Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker

 Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T₀) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected 35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T₀ and T₁ transgenic plants. Mendelian segregation of herbicide resistance was observed in some T₁ progenies. Interestingly, a majority of the T₁ progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated in T₁ transgenic wheat plants. Received: 12 May 1998 / Accepted: 15 May 1998     

Stability of transgene expression, field performance and recombination breeding of transformed barley lines

Stable inheritance of the transgene, consistent expression and competitive agronomic properties of transgenic crops are important parameters for successful use of the latter. These properties have been analyzed with 18 homozygous transgenic barley lines of the cultivar Golden Promise. The lines originated from three independent primary transformants obtained by the biolistic method with three plasmids containing respectively, the bar gene, the uidA gene and the gene for a protein-engineered heat-stable (1,3–1,4)-β-glucanase. Three production levels of recombinant β-glucanase were identified in homozygous transgenic T₃ plants, and these remained constant over a 3-year period. In micro-malting experiments, the heat-stable enzyme reached levels of up to 1.4 μg·mg⁻¹ protein and survived kiln drying at levels of 70–100%. In the field trials of 1997 and 1998 the transgenic lines had a reduced 1000-grain weight as well as variable yield depressions compared to the Golden Promise progenitor. In 1999 large-scale propagations of the lines with the highest recombinant enzyme synthesis during germination and of Golden Promise were studied at three different locations. In an irrigated field transgenic lines yielded approximately 6 t·ha⁻¹ and Golden Promise 7.7 t·ha⁻¹. Cross-breeding was carried out to transfer the transgene into a more suitable genetic background. Crosses of the semi-dwarf ari-e mutant Golden Promise gave rise to the four morphological phenotypes nutans, high erect, erect, and ari-e. Two improvements were achieved: (1) F₃ lines homozygous for the expression of heat-stable (1,3−1,4)-β-glucanase were found among lines that were homozygous for each of the four morphological phenotypes; (2) improved 1000-grain weights and yields with respect to those of the original transformants were observed in some F₄ lines homozygous for the morphological phenotypes and for the transgene. In the case of a homozygous nutans line, the transgenic plants had a higher 1000-grain weight than those lacking the transgene. Like mutants providing useful output traits, transgenic plants will often have to be improved by relocating the gene into more suitable genotypes. Received: 6 March 2000 / Accepted: 14 April 2000     

Pistil drip following pollination: a simple <Emphasis Type="Italic">in planta</Emphasis><Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in cotton

Transgenic cotton plants were developed by pistil drip inoculation in a solution containing Agrobacterium carrying a gene for resistance to the herbicide Basta (bar), 10% (w/v) sucrose, 0.05% (v/v) Silwet L-77 and 40 mg acetosyringone l⁻¹. Pistil drip during 17:00–19:00 on the first day of flowering resulted in 0.07–0.17% Basta-resistant plants/number of viable seeds generated, and stigma excision prior to pistil drip during this time period gave rise to a transformation efficiency of 0.46–0.93%, in contrast with 0.04–0.06% generated from pistil drip during 9:00–11:00 on the second day of flowering. PCR and Southern blot analysis confirmed the integration of the bar gene into the cotton genome, and a T1 and T2 generation herbicide resistance test consistently revealed expression and stable heritability of the bar gene in the two generations.     

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     

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     

Effect of ploidy and homozygosity on transgene expression in primary tobacco transformants and their androgenetic progenies

Expression of a transgene is rarely analysed in the androgenetic progenies of the transgenic plants. Here, we report differential transgene expression in androgenetic haploid and doubled haploid (DH) tobacco plants as compared to the diploid parental lines, thus demonstrating a gene dosage effect. Using Agrobacterium-mediated transformation, and bacterial reporter genes encoding neomycin phosphotransferase (nptII) and β-glucuronidase (uidA/ GUS), driven respectively by the mas 1′ and mas 2′ promoters, we have generated more than 150 independent transgenic (R₀) Nicotiana tabacum plants containing one or more T-DNA copies. Transgene analyses of these R₀, their selfed R₁ lines and their corresponding haploid progenies showed an obvious position effect (site of T-DNA insertion on chromosome) on uidA expression. However, transgene (GUS) expression levels were not proportional to transgene copy number. More than 150 haploids and doubled haploids, induced by treatment with colchicine, were produced from 20 independent transgenic R₀ plants containing single and multiple copies of the uidA gene. We observed that homozygous DH plants expressed GUS at approximately 2.9-fold the level of the corresponding parental haploid plants. This increase in transgene expression may be attributed mainly to the increase (2-fold) in chromosome number. Based on this observation, we suggest a strong link between chromosome number (ploidy dosage effect) and transgene expression. In particular, we demonstrate the effect on its expression level of converting the transgene from the heterozygous (in R₀ plants) to the homozygous (DH) state: e.g. an increase of 50% was observed in the homozygous DH as compared to the original heterozygous diploid plants. We propose that ploidy coupled with homozygosity can result in a new type of gene activation, creating differences in gene expression patterns. Received: 27 April 1998 / Accepted: 12 August 1998     

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.     

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.     

Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.)

 An Agrobacterium tumefaciens-mediated transformation method has been developed for onions (Allium cepa L.) using immature embryos as the explant source. Transgenic plants were recovered from the open-pollinated onion cultivar Canterbury Longkeeper at a maximum transformation frequency from immature embryos of 2.7%. The method takes between 3–5 months from explant to primary regenerant entering the glasshouse. Multiple-shoot formation from primary transgenic material made possible the clonal multiplication of transformants. The binary vector used carried the nptII antibiotic resistance gene and the m-gfp5-ER reporter gene. Transgenic cultures were initially screened for their ability to fluoresce and to grow in the presence of geneticin (5–25 mg/l). The transgenic nature of individual plants was confirmed by Southern blot analysis. Received: 12 October 1998 / Revision received: 17 May 1999 Accepted: 14 June 1999     

Analysis and optimisation of DNA delivery into wheat scutellum and Tritordeum inflorescence explants by tissue electroporation

Wheat scutella and tritordeum inflorescences were transformed by tissue electroporation with plasmid DNA containing a β-glucuronidase (GUS) gene (gus A) under the control of the rice actin1 promoter. Factors affecting electroporation efficiency were analysed. Important factors were electroporation voltage and pulse length, the volume of electroporation buffer, the osmoticum of electroporation buffer and medium, the osmoticum of pre-electroporation culture medium, and pre-electroporation incubation time and temperature. Maximum transient gene expression was obtained with a single pulse of 550 V/cm from a 960-μF capacitor, using 200 μl of electroporation buffer, after 2–3 h culture on media with 357 mOsm for wheat scutella or 1 day on media with 222 mOsm for tritordeum inflorescences, and 0.5–1 h pre-electroporation incubation with DNA at 24 °C. Under these conditions, up to 90% of the explants showed GUS expression, and up to 149 expression signals were recorded per replicate. Electroporated explants showed high rates of survival and retained the ability to regenerate plants via somatic embryogenesis. Received: 9 August 1996 / Revision received: 11 September 1997 / Accepted: 2 July 1998     

Transformation of Saccharopolyspora erythraea by electroporation of germinating spores: construction of propionyl Co-A carboxylase mutants

The introduction of plasmid DNA into germinating spores of an industrially improved strain of Saccharopolyspora erythraea was accomplished by electroporation. Various parameters affecting the efficiency of electroporation were examined. The most critical factor was the extent of spore germination. Electrocompetence was limited to a 4-h period following the initial emergence of the germ tube. Electroporation efficiencies as high as 2 × 10⁵ CFU μg⁻¹ of plasmid DNA were obtained using electrocompetent germlings. The optimal field strength was 12–14 kV cm⁻¹ with a pulse duration of 15–20 ms. Electrocompetent germlings were stored at −80°C without a significant decrease in transformation efficiency. The utility of this protocol was demonstrated by isolating a propionyl-CoA carboxylase mutant through targeted gene disruption and replacement. Received 3 April 1998/ Accepted in revised form 28 September 1998     

Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos

To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests – corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta^TM contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression. Received: 25 September 1998 / Accepted: 27 October 1998     

Agrobacterium-mediated transformation of Brassica campestris ssp. Parachinensis with synthetic Bacillus thuringiensis cry1Ab and cry1Ac genes

 An effective plant regeneration procedure and a gene transfer system via Agrobacterium tumefaciens were developed in Brassica campestris ssp. parachinensis. Hypocotyls from 5-day-old seedlings with 2 days pre-culture were infected with Agrobacterium strain MOG301 harboring a binary vector containing a synthetic Bacillus thuringiensis (B.t.) cry1Ab or cry1Ac gene with full codon-modification. After culture and selection on MS medium supplemented with 4.0 mg/l BAP, 2.0 mg/l NAA, 70 μM AgNO₃ and 50 mg/l kanamycin, a number of kanamycin-resistant plantlets were regenerated. PCR and Southern blotting analysis were used to identify and characterize the transgenic plants with the integrated cry1Ab or cry1Ac gene. Western blotting analysis of the transgenic plants confirmed the expression of insecticidal proteins encoded by cry1Ab or cry1Ac. Subsequent bioassay with larvae of the Diamondback moth, Plutella xylostella, demonstrated that the transgenic plants were resistant to feeding damage. Received: 22 February 1999 / Revision received: 14 April 1999 / Accepted: 26 April 1999     

An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration

 A method for producing large numbers of transgenic wheat plants has been developed. With this approach, an average of 9.7% of immature embryo explants were transformed and generated multiple self-fertile, independently transformed plants. No untransformed plants, or escapes, were regenerated. This transformation procedure uses morphogenic calli derived from scutellum tissue of immature embryos of Triticum aestivum cv. Bobwhite co-bombarded with separate plasmids carrying a selectable marker gene (bar) and a gene of interest, respectively. Transformed wheat calli with a vigorous growth phenotype were obtained by extended culture on media containing 5.0 mg/l bialaphos. These calli retained morphogenic potential and were competent for plant regeneration for as long as 11 months. The bar gene and the gene of interest were co-expressed in T0 progeny plants. This wheat transformation protocol may facilitate quantitative production of multiple transgenic plants and significantly reduce the cost and labor otherwise required for screening out untransformed escapes. Received: 15 June 1998 / Revision received: 6 April 1999 / Accepted: 26 April 1999     

Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l^–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants. Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998     

Generation of large numbers of transgenic Kentucky bluegrass (Poa pratensis L.) plants following biolistic gene transfer

A very efficient transformation system, using biolistic bombardment, has been developed for the production of transgenic plants of Kentucky bluegrass (Poa pratensis L.). Embryogenic calli, initiated from immature embryos, were transformed either with pAct1IHPT-4 containing the hygromycin phosphotransferase (hpt) gene or with pDM803 containing the phosphinothricin acetyltransferase (bar) gene and the β-glucuronidase (uidA) gene. In total 119 independent transgenic plants were recovered from 153 hygromycin-resistant lines. Bialaphos selection yielded a total of 99 bialaphos-resistant lines and from these 34 independent transgenic plants were recovered. Southern blot analysis demonstrated the independent nature of the transgenic plants and also revealed a complex transgene integration pattern with multiple insertions. The first two author contributed equally to this work     

Improving antioxidant activity in transgenic <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> plants via overexpression of the γ-tocopherol methyltransferase (γ-<Emphasis Type="Italic">tmt)</Emphasis> gene

Codonopsis lanceolata Trautv (Companulaceae) is a folk medicine in Korea. To shift the content of tocopherol and enhance its antioxidant properties, we overexpressed the γ-tocopherol methyltransferase (γ-tmt) gene in C. lanceolata. The antioxidant activity of methanolic crude extracts of the transgenic plants was compared to that of control plants using the 1,1-diphenyl-2-picrylhydrazyl radical scavenging method, with α-tocopherol and butylated hydroxy toluene as standard antioxidants. The antioxidant activity of the leaf and root extracts of transgenic plants was higher (IC ₅₀ 12–17.33 and 408–524 μg/ml, respectively) than that of control plant leaf and root extracts (18 and 529 μg/ml, respectively). High-performance liquid chromatography analysis of phenolic compounds confirmed an increase in the levels of 12 major phenolic acids and flavonoids in the leaf and root extracts of transgenic plants compared to control plants. We also found that the rate of photosynthesis was 48% higher in transgenic plants than in control plants. Based on these results, we suggest that increases in the α-tocopherol level in transgenic C. lanceolata plants may result in increases in the photosynthetic performance and antioxidant metabolism of these plants.     

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     

Overexpression of glutathione S-transferase gene increases salt tolerance of arabidopsis

The Suaeda salsa glutathione S-transferase gene (GST) was introduced into arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. Southern and northern blot analyses confirmed that GST was transferred into the arabidopsis genome, and the GST and GPX activities in transgenic plants (GT) were much higher than in wild-type plants (WT). There were no obvious morphological or developmental differences between transgenic and wild-type plants. One transgenic homozygous line (GT_6–8) and WT plants were evaluated for salt tolerance and gene expression. Seed germination and seedling salt tolerance were improved after overexpression of GST in arabidopsis; the photosynthesis rate and the fresh weight of the GT_6–8 line were distinctly higher than those of WT plants after NaCl treatment. Glutathione content increased substantially in salt-stressed arabidopsis plants of both genotypes, and the glutathione pool in GT_6–8 plants was more oxidized than in WT plants under both control and stressful conditions. The MDA content, an indicator of lipid peroxidation, increased in WT plants but was not affected distinctly in GT_6–8 seedlings after NaCl treatment. Results from different tests indicated that the expression of the GST gene promoted a higher level of salt tolerance in vivo in transgenic arabidopsis plants.     

Development and evaluation of transgenic rice seeds accumulating a type II-collagen tolerogenic peptide

Type II collagen (CII) in joint cartilage is known to be a major auto-antigen in human rheumatoid arthritis. Several animal model- and clinical-studies on tolerance-based immunotherapy for the arthritis have been conducted by administrating synthetic immunodominant peptides through an oral route. In the present study, to produce a tolerogenic peptide with therapeutic potential in transgenic rice plants, a gene construct producing glutelin fusion protein with tandem four repeats of a CII_250–270 peptide (residues 250–270) (GluA-4XCII_250–270) containing a human T-cell epitope was introduced with a selection marker, hygromycin phosphotransferase gene (hygromycin-resistance gene) (hph), by co-transformation. Several transgenic plants with high and stable expression of gluA-4XCII _250–270 , but no hph, were selected based on both DNA and protein analyses. The GluA-4XCII_250–270 fusion proteins were detected as both precursor and processed forms mainly in a glutelin fraction of rice endosperm protein extracts and in protein-body rich fractions prepared by density gradient ultracentrifugation. The amount of accumulated CII_250–270 peptide was immunochemically estimated to be about 1 μg per seed. Feeding DBA/1 mice the transgenic rice seeds (25 μg of the peptide per mouse a day) for 2 weeks showed tendencies lowering and delaying serum specific-IgG2a response against subsequent and repeated intraperitoneal-injection of type II collagen. Taken these together, the CII-immunodominant peptide could effectively be produced and accumulated as a glutelin-fusion protein in the transgenic rice seeds, which might be useful as pharmaceutical materials and functional food for prevention and therapy for anti-CII autoimmune diseases like human rheumatoid arthritis.