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.     

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.     

Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation

A simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos-resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion.     

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.     

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.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.     

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.     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Natural glufosinate resistance of soil microorganisms and GMO safety

Bacteria and fungi from pristine soil, never exposed to glufosinate herbicide, were isolated and analyzed for glufosinate tolerance. Seven of the 15 tested isolates were sensitive to 1 mM glufosinate (an active ingredient of many nonselective contact herbicides), 5 were resistant to 4 mM glufosinate and 3 even to 8 mM glufosinate in liquid medium. None of the isolated microorganisms carried the gene for glufosinate resistance bar (bialaphos resistance) in its genome and at least in some of glufosinate-resistant isolates the increased glutamine synthetase level was detected as a possible resistance mechanism. The transfer of the bar glufosinate resistance gene from transgenic maize Bt 176 into glufosinate-sensitive soil bacterium Bacillus pumilus S1 was not detected under the laboratory conditions by a classical plate count method and PCR. The ecological risk of potential bar gene transfer from genetically modified plants into soil microcosms under natural circumstances is discussed.     

Insect- and herbicide-resistant transgenic eucalypts*

Transgenic Eucalyptus camaldulensis containing both the insecticidal cry3A gene and the bar gene (conferring tolerance to the herbicide glufosinate ammonium) have been produced by Agrobacterium tumefaciens-mediated transformation of seedling explants. Transgenic plants from two lines tested were resistant to first instars of chrysomelid beetles that are important pests of commercial Australian eucalypt plantations. Both lines also exhibit tolerance to the broad-spectrum herbicide Liberty® at 6 l/ha (1.2 kg active ingredient per hectare), twice the field application rate. Transgenic insect- and herbicide-resistant eucalypts like these are likely to provide better insect and weed control options in plantations, particularly during the vulnerable establishment phase, provided that any adverse ecological impacts of releasing transgenic trees into the environment can be assessed and minimized.     

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.     

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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Cry1C,Cry2A</Emphasis> and <Emphasis Type="Italic">Cry9C</Emphasis> genes into <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> and plant regeneration

Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation.     

Generation of marker‐free transgenic hexaploid wheat via an Agrobacterium‐mediated co‐transformation strategy in commercial Chinese wheat varieties

Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker‐free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium‐mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker‐free transgenic wheat plants from various commercial Chinese varieties and their F₁ hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T‐DNA regions. The average co‐integration frequency of the gus and the bar genes located on the two independent T‐DNA regions was 49.0% in T₀ plants. We further found that the efficiency of generating marker‐free plants was related to the number of bar gene copies integrated in the genome. Marker‐free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T₁ positive plants, but the gus gene was never found to be silenced in T₁ plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in 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.     

Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment

An Agrobacterium-mediated transformation protocol for Japonica rice (cv. R321), using conventional genetic vectors and explants pretreated with antinecrotic compounds is presented. We evaluated the effect of two compounds with known antioxidant activity (ascorbic acid and cysteine) and silver nitrate on the viability of stem sections taken from in vitro rice plantlets, and on their interaction with Agrobacterium tumefaciens (At 2260) containing a shuttle vector bearing the gusand bar genes. After co-culture, calli formed on the callus-induction medium were supplemented with phosphinotricin and cefotaxime; putative transgenic plants were recovered on the regeneration medium after three months. All recovered plants were challenged with the herbicide BASTA under greenhouse conditions, and some resistant individuals were analyzed using PCR and a histochemical GUS test. Southern blot analysis of several R₁ transgenic plants indicated the presence of at least two intact bar gene copies per genome. Inheritance of the bar gene at the R₂ generation was confirmed. Antinecrotic pretreatment of the explants provides an adequate environment for the interaction of A. tumefacienswith the plant cells, thus allowing satisfactory transformation performance without the need of super-binary vectors and hyperinfective A. tumefaciens strains. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hygromycin resistance as an efficient selectable marker for wheat stable transformation

Summary A highly efficient method for stable wheat transformation using hygromycin resistance as a selectable marker is described. Young embryogenic calli growing from immature wheat embryos were transformed using a gunpowder-driven microparticle accelerator. Transgenic wheat plants were determined by PCR amplification of transgene fragments and confirmed by Southern hybridization, activity of the transgene expression and by analysis of the progeny. The hpt gene was as good as or a better selectable marker than the bar gene with an average efficiency (number of transgenic plants relative to the number of bombarded calli) of 5.5% compared with 2.6% for the bar gene.     

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     

Comparison of three selectable marker genes for transformation of tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) plants by particle bombardment

A variety of selection systems have been developed for transformation of forage crops. To compare the most frequently used systems, we tested three selectable marker genes for their selection efficiency under four selection procedures for the production of transgenic tall fescue. Embryogenic calluses initiated from mature embryos were bombarded with three constructs containing either the phosphinothricin acetyltransferase (bar) gene, the hygromycin phosphotransferase (hpt) gene or the neomycin phosphotransferase II (nptII) gene. Transformation efficiency was strongly influenced by the selectable marker gene, selection procedure and genotype. The highest transformation efficiency was observed using the bar gene in combination with bialaphos. Average transformation efficiencies with bialaphos, phosphinothricin (glufosinate), hygromycin and paromomycin selection across the two callus lines used in the experiments were 9.4%, 4.4%, 5.2% and 1.6%, respectively. Southern blot analysis revealed the independent nature of the tested transgenic plants and a complex transgene integration pattern with multiple insertions.