Homology-dependent DNA Transfer from Plants to a Soil Bacterium Under Laboratory Conditions: Implications in Evolution and Horizontal Gene Transfer

DNA transfer was demonstrated from six species of donor plants to the soil bacterium, Acinetobacter spp. BD413, using neomycin phosphotransferase (nptII) as a marker for homologous recombination. These laboratory results are compatible with, but do not prove, DNA transfer in nature. In tobacco carrying a plastid insertion of nptII, transfer was detected with 0.1 g of disrupted leaves and in oilseed rape carrying a nuclear insertion with a similar quantity of roots. Transfer from disrupted leaves occurred in sterile soil and water, without the addition of nutrients. It was detected using intact tobacco leaves and intact tobacco and Arabidopsis plants in vitro. Transfer was dose-dependent and sensitive to DNase, and mutations in the plant nptII were recovered in receptor bacteria. DNA transfer using intact roots and plants in vitro was easily demonstrated, but with greater variability. Transfer varied with plant genome size and the number of repeats of the marker DNA in the donor plant. Transfer was not detected in the absence of a homologous nptII in the receptor bacteria. We discuss these results with reference to non-coding DNA in plant genomes (e.g., introns, transposons and junk DNA) and the possibility that DNA transfer could occur in nature.     

Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA

We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences. These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy.     

Transformation of Acinetobacter sp. Strain BD413(pFG4ΔnptII) with Transgenic Plant DNA in Soil Microcosms and Effects of Kanamycin on Selection of Transformants

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 10⁹ CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.     

Nucleotide substitutions in <Emphasis Type="Italic">rolC</Emphasis> and <Emphasis Type="Italic">nptII</Emphasis> gene sequences during long-term cultivation of <Emphasis Type="Italic">Panax ginseng</Emphasis> 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.     

Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5′ deleted nptII gene can restore a Kmr phenotype in tobacco

Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation. In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene. Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Km^r) phenotype. The gene targeting frequency was 1×10^–5. In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation. A total of 1.35×10⁸ protoplasts were transformed with the repair construct. Out of nearly 221 000 transformed cells 477 Km^r calli were selected. Screening the Km^r calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Km^r calli in which gene targeting had not occurred we analysed plants regenerated from 24 Km^r calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame. The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments.     

Inducible Excision of Selectable Marker Gene from Transgenic Plants by the Cre/<Emphasis Type="Italic">lox</Emphasis> Site-specific Recombination System

In a plant transformation process, it is necessary to use marker genes that allow the selection of regenerated transgenic plants. However, selectable marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and selectable marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This autoexcision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a selectable marker gene, is easy to adopt and provides a promising approach to generate marker-free transgenic plants.     

Use of a simple semiquantitative method for appraisal of green fluorescent protein gene expression in transgenic tobacco plants

We have applied a simple method for evaluation of gfp gene expression in plants using a CCD camera and computerized processing of images. Transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transfer of plasmid T-DNA bearing a m-gfp5-ER sequence governed by the 35S promoter together with the nptII selectable marker gene. Presence of the gfp gene in plants was confirmed by a polymerase chain reaction method. Mean brightness values measured using image analysis software showed differences between transgenic and control plants and suggest the possibility of rapid selection of transgenic individuals among regenerants and their progenies.     

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.     

Removal of the Selectable Marker Gene from Transgenic Tobacco Plants by Expression of Cre Recombinase from a Tobacco Mosaic Virus Vector through Agroinfection

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus–host plant systems.     

Green fluorescent protein as a vital elimination marker to easily screen marker-free transgenic progeny derived from plants co-transformed with a double T-DNA binary vector system

We investigated the potential of a novel double T-DNA vector for generating marker-free transgenic plants. Co-transformation methods using a double T-DNA vector or using mixture of two Agrobacterium tumefaciens strains were compared, and showed that the double T-DNA vector method could produce marker-free transgenic tobacco (Nicotiana tabacum L.) plants more efficiently. A dual marker double T-DNA vector was then constructed by assembling the green fluorescent protein (GFP) gene mgfp5 and the neomycin phosphotransferase gene nptII into the same T-DNA. The frequency of co-transformants produced by this vector was 56.3%. Co-expression of mgfp5 and nptII was found in 28 out of 29 T₁ lines, and segregation of the reporter -glucuronidase gene, gusA, from mgfp5 to nptII was found in 12 out of 29 T₁ lines. Therefore, GFP could be used as a vital marker to improve the transformation efficiency and to easily monitor the segregation of marker genes, thus facilitating screening of marker-free progeny.     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

Transfer of hygromycin resistance into Brassica napus using total DNA of a transgenic B. nigra line

The successful transfer of a marker gene (hpt gene) from Brassica nigra into B. napus via direct gene transfer was demonstrated. Total DNA was isolated from a hygromycin-resistant callus line, which contained three to five copies of the hpt gene. This line had been produced via direct gene transfer with the hygromycin resistance-conferring plasmid pGL2. The treatment of B. napus protoplasts with genomic DNA of B. nigra (Hyg^R) resulted in relative transformation frequencies of 0.1–0.4%. Similar transformation rates were obtained in direct gene transfer experiments using B. napus protoplasts and plasmid pGL2.     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.     

Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake

Summary Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes.     

Generation of marker free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters

Despite the advances in transgenesis, transformation technologies still rely on the introduction of a selectable marker gene to identify cells and tissues that have integrated the gene of interest in their genome. The continuous presence of the marker genes in the transgenics is often controversial as it can potentially have multiple undesirable impacts. The present study employed the self-excising Cre-loxP system to generate marker-free Arabidopsis thaliana expressing the agronomically important glyoxalase I (glyI) gene from Brassica juncea to confer salt stress tolerance. A binary vector was constructed wherein the salt-inducible rd29A promoter was used to drive the expression of the glyI gene and the transformants of A. thaliana were recovered using kanamycin resistance as the selectable marker. The neomycin phosphotransferase II (nptII) gene was flanked by the loxP sites followed by the introduction of a heat-inducible Cre-recombinase in between the loxP sites. The kanamycin-resistant transgenic lines of A. thaliana using this vector showed an ability to withstand stress imposed by 150 mM NaCl. The exposure of the T2 transgenic lines to a mild heat shock (37°C) resulted in the recovery of salt-tolerant, kanamycin-sensitive T3 progeny. Molecular analyses of the T3 transgenic lines following the heat shock treatment confirmed the excision of the nptII gene and the completion of their life cycle in the presence of 150 mM NaCl-induced stress.     

Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia

Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. -glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line.     

Organelle transfer by microfusion of defined protoplast-cytoplast pairs

Summary Defined cybridization was performed by one-to-one electrofusion (microfusion) of preselected protoplast-cytoplast pairs of male-fertile, streptomycin-resistant Nicotiana tabacum and cytoplasmic male-sterile, streptomycin-sensitive N. tabacum cms (N. bigelovii), followed by microculture of the fusion products until plant regeneration. Dominant selectable markers, namely, kanamycin resistance (nptII) and hygromycin B resistance (hpt) genes had been previously integrated in the nuclear genomes of the otherwise almost fully isogenic parental strains using direct gene transfer to protoplasts. In addition to chromosome counts indicating the expected allotetraploid tobacco count of 48, the absence of the nucleus from the cytoplast donor line was confirmed by Southern blot hybridization using nptII and hpt probes, as well as by an in vitro selection test with leaf expiants and the corresponding enzyme assays for 30 cybrids. The cytoplasmic composition of the cybrids obtained was analyzed for chloroplast type using the streptomycin resistance/sensitivity locus. The fate of mitochondria in cybrids was checked by species-specific patterns in Southern analysis of restriction endonuclease digests of total DNA with N. sylvestris mitochondrial DNA probes.     

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.     

A co-transformation system to produce transgenic grapevines free of marker genes

A co-transformation system was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. ‘Thompson Seedless’ somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bi-directional dual promoter complex. Our technique included a short positive selection phase on medium containing 100 mg l⁻¹ kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg l⁻¹ 5-fluorocytosine. We regenerated 25 stable EGFP expressing transgenic lines. PCR analysis confirmed 18 lines contained only the egfp gene, whereas the remaining contained both egfp and codA/nptII genes. Presumably, the 18 monogenic lines arose through cross protection by being in close proximity to cells that expressed nptII and thus detoxified kanamycin in the immediate vicinity. This is the first report for grapevine using a combination of positive and negative selection to produce transgenic plants that do not contain marker genes.