Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.     

Agrobacterium rhizogenes-mediated transformation of Echinacea purpurea

Summary Echinacea purpurea seedlings were inoculated with several Agrobacterium rhizogenes strains in order to obtain hairy roots. Infection with A. rhizogenes strains LMG63 and LMG150 resulted in callus formation. Upon infection with strains ATCC 15834 and R1601 hairy roots were obtained. Opine detection confirmed transformation of E. purpurea. Comparative HPLC fingerprint analysis of the alkamides from natural plant source, control tissues, and transformed callus and roots indicated that transformed callus and hairy roots might be a promising source for continuous and standardized production of the dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide and related amides.Abbreviations HPLC high-pressure liquid chromatography - MS Murashige and Skoog culture medium     

Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum

Background

Plants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood.

Methodology/Principal Findings

We developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6-dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation.

Conclusions

We provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocols described here will allow functional analysis of genes involved in plant parasitism.     

Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species

Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species. Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism. Spontaneous bud initiation occurred in roots of G. cruciata. Roots of G. lutea, G. acaulis and G. purpurea were cultured on media with high kinetin concentration, which induced the formation of friable callus tissues. Only in G. purpurea were these calluses organogenic. Regenerated shoots of G. cruciata and G. purpurea gave rise to plants, that displayed the typical phenotypes of A. rhizogenes-transformed plants: short internodes and rolled leaves. In the roots of G. acaulis and G. cruciata, transformed with A. rhizogenes A4M70GUS, a positive reaction with X-gluc indicated the activity of β-glucuronidase. The DNA extracted from hairy roots and from the roots of transgenic plants hybridized with the appropriate genomic probes in Southern blotting. This is taken as evidence of the stable genetic transformation in the 4 Gentiana species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Artemisia tilesii Ledeb hairy roots establishment using Agrobacterium rhizogenes-mediated transformation

An efficient and rapid protocol for the establishment of Artemisia tilesii “hairy” root culture is reported. Leaf explants of aseptically growing plants were cocultured with Agrobacterium rhizogenes A4 wild strain or A. rhizogenes carrying the plasmids with nptII and ifn-α2b genes. Root formation on the explants started in 5–6 days after their cocultivation with bacterial suspension. Prolongation of explant cultivation time on the medium without cefotaxime led to stimulation of root growth. The effects of sucrose concentration as well as of the levels of synthetic indole-3-butyric acid (IBA) and native growth regulator Emistim on the stimulation of A. tilesii “hairy” root growth were studied. Maximum stimulating effect both for the control and for transgenic roots was observed in case of root cultivation on the media supplemented with IBA—up to 7.95- and 9.1-fold biomass increase, respectively. Cultivation on the medium with 10 μl/L Emistime has also led to the control roots growth stimulation (up to 2.75-fold). Emistime at 5 μl/L concentration led to 5.46-fold mass increase in only one “hairy” root line. Higher sucrose content (40 g/L) stimulated growth of two hairy root lines but had no effect on growth of the control roots.     

Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters

Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834. Root explants produced transformed roots at a higher frequency (76.5±3.5%) as compared to stem (32.7±4.8%) or cotyledon (16.2±5.7%). Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium. The competence of plant regeneration from non-transformed adventitious roots and transformed hairy roots was compared. The frequency of adventitious shoot formation from transformed roots was much higher (88.5±9.8%) than that of non-transformed roots (31.7 ±9.5%) on hormone-free medium. Rooting of hairy root-derived adventitious shoots occurred easily on growth regulator-free medium but no rooting was observed on non-transformed shoots. The stable introduction of rol genes into Taraxacum plants was confirmed by PCR and Southern hybridization. Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type (non-transgenic) plants. Plantlets formed from transformed roots had numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants. The differences observed may reflect the modification of morphological root characters by introduction of rol genes.Communicated by M.R. Davey     

Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng

 Hairy roots of Panax ginseng were obtained after root disks were infected with wild-type strain Agrobacterium rhizogenes 15834. Three lines of hairy roots with different pigmentation were selected. Embryogenic callus was induced on Murashige and Skoog medium containing 1.0 mg/l 2,4-D. The frequency of embryogenic callus formation was 37.4% in a line with red pigmentation. Somatic embryo development from embryogenic callus was efficiently achieved by lowering the concentration of 2,4-D (0.5 mg/l). After the germination of somatic embryos on medium with 10 mg/l GA₃, the embryos were transferred to 1/2-MS medium without GA₃. The transformed ginseng plantlets had an actively growing root system with abundant lateral roots. The phenotypical alteration of transformed ginseng plants might be valuable character for increasing root yield. Received: 27 March 1999 / Revision received: 18 May 1999 / Accepted 8 July 1999     

Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species. CIMAP Publication No.: 2007-28J     

Agrobacterium rhizogenes-mediated genetic transformation and regeneration of a conifer:Larix decidua

Summary Gene transfer and plant regeneration systems have been developed for European larch (Larix decidua Mill.) in our laboratory. Aseptically germinated young seedlings were hypocotyl wound-inoculated withAgrobacterium rhizogenes strains 11325 containing a wild-type Ri (root-inducing) plasmid. Swollen stems appeared at infected wounds followed by either abundant hairy roots or adventitious shoot buds that developed within 3 to 4 wk after inoculation. No symptoms were seen on wounded but uninoculated seedlings. We demonstrated agrobacteria attached to larch cells by examination of scanning electron micrographs. Subsequently, calli derived from symptomatic tissues exhibited phytohormone autotrophic growth. Adventitious buds were elongated and rooted in vitro before being transferred to the greenhouse where the transformed whole plants grew normally. Transformants tested positive for opine production and transformation was further confirmed by Southern blot analysis with larch genomic DNAs isolated from both proliferated calli and needle tissue of transgenic plants.     

Agrobacterium rhizogenes-mediated transformation and the regeneration of transformants in Alhagi pseudalhagi]

The regenerated shoot segments of Alhagi pseudalhagi were sliced and infected with Agrobacterium rhizogenes strain A4. The hairy roots and transformed calli were obtained through selection on hormone free MS medium. The transformants were cultured on MS medium with 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5-1 mg/L 6-benzylaminopurine (6-BA) to induce calli. 3 mg/L 6-BA and 0.5 mg/L naphthalene acetic acid (NAA) were applied for shoot differentiation. Shoots were planted on MS medium with 2 mg/L indole-3-butyric acid (IBA) and produced roots. Opine analysis proved the integration and expression of T-DNA in over 95% hairy roots, 75% transformed calli and transformed plantlets respectively. The 81% hairy root cells had normal chromosome numbers (2n = 18). The alterations of chromosome number were observed. After one year of subculturing, the regeneration ability of transformants was maintained.     

Agrobacterium rhizogenes-mediated transformation of a high oil-producing fil a m en to us fungusUmbelopsis isabellina

TheAgrobacterium rhizogenes-mediated transformation procedure was developed by using the hygromycin B phosphotransferase gene (hph) as a selective marker for the oil-producing fungusUmbelopsis isabellina. Different conditions were combined to increase the transformation efficiency. The highest efficiency was obtained by usingA. rhizogenes strain R105 and a vector with zygomycete promoter. Southern blot analysis demonstrated that 71 % of transformants contained random integrations of T-DNA sequences under optimal conditions. We randomly selected 115 positive transformants resistant to hygromycin to analyze the amount of total fatty acid and gamma-linolenic acid (GLA). Six transformants produced a higher amount of total fatty acids than the wild strain, and one transformant also produced a higher level of GLA than the wild strain in gas chromatography analysis. This is the first report about usingA. rhizogenes strain R105 and germinated conidia to transform successfully the recalcitrant zygomycetes and to obtain transformants with a stable phenotype.     

A novel Agrobacterium rhizogenes-mediated transformation method of soybean [Glycine max (L.) Merrill] using primary-node explants from seedlings

A novel Agrobacterium rhizogenes-mediated transformation method using a primary-node explant from Dairyland cultivar 93061 was developed for soybean using the disarmed Agrobacterium strain SHA17. Transformed plants regenerated from explants inoculated with SHA17 were fertile and phenotypically normal. In a comparative experiment, regeneration frequencies were not significantly different between explants inoculated with A. rhizogenes strain SHA17 and Agrobacterium tumefaciens strain AGL1; however, a 3.5-fold increase in transformation efficiency [(number of Southern or TaqMan-positive independent events/total number of explants inoculated) × 100] was found for explants cocultured with SHA17 compared to AGL1 (6.6 and 1.64%, respectively). Southern analysis of 48 T₀ plants suggested that 37.5, 23, and 39.6% of the T₀ plants contained 1, 2, and 3 or more T-DNA fragments integrated into the genome, respectively. Additionally, T₁ progeny analysis of 8 independent events resulted in typical Mendelian inheritance of T-DNA genes. Of seven T₀ plants that had two or more T-DNA fragments, six contained multiple loci segregating in T₁ progenies. Further analysis of four lines confirmed the presence of PAT, GUS, and/or DsRED2 proteins in transgenic plants that were encoded on the T-DNA into the T₂ generation.     

Sonication-assisted Agrobacterium rhizogenes-mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation

An efficient protocol for the establishment of transformed root culture of Verbascum xanthophoeniceum using sonication-assisted Agrobacterium rhizogenes-mediated transformation is reported. Only 10 days after the inoculation with A. rhizogenes ATCC 15834 and 45 s ultrasound exposure, hairy roots appeared on 75% of the Verbascum leaves. Ten hairy root lines were isolated, although only half of them were free of bacterial contamination and started growing when excised from mother explants. The transgenic nature of the most vigorously growing hairy root clones (VX1 and VX6) was confirmed by polymerase chain reaction. Under submerged cultivation both hairy root clones accumulated high biomass amounts (12.8 and 14.3 g L⁻¹, respectively) and significant amounts of bioactive phenylethanoid glycoside verbascoside (over 6-times more than in mother plant leaves). LC-APCI-MS analyses confirmed verbascoside accumulation in hairy root clones along with three other phenylethanoid glycosides (forsythoside B, leucosceptoside B and martynoside) and an iridoid glycoside aucubin. This is the first report on the induction of hairy roots of Verbascum plants.     

Physical and biochemical differences in Agrobacterium rhizogenes-mediated transgenic hairy root lines of Echinacea purpurea

In Vitro Cellular & Developmental Biology - Plant - Echinacea (Echinacea purpurea L.), also known as purple coneflower, is one of the important medicinal plants commonly used for respiratory...     

Agrobacterium rhizogenes-mediated transformation of Rubia peregrina L.: in vitro accumulation of anthraquinones

An Agrobacterium rhizogenes-mediated transformation system for Rubia peregrina L. has been established by co-cultivation of callus cultures or by direct infection of explants with A. rhizogenes LBA 9402 harbouring the binary vector pMON 9703 containing gus and npt-II genes as markers. The putative transformed roots were selected on medium containing kanamycin (25 mg l^-1). Antibiotic resistant root clones were subjected to histochemical analysis for the localisation of -glucuronidase activity. Polymerase chain reaction was used to confirm the presence of gus, npt-II and T _L border sequences in the transformed root clones. Spontaneous regeneration of shoots was observed from 30 day-old transgenic roots. Total anthraquinone and alizarin contents of transgenic root cultures were measured by spectrophotometry and by high performance liquid chromatography. The accumulation of total anthraquinones in transformed roots was found to be approximately 2-fold higher than that found in one year-old field grown roots (2.12±0.12 and 1.23±0.12 mg g^-1 dry weight, respectively). Alizarin was found to be the major anthraquinone in transformed root cultures and was found to be approximately 3-fold higher than in field grown roots.Abbreviations BA 6-benzyladenine - B5 Gamborg B5 medium - gus -glucuronidase gene - GUS -glucuronidase - HPLC high performance liquid chromatography - MS Murashige and Skoog medium - NAA -naphthalene acetic acid - npt-II neomycin phosphotransferase II gene - OD₆₀₀ optical density at 600 nm - PCR polymerase chain reaction - T _L left border sequence of T-DNA - vir D1 virulence D1 gene - YMB yeast mannitol broth     

A novel method based on combination of semi-in vitro and in vivo conditions in Agrobacterium rhizogenes-mediated hairy root transformation of Glycine species

Despite numerous advantages of the many tissue culture-independent hairy root transformation protocols, the process is often compromised in the initial in vitro culture stage where inability to maintain high humidity and the delivery of nourishing culture medium decrease cellular morphogenesis and organ formation efficiency. Ultimately, this influences the effective transfer of produced plantlets during transfer from in vitro to in vivo conditions, where low survival rates occur during the acclimation period. We have developed an intermediate protocol for Agrobacterium rhizogenes transformation in Glycine species by combining a two-step in vitro and in vivo process that greatly enhances the efficiency of hairy root formation and which simplifies the maintenance of the transformed roots. In this protocol, cotyledonary nodes of Glycine max and Glycine canescens seedlings were infected by A. rhizogenes K599 carrying a reporter gene construct constitutively expressing green fluorescent protein (GFP). Glass containers containing sand and nutrient solution were employed to provide a moist clean microenvironment for the generation of hairy roots from inoculated seedlings. Transgenic roots were then noninvasively identified from nontransgenic roots based on the detection of GFP. Main roots and nontransgenic roots were removed leaving transgenic hairy roots to support seedling development, all within 1 mo of beginning the experiment. Overall, this protocol increased the transformation efficiency by more than twofold over traditional methods. Approximately 88% and 100% of infected plants developed hairy roots from G. max and G. canescens, respectively. On average, each infected plant produced 10.9 transformed hairy roots in G. max and 13–20 in G. canescens. Introduction of this simple protocol is a significant advance that eliminates the long and genotype-dependent tissue culture procedure while taking advantage of its optimum in vitro qualities to enhance the micropropagation rate. This research will support the increasing use of transient transgenic hairy roots for the study of plant root biology and symbiotic interactions with Rhizobium spp.     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。