Single-copy T-DNA insertions in Arabidopsis are the predominant form of integration in root-derived transgenics,whereas multiple insertions are found in leaf discs

Different patterns of T-DNA integration in Arabidopsis were obtained that depended on whether a root or a leaf-disc transformation method was used. An examination of 82 individual transgenic Arabidopsis plants, derived from 15 independent Agrobacterium-mediated transformations in which different cointegrate and binary constructs were used, indicated that the transformation method had a significant influence on the type and copy number of T-DNA integration events. Southern hybridizations showed that most of the transgenic plants produced by a leaf-disc method contained multiple T-DNA insertions (89%), the majority of which were organized as right-border inverted repeat structures (58%). In contrast, a root transformation method mostly resulted in single T-DNA insertions (64%), with fewer right-border inverted repeats (38%). The transformation vectors, including cointegrate and binary types, and the plant selectable markers, hygromycin phosphotransferase and dihydrofolate reductase, did not appear to influence the T-DNA integration patterns.     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

Rapid transformation ofMedicago truncatula: regeneration via shoot organogenesis

Summary A rapid transformation and regeneration system has been developed forM. truncatula cv Jemalong (barrel medic) by which it is possible to obtain transgenic plants within 2.5 months. The procedure involvesAgrobacterium-mediated transformation of cotyledon explants coupled with the regeneration of transformed plants via direct organogenesis. To develop the procedure,M. truncatula explants were transformed with the binary plasmid pSLJ525 which carries thebar gene. Thebar gene encodes phosphinothricin acetyl transferase, and transformed plants were selected on media containing phosphinothricin (Ignite, AgrEvo). Transformed plants show phosphinothricin acetyl transferase activity and Southern blot analysis indicates that they carry thebar gene integrated into their genomes. The resistance to phosphinothricin is stable and is inherited by the R₁ progeny as a single dominant Mendelian trait. The transgenic plants are highly resistant to the broad spectrum herbicide, Ignite and therefore may also have commercial applications.     

Production of herbicide-resistant sweet potato plants transformed with the <Emphasis Type="Italic">bar</Emphasis> gene

Herbicide-resistant sweet potato plants were produced through biolistics of embryogenic calli derived from shoot apical meristems. Plant materials were bombarded with the vectors containing the β-glucuronidase gene (gusA) and the herbicide-resistant gene (bar). Selection was carried out using phosphinothricin (PPT). Transformants were screened by the histochemical GUS and Chlorophenol Red assays. PCR and Southern-blot analyses indicated the presence of introduced bar gene in the genomic DNA of the transgenic plants. When sprayed with Basta, the transgenic sweet potato plants was tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to sweet potato.     

Efficient regeneration and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated stable transformation of perennial ryegrass

We utilized gene transfer technology for genetic perennial ryegrass improvement, efficient regeneration, and Agrobacterium-mediated transformation of phosphinothricin acetyltransferase gene (bar). Four growth regulator combinations were compared and intact seeds of six turf-type cultivars as mature embryo sources were tested to optimize the regeneration conditions. Callus formation and regeneration were observed in all seeds. The highest callus formation frequency was observed in the seeds cultured on MS medium supplemented with 9 mg/l 2,4-D, without benzyladenine. Cv. TopGun revealed the highest callus induction and regeneration frequencies of 96 and 48.9%, respectively. By using an optimized regeneration system, embryogenic calli were transformed by an Agrobacterium strain LBA4404 containing the plasmid pCAMBIA3301. After the selection of the potentially transgenic calli with phosphinothricin, a herbicide, 22 transgenic resistant plants were regenerated. With PCR, Southern-blot hybridizations, and GUS expression techniques, we confirmed that some regenerants were transgenic. Two of the tested transgenic plants showed herbicide resistance. Our results indicated that embryogenic calli from mature seeds can be directly used for perennial ryegrass efficient regeneration and transformation and this protocol is applicable for genetic engineering of herbicide-resistant plants. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 590–596. The text was submitted by the authors in English.     

Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system

To increase expression level of cholera toxin B subunit (CTB) in lettuce plants, synthetic CTB (sCTB) gene based on the optimized codon usage was fused with an endoplasmic reticulum retention signal, KDEL. The sCTB gene was introduced into a plant expression vector and transformed to lettuce plants using Agrobacterium-mediated transformation system. As a selection marker, a bialaphos resistance (bar) gene that encodes phosphinothricin acetyltransferase (PAT), conferring tolerance to the herbicide phosphinothricin (PPT), was used. PCR amplification of genomic DNA confirmed the presence of the sCTB gene in the transgenic lettuce plants. Expressions of mRNA and protein of sCTB were observed by Northern and Western blot analyses, respectively. The sCTB synthesized in the transgenic lettuce showed strong affinity for GM₁-ganglioside suggesting that the sCTB conserved the antigenic sites for binding and proper folding of pentameric CTB structure. The expression level of CTB was relatively high, reaching total soluble protein (TSP) levels of 0.24% in transgenic lettuce.     

Generation of large numbers of transgenic Kentucky bluegrass (Poa pratensis L.) plants following biolistic gene transfer

A very efficient transformation system, using biolistic bombardment, has been developed for the production of transgenic plants of Kentucky bluegrass (Poa pratensis L.). Embryogenic calli, initiated from immature embryos, were transformed either with pAct1IHPT-4 containing the hygromycin phosphotransferase (hpt) gene or with pDM803 containing the phosphinothricin acetyltransferase (bar) gene and the β-glucuronidase (uidA) gene. In total 119 independent transgenic plants were recovered from 153 hygromycin-resistant lines. Bialaphos selection yielded a total of 99 bialaphos-resistant lines and from these 34 independent transgenic plants were recovered. Southern blot analysis demonstrated the independent nature of the transgenic plants and also revealed a complex transgene integration pattern with multiple insertions. The first two author contributed equally to this work     

Comparison of three selectable marker genes for transformation of tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) plants by particle bombardment

A variety of selection systems have been developed for transformation of forage crops. To compare the most frequently used systems, we tested three selectable marker genes for their selection efficiency under four selection procedures for the production of transgenic tall fescue. Embryogenic calluses initiated from mature embryos were bombarded with three constructs containing either the phosphinothricin acetyltransferase (bar) gene, the hygromycin phosphotransferase (hpt) gene or the neomycin phosphotransferase II (nptII) gene. Transformation efficiency was strongly influenced by the selectable marker gene, selection procedure and genotype. The highest transformation efficiency was observed using the bar gene in combination with bialaphos. Average transformation efficiencies with bialaphos, phosphinothricin (glufosinate), hygromycin and paromomycin selection across the two callus lines used in the experiments were 9.4%, 4.4%, 5.2% and 1.6%, respectively. Southern blot analysis revealed the independent nature of the tested transgenic plants and a complex transgene integration pattern with multiple insertions.     

Alternative selectable markers for potato transformation using minimal T-DNA vectors

Alternative selection systems for plant transformation are especially valuable in clonal crops, such as potato (Solanum tuberosum L.), to pyramid transgenes into the same cultivar by successive transformation events. We have modified the pGPTV series of binary vectors to construct pMOA1 to pMOA5, resulting in a series of essentially identical binary vectors except for the presence of different selectable marker genes. These selectable marker genes are tightly inserted between the left and right T-DNA borders and confer resistance to kanamycin (nptII), hygromycin (hpt), methotrexate (dhfr), phosphinothricin (bar), or phleomycin (ble). The T-DNA of all the vectors is based on the minimal features necessary for plant transformation, with no extraneous DNA segments that may be unacceptable to regulatory authorities for general release of transgenic plants. A series of unique restriction sites exists between the right border and each selectable marker gene for subsequent insertion of useful genes. We have also developed improved culture procedures for potato transformation and used the pMOA1 to pMOA5 binary vectors to define stringent selection conditions for each marker gene. Combining these advances improved the frequency of recovering transformed potato plants while maintaining a low frequency of escapes. The relative efficiency of recovering transgenic potato lines with each selectable marker gene can be summarised as: kanamycin resistance>hygromycin resistance>phosphinothricin resistance>phleomycin resistance>methotrexate resistance.     

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     

TransgenicArabidopsis tester lines with dominant marker genes

The map positions of a set of eight T-DNA insertions in theArabidopsis genome have been determined by using closely linked visible markers. The insertions are dispersed over four of the five chromosomes. Each T-DNA insert contains one or more of the chimeric marker genes neomycin phosphotransferase (neo), hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar),-glucuronidase (gusA) and indole-3-acetamide hydrolase (iaaH). Theneo, hpt andbar marker genes are dominant in a selective germination assay or when used as DNA markers in a polymerase chain reaction. These dominant markers will allow recombinants to be discerned in a germinating F2 population, one generation earlier than with a conventional recessive marker. The transgenic marker lines will speed up and simplify the isolation of recombinants in small genetic intervals, a rate-limiting step in positional cloning strategies. The transgenic lines containing thehpt marker will also be of interest for the isolation of deletion mutants at the T-DNA integration sites.     

Characterization of the potato <Emphasis Type="Italic">upreg1</Emphasis>gene,encoding a mutated ADP-glucose pyrophosphorylase large subunit,in transformed rice

The potato upreg1, which encodes a mutated ADP-glucose pyrophosphorylase (AGPase) large subunit, was introduced into rice to evaluate its potential to enhance sink-driven yield productivity in this crop. We also wished to elucidate the activities of the up-regulated allosteric variants of potato AGPase large subunit gene in rice. A T-DNA vector containing the upreg1 gene under the control of the rice glutelin promoter was constructed with a MAR sequence and transformed into rice using Agrobacterium-mediated transformation. Transgenic plants were selected on medium supplemented with phosphinothricin and confirmed by the application of herbicide. A total of 38 transgenic plants were subsequently obtained in which the integration upreg1 into the rice genome was confirmed by Southern blotting. The exogenous AGPase in transgenic rice plants showed a high affinity for 3-phosphoglycerate activator and a low affinity for the orthophosphate inhibitor, as observed in lettuce. The transgenic rice also showed increases in the number of grains per particle, the number of panicles per plant, and also in the fresh weight of the above-ground mass of plant which was about 15% higher than non-transgenic ‘Nak-dong’. The number of seeds per tiller was also found to be about 10% higher in the transgenic plants. However, the net photosynthesis rate showed very little difference in the transgenic rice, and we could not therefore confirm any linkage with the deregulation of allosteric effects. Based on these results, upreg1 mutant genes can be used for the genetic improvement of plant AGPases other than potato and to effectively increase crop yield productivity.     

Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 μM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 μM benzylaminopurine, 2.6 μM indole-3-acetic acid, 1 g l⁻¹ activated charcoal, 1.5 mg l⁻¹ phosphinothricin, and 300 mg l⁻¹ cefotaxime. Phosphinothricin at 1.5 mg l⁻¹ was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 μM 6-benzylaminopurine and 1.5 mg l⁻¹ phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l⁻¹ phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta.     

Cross-protection and selectable marker genes in plant transformation

Summary Selectable marker genes play an important role in plant transformation. The level of selection pressure is generally established by generating a kill curve for the selectable marker. In most cases, the lowest concentration which kills all explants is used. This study examined two selectable marker genes, phosphinothricin acetyl transferase (PAT) and hygromycin phosphotransferase (HPT), in transformation of tobacco leaf disks. Experiments to determine the lethal level of the herbicide, glufosinate-ammonium (phosphinothricin) (PPT) using a leaf-disk regeneration assay established that no shoots regenerated at 2 to 4 mg PPT per 1. Likewise with the antibiotic, hygromycin (HYG), no plants regenerated at 50 mg hygromycin per 1. In contrast, after cocultivation of the leaf disks withAgrobacterium tumefaciens containing either the PAT or HPT gene in combination with a Bt gene for insect resistance, plants were successfully regenerated from leaf disks at 2 to 4 mg PPT per 1 and 50 mg hygromycin per 1. However, most plants regenerated at 2 and 3 mg PPT per 1 were found to be nontransformed (95–100% escapes) by i) Southern-blot analysis, ii) herbicide application test, and iii) insect feeding bioassay. On the other hand, plants that regenerated on 50 mg hygromycin per 1 and 4 mg PPT per 1 were transgenic as determined by Southern analysis, leaf assay for PPT or HYG resistance, and death of tobacco budworms feeding on these leaves. This study showed a significant level of cross-protection and/or transient expression of the PAT selectable marker gene allowing escapes (95–100%) at selection levels of 2 and 3 mg PPT per 1 which completely kill controls. On the other hand, the HPT gene at 50 mg is efficient in selecting for T-DNA integration.     

Transgenic <Emphasis Type="Italic">Cucumis sativus</Emphasis> Expressing the Hepatitis B Surface Antigen

A DNA fragment encoding the hepatitis B virus surface antigen was amplified from a positive blood (hepatitis B) sample and introduced into the pET 32c prokaryotic expression vector. The gene encoding the HBV surface protein antigen was introduced into pCAMBIA 3300, and immobilized into Agrobacterium tumefaciens strain LBA4404. Cotyledonary leaf sections of Cucumis sativus (cucumber) cv ‘Swarnamukhi’ were cocultivated with Agrobacterium harboring the binary vector pCAMBIA 3300 carrying the HBV surface antigen gene driven by the CaMV35S promoter and the herbicide resistance gene phosphinothricin. Putative transformed shoots were induced on a Murashige and Skoog (MS) medium containing phosphinothricin, and these were then rooted on MS basal medium supplemented with 1 mg/L Indole 3-butyric acid (IBA). Integration of the T-DNA into in putative transgenic plants was confirmed by PCR and Southern blot analyses. RT-PCR and Northern blot analyses were conducted to determine RNA expression. Levels of expression in transgenic plants were confirmed by Western blot analysis, and quantification of the protein was determined by enzyme linked immuno assay (ELISA). Molecular mass of the recombinant protein was measured by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) Mass Spectrometry.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.     

T-DNA tagging in Brassica napus as an efficient tool for the isolation of new promoters for selectable marker genes

A simple strategy to identify and isolate new promoters suitable for driving the expression of selectable marker genes is described. By employing a Brassica napus hypocotyl transformation protocol and a promoterless gus::nptII tagging construct, a series of 20 kanamycin-resistant tagged lines was produced. Most of the regenerated plants showed hardly any GUS activity in leaf, stem and root tissues. However, expression was readily restored in callus tissue induced on in vitro leaf segments. Genomic sequences upstream of the gus::nptII insertions were isolated via plasmid rescue. Three clones originating from single copy T-DNA lines were selected for further evaluation. The rescued plasmids were cloned as linear fragments in binary vectors and re-transformed to Brassica napus hypocotyl and Solanum tuberosum stem segments. The new sequences maintained their promoter activity, demonstrated by transient and stable GUS activity after transformation. Furthermore, the promoters provided sufficient expression of the nptII gene to yield transgenic plants when using kanamycin as selective agent. Database searching (BLASTN) revealed that the promoters have significant homology with three Arabidopsis BAC clones, one Arabidopsis cDNA and one Brassica napus cDNA. The results presented in this paper illustrate the strength of combined methods for identification, isolation and testing of new plant promoters.     

Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation

A simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos-resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion.     

Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells

Summary Suspension cells of Oryza sativa L. (rice) were transformed, by microprojectile bombardment, with plasmids carrying the coding region of the Streptomyces hygroscopicus phosphinothricin acetyl transferase (PAT) gene (bar) under the control of either the 5 region of the rice actin 1 gene (Act1) or the cauliflower mosaic virus (CaMV) 35S promoter. Subsequently regenerated plants display detectable PAT activity and are resistant to BASTA^TM, a phosphinothricin (PPT)-based herbicide. DNA gel blot analyses showed that PPT resistant rice plants contain a bar-hybridizing restriction fragment of the expected size. This report shows that expression of the bar gene in transgenic rice plants confers resistance to PPT-based herbicide by suppressing an increase of ammonia in plants after spraying with the herbicide.     

Expression and regulation of theRSI-1 gene during lateral root initiation

The tomato geneRSI-1 was previously identified as a molecular marker for auxin-induced lateral root initiation. We have further characterized the expression mode of theRSI-1 gene in tomato andArabidopsis thaliana. Northern blot analyses revealed that the gene was induced specifically by auxin in tomato roots and hypocotyls. For experiments with transgenic plants, the 5′ flanking region of theRSI-1 gene was linked to a GUS reporter gene, then transformed into tomato andArabidopsis. In these transgenic tomato plants, GUS activity was detected at the sites of initiation for lateral and adventitious roots. Expression of the fusion gene was auxin-dependent and tissue-specific. This was consistent with results from the northern blot analyses. In transgenicArabidopsis, the overall expression pattern of theRSI-GUS gene, including tissue specificity and auxin inducibility, was comparable to that in transgenic tomato seedlings. These results indicate that an identical regulatory mechanism for lateral root initiation might be conserved in both plants. Thus, the expression mode of theRSI-CUS gene inArabidopsis mutants defective in lateral root development should be investigated to provide details of this process.