Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene

The possibility of controlling wheat scab (caused by Fusarium graminearum Schw.) was explored by engineering wheat plants for constitutive expression of pathogenesis-related (PR) protein genes. A rice thaumatin-like protein (TLP) gene (tlp) and a rice chitinase gene (chi11) were introduced into the spring wheat cultivar ’Bobwhite’ by co-transformation of the plasmids pGL2ubi-tlp (ubiquitin/tlp//CaMV 35S/hpt) and pAHG11 (CaMV 35S/chi11//ubiquitin/bar). The transformation was by biolistic bombardment. Bialaphos was used as the selection reagent. The integration and expression of the tlp, bar, chi11 and hpt genes were analyzed by Southern, Northern and Western blot analyses. The four transgenes co-segregated in the T₁ progeny of the transgenic plant and were localized at the telomeric region of the chromosome 6A long arm by sequential N-banding and fluorescent in situ hybridization (FISH) using pAHG11 or pGL2ubi-tlp as the probes. Only the transgenes tlp and bar, under the control of the ubiquitin promoter-intron, were expressed. No expression of the chi11 and hpt genes, controlled by the CaMV 35S promoter, was detected in T₁ plants. After inoculation with conidia of F. graminearum, the symptoms of scab developed significantly slower in transgenic plants of the T₁, T₂ and T₃ generations expressing the tlp gene than in non-transformed control plants. This is the first report of enhanced resistance to F. graminearum in transgenic wheat plants with constitutive expression of TLP. Received: 15 December 1998 / Accepted: 30 January 1999     

Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance

 Transformation of barley and wheat via particle bombardment with a gene derived from Vitis vinifera L. (Vst1 gene) resulted in the expression of the foreign phytoalexin, resveratrol, in the transformed plants. Transgenic barley plants were regenerated from microspores and transgenic wheat plants from immature embryos were both selected on Basta. Stable integration of the gene in the genomes of transgenic barley and wheat plants, as well as their progeny, was analysed by Southern-blot analysis. The induction of the stilbene synthase promoter and the transient expression of stilbene synthase-specific mRNA after induction by wounding and infection were proofed in T₁ and T₂ progeny plants. An enhanced expression of the Vst1 gene under control of the stilbene synthase promoter was observed with enhancer sequences from the cauliflower mosaic virus 35s (CaMV 35s) promoter. The enzyme activity of the stilbene synthase was analysed in T₁ progeny plants. The first pathological results indicated an increased resistance of transgenic barley plants to Botrytis cinerea used as a model experimental system. Received: 5 November 1997 / Accepted: 11 November 1997     

Stable transformation ofArabidopsis with thebar gene using particle bombardment

A plasmid pARK 22 harbouring thebar gene encoding phosphinothricin acetyltransferase (PAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator was constructed and introduced into root sections ofArabidopsis thaliana using the pneumatic particle gun. The root sections that had been bombarded with this plasmid gave four to eight times higher yield of drug-resistant calluses than those sections bombarded with pCaMVNEO or pCH, which respectively contain the neomycin phosphotransferase and hygromycin phosphotransferase genes. Among a number of primary transformant (T₀) plants obtained from independent bialaphos-resistant calluses, three were studied by Southern blot hybridization and PAT enzyme activity analyses, confirming the stable integration of the foreign gene into theArabidopsis genome and its expression in plants. The progeny analysis showed transmission of the foreign gene and its expression in up to the T₂ generation. Some of the T₁ progeny showed morphological abnormalities. Thus, thebar gene can be used effectively to allow selection of transgenicA. thalianna plants.     

Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells

Summary Suspension cells of Oryza sativa L. (rice) were transformed, by microprojectile bombardment, with plasmids carrying the coding region of the Streptomyces hygroscopicus phosphinothricin acetyl transferase (PAT) gene (bar) under the control of either the 5 region of the rice actin 1 gene (Act1) or the cauliflower mosaic virus (CaMV) 35S promoter. Subsequently regenerated plants display detectable PAT activity and are resistant to BASTA^TM, a phosphinothricin (PPT)-based herbicide. DNA gel blot analyses showed that PPT resistant rice plants contain a bar-hybridizing restriction fragment of the expected size. This report shows that expression of the bar gene in transgenic rice plants confers resistance to PPT-based herbicide by suppressing an increase of ammonia in plants after spraying with the herbicide.     

Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†

A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R₀ and R₁ progeny plants. Segregation of the PAT activity and herbicide resistance in R₁ progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R₁ progeny.     

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     

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.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ri binary vector

Summary Transgenic herbicide-resistant Scoparia dulcis plants were obtained by using an Ri binary vector system. The chimeric bar gene encoding phosphinothricin acetyltransferase flanked by the promoter for cauliflower mosaic virus 35S RNA and the terminal sequence for nopaline synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching young plants. Hairy roots resistant to bialaphos were selected and plantlets (R0) were regenerated. Progenies (S1) were obtained by self-fertilization. The transgenic state was confirmed by DNA-blot hybridization and assaying of neomycin phosphotransferase II. Expression of the bar gene in the transgenic R0 and S1 progenies was indicated by the activity of phosphinothricin acetyltransferase. Transgenic plants accumulated scopadulcic acid B, a specific secondary metabolite of S. dulcis, in amounts of 15–60% compared with that in normal plants. The transgenic plants and progenies showed resistant trait towards bialaphos and phosphinothricin. These results suggest that an Ri binary system is one of the useful tools for the transformation of medicinal plants for which a regeneration protocol has not been established.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

Combined expression of chitinase and β-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani

Agrobacterium-mediated transformation of rice was done using the binary vector pNSP3, harbouring the rice chitinase (chi11) gene under maize ubiquitin promoter and the tobacco β-1,3-glucanase gene under CaMV 35S promoter in the same T-DNA. Four of the six T₀ plants had single copies of complete T-DNAs, while the other two had complex integration patterns. Three of the four single-copy lines showed a 3:1 segregation ratio in the T₁ generation. Northern and western blot analyses of T₁ plants revealed constitutive expression of chitinase and β-1,3-glucanase genes. Homozygous T₂ plants of the single-copy lines CG20, CG27 and CG53 showed 62-, 9.6- and 11-fold higher chitinase activity over the control plants. β-1,3-Glucanase activity was 1.1- to 2.5-fold higher in the transgenic plants. Bioassay of homozygous T₂ plants of the three single-copy transgenic lines against Rhizoctonia solani revealed a 60% reduction in sheath blight Disease Index in the first week. The Disease Index increased from 61.8 in the first week to 90.6 in the third week in control plants, while it remained low (26.8–34.2) in the transgenic T₃ plants in the corresponding period, reflecting the persistence of sheath blight resistance for a longer period.     

Transgenic herbicide-resistant Atropa belladonna using an Ri binary vector and inheritance of the transgenic trait

Summary Transgenic Atropa belladonna conferred with a herbicide-resistant trait was obtained by transformation with an Ri plasmid binary vector and plant regeneration from hairy roots. We made a chimeric construct, pARK5, containing the bar gene encoding phosphinothricin acetyltransferase flanked with the promoter for cauliflower mosaic virus 35S RNA and the 3 end of the nos gene. Leaf discs of A. belladonna were infected with Agrobacterium rhizogenes harboring an Ri plasmid, pRi15834, and pARK5. Transformed hairy roots resistant to bialaphos (5 mg/l) were selected and plantlets were regenerated. The integration of T-DNAs from pRi15834 and pARK5 were confirmed by DNA-blot hybridization. Expression of the bar gene in transformed R₀ tissues and in backcrossed F1 progeny with a nontransformant and self-fertilized progeny was indicated by enzymatic activity of the acetyltransferase. The transgenic plants showed resistance towards bialaphos and phosphinothricin. Tropane alkaloids of normal amounts were produced in the transformed regenerants. These results present a successful application of transformation with an Ri plasmid binary vector for conferring an agronomically useful trait to medicinal plants.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease

 A 1.1-kb DNA fragment containing the coding region of a thaumatin-like protein (TLP-D34), a member of the PR-5 group, was cloned into the rice transformation vector pGL2, under the control of the CaMV 35S promoter. The Indica rice cultivars, ‘Chinsurah Boro II’, ‘IR72’, and ‘IR51500’ were transformed with the tlp gene construct by PEG-mediated direct gene transfer to protoplasts and by biolistic transformation using immature embryos. The presence of the chimeric gene in T₀, T₁, and T₂ transgenic plants was detected by Southern blot analysis. The presence of the expected 23-kDa TLP in transgenic plants was confirmed by Western blot analysis and by staining with Coomassie Brilliant Blue. Bioassays of transgenic plants challenged with the sheath blight pathogen, Rhizoctonia solani, indicated that over-expression of TLP resulted in enhanced resistance compared to control plants. Received: 11 August 1998 / Accepted: 26 August 1998     

Stable transformation of Eustoma grandiflorum by particle bombardment

Explants (7.5±2.5 mm) cut from stems and roots of 3-week-old Eustoma grandiflorum Grise, (lisianthus) cv. Glory White seedlings were bombarded with plasmid pBI221, which harbors the uidA gene encoding β-glucuronidase (GUS) driven by the cauliflower mosaic virus (CaMV) 35S promoter. More than 800 blue spots of GUS-expressing cells were observed per 90 explants. Explants bombarded with pARK22 harboring the bar gene encoding phosphinothricin acetyltransferase driven by the CaMV 35S promoter were selected for bialaphos resistance. Putative transgenic plants were obtained about 3 months after bombardment. Southern blot analysis of putative transgenic plants revealed the presence of the bar gene in their genome. Received: 10 April 1996 / Revision received: 7 November 1997 / Accepted: 22 November 1997     

Engineering herbicide resistance in plants by expression of a detoxifying enzyme

Phosphinothricin (PPT) is a potent inhibitor of glutamine synthetase in plants and is used as a non-selective herbicide. The bar gene which confers resistance in Streptomyces hygroscopicus to bialaphos, a tripeptide containing PPT, encodes a phosphinothricin acetyltransferase (PAT) (see accompanying paper). The bar gene was placed under control of the 35S promoter of the cauliflower mosaic virus and transferred to plant cells using Agrobacterium-mediated transformation. PAT was used as a selectable marker in protoplast co-cultivation. The chimeric bar gene was expressed in tobacco, potato and tomato plants. Transgenic plants showed complete resistance towards high doses of the commercial formulations of phosphinothricin and bialaphos. These data present a successful approach to obtain herbicide-resistant plants by detoxification of the herbicide.     

Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris alpha-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker

Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and α-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris α-amylase inhibitor-1 (αAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris α-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T₁ lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T₁ plants. Transgenic plants could be recovered after 8–10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved.     

Construction of a GFP-BAR plasmid and its use for switchgrass transformation

 A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by replacing the uidA (β-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l^–1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red to yellow. Basta tolerance was observed in T₁ plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene. Received: 11 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000