Homologous recombination between plasmid DNA molecules in maize protoplasts

Summary The requirements for homologous recombination between plasmid DNA molecules have been studied using the PEG (polyethylene glycol)-mediated transformation system of maize (Zea mays L.) protoplasts coupled with the transient expression assay for -glucuronidase (GUS). Two plasmids were introduced into maize protoplasts; one plasmid (pB×26) contained a genomic clone of the Adh1 maize gene; the other plasmid (piGUS) was a promoterless construction containing part of intron A of the Adhl gene fused to the gusA coding sequence. Thus, the two vectors shared an effective homologous region consisting of a 459 by (Hindlll—PvuII) fragment of the yAdh1 intron A sequence. An active gusA fusion gene would result upon homologous recombination between the plasmids within the intron A sequence, and indeed GUS activity was observed in extracts following co-transformation of maize protoplasts with the two plasmids. The presence of recombinant DNA molecules in protoplast DNA isolated 1 day after co-transformation was verified using polymerase chain reactions (PCR) and Southern blots. For efficient homologous recombination, both plasmids had to be linearized. The recombination reaction was induced by restriction of the plasmid molecules either inside the effective homologous region or at the borders of the intron sequence. However, the presence of even small, terminal, nonhomologous sequences at the 3 end of the pB×26 fragment inhibited the recombination reaction. Also, both ends of the linearized piGUS DNA molecules were involved in the recombination reaction. The results revealed some features of homologous recombination reactions occurring in plant cells which cannot be accommodated by mechanisms postulated for similar reactions in animal system and in lower eukaryotes.     

Transformation of tobacco protoplasts with DNA entrapped in pH-sensitive liposomes

A system for the transformation of tobacco mesophyll protoplasts using pH-sensitive liposomes was developed. Plasmid DNA (plGVneo23) encoding the NPT-II gene for kanamycin resistance was entrapped in pH-sensitive liposomes composed of dioleolphosphatidylethanolamine, cholesterol and oleic acid. These liposomes release their contents at low pH and are capable of delivering their contents into the cytoplasm of protoplasts. Kanamycin-resistant colonies were reproducibly recovered from transformed protoplasts at an average frequency of 1.62×10^-4 at pH 7.5. Plants regenerated from transformed cell lines were normal in appearance and were fertile. NPT-II activity was detected in leaf extracts of transformed, kanamycin-resistant plants and the presence of NPT-II DNA in the tobacco genome was shown by Southern blots. Analysis of self-pollinations and reciprocal crosses to non-transformed plants indicated that kanamycin resistance segregated as a dominant nuclear marker. Co-transformation of protoplasts with liposomes containing two selectable markers indicated that co-transformation occurred with a frequency of approximately 23%.Abbreviations DOPE dioleoylphosphatidylethanolamine - DOPC dioleoylphosphatidylcholine - Chol cholesterol - OA oleic acid - PEG polyethylene glycol 6000 - NAA -napthaleneacetic acid - BAP 6-benzylaminopurine     

Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.)

We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 21 or 11 ratio, led to 0.8 × 10^–5 and 1.6 × 10^–5 resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and -glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.Abbreviations PEG polyethylene glycol - T₀ regenerated transgenic plant - GUS -glucuronidase - CaMV cauliflower mosaic virus - ARE anaerobic responsive element - OCS octopine synthase - T₁ first generation progeny of transgenic plants     

Optimization of polyethylene glycol mediated transient gene expression in pea protoplasts

We have investigated factors influencing polyethylene glycol mediated DNA uptake and ß-glucuronidase expression in pea (Pisum sativum L.) protoplasts. It was found that for optimal \-glucuronidase expression the molecular weight and concentration of polyethylene glycol should be 4000 and 20%, respectively. The amount of plasmid DNA should be 25 g per 5×10⁵ protoplasts in each treatment, and the concentration of Mg²⁺ in the transformation buffer should be 15 mM. The optimized protocol was applicable to all four pea cultivars tested.Abbreviations FDA fluorescein diacetate - GUS ß-glucuronidase - MU 4-methylumbelliferone - MUG 4-methyl umbelliferyl glucuronide - MW molecular weight - PEG polyethylene glycol - X-Gluc 5-bromo-4-chloro-3-indolyl glucuronide     

Transformation of sugarcane protoplasts by direct uptake of a selectable chimaeric gene

Sugarcane protoplasts were transformed to kanamycin resistance at a frequency of approximately 8 in 10⁷ following PEG-induced uptake of Sma1 linearised pABD1 plasmid. DNA-treated protoplasts were cultured in agarose droplets, and protoplast-derived transformants selected on 80 g ml^–1 kanamycin. Transformed tissues maintained on this level of antibiotic expressed APH(3)II activity, and contained DNA that hybridised to a probe with the APH(3)II gene.Abbreviations APH(3)II aminoglycoside phosphotransferase - PEG polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid     

Transformation ofStreptomyces avermitilis by plasmid DNA

Summary Polyethylene glycol (PEG) efficiently mediated the transformation ofStreptomyces avermitilis protoplasts by plasmid DNA to yield 10⁷ transformants per g of plasmid DNA. Under conditios in which the maximum transformation frequency was observed, the cotransformation frequency exceeded 10%. The number of transformants increased linearly with the amount of DNA and number ofS. avermitilis protoplasts. Relaxed and supercoiled, but not linear DNA transformed protoplasts efficiently. Dimethyl sulfoxide (DMSO)-mediated transformation of protoplasts was 1000-fold less efficient. PEG and, less efficiently, DMSO also mediated the transformation of whole cells ofS. avermitilis by DNA.     

Electric field mediated stable transformation of carrot protoplasts with naked DNA

We have developed an electroporation procedure for the transformation of carrot protoplasts with Ti-plasmid DNA from Agrobacterium tumefaciens. The uptake of pTiC58 into carrot protoplasts was mediated by high voltage electrical pulses at field strengths from 0.5 to 3.8 kV/cm. Protoplast regeneration, somatic embryogenesis and plantlet regeneration were unaffected by the electroporation conditions selected for DNA uptake. Uptake of plasmid pTiC58 resulted in hormone independent regeneration of carrot protoplasts. Transformed somatic embryos were detected in carrot cultures 45 days after electroporation. The transformed somatic embryos developed into teratomas which synthesized nopaline. Hybridization was obtained between a labeled T-DNA fragment from pTiC58 and DNA fragments from 4 month old teratomas regenerated from electro-transformed protoplasts. Based on the number of somatic embryos regenerated after electro-transformation, a frequency of 1.6×10² transformants/10⁴ somatic embryos/g pTiC58 DNA was obtained.Abbreviations PEG polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - MES morpholinoethane sulfonic acid - DMSO dimethyl sulfoxide - HSV Herpes Simplex virus - TK thymidine kinase     

Mechanism and optimized conditions for PEG mediated DNA transfection into plant protoplasts

Experimental conditions influencing DNA uptake efficiency by maize protoplasts in polyethyleneglycol (PEG) mediated transfection experiments have been studied systematically. The data provide evidence that the extracellular DNA is precipitated efficiently by combined action of PEG together with divalent cations and DNA is taken up by the plant protoplasts in the precipitated form. The particle size is strongly effected by the pH of the PEG solution. At optimal pH 6– 6.5 a very fine and homogenous precipitate forms in presence of Ca²⁺ and Mg²⁺ ions and is efficiently incorporated by maize and rice protoplasts.Abbreviations CAT Chloramphenicolacetyl transferase - DAPI 4,6-Diamidino-2-Phenylindole - HEPES N-2-Hydroxyethylpiperazine-N-2- ethanesulfonic acid - MES 2(N-Morpholino)ethanesul fonic acid - PEG Polyethyleneglycol - SW seawater     

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 10⁵ protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MU methylumbelliferone - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid     

Sid: a Mendelian locus controlling thylakoid membrane disassembly in senescing leaves of Festuca pratensis

Summary A spontaneous mutation arising in Festuca pratensis has the effect of stabilizing the pigmentproteolipid complexes of thylakoid membranes so that leaf tissue does not turn yellow during senescence. Inheritance of the non-yellowing character was analysed in crosses between the wild-type cultivar Rossa and a mutant line Bf 993. Electrophoretic variants of cytoplasmic phosphoglucoisomerase coded by alleles of the nuclear gene Pgi-2 were used to identify hybrids during intercrossing. About 96% of the F₁ progeny were heterozygous and all were phenotypically yellowing. In the F₂ generation yellow green segregated in a ratio of 2.141, not significantly different from 31. In the backcross between F₁ and Bf 993 the ratio was 11 yellow green. There was no indication of linkage to Pgi-2. Senescence of detached Bf 993 and Rossa leaves was compared with that of the F₁ hybrid. The hybrid behaved in an essentially identical fashion to the wildtype parent, and in marked contrast to the mutant, in all aspects of the senescence syndrome investigated, including loss of chlorophyll, carotenoids and the light-harvesting chlorophyll-protein of thylakoid membranes, and elevation of the particulate protein chlorophyll ratio in the terminal stages. It is concluded that there exists in Festuca pratensis a nuclear gene, designated Sid (senescence-induced degradation) which regulates turnover of hydrophobic components of photosynthetic membranes in ageing leaf tissue and which occurs in at least two allelic forms, y (yellow) dominant over g (green).Abbreviations PGI phosphoglucoisomerase - Pgi nuclear gene coding for PGI - TSH tris buffer containing 2-mercaptoethanol - SDS sodium dodecyl sulphate - Chl chlorophyll - LHCP light-harvesting chlorophyll a/b binding protein - ELISA enzyme-linked immunosorbent assay     

Efficient transient expression and transformation of PEG-mediated gene uptake into mesophyll protoplasts of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

PEG-mediated transformation was used for gene delivery and evaluation of various parameters affecting the transient expression of a gene for ß-glucuronidase (gus) in mesophyll protoplasts of Capsicum annuum. Transient expression was found to be dependent on PEG concentration and exposure time of plasmid DNA to protoplasts as well as the amount of plasmid DNA. Maximum GUS activity was obtained when protoplasts were applied to 40% concentration and molecular weight was 6,000 of PEG solution with 30 min of exposure time. Protoplasts of pepper were transformed with a vector, pCAMBIA::Ac, which contained a pCAMBIA1302 T-DNA vector carrying a maize transposable element, Ac (activator), a selection marker HPT (hygromycin phosphotransferase), and a GFP-coding region driven by the 35S promoter in the presence of PEG. Approximately 30% of the protoplasts expressed GFP. Visibly transformed colonies were obtained from protoplasts after 2 months of culture and GFP was expressed. Southern hybridization confirmed the presence of Ac in the pepper genome.     

Restriction fragment length polymorphism (RFLP) analyses of plants produced by in vitro anther culture of Solanum chacoense Bitt

Summary Green mesophyll protoplasts of the dihaploid potato line 1982 (Solanum tuberosum L.) were fused with herbicide-bleached mesophyll protoplasts of the dihaploid potato line 679 using a polyethylene glycol protocol. Heterokaryons were identified under a fluorescence microscope using the dual fluorescence of carboxyfluorescein-stained, herbicide-bleached protoplasts and the autofluorescence of green mesophyll protoplasts. About 20% of the protoplasts survived the fusion treatment, and the fusion frequency was 3%–4%. Unfused and fused protoplasts were mass cultured for 6 weeks after which vigorously growing calli were selected and transferred to shoot regeneration medium. Somatic hybrids were identified by a combination of five isozyme markers, and the ploidy level was determined by flow cytometry. Out of 15 calli that regenerated shoots, 6 plants derived from 2 different calli were identified as hexaploid somatic hybrids, while one morphologically deviant plant from a third callus was identified as a mixoploid that had lost some enzyme markers after 4 months of culturing.     

Transient gene expression in electroporated banana (Musa spp., cv. ‘Bluggoe’, ABB group) protoplasts isolated from regenerable embryogenetic cell suspensions

Summary Electroporation conditions were established for transient expression of introduced DNA in banana (Musa spp., cv. Bluggoe) protoplasts isolated from regenerable embryogenic cell suspensions. The following parameters were found to be highly influential: electroporation buffer, polyethylene glycol treatment and its duration before electroporation, use of a heat shock, and chimaeric gene constructs. The maximum frequency of DNA introduction as detected by an in situ assay for transient expression of the uidA gene, amounted to 1.8% of total protoplasts. Since plants have recently been regenerated from banana protoplasts at a high frequency, the present results may contribute to the production of transgenic banana.Abbreviations AMV alfalfa mosaic virus - CaMV cauliflower mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - EGTA ethylene glycol-O-O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid - GUS glucuronidase - HEPES 4-(2-nydroxyethyl)piperazine-1-etnanesulfonic acid - MES 2-morpholinoethanesulfonic acid - MS Murashige-Skoog - NOS nopaline synthase - NFTII neomycin phosphotransferase - PEG polyethylene glycol - TGE transient GUS expression - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronic acid     

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using Onozuka cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of -amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley -amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.     

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     

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.     

PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca

ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - NOS nopaline synthase - CCC covalently closed circular DNA - L linear DNA - PEG polyethylene glycol - HS herring sperm DNA - P protoplasts - PCM protoplast culture medium - MES morpholinoethane-sulfonic acid - Cm chloramphenicol - Ac acetylated - MUG 4-methyl umbelliferyl ß-D-glucuronide - TLC thin layer chromatography     

Simplified procedure for transient transformation of plant protoplasts using polyethylene glycol treatment

A modification of the polyethylene glycol-mediated transformation procedure which eliminates the manual polyethylene glycol dilution step is presented. A transformation mixture of protoplasts, DNA and polyethylene glycol was plated directly onto agarose blocks after incubation. The procedure was simple and fast, thereby suitable for screening the gene activity of large numbers of plasmid constructions. It has been tested for both maize and rice protoplasts.