Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers

Novel ‘super-binary’ vectors that carried two separate T-DNAs were constructed. One T-DNA contained a drug-resistance, selection-marker gene and the other contained a gene for β-glucuronidase (GUS). A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tume-faciens LBA4404 that carried the vectors. Frequency of co-transformation with the two T-DNAs was greater than 47%. GUS-positive, drug-sensitive progeny were obtained from more than half of the co-transformants. Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T-DNAs. Thus, the non-selectable T-DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug-resistant transformants. Since various DNA fragments may be inserted into the non-selectable T-DNA by a simple procedure, these vectors will likely be very useful for the production of marker-free transformants of diverse plant species. Delivery of two T-DNAs to plants from mixtures of A. tumefaciens was also tested, but frequency of co-transformation was relatively low.     

Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418.

A protocol that allows the rapid isolation and growth of large numbers of independent G418-resistant Dictyostelium discoideum transformant colonies on the surface of agar media with live bacteria was developed. Transformants grown under these conditions form normal fruiting bodies. Discovery that aggregation of nontransformants was inhibited at a nonselective level of G418 (25 to 35 micrograms/ml) led to the development of a vector maintenance assay. Using this assay we examined the stability of recombinant plasmids derived from the D. discoideum native plasmids Ddp1 and Ddp2. We conclude that the origin of replication of plasmid Ddp1 does not alone confer stable maintenance and thus, Ddp1 must bear additional sequences required for its own maintenance. Analysis of the maintenance of vectors derived from Ddp2 showed that autonomously replicating shuttle vectors that contained bacterial plasmid DNA and from which one element of the Ddp2 inverted repeat was removed were much less stable than vectors that contained a complete inverted repeat or that did not carry a bacterial plasmid. Sequences between the 3' end of the rep gene and the inverted repeat appear to play a role in plasmid maintenance. An intact rep gene and one copy of the inverted repeat element were required for extrachromosomal replication. Maintenance of extrachromosomal vectors was found to be strain dependent. Four traits distinguishing integrating vectors from those capable of autonomous replication were identified.     

Kinetoplast DNA minicircles: High-copy-number mitochondrial plasmids

The kinetoplast DNA of trypanosomes is a highly unusual network of catenated DNA circles of two kinds: maxicircles, the equivalent of conventional mitochondrial DNA, and minicircles, high-copy-number mitochondrial plasmids with no known function. Kinetoplast minicircles share many features with bacterial plasmids and represent a novel model system for the study of the mechanisms and regulation of DNA replication in eukaryotic organisms.     

Biolistic co-transformation of the nuclear and plastid genomes

Particle gun-mediated (so-called 'biolistic') transformation represents a universal genetic transformation technology that is widely applied in nearly all groups of organisms. The mechanism of how accelerated DNA-coated particles, after their entry into the cell, deliver the foreign DNA to the target compartment is not known. Here we have studied this process in plants by performing co-transformation experiments with vectors targeted to two different cellular compartments, the nucleus and the plastids (chloroplasts). We find that coating of particles with both plastid and nuclear transformation vectors can result in co-transformation of chloroplasts and the nucleus. In contrast, mixing of particles coated individually with the vectors does not produce co-transformed plants. Our data suggest that a single DNA-coated particle can transform more than one compartment of the plant cell, opening up the possibility to generate doubly transgenic plants in one step. Importantly, co-transformation can also be obtained in the absence of selection, thus providing a method to produce marker-free transgenic genomes. In addition, our findings raise the possibility of occasional inadvertent co-transformation of two genomes and, therefore, have important implications for the molecular characterization and regulation of transgenic plants.     

High-copy-number derivatives of the plasmid cloning vector pBR322

A stable copy-number mutant of a pBR322-derived plasmid was isolated. The mutation was found to be a single G → T transversion located near the 3' end of a DNA segment coding for the regulatory RNA I. The resulting copy number for this plasmid is approx. 1000 per cell or 65 % of total cellular DNA. Several cloning vectors have been constructed from this copy-number mutant and their practical application is discussed.     

High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection

Three types of alpha-complementation plasmid vectors were constructed which contain a chloramphenicol- or kanamycin-resistance (CmR or KmR) gene and polylinker cloning sites within the coding region of lacZ'. These vectors are essentially based on high- or low-copy-number replicons. The low-copy-number vectors, 3.61 kb in size, confer CmR and contain the pSC101 replicon and pUC8-/pUC9-type polylinker. On the other hand, the high-copy-number vectors, 2.21 to 2.68 kb in size, confer either CmR or KmR, and contain the pBR322 replicon and pUC18-/pUC19-type or other modified polylinkers. All cloning sites except HindIII and SmaI sites in the KmR vectors are unique in each plasmid. Since almost all frequently used plasmid vectors confer ampicillin resistance, these vectors may be useful to simplify the subcloning/DNA joining experiments due to unnecessity of radioisotope labelling, size fractionation and purification of foreign DNA segments.     

Stable co-transformation of maize protoplasts with gusA and neo genes

An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 × BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing -glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 × 10^–4 (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.     

Characterization of Agrobacterium-mediated co-transformation events in rice using green and red fluorescent proteins

Molecular Biology Reports - Biotechnologists seeking to develop marker-free transgenic plants have established co-transformation methods. For co-transformation using mixed Agrobacterium strains,...     

Expression of beta-lactamase in Coxiella burnetii transformants

Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding beta-lactamase. However, non-plasmid emergence of resistance to ampicillin also develops. To validate the usefulness of the bla gene marker for selection and detection, transformed C. burnetii were examined for beta-lactamase expression by use of immunoblotting after SDS-PAGE. The 29-kDa mature form of the beta-lactamase protein was detected in C. burnetii lysates. Quantitation of these immunoblot signals showed that C. burnetii surprisingly expressed low levels of beta-lactamase. The results validate the use of plasmid-encoded ampicillin resistance as a means for selecting C. burnetii transformants; they also suggest that levels of ampicillin used for selection pressure should be empirically determined and that detection of beta-lactamase by antibody blotting done to confirm transformants.     

Plasmid copy number and stability determination in Clostridium perfringens transformants

The copy number of the streptococcal plasmid pAM beta 1 (26.5 kb), and its deletion derivatives, pVA1 (11 kb) and pVA677 (7.6 kb) contained in Clostridium perfringens 3624A transformants was determined by incorporation of [methyl-3H]thymidine (4 muCi/ml) into chromosomal and plasmid DNA and sizing of the C. perfringens genome using transverse alternating field electrophoresis. Plasmids pAM beta 1, pVA1, and pVA677 were found to be present at 1.0, 97, and 216 copies/cell, respectively. 10.2, 54, and 96% of the initial pAM beta 1-, pVA1- and pVA677-containing transformants, respectively, remained resistant to erythromycin over 220 generations of growth. The results indicate a size-dependent relationship between plasmid stability and plasmid copy number in C. perfringens.     

Recombinant plasmids carrying multiple markers: isolation during yeast co-transformation

Cotransformants of yeast cells by two partially homologous plasmids, one of which is incapable of autonomous replication, has been used to construct multiply marked recombinant plasmids. Only simultaneous elimination of three yeast markers was registered when episomal plasmid, carrying Ade2 gene, and integrative plasmid, carrying yeast genes LEU2 and URA3, were cotransformed. Transformants, in which yeast genes LEU2, URA3 and HIS3 are linked, have been isolated by analogous technique. The genetic analysis has confirmed existence of plasmid cointegrates in the transformant cells, which carry three yeast genes, bacterial DNA fragment and 2 micrometers DNA fragment, coding for replicative functions. Recombination in the region of bacterial plasmid pBR322 might have resulted in formation of such plasmids. Plasmid recombination in cotransformants has been used to construct multiply marked circular chromosomes, having included yeast genes LEU2, URA3 and TRP1, centromere of the IV yeast chromosome and the sequence coding for their replication in yeast as well as in E. coli cells.     

Morphological changes and induced sporulation in HmBR transformants ofCochliobolus lunatus

The phenomena following the transformation of the fungusCochliobolus lunatus by plasmid-encoded HmB resistance were investigated. All of the 16 tested transformants had markedly altered morphology. Unlike the untransformed fungus, the transformants produced both conidia and arthrospores, did not excrete slime, lost their purple color, and had an altered progesterone-bioconverting pathway.     

Characterization of polygalacturonase-overproducing Aspergillus niger transformants

Summary Aspergillus niger pyrA co-transformants with multiple copies of the gene (pgaII) encoding the major endopolygalacturonase were characterized in detail. Typically, these transformants produced tenfold or more polygalacturonase from plasmids that had integrated in most cases at ectopic sites, in comparison to the untransformed strain. Some mitotic instability was observed upon application of a positive selection procedure for reversion of the pyrA marker. Analysis of these strains indicated that the most frequent event involved is the excision of part of the array of tandemly integrated plasmids, without scrambling of the plasmids remaining in the chromosome. From promoter deletion analysis it was concluded that the pgaII gene is subject to positive control. The putative positive regulatory protein appears not to be limiting for overexpression of the pgaII gene. Correspondence to: J. Visser     

Homologous recombination and allele replacement in transformants of Fusarium fujikuroi

The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss was due to gene conversion in some cases and to recombination between repeated sequences in others. Received: 5 November 1999 / Accepted: 16 March 2000     

Recovery of primary transformants of soybean

Three transformants of soybean, Glycine max (L.) Merr., have been recovered among a total of 18 plants regenerated by somatic embryogenesis from immature cotyledon tissues after cocultivation with Agrobacterium strains carrying a 15 kD zein gene (pH5PZ3D). DNA from upper leaves hybridized to a synthetic RNA probe specific for the zein sequence at a level equivalent to at least one copy per haploid genome. Hybridization to a vir G/C probe, however, was negligible, indicating that sequestration of whole bacteria or even persistence of plasmids within the tissues could not account for the zein hybridization signals. Progeny of all plants were uniformly untransformed. Since most somatic embryos have a multicellular origin in the regeneration system used, it is believed that the primary transformants were chimeric. The results indicate that somatic embryogenesis may be adaptable to Agrobacterium-mediated transformation in soybean, but that greater numbers of mitotic cycles under selection before embryo initiation will be required if somatic embryogenesis is to be used efficiently for production of plants with transformed germ-line cells.Abbreviations NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid This paper (No. 88-3-267) is published with the approval of the Director of the Agricultural Experiment Station.     

青色荧光蛋白标记的禾谷镰孢转化子的构建

【背景】近年来玉米茎腐病在我国大部分玉米产区普遍重度发生,其中镰孢菌茎腐病不仅造成了重大的经济损失,而且镰孢菌产生的毒素给人体和动物的健康也带来严重威胁。【目的】玉米茎腐病的病原组成复杂,禾谷镰孢是其中的主要病原之一,该病原菌侵染寄主导致发病的机制急需深入研究。【方法】以pCAMBIA1300质粒为骨架,利用重叠PCR的方法构建表达青色荧光蛋白的质粒pCAMBIA1300-CFP-Kan,通过农杆菌介导的遗传转化技术,将青色荧光蛋白的编码基因整合到禾谷镰孢基因组中。【结果】经过PCR鉴定和荧光显微观察,确定获得了31株青色荧光标记的禾谷镰孢菌。【结论】侵染试验结果显示,激光共聚焦显微镜下禾谷镰孢在玉米茎秆组织中的定殖位置清晰可见,该结果为进一步研究不同镰孢菌在寄主中的定殖规律奠定了基础。