Field study results on the probability and risk of a horizontal gene transfer from transgenic herbicide-resistant oilseed rape pollen to gut bacteria of bees

Bees are specifically subjected to intimate contacts with transgenic plants due to their feeding activities on pollen. In this study, the probability and ecological risk of a gene transfer from pollen to gut bacteria of bees was investigated with larvae of Apis mellifera (honeybee), Bombus terrestris (bumblebee), and Osmia bicornis (red mason bee), all collected at a flowering transgenic oilseed rape field. The plants were genetically engineered with the pat-gene, conferring resistance against glufosinate (syn. phosphinothricin), a glutamine-synthetase inhibitor in plants and microorganisms. Ninety-six bacterial strains were isolated and characterized by 16S rRNA gene sequencing, revealing that Firmicutes represented 58% of the isolates, Actinobacteria 31%, and Proteobacteria 11%, respectively. Of all isolates, 40% were resistant to 1 mM glufosinate, and 11% even to 10 mM. Resistant phenotypes were found in all phylogenetic groups. None of the resistant phenotypes carried the recombinant pat-gene in its genome. The threshold of detecting gene transfer in this field study was relatively insensitive due to the high background of natural glufosinate resistance. However, the broad occurrence of glufosinate-resistant bacteria from different phylogenetic groups suggests that rare events of horizontal gene transfer will not add significantly to natural bacterial glufosinate resistance.     

Regeneration of fertile transgenic tall fescue plants with a stable highly expressed foreign gene

One hundred and seventeen green tall fescue plants and 37 albino plants were regenerated from a glufosinate ammonium resistant callus clone co-transformed with the bar gene and the gusgene, both driven by the rice actin 1 promoter. The gus gene was not detectable in regenerated plants but the presence of the bar gene in these plants was detected by the polymerase chain reaction and integration of the bar gene into the genome by Southern blot hybridization. A high and stable expression of the bar gene was evident from the assay for phosphinothricin-N-acetyltransferase activity and from spraying plants with glufosinate ammonium herbicide. There was no detectable variation with respect to the level of bar gene expression among these plants. However, no inheritance of the bar gene was found in two populations of outcrossed progenies. This revised version was published online in June 2006 with corrections to the Cover Date.     

A simple model for selection and rapid advancement of transgenic progeny in sorghum

To select agronomically useful transgenic plants, a large number of transgenic events are initially produced, gene transfer confirmed, and advanced to obtain homozygous lines for testing in field trials. Direct in planta assays for identifying the transgene carriers in the segregating progeny are based on the activity of selectable marker gene and are easy, simple and inexpensive. For this purpose, expression of bar gene as measured by tolerance to damage by glufosinate ammonium, the active ingredient in the herbicide BASTA, was investigated. Dose damage curves were generated by leaf paint tests with BASTA on four genotypes of sorghum. Transgenic plants were characterized in terms of sensitivity to the concentration of glufosinate ammonium. In transgenics, symptoms of BASTA swab tests at different growth stages and PCR analysis for cry1B were carried out and correlated. Germination tests could not be employed for large scale evaluation of transgenic progeny because of mortality of tolerant seedlings after transplantation to soil. Based on the above findings, a simple, inexpensive, time-saving, two-step scheme for effective evaluation of transgenics and their progeny containing bar gene as selection marker using BASTA swab tests is described.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformed transgenic triploid bermudagrass (<Emphasis Type="Italic">Cynodon dactylon</Emphasis> × <Emphasis Type="Italic">C. transvaalensis</Emphasis>) plants are highly resistant to the glufosinate herbicide Liberty

Glufosinate resistance gene isolated from Streptomyces hygromicinroscopicus (bar) that confers the resistance of herbicide Liberty, a broad-spectrum grass and broadleaf contact herbicide widely used for weed control, was introduced into triploid bermudagrass by Agrobacterium-mediated transformation. Embryogenic calluses derived from stolonous nodal segment were co-cultured with the disarmed strain EHA105 harboring the binary vector pBG1300H containing the bar gene under the control of adh-1 promoter. A total of 18 independent transgenic lines were obtained. The integration of bar gene into plant genome was confirmed by the GUS histochemical staining assay, PCR amplification, and Southern blotting. Herbicide bioassay indicated that the bar-expressing transgenic plants exhibited greater herbicide resistance than the wild type and the non-transformed tissue culture-derived plants.     

Transgenic <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis> expressing the <Emphasis Type="Italic">bar</Emphasis> gene are resistant to the herbicide Basta<Superscript>®</Superscript>

Lisianthus (Eustoma grandiflorum) is a cut or ornamental flower that is popular all over the world. This ornamental crop, however, lacks an effective weed control method due to its susceptibility to herbicide. In this study, transgenic plants of a lisianthus cultivar were produced using Agrobacterium-mediated delivery of the plasmid pCAMBIA3300, which carried the bialaphos resistance (bar) gene under driven by the CaMV 35S promoter. The transgenic calli were derived from wounded edges of the leaves grown on a shoot regeneration medium containing 100 mg l^?1 cefotaxime and 2 mg l^?1 glufosinate ammonium for 4 weeks. The callus that was detached from the wounded edge of the leaf was transferred to the shoot regeneration medium with 100 mg l^?1 cefotaxime and 5 mg l^?1 glufosinate ammonium for 4 weeks for shoot regeneration. The bar gene integration and expression in the transgenic plants were confirmed by Southern and Northern blot analyses, respectively. Subsequently, the transgenic lines were assessed in vitro and under greenhouse conditions for their resistance to the commercial herbicide Basta^®, which contains glufosinate ammonium as the active component. Six transgenic lines showed high percentages (67–80%) of survival in vitro under the selection condition with glufosinate ammonium (up to 216 mg l^?1). Under greenhouse conditions, the plants from these six lines remained healthy and exhibited a normal phenotype after spraying with glufosinate ammonium (up to 1,350 mg l^?1). This is the first paper to provide a detailed survey of transgenic lisianthus expressing the bar gene and exhibiting herbicide-resistance under greenhouse conditions.     

Transformation of oat and inheritance of bar gene expression

Fertile transgenic plants of oat (Avena sativa L. var. Melys) were produced following microprojectile bombardment of primary embryogenic calli from immature embryos with two plasmids containing the bar gene or the β-glucuronidase (uidA) gene, after selection with glufosinate ammonium. Eleven plants were regenerated from phosphinothricin resistant callus, with three of the eleven plants containing either intact or rearranged copies. No plants co-transformed with the non-selected uidA gene were detected. Stable transmission and expression of the bar gene in the T₁ inbred progenies occurred in a Mendelian manner in one line, which contained an intact bar gene, and in all six T₂ lines tested from this transformant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Agrobacterium-mediated transformation in Citrullus lanatus

Agrobacterium tumefaciens-mediated transformation was used to produce transgenic watermelon. Cotyledonary explants of Citrullus lanatus Thumb (cv. Daesan) were co-cultivated with Agrobacterium strains (LBA4404, GV3101, EHA101) containing pPTN289 carrying with bar gene and pPTN290 carrying with nptII gene, respectively. There was a significant difference in the transformation frequency between bacteria strains and selective markers. The EHA101/pPTN289 showed higher transformation frequency (1.16 %) than GV3101/pPTN289 (0.33 %) and LBA4404/pPTN289 or /pPTN290 (0 %). The shoots obtained (633 and 57 lines) showed some resistance to glufosinate and paromomycin, respectively. Of them, the β-glucuronidase positive response and PCR products amplified by bar and nptII specific primers showed at least 21 plants resistant to glufosinate and at least 6 plants to paromomycin. Southern blot analysis revealed that the bar gene integrated into genome of transgenic watermelon. Acclimated transgenic watermelons were successfully transplanted in the greenhouse and showed no phenotypic variation.     

Development of an expression vector for Metarhizium anisopliae based on the tef-1α homologous promoter

The high-conserved translation elongation factor 1 α (tef-1α) gene from the enthomopathogenic fungus Metarhizium anisopliae was characterized to select the promoter region. A 640-bp DNA fragment upstream to the start codon was employed to drive the expression of the reporter protein sGFP or a dominant selectable marker, the gene bar (resistance to ammonium glufosinate). Transformants carrying this homologous promoter system showed no difference in virulence bioassays against the cattle tick Boophilus microplus comparing to the M. anisopliae wild-type strain. Moreover, GFP fluorescence was detected during tick infection bioassay.     

Increased stable inheritance of herbicide resistance in transgenic lettuce carrying a petE promoter-bar gene compared with a CaMV 35S-bar gene

Inheritance of resistance to herbicide (300 mg/l glufosinate ammonium) up to the third (T3) seed generation was compared in two populations of transgenic lettuce (Lactuca sativa L. cv ’Evola’) harbouring a T-DNA containing the bar gene, linked to either the Cauliflower Mosaic Virus (CaMV) 35S promoter, or a –784-bp plastocyanin promoter from pea (petE). Only 2.5% (4/163) of CaMV 35S-bar plants, selected by their kanamycin resistance(T0 generation), transmitted herbicide resistance at high frequency to their T3 seed generation compared with 97% (29/30) for kanamycin resistant petE-bar plants. In the case of 35S-bar transformants, only 16% (341/2,150) of the first seed generation (T1) plants, 22% (426/1,935) T2 plants and 11% (1,235/10,949) T3 plants were herbicide-resistant. In contrast, 63% (190/300) T1 plants, 83% (2,370/2,845) T2 plants and 99% (122/123) T3 petE-bar transformed plants were resistant to glufosinate ammonium. The T-DNAs carrying the petE-bar and CaMV 35S-bar genes also contained a CaMV 35S-neomycin phosphotransferase (nptII) gene. ELISA showed that NPTII protein was absent in 29% (45/156) of the herbicide-resistant T2 plants from 8/19 herbicide-resistant petE-bar lines. This indicated specific inactivation of the CaMV 35S promoter on the same T-DNA locus as an active petE promoter. The choice of promoter and T-DNA construct are crucial for long-term expression of transgenes in lettuce. Received: 13 November 1998 / Accepted: 20 February 1999     

Bar-expressing peppermint (Mentha × Piperita L. var. Black Mitcham) plants are highly resistant to the glufosinate herbicide Liberty

Weed control is a substantial economic input for production of mint oils, the most commercially important of which are obtained from peppermint. The objective of this research is to obtain peppermint plants resistant to the broad-spectrum herbicide glufosinate, which can be used for development of economically efficacious weed control strategies and, perhaps, serve as a paradigm in perennial crops. The bar gene, which encodes phosphinothricin acetyltransferase (PAT) which inactivates glufosinate-ammonium or phosphinothricin (PPT), was constructed into Agrobacterium tumefaciens binary vectors under the nopaline synthase (NOS) or a chimeric promoter containing a trimer of the OCS-upstream-activating sequence (UAS) to a MAS promoter/activator region[(OCS) ₃ MAS]. A total of 142 independent transgenic peppermint (cv. Black Mitcham) plants were obtained (107 and 35 were obtained with pGPTV (and pCAS1) and pATC940 vectors, respectively) and evaluated for herbicide resistance in the greenhouse after foliar application of glufosinate herbicide Liberty as the commercial product. All transgenic plants exhibited substantially less herbicide symptom development than non-transgenic Black Mitcham or untransformed tissue cultured-derived plants, albeit variation for herbicide resistance occurred amongst the transformed lines. Plants from 35 of the 142 lines were selected at random and all were PCR-positive for the presence of bar. Five lines, that were least affected, exhibited no injury symptoms to Liberty concentrations that are 4 times the standard level for control of weeds in peppermint fields. The most resistant transgenic plants had the greatest steady-state PAT mRNA levels and PAT activities. No experimental difference in herbicide resistance was evident between plant populations obtained with pGPTV (pCAS1)-bar or pATC940-bar vector. However, 4 of 35 lines transformed with (ocs) ₃ MAS-bar exhibited maximal resistance while only 1 of 107 NOS-bar lines has comparable resistance. These herbicide resistant peppermint plants will facilitate development of post-emergent herbicide control strategies that use newer generation herbicides, like glufosinate, which have reduced environmental and product residual because of metabolism by microbes and the transgenic plants.     

Transformation of bahiagrass (<Emphasis Type="Italic">Paspalum notatum</Emphasis> Flugge)

Bahiagrass (Paspalum notatum Flugge), a forage species widely used in the southeastern United States, and from Central Mexico to Argentina, was targeted for improvement through genetic engineering. Embryogenic callus, initiated from germinating seedlings, was bombarded with a vector containing the bar selectable marker/reporter gene that confers resistance to phosphinothricin (glufosinate) herbicide (trade names Liberty, Ignite and Finale). Thirty-two transgenic plants were recovered. These plants were identified by the polymerase chain reaction (PCR) and verified by Southern analysis. Transgenic plants with bar, as well as non-transgenic plants without bar, regenerated from bombarded callus and selected with glufosinate, developed strong and stable resistance to glufosinate during selection. This unusual resistance in non-transgenic plants has persisted for over a year and is passed on to new tillers. The development of resistance in non-transgenic cells reduced the herbicide selection efficiency and made it necessary to identify transgenic plants by PCR where the 32 transgenic plants were recovered from 674 glufosinate-resistant plants, giving a very low selection efficiency.     

Controlling Transgene Escape in GM Creeping Bentgrass

Trait improvement of turfgrass through genetic engineering is important to the turfgrass industry and the environment. However, the possible transgene escape to wild and non-transformed species raises ecological and commercial concerns. Male sterility provides an effective way for interrupting gene flow. We have designed and synthesized two chimeric gene constructs consisting of a rice tapetum-specific promoter (TAP) fused to either a ribonuclease gene barnase, or the antisense of a rice tapetum-specific gene rts. Both constructs were linked to the bar gene for selection by resistance to the herbicide glufosinate. Agrobacterium-mediated transformation of creeping bentgrass (cv Penn A-4) with both constructs resulted in herbicide-resistant transgenic plants that were also 100% pollen sterile. Mendelian segregation of herbicide resistance and male sterility was observed in T1 progeny derived from crosses with wild-type plants. Controlled self- and cross-pollination studies showed no gene transfer to non-transgenic plants from male-sterile transgenic plants. Thus, male sterility can serve as an important tool to control transgene escape in bentgrass, facilitating the application of genetic engineering in producing environmentally responsible turfgrass with enhanced traits. It also provides a tool to control gene flow in other perennial species using transgenic technology.     

Stable transformation of mature zygotic embryos and regeneration of transgenic plants of chir pine (Pinus roxbughii Sarg.)

A particle inflow gun was used to transfer the plasmid pAHC25 containing the bar gene conferring resistance to glufosinate and the gusA reporter gene, each driven by the maize ubiquitin promoter, to mature embryos of Pinus roxburghii (chir pine). High levels of transient expression were obtained when embryos were cultured for 6 days on 10 μM benzyl adenine-containing medium and then exposed to high osmoticum (0.5 M sucrose) before and after bombardment. Selection on medium containing Basta enabled recovery of stably transformed shoots, both from the epicotyl and from adventitious buds. The primary transformed shoots from the epicotyl were multiplied via axillary shoots. Transformation was confirmed by histochemical staining for β-glucuronidase (GUS) activity, by polymerase chain reaction (PCR) amplification of fragments of gusA and nos terminator, and by the resistance of needles to Basta.     

Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene

Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat‐growing areas in China. Because it is vectored by the fungus‐like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co‐transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12‐1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12‐1 showed broad‐spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild‐type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus‐derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad‐spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

Distribution of Tetracycline and Streptomycin Resistance Genes and Class 1 Integrons in Enterobacteriaceae Isolated from Dairy and Nondairy Farm Soils

The prevalence of selected tetracycline and streptomycin resistance genes and class 1 integrons in Enterobacteriaceae (n = 80) isolated from dairy farm soil and nondairy soils was evaluated. Among 56 bacteria isolated from dairy farm soils, 36 (64.3%) were resistant to tetracycline, and 17 (30.4%) were resistant to streptomycin. Lower frequencies of tetracycline (9 of 24 or 37.5%) and streptomycin (1 of 24 or 4.2%) resistance were observed in bacteria isolated from nondairy soils. Bacteria (n = 56) isolated from dairy farm soil had a higher frequency of tetracycline resistance genes including tetM (28.6%), tetA (21.4%), tetW (8.9%), tetB (5.4%), tetS (5.4%), tetG (3.6%), and tetO (1.8%). Among 24 bacteria isolated from nondairy soils, four isolates carried tetM, tetO, tetS, and tetW in different combinations; whereas tetA, tetB, and tetG were not detected. Similarly, a higher prevalence of streptomycin resistance genes including strA (12.5%), strB (12.5%), ant(3″) (12.5), aph(6)-1c (12.5%), aph(3″) (10.8%), and addA (5.4%) was detected in bacteria isolated from dairy farm soils than in nondairy soils. None of the nondairy soil isolates carried aadA gene. Other tetracycline (tetC, tetD, tetE, tetK, tetL, tetQ, and tetT) and streptomycin (aph(6)-1c and ant(6)) resistance genes were not detected in both dairy and nondairy soil isolates. A higher distribution of multiple resistance genes was observed in bacteria isolated from dairy farm soil than in nondairy soil. Among 36 tetracycline- and 17 streptomycin-resistant isolates from dairy farm soils, 11 (30.6%) and 9 (52.9%) isolates carried multiple resistance genes encoding resistance to tetracycline and streptomycin, respectively, which was higher than in bacteria isolated from nondairy soils. One strain each of Citrobacter freundii and C. youngae isolated from dairy farm soils carried class 1 integrons with different inserted gene cassettes. Results of this small study suggest that the presence of multiple resistance genes and class 1 integrons in Enterobacteriaceae in dairy farm soil may act as a reservoir of antimicrobial resistance genes and could play a role in the dissemination of these antimicrobial resistance genes to other commensal and indigenous microbial communities in soil. However, additional longer-term studies conducted in more locations are needed to validate this hypothesis.     

The use of the two T-DNA binary system to derive marker-free transgenic soybeans

Summary A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101. Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T₀) representing 10 independent events were characterized. Seven of the 10 independent T₀ events co-expressed GUS. Progeny analysis was conducted by sowing the T₁ seeds and monitoring the expression of the GUS gene after 21 d. Individual T₁ plants were subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mgl⁻¹ solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Phenotypic and genotypic characterization of tetracycline and minocycline resistance in Clostridium perfringens

The aim of this study was to determine the incidence of tetracycline resistance and the prevalence of tetracycline-resistance genes in strains of Clostridium perfringens isolated from different sources between 1994 and 2005. Susceptibility to tetracycline and minocycline in strains from humans (35 isolates), chickens (15 isolates), food (21 isolates), soil (16 isolates) and veterinary sources (6 isolates) was determined, and tetracycline-resistance genes were detected. Resistance was most common in strains isolated from chickens, followed by those from soils, clinical samples and foods. The most highly resistant strains were found among clinical and food isolates. tetA(P) was the most common resistance gene, and along with tetB(P) was found in all resistant strains and some sensitive strains. One tetracycline-resistant food isolate had an intact tet(M) gene. However, PCR fragments of 0.4 or 0.8 kb with high degrees of identity to parts of the tet(M) sequences of other bacteria were found, mainly in clinical isolates, and often in isolates with tetB(P). No correlation between level of sensitivity to tetracycline or minocycline and the presence of tetA(P), tetB(P) or part of tet(M) was found. The presence of part of tet(M) in some strains of C. perfringens containing tetB(P) may have occurred by recent gene transfer.