Elite Indica Transgenic Rice Plants Expressing Modified Cry1Ac Endotoxin of Bacillus Thuringiensis Show Enhanced Resistance to Yellow Stem Borer (Scirpophaga Incertulas)

Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic of Agrobacterium-mediated approaches. A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used. Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system. Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher ( 9%) for Agrobacterium. An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt-transformants and reduced the number of escapes. Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium. In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert. Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India. Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays. However, there was an occasional white head even among such plants when assayed at the reproductive stage. Results are discussed in the light of resistance management strategies for deployment of Bt-rice.     

Genotype x culture media interaction effects on regeneration response of three indica rice cultivars

Interactive effects of genotypes with callus induction and regeneration media combinations on green plantlet regeneration response were studied for three indica rice (Oryza sativa L.) cultivars, IR-72, IR-54 and Karnal Local. Isolated mature-embryos were used to derive scutellar callus and fifteen media combinations involving MS, N6, R2, SK1 and some modifications were tested. Regeneration percentage as well as the shoot-bud induction frequency were influenced by genotype, callus induction medium, regeneration medium, interaction between genotype and the two media (callus induction and regeneration) as well the interaction between the callus induction medium and regeneration medium. Basal media combination of SK1m (callusing) and MS (regeneration) was found to be the best for cv. Karnal Local in which regeneration frequency of 88% and shoot-bud induction of 233% was observed. In IR-72, the highest regeneration frequency of 47.5% and shoot-bud induction frequency of 77% was obtained on MS-MS combination. In IR-54, highest regeneration frequency (25%) was recorded on MMS(N)-MMS(N) combination, whereas, highest frequency of shoot-bud induction (50%) was observed on MMS(S)-MS combination. Although genotype and the composition of the callus induction basal medium were the major determinants of regeneration response, an overall analysis of variation also revealed a significant interaction between the media used for de-differentiation (callusing) and re-differentiation (plantlet regeneration). This revised version was published online in June 2006 with corrections to the Cover Date.     

An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars

Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of l-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD_600 nm?=?0.5–0.8) promoted the highest frequency of transformation (83.04 %) in medium containing l-cysteine (400 mg l^?1). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of l-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.     

Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event

Elite white maize lines W506 and M37W were transformed with a selectable marker gene (bar) and a reporter gene (uidA) or the polygalacturonase-inhibiting protein (pgip) gene after bombardment of cultured immature zygotic embryos using the particle inflow gun. Successful transformation with this device did not require a narrow range of parameters, since transformants were obtained from a wide range of treatments, namely pre-culture of the embryos for 4-6 days, bombardment at helium pressures of 700-900 kPa, selection-free culture for 2-4 days after bombardment and selection on medium containing bialaphos at 0.5-2 mg l^-1. However, bombardments with helium pressures below 700 kPa yielded no transformants. The culture of immature zygotic embryos of selected elite white maize lines on medium containing 2 mg l^-1 2,4-dichlorophenoxyacetic acid and 20 mM L-proline proved to be most successful for the production of regenerable embryogenic calli and for the selection of putative transgenic calli on bialaphos-containing medium after transformation. Transgenic plants were obtained from four independent transformation events as confirmed by Southern blot analysis. Transmission of the bar and uidA genes to the T₄ progeny of one of these transformation events was demonstrated by Southern blot analysis and by transgene expression. In this event, the transgenes bar and uidA were inserted in tandem.     

Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO₃ was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed.     

High-frequency embryogenesis and plantlet regeneration from isolated microspores of indica rice

We report high-frequency embryogenesis and plantlet development from microspores isolated from anthers of two indica (IR-43, IR-54) and a japonica (T-309) rice cultivars, without prior nutrient preculture of anthers. Pretreatment stress of anthers with mannitol or a sugar-starvation medium, and use of maltose as the carbohydrate source in the microspore culture medium were found to be critical. Co-culture of microspores with rice ovaries was found beneficial but not essential. More than 60% of the microspores of the japonica variety Taipai-309 and 25–45% of the indica cultivars IR-54 and IR-43 showed induction of non-gametophytic development. Consequently, in the best treatments for IR-43 and T-309, more than 500 microspore-derived embryos could be obtained from a single dish (35 mm) containing about 80,000 microspores. Among the indica cultivars, the maximum response was obtained in the basal medium M-019. Plantlet regeneration occurred in about 9% (T-309), 7% (IR-43) and 2% (IR-54) of the transferred embryo-like structures. Received: 6 November 1996 / Revision received: 18 June 1997 / Accepted: 20 August 1997     

The development of herbicide-resistant maize: stable Agrobacterium-mediated transformation of maize using explants of type II embryogenic calli

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for 4 × 14 days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter–bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with 4 mg l^?1 phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the R₁ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Avocado globular somatic embryos were transformed with three binary vectors, pK7FNF2, pK7RNR2 and pK7S*NF2, harboring the marker genes gfp, DsRed and a gfp-gus fusion gene, respectively. GFP and DsRed fluorescence was detected in embryogenic lines growing in selection medium 2 months after Agrobacterium inoculation. The fluorescence signal was maintained thereafter in transgenic calli, as well as in mature somatic embryos. Red fluorescence in pK7RNR2 transgenic lines was higher and more easily observable than GFP fluorescence. Furthermore, calli transformed with pK7S*NF2, harboring gfp-gus, showed higher level of fluorescence than those transformed with pK7FNF2, containing two gfp. To improve plant recovery, maturated transgenic embryos that failed to germinate or showed an underdeveloped shoot were cultured for 4 weeks in a medium with 1 mg l^?1 TDZ and 1 mg l^?1 BA after partial removal of cotyledons. A 50% of embryos developed one or several shoots on the cut surface. These embryos were cultured for 4 additional weeks in a medium with 1 mg l^?1 BA for shoot elongation and then, shoots were grafted in vitro onto seedling rootstocks. Culture of micrografts in solid MS medium supplemented with 1 mg l^?1 BA allowed a 60–80% success rate. Young leaves from transgenic plants showed GFP or DsRed fluorescence located in the nucleus. The results obtained indicate that fluorescent marker genes, especially DsRed, could be useful for early selection of transgenic material and optimization of the transformation parameters in avocado. Furthermore, the protocol established allowed the successful recovery of transgenic plants, one of the main limiting steps in avocado transformation.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

High-efficiency Agrobacterium-mediated transformation in Quercus robur: selection by use of a temporary immersion system and assessment by quantitative PCR

An efficient protocol for genetic transformation of somatic embryos of Quercus robur by selection in a temporary immersion system is reported. The transformation frequency was 5 times higher than achieved by conventional culture on semi-solid medium, ranging between 6 and 26 % for the four genotypes evaluated. Clumps of globular or torpedo somatic embryos were precultured for 7–10 days, inoculated with Agrobacterium tumefaciens strain EHA105:p35SGUSINT and cocultivated for 4 days before being cultured for 4 weeks on semi-solid selection medium supplemented with 25 mg L^?1 kanamycin. Explants were transferred to RITA^® bioreactors and subjected to a two-step selection protocol involving immersion in liquid medium supplemented with 25 mg L^?1 kanamycin, for 18 weeks, and then with 75 mg L^?1 kanamycin. Putatively transformed explants appeared after serial transfer to selection medium over 12–16 weeks. The presence of neomycin phosphotransferase II and β-glucuronidase genes in the plant genome was confirmed by histochemical and molecular analysis, and the copy number was determined by Southern blotting and real-time quantitative polymerase chain reaction. Transformed somatic embryos were germinated and transferred to soil for acclimatization, approximately 8 months after inoculation of the original tissue with bacteria. As the limiting factor for recovery of plants from oak embryogenic lines is the low embryo conversion rate, axillary shoot lines were established from transformed germinated embryos. Transformed embryos and shoots were cultured in medium with or without kanamycin and the responses to several morphogenetic processes (recovery after cryopreservation, germination, shoot proliferation, and rooting) were evaluated.     

Developing phosphinothricin-resistant transgenic sunflower (Helianthus?annuus L.) plants

Most investigations on genetic transformations of sunflower have used the neomycin transferase (nptII) gene as the selectable marker. We previously reported a PPT-based selection system for sunflower transformation that uses the bialaphos resistance (bar) gene as the selectable marker and 20 mg/l of phosphinothricin (PPT) as the selective agent. Sunflower (Helianthus annuus L.) variety Skorospeliy 87 was genetically transformed via Agrobacterium tumefaciens strain EHA 105 harbouring the binary plasmid vector pBAR. Two-day-old explants from mature embryos competent for direct shooting were used. Southern blot and ELISA experiments confirmed the stability of expression in two generations of transgenic plants. Transformed plants transferred to soil in the greenhouse exhibited resistance to the herbicide Basta^? at 3 l/ha.     

Recovery of transgenic peanut (Arachis hypogaea L.) plants from elite cultivars utilizing ACCELL® technology

Transgenic plants of Florunner and Florigiant, two of the most widely cultivated peanut cultivars in the USA, have been developed using the ACCELL® gene delivery method. Shoot meristems of mature embryonic axes were bombarded with gold beads coated with DNA encoding β-glucuronidase (gus), phosphinothricin acetyl transferase (bar), and tomato spotted wilt virus-nucleocapsid protein (tswv-np) genes. Transgenic shoots were identified by screening for GUS activity, and independent transformants were recovered from both cultivars. Molecular analysis of two of these transformants in R₀ and R₁ generations demonstrated the stable integration of the foreign genes into the plant genome. One transgenic plant had one to two copies of the genes integrated into the genome of its progeny, whereas the other had multiple copies. Gus and bar genes exhibited predictable segregation ratios in the R₁ and R₂ generations and were genetically linked. Integration of the bar gene conferred resistance to BASTA^TM, a wide-spectrum herbicide, applied at 500 p.p.m. of active ingredient. Resistance of the transgenic plants to tomato spotted wilt virus is currently being tested under greenhouse conditions. The ACCELL® particle bombardment system is expected to be effective for transformation of a wide variety of commercial peanut cultivars.     

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.     

Callus Formation and Plant Regeneration from Cultured Embryos of Diploid and Tetraploid Winter Ryes (Secale cereale L)

The ability of immature, mature and endosperm-supported mature embryos of diploid and tetraploid winter ryes (Secale cereale L) was tested to compare the callus induction and plant regeneration. Immature embryos were obtained from field grown rye. Immature embryos were aseptically excised and placed, with the scutellum upwards, on the callus culture medium consisted of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised the imbibed seeds and placed, scutellum up, on MS medium supplement with 2 mg l^?1 2,4-D. Endosperm-supported mature embryos were moved slightly (not set free) in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l^?1 2,4-D for callus induction. The developed calli and regenerated plant were maintained on hormone free MS medium. Comparison of the responses of the three explants used indicated that endosperm-supported mature embryo was the most useful explant for plant regeneration in both diploid and tetraplold ryes. This is the first report of winter ryes plants having been regenerated from endosperm-supported mature embryos.     

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l^?1 citric acid, 100 µM acetosyringone, and 100 mg l^?1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l^?1 hygromycin and the embryos were germinated in basal medium containing 20 mg l^?1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.     

Efficient in vitro plant regeneration and generation of transgenic plants in barley (Hordeum vulgare L.) using particle bombardment

Plant regeneration from immature embryos of 15 Indian barley genotypes (Hordeum vulgare L.) was examined. Highest percent callus induction and number of regenerated plantlets were obtained in cultivars BL 2 (94.4 %; 12.1), RD 2668 (92.6 %; 9.1) and RD 2552 (90.8 %; 7.8). The highly responding cultivar BL 2 was selected for further development of transformation protocol. The plasmid DNA isolated from pCAMBIA1381 harbouring hptII gene as selectable marker and gusA gene as reporter was used. Particle bombardment was used for transformation of immature embryos and regeneration of transgenic plants in Indian barley genotype for the first time. Transformation experiments were carried out using different parameters and optimum conditions for DNA delivery was standardized. The transient expression of gusA gene was investigated as a preliminary test of optimum DNA delivery and for selecting the most appropriate bombardment parameters. The optimum conditions were: gold microparticles (diameter 1.0 μm) shot with 1,100 psi rupture disc pressure. The 3 cm distance between rupture disk and macrocarrier and 9 cm target tissue distance yielded high transient GUS expression. The immature embryos were bombarded twice to increase area for efficient gene delivery. Osmotic medium optimization with 0.4 M sorbitol and preculture of immature embryos for 5 days prior to bombardment resulted into efficient gene transfer in barley. Selection of transformed tissue was performed after 7 days resting step on selection medium containing 50 mg?l^?1 hygromycin. After two more selection steps, green shoots were rooted on MSB₅ medium with 50 mg?l^?1 hygromycin. PCR analysis using primers specific for hptII and gusA genes and Southern blot analysis with hptII probes confirmed the stable integration of transgene in barley genome. Molecular analysis of T₁ generation plantlets revealed the amplification of selectable marker hptII gene in the progeny.     

Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojecticle bombarded leaf cells

Young leaf tissue of orchardgrass (Dactylis glomerata L.) was placed on Schenk and Hildebrandt medium containing 30 μM dicamba. Microprojectiles coated with DNA containing the selectable bar gene (Basta? tolerance) and the reporter gene uidA coding for β-glucuronidase (GUS), both driven by the maize ubiquitin promoter (Ubi1), were propelled into the tissue with a particle inflow gun. Transient GUS expression was observed as blue spots of various sizes on leaf segments. Somatic embryos staining entirely blue were also produced, and embryos germinated on medium containing 3.0 mg 1^–1 bialaphos. Leaves of 67 putative transformed plants were painted with 0.1% Basta. Ten showed no reaction, and 6 showed only a localized response. Cultured leaf segments from tolerant plants also produced somatic embryos that expressed GUS. The genetic transformation was confirmed by Southern blot hybridization and PCR analyses of T₀ plants and by PCR analyses of somatic embryos produced from T₀ plants. Received: 9 April 1997 / Revision received: 11 May 1997 / Accepted: 3 June 1997     

Fertile transgenic Lotus corniculatus resistant to the non‐selective herbicide phosphinothricin

Resistance to the non‐selective herbicide dl ‐phosphinothricin (PPT) was introduced into commercial Lotus corniculatus cv. Bokor by co‐cultivation of cotyledons with Agrobacterium tumefaciens AGL1 harbouring the binary vector pDM805 which contains the bialaphos resistance gene (bar) from Streptomyces hygroscopicus encoding phosphinothricin acetyltransferase (PAT) and the uidA gene encoding β‐glucuronidase. The half‐cotyledon explants were precultured on regeneration Murashige and Skoog's (MS) medium supplemented with 6‐benzyladenine (BA) and 1‐naphthaleneacetic acid (NAA) at 0.5 mg L^?1 each, 3 days prior to infection. Upon co‐cultivation, the explants were cultured on PPT‐free regeneration medium for 10 days, and then subcultured on regeneration/selection media with increasing PPT concentrations (5–7 mg L^?1) for about 18 weeks. Out of 480 initially co‐cultivated explants, 272 regenerated shoots survived the entire PPT selection procedure. Resistant shoots were grown further, multiplied by tillering that was additionally promoted by PPT and rooted on hormone‐free MS medium containing 5 mg L^?1 PPT. Established shoot cultures, continuously maintained on the same medium, have preserved PPT resistance up to now (more than 2 years). Transformed plants assessed in vitro and in a greenhouse were tolerant to the herbicide PPT at 300 mg L^?1 equivalent to more than twofold the recommended field dosage for weed eradication. Applied PPT treatment did not affect the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH‐dependent glutamate dehydrogenase (NADH‐GDH; EC 1.4.1.2) in transformed plants. However, PPT did increase the mobility of glutamine synthetase isoforms GS1 and GS2 as well as the inhibition of an additional high mobility GS (hmGS) activity. In untransformed plants, PPT treatment reduced total GS activity by 4.4‐fold while contrary the activity of NADH‐GDH was increased by ninefold. All transformed herbicide‐resistant plants were phenotypically normal and exhibited genomic stability, as were the untransformed plants analysed by flow cytometry. Under greenhouse conditions, they grew to maturity, flowered and set seeds. Stable integration and expression of the bar gene in T0 and T1 plants were confirmed by Southern and Western blot analysis, while integration of the reporter uidA gene did not occur. The bar gene was inherited in a Mendelian fashion by the progeny, as detected by PPT resistance. The production of PPT‐resistant plants may have significant practical applications in weed control in fields of L. corniculatus.     

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.