Electroporation of rapeseed protoplasts – transient and stable transformation

Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β-glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead-type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10⁻⁵ and a relative transformation frequency of 0.3% could be achieved. X-ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.     

Ketoisophorone transformation by Marchantia polymorpha and Nicotiana tabacum cultured cells

Stereospecific olefin (C=C) and carbonyl (C=O) reduction of the readily available prochiral compound ketoisophorone (2,2,6-trimethyl-2-cyclohexene-1,4-dione) (1) by Marchantia polymorpha and Nicotiana tabacum cell suspension cultures produce the chiral products (6R)-levodione (2), (4R,5S)-4-hydroxy-3,3,5-trimethylcyclohexanone (3), and (4R,6R)-actinol (4) as well as the minor components (4R)-hydroxyisophorone (5) and (4S)-phorenol (6).     

High frequency transformation of Streptomyces niveus protoplasts by plasmid DNA

H.A. HUSSAIN AND D.A. RITCHIE. 1991. A procedure has been developed for transforming protoplasts of the novobiocin producing strain Streptomyces niveus at high frequency. This required the isolation of strains LH13 and LH20 defective in DNA restriction from the wild type (ATCC 19793) which is transformed at very low frequencies. The LH13 and LH20 derivatives were obtained by curing pIJ702 DNA from the few S. niveus transformed protoplasts obtained by transformation of the wild type with high concentrations of pIJ702 DNA. Protoplasts of S. niveus strains LH13 and LH20 produced about 10⁶ transformants/μg DNA with modified pIJ702 DNA derived by replication in S. niveus. Unmodified DNA (derived from replication in S. lividans ) from a series of pIJ101, SCP2 and pSN2-based derivatives, gave transformation frequencies in the range of 10²-10³ transformants/μg DNA. Optimal conditions for the formation and transformation of S. niveus protoplasts are described.     

Transformation of barley by microinjection into isolated zygote protoplasts

Barley zygote protoplasts were mechanically isolated, embedded in agarose droplets, and microinjected with a rice actin promoter Act1–gusA-nos gene construct. On average 62% of the cells survived the injection and of these 55% continued development into embryo-like structures and eventually to plants. PCR screening for the presence of a 307-bp fragment in the middle of the gusA gene showed that on average 21% of the derived structures contained this fragment. However, among the hundreds of injected zygotes, derived structures and regenerants we only found significant GUS expression in two cases (embryo-like structures nine days after injection). Two lines of green plants, derived from zygotes microinjected with linearized plasmid (line A147-1) or an isolated Act1–gusA-nos gene cassette (line A166-h) proved to be transgenic. Line A147-1 appeared to contain a single and intact copy of the expression cassette but a PCR based progeny analysis indicated the presence of additional shorter fragments of the cassette. Line A166-h appeared to contain a single fragment of the gusA gene that was transferred to the progeny as a single Mendelian trait. One additional fragment of the gusA gene was identified in this line. The present data show that transformation of barley by microinjection of DNA into isolated zygotes is feasible but also that gene expression rarely is achieved, possibly due to degradation of the introduced DNA.     

High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA.

Summary A highly efficient method for transformation of Bacillus subtilis by plasmid DNA is reported. The procedure, which involves polyethylene glycolinduced DNA uptake by protoplasts and subsequent regeneration of the bacterial cell wall, yields up to 80% transformants with an efficiency of 4x10⁷ transformants per g of supercoiled DNA. Plasmids constructed by in vitro ligation or endonuclease-generated fragments of linear plasmid DNA can also transform PEG-treated protoplasts, but at a lower frequency.     

High frequency transformation of Streptomyces niveus protoplasts by plasmid DNA.

A procedure has been developed for transforming protoplasts of the novobiocin producing strain Streptomyces niveus at high frequency. This required the isolation of strains LH13 and LH20 defective in DNA restriction from the wild type (ATCC 19793) which is transformed at very low frequencies. The LH13 and LH20 derivatives were obtained by curing pIJ702 DNA from the few S. niveus transformed protoplasts obtained by transformation of the wild type with high concentrations of pIJ702 DNA. Protoplasts of S. niveus strains LH13 and LH20 produced about 10(6) transformants/micrograms DNA with modified pIJ702 DNA derived by replication in S. niveus. Unmodified DNA (derived from replication in S: lividans) from a series of pIJ101, SCP2 and pSN2-based derivatives, gave transformation frequencies in the range of 10(2)-10(3) transformants/micrograms DNA. Optimal conditions for the formation and transformation of S. niveus protoplasts are described.     

High yield and stable transformation of the unicellular green alga Acetabularia by microinjection of SV40 DNA and pSV2neo

SV40 DNA and pSV2neo were microinjected into isolated nuclei of Acetabularia mediterranea. The injected nuclei were implanted into anucleate cell fragments of the same species. Such combinations not only survived but also formed progeny. The F₁, F₂ and F₃ generations of these combinations were analyzed. In the case of SV40-treated cells T-antigen was expressed and accumulated in the nuclei of all three generations studied as shown by indirect immunofluorescence. Nuclear exchange experiments revealed expression of the T-antigen only if a transformed nucleus but not if only a transformed cytoplasm was involved. Transformation by pSV2neo, a chimeric gene with a selectable marker was demonstrated by the induction of G-418 resistance as well as immunofluorescence. Genomic DNA was isolated from gametes, originating in cysts from the F₁, F₂ and F₃ generations of injected cells, and subjected to Southern analysis. These experiments demonstrated that both types of DNA are integrated into the host genome.     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

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     

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg^-1 H₂O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca²⁺ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP bromocresol purple - FDA fluoresceindiacetate - MES 2-(N-morpholino) ethanesulfonic acid - PEG polyethyleneglycol     

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     

Electroporation: Mechanism and transient expression, stable transformation and biological effects in plant protoplasts

The present state of knowledge about the mechanistic and theoretical aspects of electroporation is summarized. Parameters affecting the efficiency of transient expression and stable transformation of electroporated plant protoplasts are rewieved. Biological effects of electroporation on plant protoplasts are described.     

Factors influencing stable transformation of maize protoplasts by electroporation

Two chimaeric genes, containing the promoter of the 35S gene of cauliflower mosaic virus coupled to neomycin phosphotransferase (35S-NPT-NOS) or to hygromycin phosphotransferase (35S-HPT-NOS) have been stably transferred to maize (Zea mays cv. Black Mexican sweet) cells by electroporation. Transformation frequencies of 7.6×10^-4 and 8×10^-4, respectively, (based on the number of surviving cells that divided) were obtained with four pulses of 1 ms duration using 400 V capacitive discharge. Cells transformed to kanamycin-resistance and hygromycin-resistance subsequently multiplied to form callus. Southern blot analysis demonstrated the integration of the selectable marker genes, neomycin or hygromycin phosphotransferase, with single or multiple copy numbers. The blots from DNA of hygromycin-resistant calli also suggested the formation of plasmid concatemers.     

Gene regulation in intertypic heterokaryons of Solanum tuberosum and Nicotiana tabacum tissue protoplasts

Activities of the ß-glucuronidase (GUS) reporter enzyme were evaluated in transgenic plants, protoplasts, and intertypic heterokaryons of Solanum tuberosum and Nicotiana tabacum. With GUS under control of the promoter of the cauliflower-mosaicvirus 35S RNA gene (CaMV), activities of the enzyme were nearly evenly distributed over the tissues of plants grown in vitro. Activities in microtubers of potato plants with the reporterenzyme under control of the promoter of granule-bound starch synthase (GBSS) from S. tuberosum were higher than in leaves. The CaMV-GUS construct present in leaf protoplasts showed an increased expression in biochemical and cytochemical assays after fusion with wildtype-tuber protoplasts. Such an increase was not observed in the case of the GBSS-GUS constructs. It was concluded that nuclei in plant heterokaryons are being influenced by the fusion partner, but that repressive, trans-acting regulation factors in leaf protoplasts possibly prevent an increase of the expression of the chimeric GBSS-gene in heterokaryons.     

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.