Mechanisms of intermolecular homologous recombination in plants as studied with single- and double-stranded DNA molecules.

To elucidate the mechanism for intermolecular homologous recombination in plants we cotransformed Nicotiana tabacum cv Petit Havana SR1 protoplasts with constructs carrying different defective derivatives of the NPTII gene. The resulting kanamycin resistant clones were screened for possible recombination products by PCR, which proved to be a valuable technique for this analysis. Our results show that the double-stranded circular DNA molecules used in this study recombine predominantly via a pathway consistent with the single-strand annealing (SSA) model as proposed for extrachromosomal recombination in mammalian cells. In the remaining cases recombination occurred via a single reciprocal recombination, gene conversion and possibly double reciprocal recombination. Since single-stranded DNA is considered to be an important intermediate in homologous recombination we also established the recombination ability of single-stranded DNA in intermolecular recombination. We found that single-stranded DNA enters in recombination processes more efficiently than the corresponding double-stranded DNA. This was also reflected in the recombination mechanisms that generated the functional NPTII gene. Recombination between a single-stranded DNA and the complementing DNA duplex occurred at similar rates via a single reciprocal recombination and the SSA pathway.     

Single-stranded DNA transforms plant protoplasts

Summary The transfer of the Agrobacterium T-DNA to plant cells involves the induction of the Ti plasmid virulence genes. This induction results in the generation of linear single-stranded (ss) copies of the T-DNA inside Agrobacterium and such molecules might be directly transferred to the plant cell. A central requirement of this ss transfer model is that the plant cell must generate a second strand and integrate the resulting double-stranded (ds) molecule into its genome. Here we report that incubating plant protoplasts with ss or ds DNA under conditions favouring DNA uptake results in transformation. The frequencies of transformation are similar and analysis of ss transformants suggests that the introduced DNA becomes double stranded and integrated. Analysis of transient expression from introduced ss DNA suggests that generation of the second strand is rapid and extrachromosomal.     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts

Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)⁺ RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.     

Transformed calli obtained by direct gene transfer into sunflower protoplasts

Helianthus annuus protoplasts were transformed with the plasmid pCaMVNEO (Frommet al. 1986) conferring kanamycin resistance to plant. Transformed calli were selected with a frequency of 4 calli for 10⁶ treated protoplasts. DNA was extracted from kanamycin resistant calli. Analysis of this DNA shows the presence of the NPTII gene.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA 1-naphtalenoacetic acid - NPT Neomycin phosphotransferase - PEG Polyethyleneglycol     

PEG- and electroporation-induced transformation in Nicotiana tabacum: influence of genotype on transformation frequencies

Summary Experimental parameters for direct gene transfer with recombinant DNA encoding neomycin phosphotransferase II (NPTII) under control of eukaryotic expression signals were established. The introduced gene was shown by the growth of transformants on media containing kanamycin, by genomic blotting and by assaying NPTII activity. Leaf protoplasts from three green genotypes of varieties xanthii and petit havanna, and from four plastome-encoded albino genotypes of Nicotiana tabacum were analyzed with respect to cell division kinetics and yield of kanamycin-tolerant colonies after direct gene transfer. No clear correlation was found between the time of onset of cell division and transformation frequency.     

Fate of plasmid DNA in transformation of Bacillus subtilis protoplasts

Summary Polyethylene glycol-treated protoplasts of B. subtilis can be transformed by plasmid DNA at very high frequencies (Chang and Cohen 1979). From analysis of plasmid mediated transformation of transformation-deficient mutants it appeared that mutants, reduced in the transformation by plasmid DNA in the competent state, were plasmid transformation-proficient when transformed as protoplasts. By means of CsCl-gradient centrifugation of re-extracted plasmid DNA it could be demonstrated that plasmid DNA enters the protoplasts in the double-stranded form. In addition, sucrose gradient centrifugation of the re-extracted plasmid DNA showed that the entered DNA is predominantly present as covalently closed circular DNA. The efficiency of plasmid transformation in protoplasts was found to be close to one (each plasmid molecule having entered into the protoplasts gives rise to a transformed cell). This is in good agreement with the observation that little, if any, damage is done to this DNA during or after entry into protoplasts.     

Factors affecting transient gene expression in electroporated Glycine max protoplasts

Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 F and a protoplast concentration of 1–3 × 10⁶/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - PEG polyethylene glycol     

Plasmid transformation in Bacillus subtilis: Fate of plasmid DNA

Summary Only multimeric, and not monomeric forms of B. subtilis plasmids can transform B. subtilis cells (Canosi et al. 1978). This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation. Competent cells of B. subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA. Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation. No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA, Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products. However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA.We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure. Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.Some of these results were presented at the 5th European Meeting on Bacterial Transformation and Transfection, September 1980, Florence     

Effect of pol III dependent elements on CAT reporter gene transcription in Nicotiana tabacum protoplasts

ABSTRACT

The effect of RNA polymerase III promoter elements on the expression of RNA polymerase II dependent genes was evaluated. The influence of tRNA(Tyr) gene, derived from Nicotiana rustica, on the expression of CAT gene, driven by CaMV35S, was tested in transformation experiments using Nicotiana tabacum protoplasts. Five “tRNA(Tyr) gene-CaMV35S?d combinations, differing in the position and orientation of the RNA polymerase III dependent gene, were utilised. Transient gene expression was evaluated by HPLC analysis. CAT expression increased with only two plasmidic constructs compared with control. Our results suggest that RNA polymerase III promoter elements exert a position- and orientation-dependent enhancer effect in Nicotiana tabacum protoplasts.     

Experession and integration of genes introduced into highly synchronized plant protoplasts

Summary Chimaeric genes containing the chloramphenicol acetyltransferase (CAT) coding sequence were introduced into protoplasts of suspension-cultured tobacco cells using improved conditions of electroporation (Okada et al. 1986). CAT activity became detectable in the protoplasts within 3 h, was maximal during a period of 18–36 h after electroporation, and then declined gradually. Alpha-amanitin added to the medium abolished the transient expression of the CAT gene. The closed circular form of input DNA was as effective as the linear form for the transient expression. The suspension culture was treated with aphidicolin, and S, G₂, M and G₁ phases were identified in the highly synchronized cell cycle obtained by releasing the cells from the inhibition of DNA synthesis. When a chimacric CAT gene was introduced into M phase protoplasts prepared from the synchronized culture, the transient expression of the CAT gene was 3–4 times higher than when it was introduced into protoplasts of other cell cycle phases. The frequency of stable transformation with a chimaeric neomycin phosphotransferase II gene was studied using the same system. G-418-resistant transformants were obtained from M phase protoplasts at frequencies 2–8 times those obtained from protoplasts at other cell cycle phases. The results indicate that the absence of the nuclear membrane in mitotic cells favours delivery to the nucleus of exogenous DNA introduced into the cytoplasm.     

Production of transformed soybean plants by electroporation of protoplasts

Protoplasts obtained from immature seeds of Glycine max (L.) Merr. cv. Clark 63 (soybean) were electroporated with DNA carrying either the kanamycin or hygromycin resistance genes and the reporter genes, β-glucuronidase or opine synthesis. Antibiotic resistance could be selected for at the frequency of about one colony from 2 000 electroporated protoplasts (0.05%) and the reporter genes were expressed in from 75 to 90% of the selected colonies. Antibiotic resistance and reporter gene expression were not found in untreated protoplasts. Shoots formed within about 5 months after a number of transfers of selected portions of the callus on the regeneration medium. The shoots have been rooted to form plants which express the reporter genes and contain the transforming DNA in their leaves as shown by Southern hybridization. The reporter genes are expressed (opine synthesis) in all leaves and roots and NPTII activity was present in all leaves, indicating that the transformed plants are not chimeral. We expect these plants to set seed since untransformed plants regenerated from protoplasts did. We can obtain shoots from several of the soybean genotypes we have used so far. Thus, we should have a method for the efficient production of nonchimeral, transformed plants of the important crop plant soybean.     

Efficient transfer of base changes from a vector to the rice genome by homologous recombination: involvement of heteroduplex formation and mismatch correction

Gene targeting refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome by homologous recombination. Through a gene-targeting procedure with positive–negative selection, we previously reported the generation of fertile transgenic rice plants with a positive marker inserted into the Adh2 gene by using an Agrobacterium-mediated transformation vector containing the positive marker flanked by two 6-kb homologous segments for recombination. We describe here that base changes within the homologous segments in the vector could be efficiently transferred into the corresponding genomic sequences of rice recombinants. Interestingly, a few sequences from the host genome were flanked by the changed sequences derived from the vector in most of the recombinants. Because a single-stranded T-DNA molecule in Agrobacterium-mediated transformation is imported into the plant nucleus and becomes double-stranded, both single-stranded and double-stranded T-DNA intermediates can serve in gene-targeting processes. Several alternative models, including the occurrence of the mismatch correction of heteroduplex molecules formed between the genomic DNA and either a single-stranded or double-stranded T-DNA intermediate, are compared to explain the observation, and implications for the modification of endogenous genes for functional genomic analysis by gene targeting are discussed.     

Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation.

We determined whether T-DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination. For the detection of such recombination events different mutant versions of a NPTII construct were used. In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains. Each strain contained a different T-DNA, one carrying a 5' deleted NPTII gene and the other a NPTII gene with a 3' deletion. A restored NPTII gene was found in 1-4% of the protoplasts that had been cotransformed with both T-DNAs. Restoration of the NPTII gene could only be the consequence of homologous recombination between the two different T-DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments. In subsequent experiments was investigated the potential use of Agrobacterium for gene targeting in plants. A transgenic tobacco line with a T-DNA insertion carrying a defective NPTII gene with a 3' deletion was transformed via Agrobacterium with a T-DNA containing a defective NPTII repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTII gene integrated in their genome. In one of these lines the defective NPTII gene at the target locus had been properly restored. Our results show that in plants recombination can occur between a chromosomal locus and a homologous T-DNA introduced via A. tumefaciens. This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome.     

Rapid production of fertile transgenic barley (Hordeum vulgare L.) by direct gene transfer to primary callus-derived protoplasts

Protoplasts were isolated from primary calli of barley (Hordeum vulgare L.), and an antibiotic (G418) resistance gene was introduced into these protoplasts using a polyethylene glycol (PEG) DNA uptake method. Sixty-four G418 resistant calli were obtained in nine experiments, and two plants were regenerated from these calli. NPTII ELISA and Southern analysis indicated that the G418 resistance gene was introduced and expressed in two T₀ plants. These plants set seed and the introduced gene was transmitted to T₁ plants. These results suggest that our transformation system using primary callus-derived protoplasts is a useful method for the generation of transgenic barley. Received: 14 November 1997 / Revision received: 12 March 1998 / Accepted: 24 April 1998     

Hybrid genes in the analysis of transformation conditions. 3. Temporal/spatial fate of NPTII gene integration,its inheritance and factors affecting these processes in Nicotiana plumbaginifolia

Freshly isolated haploid mesophyll protoplasts of Nicotiana plumbaginifolia were transformed for kanamycin resistance. In 38% of the 224 transformants analysed, transmission of the NPTII gene occurred as a homozygous trait, while 62% of the transformants were heterozygous for the trait. In the first case, the foreign DNA integration predominantly (95%) resulted in monogenic inheritance. The second group was characterized by a significant (46%) proportion of multiple insertions. However, there was no clear-cut difference in the integration pattern between the two groups. Furthermore, transformation rates were increased by 4- to 10-fold when transformed diploid protoplasts were treated with UV light or with 3-aminobenzamide. The number of insertion sites was also increased by these treatments. These results shed further light on the fate of the foreign DNA in transformed plants and on means to control or manipulate the integration event(s).     

Evaluation of an ELISA assay for rapid detection and quantification of neomycin phosphotransferase II in transgenic plants

Neomycin phosphotransferase II (neo) is a selectable marker gene used extensively in plant transformation experiments. Here we evaluate immunological detection of its gene product (NPTII) as an alternative to widely used radioactive assays. We have taken a commercially available non-radioactive NPTII Enzyme linked-Immunosorbant Assay (ELISA) kit, modified the protocol for application to plant tissues, and used it to quantify levels of NPTII protein in transformed plants. The ELISA proved safe, economical and convenient to reliably screen and quantify NPTII protein in large numbers of plant samples. The sensitivity of the ELISA for NPTII detection in tobacco plants is at least an order of magnitude greater than a widely used radioactive gel assay. Using three replicates per sample, standard errors are low and the assay is highly reproducibleover time for tissue-cultured tobacco. However, background readings varied with plant species, and also with plant age for untransformed glasshouse-grown tobacco. It is therefore essential to ensure that untransformed controls are closely matched to test plant.     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40–50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kbHind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells ofEscherichia coli, and colonies were recovered that carried pGEM withHind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transformE. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning intoE. coli.     

A Protoplast Transformation System for Gene Deletion and Complementation in Sclerotinia sclerotiorum

Protoplast transformation is an important technique for establishing a mutation library and determining gene function for Sclerotinia sclerotiorum and other plant pathogenic fungi. In this study, we determined the effect of various conditions on preparation of protoplasts for transformation. These conditions included the age of the culture providing the hyphae to be digested; enzyme composition, buffer solution and concentration; and digestion time and temperature. The optimum conditions for preparing protoplasts were as follows: 10 ml of enzyme solution (1.5% lysing enzyme in 0.8 m mannitol and citric acid‐sodium citrate buffer) reacting with 0.1 g of hyphae (cultured for 36 h) at 30°C for 2.5 h. The transformation efficiency was 60–85 transformants per microgram of DNA. In addition, an expression vector for gene complementation was constructed, and an additional dominant selectable marker (neomycin) was demonstrated. To verify the reliability of the expression vector, we constructed and transformed the complementation vector of Shk1 for gene complementation based on the Shk1 deletion mutant △Shk1. The results showed that the expression level and biological phenotypes of Shk1 were restored in the complementary strain △Shk1+Shk1. The techniques and procedures described will improve our ability to study gene function in S. sclerotiorum and are likely applicable to other plant pathogens.