Spread of recombinant DNA by roots and pollen of transgenic potato plants,identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII'; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII' and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.     

Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation

We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6?×?10⁶-fold excess of plant DNA. Only 18 ng of potato DNA (2.5?×?10³ genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR).     

Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation

We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 × 10⁶-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 × 10³ genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR). Received: 20 May 1997 / Accepted: 17 October 1997     

Monitoring the Spread of Recombinant DNA from Field Plots with Transgenic Sugar Beet Plants by PCR and Natural Transformation of Pseudomonas Stutzeri

Previous studies had shown that recombinant DNA can be detected for several months in soil after the deposition of litter from transgenic (tg) plants. Here we show by PCR monitoring of field releases of tg sugar beet plants that during the growth of the plants the soil close to the plants and also plant material contains recombinant DNA, in the form of extracellular molecules. Surprisingly, the monitoring also revealed the presence of tg DNA in many field plots (30–70%) in which tg plants were never grown. These studies and the further monitoring during other tg sugar beet release experiments by PCR and a novel bioassay (measuring the transforming potential of recombinant DNA for Pseudomonas stutzeri) indicated that recombinant DNA was only detectable in the surface soil of field plots and their vicinity where flowering of the tg beet plants was allowed. Recombinant DNA was found in soil at a distance of 50 m from pollen-producing plants surrounded by a strip with hemp plants as a containment regime. It is concluded that recombinant DNA is deposited in soil during the growth of tg sugar beets and that a major mechanism of recombinant DNA spread in the environment is the dispersal of pollen which allows recombinant DNA to persist in the field plot for at least a year.     

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.     

Transformation of the tropane alkaloid-producing medicinal plant Hyoscyamus muticus by particle bombardment

We report an efficient whole plant transformation system for Hyoscyamus muticus, an important medicinal plant of the Solanaceous family. We developed a system using a plasmid carrying the nptII and gusA genes, which was delivered into leaf explants by particle bombardment. Ten percent of bombarded leaf explants formed kanamycin-resistant callus, from which putative transgenic plants were recovered. The nptII gene conferring kanamycin resistance was found to be incorporated into the genome of all transgenic plants screened. Over 50% of the kanamycin resistant plants showed strong expression of the non-selected gusA gene. The majority of transgenic plants reached maturity, could be self pollinated, and produced fertile seed. A simple and efficient whole plant transformation system for this medicinal plant is an important step in furthering our understanding of tropane alkaloid production in plants.     

An assessment of gene transfer by pollen from field-grown transgenic potatoes to non-transgenic potatoes and related species

Information on the extent of transgene dispersal by pollen to adjacent potato plots and to related weed species is an important requisite for risk assessment; a procedure followed before novel transgenic plants are evaluated under field conditions. The purpose of the investigation was to determine the frequency of cross-pollination between potato (Solanum tuberosum) plants at different distances, using a kanamycin resistnace transgene (nptII) as a selectable marker. All potato plants were from the variety Désirée. Non-transgenic potato plants, used as potential recipients of transgene-containing pollen, were planted in 12 sub-plots, at distances of 0–20 m from the nearest transgenic potato plants. Seeds harvested from the non-transgenic plants were screened for resistance to kanamycin, and molecular methods were used to confirm that resistant progeny contained thenptII gene. Where transgenic and non-transgenic potato plants were in alternate rows (leaves touching), 24% of seedlings from the non-transgenic parent plants were kanamycin-resistant. Comparable seedlings from plants at up to 3 m distance had a resistance frequency of 2%, at 10 m the frequency was 0.017% and at 20 m no resistant progeny were observed. Plants of the weed speciesS. dulcamara andS. nigrum were also planted close to the transgenic potatoes to test for evidence of hybridization, and no kanamycin-resistant seedlings were observed among progeny fromS. dulcamara andS. nigrum. This investigation provided evidence that the extent of gene dispersal from transgenic potatoes to non-transgenic potatoes falls markedly with increasing distance, and is negligible at 10 m. There was, also, no evidence of transgene movement from potato toS. dulcamara andS. nigrum under field conditions. These data will be valuable in defining genetic isolation procedures for the early field evaluation and the use of novel transgenic potato genotypes.     

Evaluation of possible horizontal gene transfer from transgenic plants to the soil bacterium Acinetobacter calcoaceticus BD413

 The use of genetically engineered crop plants has raised concerns about the transfer of their engineered DNA to indigenous microbes in soil. We have evaluated possible horizontal gene transfer from transgenic plants by natural transformation to the soil bacterium Acinetobacter calcoaceticus BD413. The transformation frequencies with DNA from two sources of transgenic plant DNA and different forms of plasmid DNA with an inserted kanamycin resistance gene, nptII, were measured. Clear effects of homology were seen on transformation frequencies, and no transformants were ever detected after using transgenic plant DNA. This implied a transformation frequency of less than 10^-13 (transformants per recipient) under optimised conditions, which is expected to drop even further to a minimum of 10^-16 due to soil conditions and a lowered concentration of DNA available to cells. Previous studies have shown that chromosomal DNA released to soil is only available to A. calcoaceticus for limited period of time and that A. calcoaceticus does not maintain detectable competence in soil. Taken together, these results suggest that A. calcoaceticus does not take up non-homologous plant DNA at appreciable frequencies under natural conditions. Received: 1 November 1996 / Accepted: 18 April 1997     

Marker Rescue Studies of the Transfer of Recombinant DNA to Streptococcus gordonii In Vitro, in Foods and Gnotobiotic Rats

A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 × 10⁻⁶ to 5.8 × 10⁻⁷ transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 × 10⁻⁹). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 × 10⁻¹⁰), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 × 10⁻¹¹. No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> -mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) using a heterologous bean chitinase gene

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T₁ plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T₀ transformants and progeny plants (T₁) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.     

Reconsideration of pollen dispersal data from field trials of transgenic potatoes

During the initial field evaluation of transgenic plants, it is usual to isolate them genetically from other plants of the same species. Several field experiments on potatoes, using transgenes as markers, have shown that transgene dispersal by pollen to other potato plants is limited and very unlikely at distances over 10 m. In a recent study in Sweden, a frequency of transgene-containing progeny of over 30% is reported from non-transgenic potato plants grown at distances of 10–1000 m from transgenic plants containing nptII and gus marker genes. Data from the Swedish study is discussed along with other relevant observations, and it is concluded that the high frequency of gene dispersal in that study results from a high frequency of false positives during PCR analysis of the nptII gene. From the data available in potato, it is concluded that a distance of 20 m is generally adequate for the initial field evaluation of transgenic potatoes containing novel gene constructs.     

Efficacy of an intron-containing kanamycin resistance gene as a selectable marker in plant transformation

In this project we have analysed the use of an intron-containing neomycin phosphotransferase II - nptII - gene. The advantage of this construct is that only eukaryotic organisms will be able to process this gene. Accordingly, the theoretical risk of horizontal gene flow of antibiotic resistance genes from transgenic plants to enteric bacteria is eliminated. The ST-LS1 intron IV2 from potato was inserted into the coding region of nptII. Transformation of Solanum tuberosum (potato) and Nicotiana tabacum (tobacco) with constructs containing the intron nptII showed similar transformation frequencies to transformation with constructs containing the normal nptII. Analysis of total DNA and RNA confirmed that the intron-containing nptII gene was present in the plants and that the mRNA was processed correctly.     

Vector integration in triple R gene transformants and the clustered inheritance of resistance against potato late blight

Genetic transformation with resistance (R) genes is expected to enhance resistance durability against pathogens, especially for potato, a vegetatively propagated crop with tetrasomic inheritance and a long-term breeding program. In this study, 128 potato transformants were analysed for the presence of vector T-DNA genes, borders and backbone sequences. They were harvested after transformation using a construct containing neomycin phosphotransferase II (nptII) and three R genes against potato late blight (Phytophthora infestans). Our analysis revealed that 45 % of the R gene-containing transformants possessed a low T-DNA copy number, without the integration of vector backbone and borders. The integration of vector backbone sequences was characterized using eight genes, and backbone gene tetA was selected for the early prediction of plants with backbone sequence integration. Three transformants, two plants harbouring one T-DNA copy and one plant harbouring three T-DNA copies, were crossed with susceptible cv. Katahdin. Based on our results, we conclude that all four T-DNA genes were inherited as one cluster and segregated in a Mendelian fashion. The three T-DNA inserts from the transformant harbouring three T-DNA copies were statistically proven to be un-linked and inherited into the offspring plants independently. All of the R genes were functionally expressed in the offspring plants as in their parental transformants. This functional gene stacking has important implications towards achieving more durable resistance against potato late blight.     

Fertile transgenic Indica rice plants obtained by electroporation of the seed embryo cells

We have obtained fertile transgenic plants of Indica rice variety IR36, by using electroporation to transfer the neomycin phosphotransferase II (nptII) gene into cells of mature embryos. Resistant calli were selected in the presence of 30 g/ml G418. Nearly thirty transgenic plants were regenerated within three months after transformation. Many of them yielded seeds following self-pollination. Data from molecular analysis and enzyme assay proved that the foreign gene was stably integrated into the genome of resistant calli, R0 and R1 plants, and also expressed. Mendelian segregation of the nptII gene was observed in R1 progeny plants.Abbreviations NOS nopaline synthase - NPTII and nptII neomycin phosphotransferase II - OCS octopine synthase - Km kanamycin     

Agrobacterium tumefaciens-mediated transgenic plant production via direct shoot bud organogenesis from pre-plasmolyzed leaf explants of Catharanthus roseus

Production of Agrobacterium tumefaciens-mediated transgenic plants, via direct shoot bud organogenesis from leaves of Catharanthus roseus, is reported. A. tumefaciens harbouring the plasmid pBI121 with GUS gene uidA and kanamycin resistance gene nptII was used. Highest transformation efficiency of 1.4 transgenic shoots/responded explant was obtained when pre-plasmolysed leaves, pre-incubated on shoot bud induction medium for 10 days, were subjected to sonication for 30 s prior to transformation. Using a selection medium containing 50 mg kanamycin l⁻¹, transformants grew into micro-shoots and formed roots on a hormone-free half strength MS medium. The transgenic nature of the regenerated plants was confirmed by PCR amplification of uidA gene and GUS histochemical assay.     

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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

An experimental assessment of the factors influencing Agrobacterium-mediated transformation in tomato

Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pTOK233, which contained the GUS reporter gene and a kanamycin-resistance gene nptII, was employed for optimizing the transformation efficiency evaluated by a GUS gene transient expression level. Eight factors including explant types, explant size and source, the concentration of cytokinin, inoculation time, pH of inoculation and cocultivation media, bacterial concentration, acetosyringone concentration, and cocultivation duration were investigated in detail. This optimized protocol was then adopted to obtain transgenic tomato plants resistant to cucumber mosaic virus (CMV) mediated by Agrobacterium tumefaciens, strain LBA4404, carrying a binary vector pR-ΔGDD containing the kanamy cin-resistance gene and CMV replicase gene with GDD deletion. The presence of the CMV-RNA2 gene was confirmed by genomic DNA Southern blot analysis in all transformants analyzed. Field spray test showed that the transgenic tomato plants were resistant to 100 mg/l kanamycin. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 280–284. The text was submitted by the authors in English.     

Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment

Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 × B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by thebar orpat genes, was more efficient than selection on kanamycin, mediated by thenptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants-transgene silencing, as well as poor transgene transmission to progeny, was observed in some plant lines in which the parent plants had expressed the transgene.