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.     

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.     

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.     

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.     

Herbicide-tolerant Transgenic Pineapple (Ananas comosus) Produced by Microprojectile Bombardment

Transformed plants of the commercially important Thai pineapple(Ananas comosus‘Phuket’) were produced followingmicroprojectile-mediated delivery of the plasmid AHC25, carryingthe ß-glucuronidase (gus) reporter gene and the bialaphosresistance (bar) gene for herbicide tolerance, into leaves ofmicropropagated shoots. Transformed plants were regeneratedfrom bombarded leaf bases on Murashige and Skoog-based mediumcontaining 0.5 mg l^-12,4-dichlorophenoxyacetic acid, 2.0 mgl^-16-benzylamino purine and 0.5 mg l^-1phosphinothricin. Integrationand expression of thebar gene in regenerated plants was confirmedby Southern analysis and RT-PCR, respectively. Regenerated plantswere assessed in vitro and under glasshouse conditions for theirtolerance to the commercial herbicide Basta^TM, containing glufosinateammonium as the active component. Plants sprayed with Basta^TMcontainingconcentrations of glufosinate ammonium up to 1400 mg l^-1remainedhealthy and retained their pigmentation. The generation of herbicide-tolerantpineapple will facilitate more efficient weed control in thiswidely cultivated tropical crop. Copyright 2001 Annals of BotanyCompany bar gene, Biolistics, herbicide tolerance, pineapple, phosphinothricin (PPT)     

Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.)

Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion.     

High frequency production of rapeseed transgenic plants via combination of microprojectile bombardment and secondary embryogenesis of microspore-derived embryos

Transgenic doubled haploid rapeseed (Brassica napus L. cvs. Global and PF₇₀₄) plants were obtained from microspore-derived embryo (MDE) hypocotyls using the microprojectile bombardment. The binary vector pCAMBIA3301 containing the gus and bar genes under control of CaMV 35S promoter was used for bombardment experiments. Transformed plantlets were selected and continuously maintained on selective medium containing 10 mg l⁻¹ phosphinothricin (PPT) and transgenic plants were obtained by selecting transformed secondary embryos. The presence, copy numbers and expression of the transgenes were confirmed by PCR, Southern blot, RT-PCR and histochemical GUS analyses. In progeny test, three out of four primary transformants for bar gene produced homozygous lines. The ploidy level of transformed plants was confirmed by flow cytometery analysis before colchicine treatment. All of the regenerated plants were haploid except one that was spontaneous diploid. High frequency of transgenic doubled haploid rapeseeds (about 15.55% for bar gene and 11.11% for gus gene) were considerably produced after colchicines treatment of the haploid plantlets. This result show a remarkable increase in production of transgenic doubled haploid rapeseed plants compared to previous studies.     

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.     

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 Method of Agrobacterium-mediated Transformation for Triticale (x Triticosecale Wittmack)

Transgenic plants of triticale cv. Wanad were obtained after transformation using three combinations of strain/vectors. Two of them were hypervirulent Agrobacterium tumefaciens strains (AGL1 and EHA101) with vectors containing bar under maize ubiquitin 1 promoter (pDM805), and both hpt under p35S and nptII under pnos (pGAH). The third one was a regular LBA4404 strain containing super-binary plasmid pTOK233 with selection genes the same as in pGAH. The efficiency of transformation was from 0 to 16% and it was dependent on the selection factor, auxin pretreatment, and the strain/vector combination. The highest number of transgenic plants was obtained after transformation with LBA4404(pTOK233) and kanamycin selection. Pretreatment of explants with picloram led to the highest number of plants obtained after transformation with both Agrobacterium/vector systems LBA4404(pTOK233) and EHA101(pGAH) and selected with kanamycin. Transgenic character of selected plants was examined by PCR using specific primers for bar, gus, nptII, and hpt and confirmed by Southern blot hybridization analysis. There was no GUS expression in T₀ transgenic plants transformed with gus under p35S. However the GUS expression was detectable in the progeny of some lines. Only 30% of 46 transgenic lines showed Mendelian segregation of GUS expressing to GUS not expressing plants. In the remaining 70% the segregation was non-Mendelian and the rate was much lower than 3:1. Factors that might effect expression of transgenes in allohexaploid monocot species are 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.     

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.     

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     

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.     

Genetic transformation of <Emphasis Type="Italic">Coffea canephora</Emphasis> by particle bombardment

Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.This research was supported by the Consorcio Brasileiro de Pesquisa e Desenvolvimento do Cafe (CBP&D-Cafe).     

Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts

Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 g/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 g/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts.Abbreviations gus -glucuronidase - PAT phosphinothricin N-acetyltransferase - PPT phosphinothricin - MS Murashige and Skoog medium     

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.     

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.     

Development of a modified transformation platform for apomixis candidate genes research in Paspalum notatum (bahiagrass)

The aim of this work was to improve existing transformation protocols and to transform specific genotypes of Paspalum notatum (bahiagrass) for functional analyses of candidate genes involved in reproduction. Three different explants were assayed for in vitro plant regeneration: mature seeds, mature embryos, and shoot meristems. Plant regeneration was achieved with all explant types, but mature seeds produced the optimal rate (78.0%) and were easiest to manipulate. A method based on serial re-induction of calli from meristems of the regenerated lines was also developed, which could be useful in plant breeding strategies pursuing somaclonal variation. Transient transformation experiments were performed on calli obtained from mature seeds using a compressed helium gene gun. Transient transformation constructs included anthocyanin-synthesis genes cloned under the CAMV 35S promoter and an enhanced green fluorescent protein gene (egfp) driven by the rice actin1 (act1) promoter. Selection curves for ammonium glufosinate were developed in order to determine the optimal selective pressure for stable transformation (1.0 mg/L). Stable co-transformation experiments were carried out with two different constructs containing: (1) the reporter egfp gene cloned under the rice act1 promoter and (2) the selector bar gene driven by the ubiquitin promoter. A total of 27 (64.2%) transgenic plants out of 42 resistant plants analyzed were obtained. The presence of the transgenes in regenerated plants was confirmed by polymerase chain reaction and DNA gel blot analysis. Gene expression was demonstrated by eGFP fluorescence detection and in vivo assays for ammonium glufosinate tolerance. This platform is being used to generate transgenic plants of P. notatum to analyze the function of apomixis-associated candidate genes.     

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method – plant development and surfactant are important in optimizing transformation efficiency

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the -glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences. The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production. Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds. The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant. Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically. Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits. These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop.