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°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.     

Analysis of transgene integration and expression following biolistic transfer of different quantities of minimal expression cassette into sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. hybrids)

Sugarcane (Saccharum spp. hybrids) is an interspecific hybrid with a highly polyploid and frequently aneuploid genome. This C4 grass accounts for nearly 70% of the global sugar production and more recently has become an important biofuel feedstock. Biolistic gene transfer of plasmid DNA is the most frequently used approach for genetic transformation of sugarcane. Minimal expression cassettes lacking vector backbone sequences (MC) have been reported to support simple transgene integration in other species. In this study, we introduced a MC of nptII into embryogenic callus derived from immature leaf whorl cross-sections by biolistic gene transfer. The precipitation equivalents of 12.5, 25 or 50 ng of the nptII MC were delivered per shot to the target tissue with 1.0 μm gold particles. A total of 203 independent putative transgenic plants were regenerated following 80 bombardments and selection on geneticin or paromomycin containing media and 176 transgenic lines were confirmed with PCR. Twenty independent transgenic lines were selected for Southern blot analysis and expression analysis by NPTII ELISA from each of the three treatments. Genomic DNA from transgenic sugarcane plants displayed two to 13 nptII hybridization signals on Southern blots. There was a trend toward reduced transgene integration complexity and reduced transgene expression levels when lower (12.5 ng) MC was used per shot. These results demonstrate that backbone free MCs can be efficiently integrated and expressed in sugarcane.     

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with<Emphasis Type="Italic">HMG I/Y</Emphasis> as an endogenous reference gene

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T₀) were calculated by comparing threshold cycle (C _T) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsC _T value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.     

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     

Genetic transformation of selected mature cork oak (Quercus suber L.) trees

A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and -glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.Abbreviations BA N⁶-Benzyladenine - GUS -Glucuronidase - MSSH Expression-proliferation medium - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase gene - uidA -Glucuronidase gene     

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).     

Unsuccessful search for DNA transfer from transgenic plants to bacteria in the intestine of the tobacco horn worm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

DNA transfer from transgenic plants to native intestinal bacteria and introduced Acinetobacter BD413 was assessed in the gut of the tobacco horn worm (Manduca sexta). The marker was kanamycin resistance gene (nptII), and tobacco carrying the nptII gene in the chloroplasts served as the donor. We detected neither whole gene transfer to native bacteria, nor transfer of fragments of nptII to Acinetobacter, using a marker exchange assay. This negative result was attributed to a heat-labile activity that degraded DNA in the feces, probably DNAase. Nevertheless, a few intact leaf cells survived transit through the gut, and DNA extracted from feces did transform Acinetobacter, albeit at lower frequencies than DNA extracted from leaves.     

Exchange of chromosomal markers by natural transformation between the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell

Both the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell, have been shown previously to be naturally transformable. This study reports the detection of genetic exchange by natural transformation between these two isolates. Transformation frequency was determined by filter transformation procedures. Three independent antibiotic resistance loci were used as chromosomal markers to monitor this exchange event: resistance to rifampicin, streptomycin, and nalidixic acid. The maximum frequencies of transformation were on the order of 3.1 to 3.8×10^-6 transformants per recipient; frequencies over an order of magnitude greater than those for spontaneous antibiotic resistance, although they are lower than those observed for soil: soil or marine: marine strain crosses. This exchange was inhibited by DNase I. Transformation was observed between soil and marine strains, both by filter transformation using purified DNA solutions and when transforming DNA was added in the form of viable donor cells. The results from this study support the close genetic relationship betweenP. stutzeri JM300 andP. stutzeri strain ZoBell. These results also further validate the utility ofP. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.     

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.     

Evaluation of dietary effects of transgenic corn pollen expressing Cry3Bb1 protein on a non-target ladybird beetle, Coleomegilla maculata

A transgenic corn event (MON 863) has been recently developed by Monsanto Company for control of corn rootworms, Diabrotica spp. (Coleoptera: Chrysomelidae). This transgenic corn event expresses the cry3Bb1 gene derived from Bacillus thuringiensis (Berliner), which encodes the insecticidal Cry3Bb1 protein for corn rootworm control. A continuous feeding study was conducted in the laboratory to evaluate the dietary effect of MON 863 pollen expressing the Cry3Bb1 protein on the survival, larval development, and reproductive capacity of the non-target species, Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae). First instar C. maculata (less than 24 h old) and newly emerging adults (less than 72 h old) were fed individually on a diet mixture containing 50% of MON 863 pollen, non-transgenic (control) corn pollen, bee pollen (a component of normal rearing diet), or potassium arsenate-treated control corn pollen. In the larval tests, 96.7%, 90.0%, and 93.3% of C. maculata larvae successfully pupated and then emerged as adults when fed on MON 863 pollen, non-transgenic corn pollen, and bee pollen (normal rearing) diets, respectively. Among the larvae completing their development, there were no significant differences in the developmental time to pupation and adult emergence among the transgenic corn pollen, non-transgenic corn pollen, and bee pollen diet treatments. All larvae fed on arsenate treated corn pollen diet died as larvae. For tests with adults, 83.3%, 80.0%, and 100% of adult C. maculata survived for the 30 days of the test period when reared on diets containing 50% of MON 863 pollen, non-transgenic corn pollen, and bee pollen respectively. While the adult survival rate on MON 863 pollen diet was significantly less than that on the bee pollen diet, there was no significant difference between the MON 863 and non-transgenic corn pollen treatments. During the period of adult testing, an average of 77, 80, and 89 eggs per female were laid by females fed on the MON 863 pollen, control corn pollen, and bee pollen, respectively; no significant differences were detected in the number of eggs laid among these treatments. These results demonstrate that when offered at 50% by weight of the dietary component, transgenic corn (MON 863) pollen expressing Cry3Bb1 protein had no measurable negative effect on the survival and development of C. maculata larvae to pupation and adulthood nor any adverse effect on adult survival and reproductive capacity. Relevance of these findings to ecological impacts of transgenic Bt crops on non-target beneficial insects is discussed.     

Comparison of genetic transformation in <Emphasis Type="Italic">Morus alba</Emphasis> L. via different regeneration systems

Three different regeneration systems, viz. direct regeneration of adventitious shoot buds from explant, regeneration through callus cultures and somatic embryos were compared to see their effect on transfer of neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) reporter gene (gus) to Morus alba clone M5, through Agrobacterium tumefaciens mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency. The highest transformation frequency of 18.6% was obtained using direct induction of adventitious shoot buds. Expression and presence of transgene were assayed histochemically and through polymerase chain reaction. Southern analysis of GUS and PCR positive transformants confirmed stable integration of transgenes with two to four copy numbers. The selected transformants showed normal phenotype under in vitro and field conditions.     

Generation of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage- and turf-type cultivars

Perennial ryegrass (Lolium perenne L.) is the most important grass species in areas with a temperate climate. Biolistic transfer of a ubiquitin promoter driven nptII expression cassette into mature or immature tissue derived calli of perennial ryegrass followed by paromomycin selection, resulted in the rapid and efficient production of fertile transgenic ryegrass plants. Transformation efficiencies after paromomycin selection in combination with the nptII selectable marker compared favourably with hygromycin selection in combination with the hph selectable marker. In total 83 independent nptII expressing plants were produced. Transformation frequency was highly affected by genotype, explant, selection regime and the duration of the callus induction period. The optimised transformation protocol for mature embryo derived calli of turf-type or forage-type cultivars resulted in an average transformation efficiency of 5.2% or 6.6% respectively. This converts into 1.7 or 2.2 independent transgenic plants per bombardment. Immature inflorescence- and immature embryo-derived calli were also successfully used as target for the gene transfer, resulting in transformation efficiencies of up to 3.7% or 11.42% respectively. Transgenic plants were transferred to soil 12 or 9 weeks after excision of mature and immature embryos or inflorescences respectively. Transgene integration and expression were confirmed by PCR and ELISA or western blot analysis. Southern blot analysis confirmed the independent nature of the transgenic lines. The majority of lines showed the integration of two to six transgene copies, while 21% of the analysed lines had a single copy insert. A short tissue culture period in comparison to recently published reports seems to be beneficial for the production of normal and fertile transgenic ryegrass plants. Consequently we report for the first time molecular evidence for sexual transgene transmission in fertile transgenic perennial ryegrass.     

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.     

Identification of plant-derived genetically modified organisms in food and feed using a hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in food and feed products. The biochip contained 22 immobilized oligonucleotide probes that were intended for (1) detection of plant DNA, (2) determination of plant species (soybean, maize, potato, and rice), and (3) identification of transgenic elements, including sequences of 35S CaMV, 35S FMV, rice actin gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, and nptII marker genes. The limit of detection was 0.5% for genetically modified (GM) soybean and maize in the analyzed samples. The tests on food and feed products using the developed approach and real-time PCR showed full agreement in determination of transgenic DNA in the samples. The proposed assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of GM component based on the identified transgene.     

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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated co-transformation of multiple genes in upland cotton

Two cotton genotypes, Simian 3 (SM 3) and WC, were co-transformed using a mixture of four Agrobacterium tumefaciens cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin, and fibroin. The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the two genotypes. However, there were no differences between the two genotypes for number of plantlets carrying multiple gene copies of different gene combinations as well as transformation frequency for different gene combinations. PCR analysis indicated that more than 80% of plantlets carried the nptII gene for kanamycin resistance. Overall, the co-transformation frequency of two or more genes was about 35%. Southern blot analysis confirmed integration of target genes into the cotton genome, and the number of copies of the transgene(s) varied from one to four. Multiple transgene expression was confirmed by RT-PCR analysis in some transgenic lines. Further analysis of T₁ plants demonstrated that multiple transgenes were inherited and expressed in progenies. Fei-Fei Li and Shen-Jie Wu are joint first authors.     

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.     

Genetic transformation of European chestnut

Stable incorporation of the nptII gene into Castanea sativa Mill. has been achieved by Agrobacterium-mediated transformation. The transformation assays were performed by infecting wounded hypocotyls with a strain of Agrobacterium tumefaciens, LBA 4404 harbouring the plasmid p35SGUSINT. Although two schemes of selection were tested, many escapes were obtained. The best strategy to avoid this problem is the introduction of higher concentrations of kanamycin in the culture medium, immediately after coculture. PCR analysis showed of the selectable nptII gene integration in the plant genome. β-Glucuronidase histochemical assay revealed the expression of the uidA gene in shoots, regenerated from transformed explants. Received: 3 December 1996 / Revision received: 4 February 1997 / Accepted: 1 March 1997