Agrobacterium Rhizogenes Strain Research Articles

Agrobacterium-Mediated Transformation in Alpinia galanga (Linn.) Willd. for Enhanced Acetoxychavicol Acetate Production

Agrobacterium-mediated transformations ensure elevated amounts of secondary metabolite accumulation with genetic and biosynthetic stability. In the present study, Alpinia galanga rich in bioactive compounds was genetically transformed using different strains of Agrobacterium rhizogenes viz. LBA 9402, A(4), 532, 2364 and PRTGus. Even though a higher growth rate was obtained with the LBA 9402 strain, maximum acetoxychavicol acetate accumulation (ACA) was seen in the PRTGus transformant. PRTGus root line has shown 10.1 fold higher ACA content in comparison to the control roots. The lowest ACA production was shown by the A(4) transformant (4.9 fold). The quantification of ACA in the transformed roots was carried out by using HPLC, which was found to be in the order of PRTGus > LBA 9402 > 2364 > 532 > A(4). The fast growth rate of hairy roots, genetic stability and their ability to synthesize more than one metabolite offer a promising system for the production of valuable secondary metabolites.

Effects of Agrobacterium rhizogenes strains and other parameters on production of isoflavonoids in hairy roots of Pueraria candollei Grah. ex Benth. var. candollei

Using several explants of Pueraria candollei Grah. ex Benth. var. candollei and two strains of Agrobacterium rhizogenes (ATCC 15834 and 43057), hairy root cultures were established. Including 100 μM acetosyringone in the culture medium enhanced frequency of hairy root induction by up to 58 %. Subsequently, effects of inoculum size (IS) and temperature on growth and production of isoflavonoids in hairy roots were determined. Conditions of 1 % IS and 32 °C promoted the highest accumulation of total isoflavonoid content, up to 31.0 ± 22.6 mg/g dry weight (DW), in hairy roots. Moreover, culture of hairy roots at 32 °C decreased browning of hairy roots. Furthermore, this temperature promoted accumulation of the secondary metabolite daidzein; whereas, hairy root cultures at the stationary phase accumulated higher amounts of the isoflavonoid puerarin rather than daidzein.

High-efficiency regeneration of Agrobacterium rhizogenes-induced hairy root in apple rootstock Malus baccata (L.) Borkh

Malus baccata is widely used as a rootstock in cold regions of the world because of its cold hardiness. In this study, a highly efficient Agrobacterium rhizogenes strain 8196 transformation system was developed using in vitro-derived stem segments of M. baccata. Approximately 37 % agro-infected explants produced hairy roots when they were incubated on Murashige and Skoog (MS) medium without plant growth regulators. A total of 95 % of hairy roots exhibited glucuronidase activity. Calli were induced from putatively-transformed hairy roots, and subsequently shoots were observed within 4 weeks of culture. The influence of 6-benzyladenine (BA), indole-3-butyric acid (IBA), thidiazuron (TDZ), and gibberellic acid 3 (GA3) on regeneration were investigated using an L9 (34) orthogonal experiment. About 73 % of shoots were regenerated when callus was incubated on MS medium along with 2.0 mg L−1 BA, 0.5 mg L−1 IBA, 0.3 mg L−1 GA3, and 0.5 mg L−1 TDZ. Moreover, hairy root regenerants showed higher rooting ability and exhibited morphological aberrations such as shortened stem, etiolated, wrinkled and clustered leaves than those of control.

Hairy root cultures of Rehmannia glutinosa and production of iridoid and phenylethanoid glycosides

Hairy root lines through the infection of Agrobacterium rhizogenes strain (A4) were established from shoot tips and leaves of Rehmannia glutinosa Libosch. Ten lines of hairy roots were selected on the basis of biomass increase in half-strength Gamborg medium (1/2 B5). Transgenic status of the roots was confirmed by polymerase chain reaction using rolB and rolC specific primers. Iridoid glycosides (catalposide, loganin, aucubin and catalpol) and phenylethanoid glycosides (verbascoside and isoverbascoside) identified using HPLC–ESI–MS, and their contents were compared with untransformed root culture and roots of 1-year-old field-grown plants of R. glutinosa by RP-HPLC. The growth and production of secondary metabolites in ten hairy root lines varied considerably as to the media. Woody plant (WP) medium displayed higher growth in terms of fresh (FW) and dry weights (DW) compared to 1/2 B5 medium. High-yielding hairy root lines produced higher amounts of loganin, catalposide, verbascoside and isoverbascoside in comparison to the untransformed root culture and roots of 1-year-old field-grown plants. The highest amounts of catalposide and loganin in transformed roots were 4.45 mg g−1 DW (RS-2 hairy root line) and 4.66 mg g−1 DW (RS-1 hairy root line), respectively. Aucubin and catalpol were detected in some lines in trace amounts. The highest amounts of verbascoside (16.9 mg g−1 DW) and isoverbascoside (3.46 mg g−1 DW) were achieved in RS-2 root line. The contents of catalposide, verbascoside and isoverbascoside in high-producing lines were several times higher than in untransformed root culture and roots of R. glutinosa plants grown in soil. Loganin and aucubin could not be detected in roots of field-grown plants. However, the levels of catalpol were much lower in the in vitro roots.

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.

Analysis of 6-methoxy podophyllotoxin and podophyllotoxin in hairy root cultures of Linum album Kotschy ex Boiss

Linum album is a herbaceous medicinal plant that contains some lignans with antiviral and anticancer properties such as podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (MPTOX). In this research, hairy root cultures of L. album were established by transformation with Agrobacterium rhizogenes strains LBA9402, A4, AR15834 and Agrobacterium tumefaciens strain C58C1 (pRiA4). The presence of PTOX and MPTOX in the hairy roots was verified by ESI/MS in positive ion mode. MPTOX was confirmed and its enantiomer determined by nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy, respectively. PTOX and MPTOX production was determined by HPLC, in different lines of hairy roots. The results showed that all obtained hairy root lines produced higher yield of lignans than mother plant roots. In addition, the lignan content in the roots derived from A. rhizogenes strain LBA9402 was higher than in those obtained from A. tumefaciens strain C58C1.

Hairy roots induction from difficult-to-transform pharmacologically important plant Eurycoma longifolia using wild strains of Agrobacterium rhizogenes

Wild strains of Agrobacterium rhizogenes were used to induce hairy roots from Eurycoma longifolia, a high value medicinal plant in South-East Asia. Among 5 wild strains of A. rhizogenes tested, 3 strains namely MAFF 210265, MAFF 301726 and MAFF 720002 exhibited the ability to induce hairy roots from E. longifolia. After many unsuccessful attempts to produce hairy roots from this recalcitrant plant, hairy roots were successfully initiated, through infecting the hypocotyls region of this plant. The generated hairy roots were observed to be thin, non-geotropic, hairy and brittle in appearance. Amplification of the deoxyribonucleic acid(DNA) fragments which is about 1100 bp using rol genes that extended between rol C and Dgene sequence by Polymerase chain reaction (PCR) method confirmed the integration of the portions of transferred deoxyribonucleic acid (T-DNA) into the transformed hairy roots. Generating hairy roots in E. longifolia will be highly beneficial mainly to the pharmaceutical industry as through the hairy root induction increased the amount of valuable secondary metabolites which is directly linked to its root differentiation can be achieved from low biomass starting material. This is the first report on hairy root induction from the medicinally eminent plant, E. longifolia using wild type strains of A. rhizogenes. Key words: Eurycoma longifolia, hairy roots, transformation, wild strains of Agrobacterium rhizogenes.

Antioxidant Profiles and Selected Parameters of Primary Metabolism in Physalis Ixocarpa Hairy Roots transformed by two Agrobacterium Rhizogenes Strains

Antioxidant Profiles and Selected Parameters of Primary Metabolism in Physalis Ixocarpa Hairy Roots transformed by two Agrobacterium Rhizogenes Strains

IN VITRO ROOTING OF ARAUCARIA EXCELSA R. BR. VAR. GLAUCA USING AGROBACTERIUM RHIZOGENES

The family Araucariaceae encompasses several evergreen forest tree species, which has a high ornamental value due to being a good specimen and having symmetrical branches. Conventional propagation of Araucaria excelsa R. Br. var. glauca by cutting has limited success because of topophysis and difficult-to-root characteristics, and grafting is accompanying incompatibility. The aim of this research was to evaluate the application of Agrobacterium rhizogenes as well as the IBA, NAA and ancillary compounds potential to increase the rooting of this plant under in vitro condition. Neither ancillary compounds such as salicylic acid, putrescine nor hydrogen peroxide affected the rooting of this recalcitrant species. Subculturing in vitro shoots to MS medium containing 7.5 μM of both IBA and NAA for 15 days before being moved to hormone-free half-strength MS medium, resulted in a 33% increase of rooting of shoots each with one or two roots. Using Agrobacterium rhizogenes strain K599 improved rooting percentage up to 40%. Green fluorescents protein (GFP) gene, as a reporter gene, was employed to verify the successful transformation.

Striga parasitizes transgenic hairy roots of Zea mays and provides a tool for studying plant-plant interactions.

BackgroundStriga species are noxious root hemi-parasitic weeds that debilitate cereal production in sub-Saharan Africa (SSA). Control options for Striga are limited and developing Striga resistant crop germplasm is regarded as the best and most sustainable control measure. Efforts to improve germplasm for Striga resistance by a non-Genetic Modification (GM) approach, for example by exploiting natural resistance, or by a GM approach are constrained by limited information on the biological processes underpinning host-parasite associations. Additionaly, a GM approach is stymied by lack of availability of candidate resistance genes for introduction into hosts and robust transformation methods to validate gene functions. Indeed, a majority of Striga hosts, the world’s most cultivated cereals, are recalcitrant to genetic transformation. In maize, the existing protocols for transformation and regeneration are tedious, lengthy, and highly genotype-specific with low efficiency of transformation.ResultsWe used Agrobacterium rhizogenes strain K599 carrying a reporter gene construct, Green Fluorescent Protein (GFP), to generate transgenic composite maize plants that were challenged with the parasitic plant Striga hermonthica. Eighty five percent of maize plants produced transgenic hairy roots expressing GFP. Consistent with most hairy roots produced in other species, transformed maize roots exhibited a hairy root phenotype, the hallmark of A. rhizogenes mediated transformation. Transgenic hairy roots resulting from A. rhizogenes transformation were readily infected by S. hermonthica. There were no significant differences in the number and size of S. hermonthica individuals recovered from either transgenic or wild type roots.ConclusionsThis rapid, high throughput, transformation technique will advance our understanding of gene function in parasitic plant-host interactions.

Production of camptothecin by hairy roots and regenerated transformed shoots of Ophiorrhiza rugosa var. decumbens

Camptothecin (CPT), the derivatives of which are used clinically for the treatment of metastatic colon cancer, is isolated from intact plants that can be subjected to environmental fluctuations. In vitro cultures may be an alternate and continuous source for year-round production of CPT. Since CPT production by undifferentiated cell cultures is low, differentiated tissues such as root cultures may be a viable alternate source for CPT production. Hairy roots were induced in Ophiorriza rugosa, a source of CPT, using Agrobacterium rhizogenes strain LBA9402. The hairy roots, when cultured in light, showed spontaneous regeneration of shoots. Analysis of CPT levels in the hairy roots and in vitro-grown transformed shoots revealed 0.009% d.w. and 0.012% d.w., respectively.

Agrobacterium rhizogenes-mediated hairy root induction in Taraxacum officinale and analysis of sesquiterpene lactones

Hairy roots were efficiently induced from leaf and petiole explants of Taraxacum officinale after infection with the Agrobacterium rhizogenes strains A4 and ATCC 15834. The highest frequency of hairy root initiation was observed after transformation of leaf explants with the A4 strain. Hairy roots developed from leaf tissue produced more biomass than non-transformed roots. A quantitative study of sesquiterpene lactones showed that A. rhizogenes induced root cultures accumulated higher levels of these compounds than non-transformed and wild plant roots. The present results demonstrate that T. officinale hairy root culture is a valuable alternative approach for the production of sesquiterpene lactones.

High-frequency transgenic plant regeneration and plumbagin production through methyl jasmonate elicitation from hairy roots of Plumbago indica L.

High-frequency transgenic plant regeneration and production of plumbagin were accomplished from hairy roots induced by Agrobacterium rhizogenes strain A4M70GUS on Plumbago indica L. Of the two types of hairy roots developed on Murashige and Skoog (MS) basal medium, Type I was long and thick with lesser branches and root hairs, and Type II was highly-branched short and slender with tufts of root hairs. Of the different lines grown in half-strength MS liquid basal media, root line 5 (R5) of the Type I yielded the highest plumbagin (0.92% DW) and was significantly different to that of the in vitro control. R5 showed a stable production of plumbagin (1.09% DW) in subsequent cultures. Elicitation of R5 with 50 μM methyl jasmonate for 48 h increased the yield of plumbagin to 5.0% DW, and was superior to 100 μM acetylsalicylic acid (3.8% DW). Plumbagin yield was five times that of twoyear-old ex vitro roots. Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the transformation. Hairy root segments cultured on MS medium containing 8.8 μM benzyladenine and 2.5 μM indole-3-butyric acid induced a mean of 9.1 shoots. Subsequent culture of the isolated shoots developed more than 50 normal shoots per culture. The root-free shoots were rooted on half-strength MS basal medium. The plantlets transferred in field conditions grew normally and exhibited 90% survival. Transgenic plant regeneration and hairy root induction in P. indica serves as reliable source of plumbagin which in turn cut off the mass destruction of the plant species.

Changes in morphological phenotypes and essential oil components in lavandin ( Lavandula × intermedia Emeric ex Loisel.) transformed with wild-type strains of Agrobacterium rhizogenes

Hairy roots were induced from leaf-derived calli of lavandin ( Lavandula × intermedia Emeric ex Loisel.) by infection with wild-type strains of Agrobacterium rhizogenes, A-5 (MAFF 02-10265) and A-13 (MAFF 02-10266). A-5-inoculated calli formed hairy roots more efficiently than A-13 ones. The transgenic shoots could be obtained from hairy root segments mediated by each Agrobacterium strain. However, different plant growth regulators were required for efficient adventitious shoot formation in each strain. In A-5, the most efficient adventitious shoot formation rate of 23.8% was observed in a medium with 4.4 × 10 −6 M of 6-benzylaminopurine. On the other hand, a significantly higher rate of 13.2% was detected in a medium with 4.0 × 10 −7 M of N-(2-chloro-4-pyridyl)- N′-phenylurea in A-13. Most of the regenerated plants showed dwarfism with closed internodes and extensive lateral branching, which were typical characteristics of ‘hairy root syndrome’. On the other hand, only nine of the 45 regenerated plants formed flower buds in early June, a delay of about one month compared with nontransgenic regenerated plants. The floral stalks and spikes of these plants were very short, resulting in a compacted form. Many regenerants showed a significantly lower productivity of essential oil than nontransgenic regenerants. Moreover, the relative percentage of the linalyl-cation-derived compounds, linalool and linalyl acetate, decreased in most of the regenerated plants. Compact plants with the ability of flower bud formation are assumed to be valuable not only for lavandin breeding, but also for clarifying the interaction between rol genes expression and essential oil production.

Genetically transformed roots as a model system for studying physiological and biochemical processes in intact roots

The sequence of operations during plant genetic transformation using wild strains of the soil bacterium Agrobacterium rhizogenes and subsequent root production capable of long-term growth on the relatively simple media free of growth compounds are described. The basic morphological, physiological, and biochemical characteristics of transformed roots are reported, and a technology of their in vitro cultivation is described. The modes of growth optimization of long-term cultivated roots of valuable plants and of the increase in the concentration of secondary metabolites synthesized by these roots are enumerated. The efficiency of the method of so-called “artificial seed” application for preservation of valuable lines of cultivated roots and a possibility of their healthy state restoration are briefly appraised. On the basis of literature and our own experiments, the main directions for the usage of genetically transformed roots in applied and fundamental studies are outlined. The terminology used for designation of genetically transformed roots by Russian researches is briefly discussed. Main materials and equipment required for plant transformation with the soil bacterium and for the maintenance of long-term growth of obtained roots are listed.

Overexpression of TaNHX2 enhances salt tolerance of ‘composite’ and whole transgenic soybean plants

Salinity is a major factor resulting in extensive loss of agricultural production. Genetic transformation has become a powerful tool for studying gene function and for improving crop salt tolerance. In this study, a TaNHX2 gene was transformed into a plant cloning vector under the control of cauliflower mosaic virus 35S promoter, and then introduced into Agrobacterium rhizogenes strain K599. Explants of soybean were transformed with A. rhizogenes and ‘composite’ plants consisting of wild-type shoots and transgenic hairy roots overexpressing TaNHX2 were produced. When exposed to salt stress, ‘composite’ plants displayed high salinity tolerance at 171 mM NaCl in vermiculite and in solid medium supplemented with up to 200 mM NaCl, whereas control plants displayed chlorosis and died within 15 days under above treatment conditions. We subsequently obtained soybean plants overexpressing TaNHX2 through A. tumefaciens-mediated transformation and studied four homozygous lines of TaNHX2. Transgenic lines displayed an enhanced salt tolerance in plant biomass and flower number per plant, compared with wild type plants grown on sand culture containing 150 mM NaCl. Furthermore, transgenic plants of line C12-11 showed longer survival, less growth inhibition and greater number of flowers than wild type plants. Taken together, these results indicated that TaNHX2 gene could enhance salt tolerance of soybean, and A. rhizogenes-mediated transformation system could be used as a complementary tool of A. tumerfaciens-mediated transformation to rapidly investigate candidate gene function in soybean.

Agrobacterium rhizogenes strains이 황금 모상근 유도와 생육에 미치는 영향

Agrobacterium rhizogenes, a gram-negative soil bacterium, is one of the most widely studied among them. A, rhizogenes can transfer T-DNA, excised from Ri (root inducing)-plasmids from the bacterial to the plant cell. It is the causal agent of ‘hairy root’ diseases in plants, and has been used for the production of hairy root cultures from a multitude of species. Five different strains of Agrobacterium rhizogenes differed in their ability to induce Scutellaria baicalensis hairy roots and also showed varying effects on the growth in hairy root cultures. A. rhizogenes R 1000 is the most effective strain for the induction (57.3%) and growth (11.9 g l?¹) in hairy root of Scutellaria baicalensis. Our results demonstrate that use of suitable strains of A. rhizogenes may allow study of the regulation of flavone biosynthesis in hairy root cultures of Scutellaria baicalensis.

Bioengineering of symbiotic systems: Creation of new associative symbiosis with the use of lectins on the example of tobacco and oil seed rape

"Barbate roots" in tobacco and colza transgenic on lectin gene were obtained with the use of a wild strain of Agrobacterium rhizogenes 15834 transformed with pCAMBIA1305.1 plasmid containing the full-size lectin gene (psl) from the Pisum sativum. Influence of expression oflectin gene on colonization oftransgenic roots with symbiont of pea (Rhizobium leguminosarum) was investigated. The number of adhered bacteria onto the roots transformed with lectin gene was 14-fold and 37-fold higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of tobacco and colza. The developed experimental approach, based on the simulation of recognition processes and early symbiotic interactions with lectins of pea plants, may, in perspective, be used for obtaining stable associations of economically valuable, nonsymbiotrophic plant species with rhizobia.

Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants

Resistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum×almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed.

Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants

We have developed an efficient transformation system for Bacopa monnieri, an important Indian medicinal plant, using Agrobacterium rhizogenes strains LBA 9402 and A4. Transformed roots induced by strain LBA 9402 spontaneously dedifferentiated to callus while excised roots induced by strain A4 spontaneously showed induction of shoot buds within 10days. PCR and RT-PCR analysis revealed the presence and expression of the rolAB and rolC genes at the transcription level in pRi A4 transformed cultures indicating that the TL-DNA was integrated retained and expressed in the A4-Ri transformed shoots. Transformed calli showed the presence of rolAB or rol A, TR and ags genes. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes. Growth and biomass accumulation was significantly higher in the transformed shoots (twofold) and roots (fourfold) than in the non-transformed (WT) plants. In pRi A4-transformed plants, the content of bacopasaponin D, bacopasaponin F, bacopaside II and bacopaside V was enhanced significantly as compared to WT plants of similar age while bacoside A3 and bacopasaponin C content was comparable with that of WT plants. Significant increase in content of five bacopa saponins could be detected in pRi 9402-transformed callus cultures. There is an overall stimulatory effect on accumulation of bacopa saponins in transformed plants and cells of B. monnieri establishing the role of endogenous elicitation by Ri T-DNA of A. rhizogenes.