Neomycin Phosphotransferase Gene Research Articles

No Unintended Impacts of Transgenic Pine (<I>Pinus radiata</I>) Trees on Above Ground Invertebrate Communities

As part of an investigation into the potential unintended ecological impacts of transgenic trees, invertebrates were sampled from a field trial of transgenic Pinus radiata D. Don carrying the expressed antibiotic resistance marker gene neomycin phosphotransferase II (nptII) along with other genes known to affect reproductive development in plants and from nontransformed control trees. Invertebrate species abundance, richness, diversity, and composition were measured and compared among trees of five different transclones and nontransformed isogenic control trees. Invertebrates were sampled at six-monthly intervals over a period of 2 yr. In total, 19,162 individuals were collected comprising 279 invertebrate recognizable taxonomic units. Total invertebrate populations as well as populations of herbivorous lepidopteran larvae and Hemiptera were compared among transgenic and control trees. The results show that the transclones had no significant unintended influence on species abundance, richness, diversity, or composition for all populations investigated.

Agrobacterium tumefaciens-mediated genetic transformation of sesame (Sesamum indicum L.)

Sesame (Sesamum indicum) is an important oil seed crop that has not yet been transformed genetically. We report herein for the first time successful recovery of fertile transgenic plants of sesame from cotyledon explants inoculated with Agrobacterium tumefaciens carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. Green shoots recovered from A. tumefaciens-infected explants on selection medium [Murashige and Skoog (MS) basal medium containing 25.0 μM benzyladenine (BA), 25.0 mg l−1 kanamycin and 400.0 mg l−1 cefotaxime] were rooted on MS basal medium containing 2.0 μM indole-3-butyric acid and 5.0 mg l−1 kanamycin. The rooted shoots were established in soil and grown to maturity to collect seeds. The presence, integration and expression of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR), Southern blot hybridization and GUS histochemical assay, respectively. GUS activity was detected in vegetative and reproductive parts of T0 and T1 plants. Presence of thiol compounds in coculture medium and kanamycin selection at shoot regeneration and at rooting stages were found to be critical for transformation. The transgenes were inherited in Mendelian fashion in T1 progeny as detected by PCR. RT-PCR analysis of T1 plants confirmed the presence of transcripts of uidA gene. The transformation frequency was 1.01%, and 22–24 weeks were required from seed to seed generation time. This protocol can be used to transfer new traits in sesame for quantitative and qualitative improvement.

Backbone-free transformation of barrel medic (Medicago truncatula) with a Medicago-derived transfer DNA

In the present work, Agrobacterium tumefaciens-mediated genetic transformation of the model legume Medicago truncatula Gaertn. (barrel medic) was carried out using the pSIM843 vector that contains a Medicago-derived transfer DNA, delineated by a 25-bp sequence homologous to bacterial T-DNA borders. The transfer DNA contains an expression cassette for the nptII (neomycin phosphotransferase) gene and is flanked by an expression cassette for the backbone integration marker gene ipt (isopentenyl transferase). Our results demonstrate that the Medicago-derived RB-like elements efficiently support DNA mobilization from A. tumefaciens to M. truncatula. Kanamycin-resistant shoots with normal phenotype and ipt-shooty lines were recovered at a frequency of 11.7 and 7.8%, respectively. Polymerase chain reaction (PCR) analyses demonstrated that 44.4% of the independent transgenic lines were backbone-free and evidenced the occurrence of backbone-transfer events.

Factors affecting Agrobacterium-mediated genetic transformation of embryogenic callus of Parthenocissus tricuspidata Planch

A protocol for Agrobacterium-mediated transformation was developed for embryogenic callus of an excellent climber species, Parthenocissus tricuspidata. A. tumefaciens strain EHA105 or C58 harboring the pCAMBIA2301 binary vector with the neomycin phosphotransferase (nptII) and β-glucuronidase (uidA) gene was used. Factors affecting the transformation efficiency, including the Agrobacterium strains, co-cultivation time, Agrobacterium concentration, and infection time, were evaluated. Strain EHA105 proved to be significantly better than C58, and 4 days of co-culture was critical for transformation. An Agrobacterium suspension at a concentration of 0.5–0.7 × 108 cells ml−1 (OD600 = 0.5–0.7) and an infection time of 40 min was optimal for transformation. By applying these optimized parameters, we recovered six independent transformed shoots that were kanamycin-resistant and contained the nptII gene, as verified by polymerase chain reaction (PCR) analysis. Southern blot analysis confirmed that T-DNA was stably integrated into the genome of three out of six PCR-positive lines. Furthermore, histochemical GUS assay revealed the expression of the uidA gene in kanamycin-resistant calli, somatic embryos, and leaves of transgenic plants.

Isolation of Transfected Adult Fibroblast Clones and SCNT Embryo Research*

Transgenesis in rabbit has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock by the combination of transgenic technology. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. 24-well cell culture plates were used to isolate cell lineages obtained from a single fibroblast clone transfected with the pRNAT-U6.1/Neo plasmid. Since single fibroblast clone does not grow well in fresh medium, the use of conditioned medium was evaluated. Meanwhile, the effect of initial cell density and linear-plasimid on transgenic fibroblast colony growth were investigated. The increasing initial cell density and lining plasmid could improve colony growth and expansion. There was a significant difference in the conditioned medium or initial cell density compared to the control group. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. When the transgenic fibroblasts were used as donor cells of nuclear transfer, the blastocyst rate were 23.5%. There was not a significant difference in the transgenic fibroblasts compared to the normal group. PCR or Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal β-actin DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production; further improvements in related technologies will facilitate the use of this method for the generation of the genetic engineering of rabbits.

Investigation of Factors in Optimizing Agrobacterium-Mediated Gene Transfer in Citrullus lanatus cv. Round Dragon

Agrobacterium tumefaciens strain LBA 4404 harboring a binary plasmid pCambar (containing Basta resistance (bar) gene, neomycin phosphotransferase (npt II) gene and s-Glucuronidase (GUS) gene) was used to optimize the transformation efficiency in Citrullus lanatus cv. Round Dragon. In this study, the ability of Agrobacterium tumefaciens to mediate a gene transfer in Citrullus lanatus was highly dependent on various transformation factors. In current investigation, we have established factors influencing gene expression including explant ages of cotyledon- (5 days old cotyledon), pre-culture condition (2 days), wounding technique (multi wired with massive wounding technique), Agrobacterium concentration (A600 nm 0.8), co-incubation period of explants with Agrobacterium (30 min) and acetosyringone concentration (200 µM) in co-cultivation medium. These factors gave maximized transformation efficiency. The expression of the foreign functional gene in the plant genome was confirmed by histochemical GUS assay activity after 3 days of co-cultivation period. By combining the best conditions from each evaluated factors, we successfully established an efficient and reproducible Agrobacterium-mediated transformation protocol for Citrullus lanatus cv. Round Dragon which yielded 100% of transgene expression with 181.18±0.57 blue spots per responding explant. In conclusion, the transformation system for Citrullus lanatus cv. Round Dragon was optimized. The transformation procedure was proved to influence the transformation efficiency in Citrullus lanatus at 100% transient expression. Present study also suggested that different plant varieties may affect the transformation rate. The optimized conditions will then be used in future research to make a transgenic plant.

Chromosomal position effects on AAV-mediated gene targeting

The effects of chromosomal position and neighboring genomic elements on gene targeting in human cells remain largely unexplored. To study these, we used a shuttle vector system in which murine leukemia virus (MLV)-based proviral targets present at different chromosomal locations and containing mutations in the neomycin phosphotransferase (neo) gene were corrected by adeno-associated virus (AAV)-mediated gene targeting. Sixteen identical target loci present in HT-1080 human sarcoma cells were all successfully corrected by gene targeting. The gene targeting frequencies varied by as much as 10-fold, and there was a clear bias for correction of one of the targets in clones containing two target sites. The targeting frequency at each site was correlated to the proximity and density of various genomic elements, and we found a significant association of higher targeting frequencies at loci near a subset of dinucleotide microsatellite repeats (r = –0.55, P < 0.05), in particular GT repeats (r = –0.87, P < 0.0001). Additionally, there was a correlation between meiotic recombination rates and targeting frequencies at the target loci (r = 0.52, P < 0.05). There was no correlation between surrounding chromosomal transcription units and targeting frequencies. Our results indicate that certain chromosomal positions are preferred sites for gene targeting in human cells.

Development of real time PCR assays for detection and quantification of transgene DNA of a Bacillus thuringiensis (Bt) corn hybrid in soil samples

Real time PCR assays were developed to detect and quantify the transgene DNA of a commercially released Bacillus thuringiensis (Bt) corn (Zea mays L.) hybrid (DKC42-23), which was derived from the event MON863 and also carried a neomycin phosphotransferase gene (the nptII gene). We applied the real time PCR assays to investigate the persistence of the transgene DNA in a field trial grown with DKC42-23 over 3years, in combination with bacterial natural transformation. The results showed that under continuous cultivation of DKC42-23, its transgene DNA was detectable in the field plots all year around. Meanwhile, when soil DNA extracts from DKC42-23 plots were used as donor in bacterial natural transformation, successful recovery of kanamycin resistant (Km(R)) transformants indicated that the nptII gene carried by DKC42-23 could be taken up and integrated into naturally competent Pseudomonas stutzeri pMR7 cells, leading to the restoration of the antibiotic resistance of P. stutzeri pMR7. However, after the cultivation of a soybean line in the same plots for the subsequent growing season, the presence of transgene DNA of DKC42-23 was reduced to undetectable levels at the end of that growing season. Therefore, existing corn-soybean crop rotation practices reduce the availability of transgene DNA in soil and thus minimize the risks that might be attributable to horizontal gene transfer. The real time PCR assays are useful for investigating the persistence of transgene DNA derived from the MON863 event in soil environments.

Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane

A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8-4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and beta-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1-3 transgene insertion events that were randomly integrated in the majority of the plants produced.

Agrobacterium-mediated transformation of mature Prunus serotina (black cherry) and regeneration of transgenic shoots

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 μM acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured on woody plant medium supplemented with 9.08 μM thidiazuron, 1.07 μM napthaleneacetic acid, 60 μM silver thiosulphate, 3% sucrose, plus 200 mg l−1 timentin in darkness for 3 weeks. Regenerating shoots were selected 27 days after initial co-culture, on Murashige and Skoog medium with 3% sucrose, 8.88 μM 6-benzylaminopurine, 0.49 μM indole-3-butyric acid, 0.29 μM gibberellic acid, 200 mg l−1 timentin, and 30 mg l−1 kanamycin for five subcultures. After 5–6 months of selection, transformation efficiencies were determined, based on polymerase chain reaction (PCR) analysis of individual putative transformed shoots relative to the initial number of leaf explants tested. The transformation efficiency was 1.2%. Southern blot analysis of three out of four PCR-positive shoots confirmed the presence of the neomycin phosphotransferase and AG genes. Transgenic shoots were rooted (37.5%), but some shoot tips and leaves deteriorated or died, making acclimatization of rooted transgenic plants difficult. This transformation, regeneration, and rooting protocol for developing transgenic black cherry will continue to be evaluated in future experiments, in order to optimize the system for several mature black cherry genotypes.

Genetic transformation of the bast fiber plant ramie (Boehmeria nivea Gaud.) via Agrobacterium tumefaciens

Hypocotyls and cotyledons of three ramie (Boehmeria nivea Gaud.) cultivars (Zhongzhu No.1, Zhongsizhu No.1 and NC01), important plants for textile fiber, were pre-cultured on callus-inducing medium for 1 day before co-cultivation for 2 days with Agrobacterium tumefaciens strain LBA4404 harboring the plasmid pGBI4ABC carrying two insect resistance (CryIA and CpTI), gus, and neomycin phosphotransferase (npt II) genes. Calli were induced from both hypocotyl and cotyledon explants grown on a kanamycin selection medium. Regenerated shoots were obtained after two cycles of culture and transferred to rooting medium. Kanamycin-resistant plantlets were rooted in 2 weeks, and then transplanted to soil. Transgenic plants were subsequently confirmed by polymerase chain reaction, Southern blot hybridization, and GUS assays. More than 100 transgenic plants carrying insect-resistance genes were produced. A transformation frequency of 8.8 to 10.3% was obtained using hypocotyls as explants, which was higher than all previously reported transformation frequencies. The whole protocol, from transformation recovery of plants grown in soil, was completed within 2–4 months. Therefore, a simple, efficient, and robust Agrobacterium tumefaciens -mediated transformation system for ramie has been developed.

Production of transgenic Podophyllum peltatum via Agrobacterium tumefaciens-mediated transformation

Transgenic Podophyllum peltatum plants were successfully produced by Agrobacterium tumefaciens-mediated transformation. Embryogenic callus was co-cultivated with Agrobacterium tumefaciens harboring a binary vector pBI 121 carrying β-glucuronidase (GUS) and neomycinphosphotransferase (NPT II) gene. GUS-histochemical analysis revealed that, 50 µM acetosyringone treatments during Agrobacterium infection and 3 d co-cultivation with Agrobacterium showed enhanced transformation efficiency. Percentage of GUS positive callus increased rapidly as the subculture time proceeded on selection medium containing 100 mg dm−3 kanamycin. Kanamycin resistant somatic embryos were formed from embryogenic callus after cultivation with 11.35 µM abscisic acid (ABA) for 3 weeks and then on hormone-free selection medium. Somatic embryos were germinated and converted into plantlets on medium containing 2.89 µM gibberellic acid (GA3). The integration of GUS and NPT II gene into transgenic plants was confirmed by polymerase chain reaction and Southern analysis.

Genetic transformation of the oilseed crop Crambe abyssinica

Engineering oilseed crops for industrial purposes requires a suitable crop that does not outcross to any food oilseed crop, thus eliminating problems of gene flow. Crambe abyssinica is such a dedicated crop as it does not hybridize with any existing food oilseed crops. However, lack of regeneration and transformation protocols has limited the use of C. abyssinica in genetic manipulation studies. In this study, efficient regeneration and transformation protocols for Crambe have been developed. Hypocotyls of C. abyssinica cv. Galactica were incubated on a Murashige and Skoog medium supplemented with various plant growth regulators (PGRs). Among the different PGR combinations tested, 10 μM thidiazuron and 2.7 μM α-naphthaleneacetic acid promoted highest frequency of regeneration, up to 60%. Among six Agrobacterium stains evaluated, each harbouring the cloning vector containing the neomycin phosphotransferase (nptII) and β-glucuronidase (gus) genes. EHA101 and AGL-1 yielded the highest transformation frequencies of 1.3 and 2.1%, respectively. Putative transgenic lines were recovered, and confirmed as transgenic by Southern blot analysis. Subsequently, Agrobacterium-mediated transformation of hypocotyls of cv. Galactica with constructs harbouring the wax synthase and fatty acid reductase genes have also successfully recovered confirmed transgenic plants carrying these transgenes.

Proanthocyanidin from Blueberry Leaves Suppresses Expression of Subgenomic Hepatitis C Virus RNA

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.

Influence of Agrobacterium rhizogenes on induction of hairy roots and benzylisoquinoline alkaloids production in Persian poppy (Papaver bracteatum Lindl.): preliminary report

Persian poppy (Papaver bracteatum Lindl.) is an important medicinal plant and source of the opium alkaloids codeine, morphine and thebaine. Transgenic root cultures of P. bracteatum Lindl. are well-defined model systems to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis. Agrobacterium rhizogenes was able to produce hairy roots on wounded Persian poppy seedlings. Excised shoots from 7-day-old Persian poppy were co-cultivated with the A. rhizogenes strain R15834 carrying the pBI121 binary vector. All media, except for the co-cultivation medium, included 40 mg l−1 paromomycin to select for pBI121 transformants and 200 mg l−1 cefotaxime to eliminate the Agrobacterium. Eight weeks after infection, paromomycin-resistant roots appeared on 45–50% of explants maintained on hormone-free medium. Isolated hairy roots were propagated in liquid medium containing 1.0 mg l−1 1-naphthaleneacetic acid to promote rapid growth. Also, callus induction and shoot regeneration of transformed Calli in vitro was achieved on B5 medium containing 1.0 mg l−1 1-naphthaleneacetic acid. Detection of the neomycin phosphotransferase gene and GUS histochemical localization confirmed the integrative transformation of root cultures. This is the first study to illustrate useful protocol to introduce foreign genes into transgenic Persian poppy hairy root cultures using A. rhizogenes strain R15834.

Nucleotide substitutions in rolC and nptII gene sequences during long-term cultivation of Panax ginseng cell cultures

It has been shown previously that the rolC gene from Agrobacterium tumefaciens gene was stably and highly expressed in 15-year-old Panax ginseng transgenic cell cultures. In the present report, we analyze in detail the nucleotide composition of the rolC and nptII (neomycin phosphotransferase) genes, which is the selective marker used for transgenic cell cultures of P. ginseng. It has been established that the nucleotide sequences of the rolC and nptII genes underwent mutagenesis during cultivation. Particularly, 1-4 nucleotide substitutions were found per sequence in the 540 and 798 bp segments of the complete rolC and nptII genes, respectively. Approximately half of these nucleotide substitutions caused changes in the structure of the predicted gene product. In addition, we attempted to determine the rate of accumulation of these changes by comparison of DNA extracted from P. ginseng cell cultures from 1995 to 2007. It was observed that the frequency of nucleotide substitutions for the rolC and nptII genes in 1995 was 1.21 +/- 0.02 per 1,000 nucleotides analyzed, while in 2007, the nucleotide substitutions significantly increased (1.37 +/- 0.07 per 1,000 nucleotides analyzed). Analyzing the nucleotide substitutions, we found that substitution to G or to C nucleotides significantly increased (in 1.9 times) in the rolC and nptII genes compared with P. ginseng actin gene. Finally, the level of nucleotide substitutions in the rolC gene was 1.1-fold higher when compared with the nptII gene. Thus, for the first time, we have experimentally demonstrated the level of nucleotide substitutions in transferred genes in transgenic plant cell cultures.

Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and beta-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were transformed in the presence of 100 microM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l(-1) was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with 13.3 microM 6-benzylaminopurine, 4.5 microM thidiazuron, 50 mg l(-1) adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse. This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash.

Agrobacterium-mediated Transformation of Buddleia Species

Two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were transformed using Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBI121 carrying the neomycin phosphotransferase gene and β-glucuronidase gene (uidA). Transgenic plants were recovered from the Agrobacterium-infected leaf tissues through organogenesis in the selection medium (woody plant medium containing 250 mg·L−1 cefotaxime plus 500 mg·L−1 carbenicillin plus 40 mg·L−1 kanamycin). The rate of shoot regeneration from transformed leaf tissues increased from 5.7% to 32% through extending cocultivation time from 3 to 9 days. Integration of marker genes was verified with polymerase chain reaction (PCR) and Southern blot analysis. Southern blot analysis confirmed that one to three copies of transgenes were integrated into the buddleia genome. This transformation system could be used for improvement of buddleia or other related species. Chemical names used: 6-benzyladenine (BA), naphthalene acetic acid (NAA), acetosyringone (AS), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (X-Glu), cefotaxime (Cef), carbenicillin (Carb), kanamycin (Km).

Elaboration of a Reliable Strategy Based on Real-Time PCR To Characterize Genetically Modified Plantlets and To Evaluate the Efficiency of a Marker Gene Removal in Grape (Vitis spp.)

We have developed an effective strategy based on real-time PCR assay for the molecular characterization of genetically modified grape and to quantify the efficiency of a marker gene removal. This research has been implemented in Vitis vinifera cv. Brachetto plantlets where exogenes were inserted during cocultures of embryogenic calli with Agrobacterium tumefaciens carrying the chemically inducible site-specific cre/loxP pX6 vector where the expression of the cre recombinase is regulated by 17-beta-estradiol. The neomycin phosphotransferase gene (nptII) for the kanamycin resistance trait was inserted as part of the gene transfer protocol, and this exogene was employed as a case study for carrying out our research. The 9-cis-epoxycarotenoid dioxygenase (nced2) and chalcone isomerase (chi) genes coding for two enzymes, involved respectively in abscisic acid and flavonoid biosynthesis, proved to be valuable reference endogenes for real-time PCR assays. Two types of duplo-target plasmids were exploited for building the standard curves: in one type (p-nptII/nced2) the nptII sequence is linked to the nced2 sequence; in the other (p-nptII/chi) it is linked to the chi. These calibrators were intended to simulate an ideal genetically modified plant carrying a homozygous single-copy exogene insertion. The repeatability test confirmed the suitability of both plasmid calibrators. Foreign gene stability can be monitored during long-term plant preservation, and the method proved to be suitable for quantifying the efficiency of nptII gene removal induced by 17-beta-estradiol.

Development and evaluation of a GFLV inverted repeat construct for genetic transformation of grapevine

Grapevine Fanleaf virus (GFLV) is one of the most damaging and widespread nepovirus in grapevine, causing the fanleaf disease. Here, we report the development of inverted repeat (IR) constructs, consisting of GFLV-derived sequences, for genetic transformation of grapevine to induce GFLV-specific silencing. The IR construct was designed with fragments of a conserved region of the GFLV movement protein (MPc) gene from a Tunisian isolate (GFLV tun) and with cassettes containing the neomycin phosphotransferase or bar gene as selectable marker. For proof of concept, the IR construct was transferred into Nicotiana benthamiana by Agrobacterium mediated transformation. Challenge inoculation of T1 transgenic plant lines with the relevant virus resulted in plants showing resistance, recovery, retarded infection and susceptibility. Northern blot analysis, using a virus-derived sequence (MPc) as probe, could detect transgene specific small interfering RNA in resistant transgenic N. benthamiana plants after GFLV inoculation. Embryogenic cells of grapevine (Vitis vinifera cv Arich dresse) were transformed with the IR construct in combination with the bar gene using A. tumefaciens strain LBA4404. After molecular characterization of regenerated putative transgenic grapevine plants, Northern blot analysis detected different levels of IR transcripts in independent transgenic grapevine lines indicating a post-transcriptional gene silencing.