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.     

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 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 of soybean to study root biology

This protocol is used to induce transgenic roots on soybean to study the function of genes required in biological processes of the root. Young seedlings with unfolded cotyledons are infected at the cotyledonary node and/or hypocotyl with Agrobacterium rhizogenes carrying the gene construct to be tested and the infection sites are kept in an environment of high humidity. When the emerged hairy roots can support the plants, the main roots are removed and the transgenic roots can be tested. Using this method, almost 100% of the infected plants form hairy roots within 1 month from the start of the experiments.     

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     

骆驼刺发根农杆菌转化系的原生质体培养和植株再生

从发根农杆菌A4转化的荒漠植物—璐驼刺毛状根愈伤组织中分离的原生质体培养的结果表明,酶解新转代7～10d的淡黄色松软愈伤组织,可获得大量有活力的原生质体。原生质体在附加有1．5mg．L-1 2,4．D、0．2mg．L-1 6．BA、0．3m01．L-1甘露醇、2％（W/V）蔗糖和500mg·L-1水解酪蛋白的DPD培养基中进行液体浅层培养可持续分裂。培养基的最适渗透压为（450±3）mOsm·kg-1,原生质体的最适植板密度为4×10^5个．mL-1。制备原生质体的愈伤组织以低温（4℃）预处理后,原生质体的产率和分裂频率均提高,分裂频率最高可达50％。原生质体分裂形成的愈伤组织转移在附加1-2mg．L-1 6-BA（或KT）和0．2mg·L-1NAA的MS培养基上培养后,可以分化并获得再生植株。纸电泳检测表明,原生质体再生的愈伤组织和分化植株仍然含有毛状根转化系的特异产物——冠瘿碱。     

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 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     

Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transformants

Seedling hypocotyls of Lithospermum erythrorhizon were infected with Agrobacterium rhizogenes (strain 15834) harboring a binary vector with an intron-bearing the β-glucuronidase (GUS) gene driven by cauliflower mosaic virus (CaMV) 35S promoter as well as the hygromycin phosphotransferase (HPT) gene as the selection marker. About 20% of the hairy roots isolated were hygromycin resistant and had co-integrated GUS and HPT genes in their Lithospermum genomic DNA. Because GUS activity was detected in almost all the hygromycin-resistant root tissues, the CaMV 35S promoter seems to be ubiquitously active in L. erythrorhizon hairy roots. In pigment production medium M9, the hairy root cultures had shikonin productivity similar to that of cell suspension cultures of Lithospermum. They also showed light-dependent inhibition of shikonin biosynthesis similar to that of Lithospermum cell cultures. These findings suggest that this hairy root system transformable with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics. Received: 2 March 1998 / Revision received: 25 May 1998 / Accepted: 8 July 1998     

Identification of genetic factors controlling the efficiency of Agrobacterium rhizogenes-mediated transformation in Brassica oleracea by QTL analysis

We have identified quantitative trait loci (QTL) for transgenic and adventitious root production using an Agrobacterium rhizogenes-mediated co-transformation system in conjunction with a Brassica oleracea double haploid (DH) mapping population. Three QTL for green fluorescent protein (GFP)-fluorescent root production and four QTL for adventitious root production were identified as accounting for 26% and 32% of the genetic variation in the population, respectively. Two of the QTL regions identified were common to both transgenic and adventitious root production. Two different methods of QTL analysis were employed (marker regression and interval mapping) and with the exception of one region on linkage group O7 for transgenic root production, both techniques detected the same regions of the genome. The regions we identified to be associated with the control of transgenic root production following A. rhizogenes-mediated transformation are the first to be detected using a QTL mapping approach. In addition, this is the first study to identify genetic regions that co-regulate both transgenic and adventitious root production within the constraints of an A. rhizogenes-mediated transformation process. We have identified plant genotypes that do not produce any transgenic roots that may be deficient for T-DNA integration via illegitimate recombination and that may also be potentially important for the development of homologous recombination protocols. Conversely, we have also identified plant genotypes with high rates of transgenic root production that will be critical in the development of high throughput transformation systems.     

Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants

Transgenic muskmelon (Cucumis melo L.) plants were produced efficiently by inoculating cotyledon explants with Agrobacterium tumefaciens strain LBA4404 bearing a Ti plasmid with the NPT II gene for kanaymcin resistance. After co-cultivation for three days, expiants were transferred to melon regeneration medium with kanamycin to select for transformed tissue. Shoot regeneration occurred within 3–5 weeks; excised shoots were rooted on medium containing kanamycin before transferring to soil. Morphologically normal plants were produced in three months. Southern blot analysis confirmed that ca. 85% of the regenerated plants contained the NPT gene. Dot blot analysis and leaf callus assay of progeny of transgenic plants verified transmission of the introduced gene(s) to the next generation. Factors affecting transformation efficiency are discussed.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - IAA indole 3 acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NPT II neomycin phosphotransferase II     

Features of lettuce transgenic plants with ifn-alpha2b gene regenerated after Agrobacterium rhizogenes-mediated transformation

"Hairy" roots of lettuce Lactuca sativa and regenerated plants with interferon-alpha2b gene had been obtained via Agrobacterium rhizogenes-mediated transformation. According to the results of PCR and rt-PCR analyses the studied plants had ifn-alpha2b gene. The regenerated plants differed from the plants of wild type by elongated internodes, early flower-bearing stem formation and purple coloration of leaves in artificial illumination conditions.     

Agrobacterium rhizogenes-mediated transformation of Brassica oleracea var. sabauda and B. oleracea var. capitata

Agrobacterium rhizogenes A4M70GUS-mediated transformation of Savoy cabbage (Brassica oleracea L. var. sabauda) and two local lines of cabbage (B. oleracea L. var. capitata) was obtained using hypocotyl and cotyledon explants. The percentage of explants which formed roots was very high in all genotypes: 92.3 % in Savoy Gg-1, 64.4 % in cabbage P₂₂I₅, and 87.2 % in P₃₄I₅. Spontaneous shoot regeneration of excised root cultures grown on the hormone-free medium occurred in all three genotypes. In cabbage lines P₂₂I₅ and P₃₄I₅ shoot regeneration was higher (9.3 and 2.6 % respectively) than in Savoy cabbage Gg-1 (1.3 %). Transgenic nature of hairy root-derived plants was evaluated by GUS histological test and PCR analysis. All the tested cabbage shoots were GUS positive whilst in a Savoy cabbage GUS expression was registered only in 55 % of tested clones. PCR analysis demonstrated the presence of the GUS gene in regenerated shoot clones and in T₁ progeny.     

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.     

Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens

Adventitious shoots formed on the proximal cut edges of different cotyledonary explants of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai; cvs. Sweet Gem and Gold Medal] cultured on Murashige and Skoog's (MS) medium with 1 mgl^-1 6-benzyladenine (BA). Light (16-h photoperiod, about 7 Wm^-2 cool-white fluorescent lamps) was essential for shoot formation. To obtain transformed plants, cotyledonary explants of Sweet Gem were cocultured with Agrobacterium tumefaciens LBA4404, a disarmed strain harboring a binary vector pBI121 carrying the CaMV 35S promoter--glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker, for 48 h on MS medium with 1 mgl^-1 BA and 200 M -hydroxyacetosyringone. After 48 h of culture, explants were transferred to medium with 1 mgl^-1 BA 250 mgl^-1 carbenicillin, and 100 mgl^-1 kanamycin and cultured in the light. Adventitious shoots formed on the explants after 4 weeks of culture. When subjected to GUS histochemical assay, young leaves obtained from the shoots showed a positive response at a frequency of up to 16%. Preculturing cotyledonary explants on MS medium with 1 mgl^-1 BA for 5 d enhanced the competence of the cells to be transformed by Agrobacterium. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants. The transformed plants were grown to maturity.