by Hammouri et al. Nonetheless, this result differs from that reported in by Shimon-Kerner et al. It is also important to note that the concentration of PTOX between days 32 and 36 at the beginning of the stationary growth phase was the highest obtained 5.
PTOX accumulation in wild plants and plant material obtained by in vitro cultures cell, callus, tissues, plantlets, transformed tissues show a different pattern. To date, the greatest supply of PTOX has been obtained from the wild plant Podophyllum hexandrum 43 mg g -1 DW [ 1 ]; however, this yield diminishes when PTOX is obtained from cell suspension cultures of this species 7.
Similarly, the concentration of PTOX in wild Podophyllum peltatum plants is similar to that obtained in cell suspension cultures 4. The accumulation of PTOX in Linum album has been reported to be higher in cell suspension cultures 8 mg g -1 DW [ 7 ] than in wild roots of the same species 0. The highest concentration of PTOX reported using in vitro cultures was obtained in hairy root cultures of Linum album 15 and 5.
The culture medium did not significantly affect biomass growth but was a determining factor for the accumulation of PTOX in the hairy roots of H. PTOX accumulation in kinetic suspension culture of H. We thank Dr. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Data Availability: All relevant data are within the paper.
Obtaining and multiplying regenerated plantlets The in vitro multiplication of plantlets was achieved by placing nodal segment explants measuring 3 cm in length under the same conditions described previously. Genetic transformation confirmation. Cytotoxic evaluation To evaluate cytotoxicity, two types of extracts obtained through the MD and chloroformic methods from ten HR lines were tested.
Effect of thiamine addition. Quantification of PTOX. Carbohydrate quantification. Results In vitro regeneration and effect of medium concentration on Hyptis suaveolens plantlets The concentration of MS [ 18 ] culture medium significantly influenced the budding and length of the regenerated plantlet roots, which grew faster on TQS MS MS Sigma.
Download: PPT. Table 1. Effect of MS culture medium concentration on the growth of Hyptis suaveolens plantlets. Cytotoxic evaluation Both extracts from the ten HR lines were evaluated for cytotoxicity using the sulforhodamine SRB method. Table 3. Fig 5.
The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites.
The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. Among different strains, R was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content.
The R, , and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin A suitable A. Overall, R was the most promising strain for hairy root induction in buckwheat. Fagopyrum tataricum Gaertn is commonly known as tartary buckwheat. Tartary buckwheat has a bitter taste. Therefore, its consumption has decreased. However, the plant has received increased attention due to the presence of significant amounts of pharmacologically important phenolic compounds, such as quercetinglycoside, kaempferolglycoside, chlorogenic acid, iso-orientin, orientin, rutin, vitexin, and quercitrin Lee et al.
In addition, it contains dietary fibers, proteins, starch, polyunsaturated fatty acids, and vitamin B and C complexes Bonafaccia et al. Phenolic compounds, such as rutin and anthocyanins, that have been identified in buckwheat showed better in vitro antioxidant activity and in vitro pharmacological functions, such as cholesterol reduction, tumor inhibition, anti-hypertension, and control of diabetes and carcinogenesis Kayashita et al.
Various strategies have been demonstrated for the synthesis and production of pharmaceutically important phytochemicals Nagella et al. These strategies include Agrobacterium -mediated gene transfer, in vitro cell line establishment, in vitro cell suspension cultures, bioreactor cultivation, shoot cultivation, organ cultivation at the bioreactor level, and in vitro hairy root cultivation Bourgaud et al.
However, among these techniques, Agrobacterium -mediated gene transfer has been widely studied as a strategy for producing hairy root lines with high yield for plant compounds Ali et al. Agrobacterium is a Gram-negative soil-borne bacterium that is able to transfer part of its DNA i.
The hairy roots obtained by infecting plants with Agrobacterium rhizogenes are unique with respect to their genetic and biosynthetic stability and have been intensively used to induce a stable and high yield production of selected plant metabolites.
Currently, modern molecular techniques are used to regulate metabolic pathway genes that are required for the production of secondary metabolites Georgiev et al. Additionally, hairy roots are considered as a potential source of new natural compounds. Li et al. Furthermore, growth regulators are not required for the hairy root cultivation, which is an important consideration because some plant hormones are toxic e. Therefore, hairy root culture provides possibilities for isolating and synthesizing new compounds with high pharmaceutical properties Kwon et al.
Many strains of A. Recently, Ooi et al. The selection of an effective Agrobacterium strain for the production of transformed root cultures significantly depends on the plant species and must be determined empirically. The differences in virulence, morphology, and growth rate are at least partially related to the variety of Ri root inducing -plasmids within each bacterial strain Park and Facchini, Among the T-DNA genes in Ri-plasmid, the rol oncogenes cause striking phenotypical and biochemical alterations in the transformed hairy root.
The rol genes are potential activators of the secondary metabolism in transformed cells from the Solanaceae, Araliaceae, Rubiaceae, Vitaceae, and Rosaceae families Bulgakov et al. It is largely unknown whether different A. In our previous study, we claimed that F. However, to date, there are no reports on the transformation of F. Therefore, in the present study, we aimed to develop a novel hairy root culture method for the production of pharmacologically important phenolic compounds.
Different A. Flavonoid pathway scheme. Phenolic compound standards for rutin, catechin, chlorogenic acid, benzoic acid, ferulic acid, and quercetin were procured from Extrasynthese Genay, France.
Cyanidin 3- O -rutinoside and cyanidin O -glucoside were purchased from Fujicco Co. Kobe, Japan. Other routine reagents and chemicals were procured from Sigma—Aldrich St. The seeds were mixed with ice-cold autoclaved sterile distilled water three to five times and completely washed five times to remove any trace elements. The surface-sterilized seeds were wiped with sterile cotton to remove water droplets and, then the seeds were transferred into a sterile culture flask containing 25 ml of autoclaved half-strength MS Murashige and Skoog, without hormones.
Wild-type A. The strains were kindly gifted by Centro de Investigacion Cientifica de Yucatan, Mexico, and were maintained in our laboratory.
The bacterial strains were cultivated in an LB medium containing 1 g of tryptone, 0. Viability of the strains was determined intermittently by growing in LB agar. Hairy root induction was performed using a slightly modified method of Thwe et al. Briefly, 5 ml of freshly cultivated A. Cell pellets were further washed with an ice-cold phosphate buffer pH 7 and re-suspended in half-strength MS liquid medium. The explants were dipped into suspensions of different A. After incubation, the hairy roots were harvested to examine the gene expression levels and to quantify the phenolic compounds.
The gels were analyzed for fragment sizes of the rol genes rol A, B, C, and D. The purity of RNA was evaluated using 1. The primers for phenylpropanoid biosynthetic genes were designed as described by Li et al. The transcript levels of mRNA were compared relative to those of the standard histone H3 gene. The variations in expression levels were calculated by comparing three replicates of each sample. The slurry was mixed intermittently every 20 min during the extraction.
After 1 h, the samples were centrifuged at 21, rcf for 10 min, and the supernatant was filtered through a 0. The operating conditions for HPLC and the separating protocols for individual phenolic compounds were according to the method of Li et al. The solutions consisted of a mixture of A MeOH:water:acetic acid All phenolic compounds were calculated by comparing the HPLC peak areas with those of authentic standards, according to the procedures of Li et al.
After proper mixing for 5 min, the samples were incubated in a sonicator for 20 min with intermittent vortexing. The collected debris-free supernatant was filtered and stored in a sterile brown bottle for HPLC analysis. Cyanidin O -glucoside and cyanidin O -rutinoside standards were used for the quantification of individual anthocyanin content in the samples. A statistical analysis system SAS version 9.
Mature seeds of tartary buckwheat were treated with different strains of A. Primary results confirmed that the induction of hairy roots in tartary buckwheat is strain-specific because different A. Among the A. Interestingly, each strain showed a similar infection type related to hairy roots and hairy root length. With respect to the hairy root length and the number of hairy roots, the strain R average, 2.
Other strains showed a moderate number of hairy roots and slightly shorter hairy roots. However, no significant variations were observed among R, R, and strains. The occurrence of necrotic explant tissues in the form of hairy roots on a MS medium was fast and clearly indicated the transformation efficiency. Furthermore, the hairy root induction was comparatively faster Figure 2. The dry weight of the developed hairy roots indicated that R was dominant 0.
Whereas, R exhibited the lowest dry weight Figure 3. Incubation period indicated the accumulation of phenolic compounds. PCR amplification of the related rol A, B, C, and D genes bp, bp, bp, and bp, respectively in all of the transformed tartary buckwheat hairy roots confirmed the successful transformation.
TABLE 2. Development of F. Expression patterns of the selected genes in the transformed roots were strain-specific.
All nine strains exhibited a wide range of gene expressions, which clearly indicated that each strain had a different gene regulation pattern in the tartary buckwheat host.
In most cases, the transcript levels were comparatively higher for the strains , A4, and R Figure 4. R showed a higher expression pattern compared with other strains. These genes are directly involved in the biosynthesis of phenolic compounds in buckwheat. However, their relative expression in strains , R, LBA, and A4 was lower compared with the other strains.
Expression levels of phenylpropanoid biosynthetic pathway genes observed using qRT-PCR at 14 days after culturing of hairy roots.
Individual content of phenolic compounds in hairy roots of tartary buckwheat is listed in Table 3. Six phenolic compounds were detected using HPLC. Irrespective of the strain, all six phenolic compounds were observed in the transformed hairy roots.
Among the phenolic compounds, rutin 7. Infection with R produced the highest rutin content TABLE 3. Cyanidin derivatives of anthocyanins, such as cyanidin 3- O -glucoside and cyanidin 3- O -rutinoside, were detected in the hairy roots of tartary buckwheat using HPLC Table 4.
TABLE 4. Plant metabolites and functional compounds have attracted interest because of their desired pharmaceutical properties and various useful applications in the medical field Banerjee et al. Among plants, buckwheat is considered as a stable healthy food worldwide because of the presence of a wide variety of phytochemicals and nutrients, such as flavonoids, phenolic compounds, amino acids, and vitamins Golisz et al. Buckwheat is a rich source of rutin Hagels et al.
Rutin accumulates to the highest extent in buckwheat and cannot be found in other grains, such as wheat, rice, and corn. Thus, buckwheat is considered to be a major dietary source of rutin.