The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants

Summary A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. Intermediate integration vectors constructed from ColE1-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. corniculatus.     

Genetic transformation of strawberry: Stable integration of a gene to control biosynthesis of ethylene

Summary Efficient methods ofAgrobacterium-mediated transformation are described for two Pacific Northwest cultivars of strawberry (Fragaria ×ananassa), Tristar and Totem. We report stable incorporation of a gene for control of ethylene biosynthesis, into strawberry (cultivar Totem) for the first time. Cultivar Tristar was transformed with disarmed strains ofAgrobacterium tumefaciens (A. tumefaciens), LBA4404 or EHA101, containing a binary vector with marker genesuidA andnptII. Cultivar Totem was transformed withA. tumefaciens strains EHA101 or EHA105 harboring binary vectors with selectable marker genesnptII orhpt and with a gene for S-adenosylmethionine hydrolase (SAMase) for control of ethylene biosynthesis. The frequency of transgenic shoots ranged from 12.5% to 58.8% of the original treated explants when using plasmids containing the gene encoding SAMase. Primary shoot regenerants obtained on selection medium were subjected to several iterations of tissue isolation and reculture on higher stringency selection medium for recovering uniformly transformed plantlets. Transgenic plants were confirmed by their ability to undergo rooting on medium with selection at 60 mg/liter kanamycin or 10 mg/liter hygromycin. About 95–100% of the transformation events from different experiments were capable of profuse rooting in the presence of selection. Insertion of the SAMase gene and its integration into the strawberry genome were confirmed by Southern hybridization. About 500 plants from 250 independent transgenic events have been successfully transferred to soil for further evaluation.     

Expression and inheritance of inserted markers in binary vector carrying Agrobacterium rhizogenes-transformed potato (Solanum tuberosum L.)

Summary Transgenic shoots were regenerated from eight diploid potato hairy root clones obtained by transformation with Agrobacterium rhizogenes harboring next to its wild-type Ri-plasmid a binary vector containing the neomycin phosphotransferase and the -glucuronidase genes. The plants exhibited the typical hairy root phenotype. Of the plants isolated, 58% were tetraploid and 38% were diploid. Flowering and tuberization was much better in the diploid than in the tetraploid plants. Transgenic plants formed a significantly larger root system when grown on kanamycin-containing medium as compared to growth on kanamycin-free medium. Direct evidence for genetic transformation was obtained by opine, neomycin phosphotransferase and -glucuronidase assays, and by molecular hybridization. Fourteen flowering diploid plants were reciprocally crossed with untransformed S. tuberosum plants, but only six were successful. Seedlings obtained from four crosses showed that all traits were transmitted to the offspring. Molecular analysis confirmed the presence of multiple integrations (copies) of both vector T-DNA and Ri-T-DNA. The genetic data, furthermore, suggest that the traits derived from Ri-T-DNA and binary vector T-DNA are linked, as no recombination between the different traits was observed.     

Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype

Summary Segments of the TL-DNA of the agropine type Ri plasmid pRi 1855 encompassing single and groups of open-reading frames were cloned in the Ti plasmid-derived binary vector system Bin 19. Leaf disc infections on Nicotiana tabacum led to transformed plants, some of which showed typical hairy root phenotypes, such as the wrinkled leaf morphology, excessive and partially non geotropic root systems and the ability of leaf explants to differentiate roots in a hormone-free culture medium. Particularly interestingly, most of these traits were shown by plants transformed with a TL-DNA segment encompassing the single ORF 11, corresponding to the rolB locus. Hairy root can be induced by this latter T-DNA segment on wounded stems of tobacco plants; hairy root induction on carrot discs requires, on the contrary, a more complex complement of TL-DNA genes.Abbreviations YMB yeast mannitol broth - MS Murashige and Skoog medium - 6-BAP 6-benzylaminopurine - NAA naphthalene acetic acid - Km kanamycin - Cb carbenicillin     

Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.)

 An Agrobacterium tumefaciens-mediated transformation method has been developed for onions (Allium cepa L.) using immature embryos as the explant source. Transgenic plants were recovered from the open-pollinated onion cultivar Canterbury Longkeeper at a maximum transformation frequency from immature embryos of 2.7%. The method takes between 3–5 months from explant to primary regenerant entering the glasshouse. Multiple-shoot formation from primary transgenic material made possible the clonal multiplication of transformants. The binary vector used carried the nptII antibiotic resistance gene and the m-gfp5-ER reporter gene. Transgenic cultures were initially screened for their ability to fluoresce and to grow in the presence of geneticin (5–25 mg/l). The transgenic nature of individual plants was confirmed by Southern blot analysis. Received: 12 October 1998 / Revision received: 17 May 1999 Accepted: 14 June 1999     

Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ri binary vector

Summary Transgenic herbicide-resistant Scoparia dulcis plants were obtained by using an Ri binary vector system. The chimeric bar gene encoding phosphinothricin acetyltransferase flanked by the promoter for cauliflower mosaic virus 35S RNA and the terminal sequence for nopaline synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching young plants. Hairy roots resistant to bialaphos were selected and plantlets (R0) were regenerated. Progenies (S1) were obtained by self-fertilization. The transgenic state was confirmed by DNA-blot hybridization and assaying of neomycin phosphotransferase II. Expression of the bar gene in the transgenic R0 and S1 progenies was indicated by the activity of phosphinothricin acetyltransferase. Transgenic plants accumulated scopadulcic acid B, a specific secondary metabolite of S. dulcis, in amounts of 15–60% compared with that in normal plants. The transgenic plants and progenies showed resistant trait towards bialaphos and phosphinothricin. These results suggest that an Ri binary system is one of the useful tools for the transformation of medicinal plants for which a regeneration protocol has not been established.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

Characterization of ornamental Datura plants transformed by Agrobacterium rhizogenes

Summary Datura arborea and D. sanguinea hairy roots were produced by cocultivation of leaf fragments with Agrobacterium rhizogenes strain NCPP 1855. Adventitious buds emerged spontaneously, without exogenous growth regulators, from seven hairy root clones of D. arborea and from one hairy root clone of D. sanguinea. Regenerated plants were successfully acclimatized in the greenhouse. The integration of the bacterial TL-DNA into the genome of the putative transformed plants was confirmed by Southern blot analysis. Transgenic plants displayed increased ability to root in vivo. Morphological traits with relevant ornamental value like plant height, leaf number, size and shape, internode number, and internode length were also affected. Transformation by wild-type Ri TL-DNA provided the chance to study plant growth and differentiation and to select improved genotypes.     

Agrobacterium-mediated transformation of cotyledonary explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis)

 A procedure for producing transgenic Chinese cabbage plants by inoculating cotyledonary explants with Agrobacterium tumefaciens strain EHA101 carrying a binary vector pIG121Hm, which contains kanamycin-resistance and hygromycin-resistance genes and the GUS reporter gene, is described. Infection was most effective (highest infection frequency) when explants were infected with Agrobacterium for 15 min and co-cultivated for 3 days in co-cultivation medium at pH 5.2 supplemented with 10 mg/l acetosyringone. Transgenic plants of all three cultivars used were obtained with frequencies of 1.6–2.7% when the explants were regenerated in shoot regeneration medium solidified with 1.6% agar. A histochemical GUS assay and PCR and Southern blot analyses confirmed that transformation had occurred. Genetic analysis of T1 progeny showed that the transgenes were inherited in a Mendelian fashion. Received: 15 December 1998 / Revision received: 2 July 1999 · Accepted: 8 July 1999     

Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting transformation efficiency are discussed.     

Morphogenesis and Auxin Sensitivity of Transgenic Tobacco with Different Complements of Ri T-DNA

Leaf explants of hairy root tobacco (Nicotiana tabacum) regenerants characteristically differentiate roots from the wound margins on hormonefree medium. The same response can be elicited on normal tobacco by culturing the explants in the presence of auxin. We show here that the spontaneous rooting of transformed plants is neither due to the activity of right T-DNA-borne auxin genes nor to a substantially altered balance of endogenous hormones. Rather, an increased sensitivity to auxin is conferred to transformed cells by the left T-DNA (TL-DNA). Analysis of the morphogenetic behavior of transgenic tobacco plants obtained by transferring segments of TL-DNA cloned in a binary vector system allowed us to pinpoint TL-DNA genes responsible for this increased auxin sensitivity of hairy root tissues. Three genes (open reading frames 10, 11, 12) are responsible for the spontaneous rooting of leaf explants and confer to transgenic plants an exaggerated response to auxin.     

High frequency production of rapeseed transgenic plants via combination of microprojectile bombardment and secondary embryogenesis of microspore-derived embryos

Transgenic doubled haploid rapeseed (Brassica napus L. cvs. Global and PF₇₀₄) plants were obtained from microspore-derived embryo (MDE) hypocotyls using the microprojectile bombardment. The binary vector pCAMBIA3301 containing the gus and bar genes under control of CaMV 35S promoter was used for bombardment experiments. Transformed plantlets were selected and continuously maintained on selective medium containing 10 mg l⁻¹ phosphinothricin (PPT) and transgenic plants were obtained by selecting transformed secondary embryos. The presence, copy numbers and expression of the transgenes were confirmed by PCR, Southern blot, RT-PCR and histochemical GUS analyses. In progeny test, three out of four primary transformants for bar gene produced homozygous lines. The ploidy level of transformed plants was confirmed by flow cytometery analysis before colchicine treatment. All of the regenerated plants were haploid except one that was spontaneous diploid. High frequency of transgenic doubled haploid rapeseeds (about 15.55% for bar gene and 11.11% for gus gene) were considerably produced after colchicines treatment of the haploid plantlets. This result show a remarkable increase in production of transgenic doubled haploid rapeseed plants compared to previous studies.     

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal -endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.     

High-frequency transformation of<Emphasis Type="Italic"> Lobelia erinus</Emphasis> L. by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated gene transfer

A highly efficient transformation procedure was developed for Lobelia erinus. Leaf or cotyledon discs were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector plasmid pIG121Hm, which contains a -glucuronidase gene with an intron as a reporter gene and both the neomycin phosphotransferase II and hygromycin phosphotransferase genes as selectable markers. The hygromycin-resistant calli produced on the selection medium were transferred to MS medium supplemented with 0.5 mg/l benzyladenine and 0.2 mg/l indole-3-acetic acid for regeneration of adventitious shoots. Transgenic plants were obtained as a result of the high regeneration rate of the transformed calli, which was as high as 83%. In contrast, no transgenic plant was obtained by the procedure of direct shoot formation following inoculation with A. tumefaciens. Transgenic plants flowered 3–4 months after transformation. Integration of the transgenes was detected using PCR and Southern blot analysis, which revealed that one to several copies were integrated into the genomes of the host plants. The transformation frequency at the stage of whole plants was very high—45% per inoculated disc.Abbreviations BA: 6-Benzyladenine - 2,4-D: 2,4-Dichlorophenoxyacetic acid - GUS: -Glucuronidase - IAA: Indole-3-acetic acidCommunicated by G.C. Phillips     

Introduction of hygromycin resistance inLotus spp. throughAgrobacterium rhizogenes transformation

The speciesLotus corniculatus andL. tenuis were transformed with anAgrobacterium rhizogenes binary vector, conferring resistance to the antibiotic hygromycin. Transgenic plants recovered from both species were tested for the ability of leaf-derived calluses to grow in a hygromycin-supplemented medium. Molecular analysis showed the integration of the Ri T-DNA and of the gene for hygromycin resistance, with a high frequency of co-transformation. Progeny analysis of the hygromycin resistance indicated this to be a single Mendelian trait in test plants. The transformed plants will be utilized in somatic hybridization experiments with lucerne for producing non-bloating genotypes with condensed tannins in leaves.     

Agrobacterium-mediated transformation of Campanula glomerata

A transformation system for Campanula glomerata 'Acaulis' based on the co-cultivation of leaf explants with Agrobacterium tumefaciens LBA4404 or EHA105 was developed. A. tumefaciens was eliminated when the explants were cultured on medium containing 400 mg/l vancomycin and 100 mg/l cefotaxime. Transgenic plants containing the uidA gene that codes for #-glucuronidase (gus) were obtained following co-cultivation with either strain of A. tumefaciens, LBA4404 or EHA105, both of which harbored the binary vector pGUSINT, coding for the uidA and neomycin phosphotransferase II (nptII) genes. While the transformation frequency (2-3%) was similar for both strains, A. tumefaciens LBA4404 was effectively eliminated from Campanula at a lower concentration of antibiotic as compared to EHA105. The concentration of individual antibiotics required to eliminate EHA105 resulted in a decreased rate (55-67%) of regeneration. The highest percentage of explants that regenerated plants (79%) and the highest regeneration rate was achieved with 100 mg/l cefotaxime combined with 400 mg/l vancomycin. Plants were also transformed with the isopentenyl transferase (ipt) gene using LBA4404 containing the 35S-ipt vector construct (pBC34).     

Hairy root induction and plant regeneration of <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis> Libosch. f. <Emphasis Type="Italic">hueichingensis</Emphasis> Hsiao via <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation

The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from hairy roots by infecting the leaf sections and stem segments of in vitro Rehmannia glutinosa Libosch. f. hueichingensis Hsiao plantlets. Hairy roots were induced from them after co-culturing with Agrobacterium rhizogenes strain 15834 at a frequency of 32 and 29.4%, respectively. The calluses were induced from hairy roots on half-strength Murashige and Skoog medium containing 0.2 mg/l kinetin and 3.0 mg/l benzyladenine at a frequency of 100%, from which transgenic shoots and plantlets were developed. Transgenic plantlets did not have differences in morphology except the shortened internodes and an increase in adventitious root formation compared to wild-type plants. PCR and Southern-blot analyses confirmed that rolB gene of TL-DNA was inserted in the genome of transformed hairy roots and plantlets. RT-PCR analysis and opine paper electrophoresis revealed that rolB gene was expressed in the transformed hairy roots and plantlets. Conclusively, transgenic hairy roots and transgenic plants of Rehmannia glutinosa Libosch. f. hueichingensis Hsiao were developed for the first time. This text was submitted by the authors in English. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 247–255.     

Agrobacterium-mediated transformation of pepino and regeneration of transgenic plants

Regeneration of pepino (Solanum muricatum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS (-D-glucuronidase) reporter gene carried on pKIWI110. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation.     

Transformation of radish (Raphanus sativus L.) via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group 3 LEA gene from B. napus

A protocol for producing transgenic radish (Raphanus sativus) was obtained by using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation. The Agrobacterium strain LBA4404 contained the binary vector pBI121-LEA (late embyogenesis abundant), which carried a Group 3 LEA gene, from Brassica napus. Among six combinations, Agrobacterium-mediated transformation assisted by a combination of 5-min sonication with 5-min vacuum infiltration resulted in the highest transformation frequency. The existence, integration and expression of transferred LEA gene in transgenic T₁ plants were confirmed by PCR, genomic Southern and Western blot analysis. Transgenic radish demonstrated better growth performance than non-transformed control plants under osmotic and salt stress conditions. Accumulation of Group 3 LEA protein in the vegetative tissue of transgenic radish conferred increased tolerance to water deficit and salt stress.     

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     

Increases in densities of the citrus red mite,Panonychus citri [Acarina: Tetranychidae], in association with the Argentine ant,Iridomyrmex humilis [Hymenoptera: Formicidae], in southern California citrus

We monitored the activities of the Argentine ant,Iridomyrmex humilis (Mayr), in 3 citrus orchards during 1984 and 1985. We also monitored densities of citrus red mite,Panonychus citri (McGregor), the adult spider mite destroyer,Stethorus picipes Casey [Col.: Coccinellidae], and the predatory mite,Euseius tularensis Congdon [Acarina: Phytoseiidae] during the same period in ant-present and ant-free plots in each orchard. I. humilis was excluded for at least 9 months from trees pruned 60 cm from the ground following a basal application of 1 or 2 % chlorpyrifos 4 EC. Autumn densities ofP. citri in these ant-free plots were significantly lower than those in the plots infested withI. Humilis. S. picipes appeared to be the most important predator, maintaining lowP. citri densities on citrus in southern California in the absence ofI. humilis activity.S. picipes densities increased numerically in response to the early spring and late autumn increases in the density ofP. citri populations. However, in the presence ofI. humilis activity, effective predation was apparently inhibited. Densities ofE. tularensis were unaffected by the presence ofI. humilis, nor didE. tularensis respond to spring or autumn increases in the density ofP. citri populations in any of the orchards.