Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP⁺) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP⁺ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP⁺ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.     

GUS expression in blueberry (Vaccinium spp.): factors influencing Agrobacterium-mediated gene transfer efficiency

Several factors were investigated for their influence on the transfer of an intron-containing β-glucuronidase (GUS) gene into blueberry (Vaccinium spp.) leaf explants during the early stages of Agrobacterium-mediated gene transfer, including days of cocultivation, strain of Agrobacterium tumefaciens, explant age and genotype. The number of GUS-expressing leaf zones and calli were counted immediately and 2 weeks after cocultivation, respectively, to evaluate the gene transfer process. Agrobacterium tumefaciens strain EHA105 (pEHA105/p35SGUS-int) was significantly more effective for transformation than strain LBA4404 (pAL4404/p35SGUSint). Four days of cocultivation with A. tumefaciens strain EHA105 yielded about 50-fold more GUS-expressing zones than 2 days of cocultivation. Significant differences among cultivars were observed for both GUS-expressing leaf zones and calli. For some cultivars, explant age influenced the number of GUS-expressing leaf zones and calli. In most cases, the number of GUS-expressing calli was highest in those cultivars where GUS expression in the leaves was high. Received: 25 May 1998 / Revision received: 29 July 1998 / Accepted: 14 August 1998     

Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting transformation efficiency are discussed.     

Transformation of microspore-derived embryos of winter oilseed rape (Brassica napus L.) by usingAgrobacterium tumefaciens

Haploid microspore-derived embryos (MDEs) constitute a unique material for the introduction of new traits into winter oilseed rape (Brassica napus). MDEs have been transformed by usingAgrobacterium tumefaciens strains EHA105 and LBA4404, both carrying the binary vector pKGIB containing theuidA gene encoding β-glucuronidase (GUS) and thebar gene as a marker of resistance to phosphinotricin. Transformed embryos expressed GUS and regenerated plants that were resistant to herbicide Basta, as confirmed by a leaf-painting test. Progeny plants of the transformant T-39 were all transgenic, as they inherited T-DNA from their doubled haploid parental plant. Southern-blot analysis confirmed the integration and transmission of T-DNA into T₁ plants. Transformation of MDEs facilitates the obtaining of winter oilseed rape homozygous for the introduced genes.     

Transformation of tomato with the <Emphasis Type="Italic">BADH</Emphasis> gene from <Emphasis Type="Italic">Atriplex</Emphasis> improves salt tolerance

Glycinebetaine is an important quaternary ammonium compound that is produced in response to salt and other osmotic stresses in many organisms. Its synthesis requires the catalysis of betaine aldehyde dehydrogenase encoded by BADH gene that converts betaine aldehyde into glycinebetaine in some halotolerant plants. We transformed the BADH gene, cloned from Atriplex hortensis and controlled by two 35S promoters of the cauliflower mosaic virus, into a salt-sensitive tomato cultivar, Bailichun, using Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBin438, and using a leaf regeneration system. Polymerase chain reaction and Southern hybridization analyses demonstrated that the BADH gene had integrated into the genome of tomato. Transgenic tomato plants showed significantly higher levels of mRNA and BADH enzyme activity than wild-type plants. Observations on rooting development and relative electronic conductivity suggested that the transgenic plants exhibited tolerance to salt stress, with these plants growing normally at salt concentrations up to 120 mM.     

Expression of vde Gene Integrated Into Tobacco Genome in Antisense and Overexpressed Ways

Violaxanthin de-epoxidase (VDE) is localised in the thylakoid lumen of chloroplasts and catalyses de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin. Tobacco vde gene was inserted into a binary vector pCAMBIA1301 with the hygromycin resistant gene for selection in antisense and overexpressed ways. Two constructs with antisense and overexpressed vde gene were introduced in tobacco (Nicotiana tabacum L.) using Agrobacterium tumefaciens strain LBA4404, PCR and Southern blot analyses demonstrated that the exogenous gene was integrated into genome of tobacco plants. VDE activity assay and HPLC analysis of pigments showed that the vde gene was expressed in the overexpressed transformants, whereas suppressed in the antisense ones. The chlorophyll fluorescence measurements proved that the contents of VDE in transgenic plants have a significant function in non-photochemical quenching.     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

The usefulness of the <Emphasis Type="Italic">gfp</Emphasis> reporter gene for monitoring <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of potato dihaploid and tetraploid genotypes

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. ‘Baltica’, respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.     

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.     

An efficient protocol for regeneration and transformation of <Emphasis Type="Italic">Symphyotrichum novi</Emphasis>-<Emphasis Type="Italic">belgii</Emphasis>

A protocol for adventitious shoot formation in Symphyotrichum novi-belgii was developed after investigating the effects of cultivar and hormone combinations. A Murashige and Skoog medium with 1.0 mg l⁻¹ 6-benzyladenine induced adventitious shoot formation in 15 out of 19 cultivars. Addition of 0.1 mg l⁻¹ indole-3-acetic acid or naphthaleneacetic acid increased the total number of shoots per explant, but not the number of shoots longer than 1 cm. Addition of dichlorophenoxyacetic acid (2,4-D) promoted callus formation, but inhibited shoot elongation. A transformation system for the two cultivars Victoria Fanny and Victoria Jane was developed by co-cultivation of leaf explants with Agrobacterium tumefaciens. Three bacterial strains (LBA 4404, A281 and C58) all carrying the binary vector, p35S-GUS-INT, and harbouring the uidA gene coding for β-glucuronidase (GUS) were used. Regeneration of transgenic plants after co-cultivation with A281 was independent of cultivar, and all explants produced callus followed by indirect shoot formation. In ‘Victoria Fanny’ shoots were formed faster and without a callus phase after co-cultivation with LBA 4404 or C58. The highest number of potentially transformed shoots was regenerated after co-cultivation of ‘Victoria Fanny’ leaf explants with LBA 4404. Integration of the transgenes in the plant genome was confirmed using PCR and Southern blot hybridisation. To verify that the transgenes could be transferred to offspring, crosses were conducted between three transgenic lines of ‘Victoria Fanny’ and two wild type pollen donors. It was demonstrated that viable seeds were produced and that the uidA gene was inherited.     

Transformation and regeneration of the self-incompatible species Nicotiana alata Link & Otto

A transformation and regeneration system has been developed for Nicotiana alata, a plant which is being intensively studied as a model of gametophytic self-incompatibility. Plantlets can be regenerated efficiently from seedling hypocotyls. Kanamycin-resistant, transformed plants have been obtained by cocultivation of regenerating hypocotyls with Agrobacterium tumefaciens strain LBA4404 containing a binary vector. The transformation frequency was low with <1% of tissue explants regenerating transformed plants. The transformed plants contained from one to three copies of the introduced DNA. In most cases, the kanamycin resistance phenotype was transmitted to the offspring as a normal Mendelian factor. In one unusual case, none of the offspring inherited the kanamycin resistance of the transformed maternal parent. This plant may have been chimeric or the kanamycin resistance gene may have been inactivated.     

Morphological changes in transgenic poplar induced by expression of the rice homeobox gene OSH1

Genetically transformed lombardy poplar (Po-pulus nigra L. var. italica Koehne) plants were regenerated after co-cultivation of stem segments with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector which included the rice gene for a homeodomain protein (OSH1) and a gene for neomycin phosphotransferase. The expression of the OSH1 gene under control of the cauliflower mosaic virus 35S promoter induced morphological abnormalities in the leaves and stems of the newly generated transgenic poplar plants. This result suggests that OSH1 can function as a regulator of morphogenesis in transgenic poplar, as it does in transgenic rice, Arabidopsis, and tobacco plants. Received: 16 October 1998 / Revision received: 27 November 1998 / Accepted: 12 December 1998     

Production of transgenic <Emphasis Type="Italic">Podophyllum peltatum via Agrobacterium tumefaciens</Emphasis>-mediated transformation

Transgenic Podophyllum peltatum plants were successfully produced by Agrobacterium tumefaciens-mediated transformation. Embryogenic callus was co-cultivated with Agrobacterium tumefaciens harboring a binary vector pBI 121 carrying β-glucuronidase (GUS) and neomycinphosphotransferase (NPT II) gene. GUS-histochemical analysis revealed that, 50 μM acetosyringone treatments during Agrobacterium infection and 3 d co-cultivation with Agrobacterium showed enhanced transformation efficiency. Percentage of GUS positive callus increased rapidly as the subculture time proceeded on selection medium containing 100 mg dm⁻³ kanamycin. Kanamycin resistant somatic embryos were formed from embryogenic callus after cultivation with 11.35 μM abscisic acid (ABA) for 3 weeks and then on hormone-free selection medium. Somatic embryos were germinated and converted into plantlets on medium containing 2.89 μM gibberellic acid (GA₃). The integration of GUS and NPT II gene into transgenic plants was confirmed by polymerase chain reaction and Southern analysis.     

Agrobacterium-mediated transformation of Campanula glomerata

A transformation system for Campanula glomerata 'Acaulis' based on the co-cultivation of leaf explants with Agrobacterium tumefaciens LBA4404 or EHA105 was developed. A. tumefaciens was eliminated when the explants were cultured on medium containing 400 mg/l vancomycin and 100 mg/l cefotaxime. Transgenic plants containing the uidA gene that codes for #-glucuronidase (gus) were obtained following co-cultivation with either strain of A. tumefaciens, LBA4404 or EHA105, both of which harbored the binary vector pGUSINT, coding for the uidA and neomycin phosphotransferase II (nptII) genes. While the transformation frequency (2-3%) was similar for both strains, A. tumefaciens LBA4404 was effectively eliminated from Campanula at a lower concentration of antibiotic as compared to EHA105. The concentration of individual antibiotics required to eliminate EHA105 resulted in a decreased rate (55-67%) of regeneration. The highest percentage of explants that regenerated plants (79%) and the highest regeneration rate was achieved with 100 mg/l cefotaxime combined with 400 mg/l vancomycin. Plants were also transformed with the isopentenyl transferase (ipt) gene using LBA4404 containing the 35S-ipt vector construct (pBC34).     

Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.     

根癌农杆菌介导的乙肝表面抗原基因对烟草的转化

构建了植物表达载体pBRSAg,该载体具有完整的植物表达元件,CaMV35S启动子、农杆菌T-DNA左右边界、植物报告基因gus和植物选择标记基因hpt,适用于农杆菌的转化;通过冻融法将重组质粒pBRSAg转入根癌农杆菌LBA4404中,利用农杆菌介导法转化烟草叶盘,经筛选培养获得烟草植株。抗性植株经GUS染色和PCR检测为阳性,初步表明乙肝表面抗原基因在烟草中得到表达。     

Stable transformation of eggplant (Solanum melongena L.) by cocultivation of tissues withAgrobacterium tumefaciens carrying a binary plasmid vector

Stable transformation of eggplant to kanamycin resistance was obtained by cocultivation of cotyledonary and young leaves with the hypervirulent, fully oncogenicAgrobacterium tumefaciens strain A281 carrying plasmid pGA472. No transformation was observed when using the disarmedA. tumefaciens LBA4404 strain carrying pGA472 or when using either strain for cocultivation with eggplant suspension cells.The NPTII enzyme and DNA dot blot assays performed on callus cells growing in the presence of kanamycin indicated both the presence and expression of the foreign gene. The highest proportion of transformed explants was obtained from intact cotyledonary leaf pieces while the highest NPTII enzyme specific activity was detected in callus cells originating from superficially wounded cotyledonary leaf pieces. Kanamycin-resistant plantlets were regenerated after six months in culture.Abbreviations CTAB Cetyltrimethylammonium Bromide - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic Acid - NAA Naphtaleneacetic Acid - 2,4-D 2,4-Dichlorophenoxyacetic Acid - NPT II Neomycin Phosphotransferase     

Obtaining of transgenic French bean plants (Phaseolus vulgaris L.) resistant to the herbicide pursuit by Agrobacterium-mediated transformation

The transgenic plants of French bean (Phaseolus vulgaris) resistant herbicide Pursuit and kanamycin have been obtained. The genetic transformation was carried out with Agrobacterium tumefaciens strain LBA4404 containing binary vector carrying mutant ahas/als and selective nptII genes. Integration of the transgenes into plant genome was confirmed by polymerase chain reaction.