Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination

 For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R. pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA ^- mutant of R. pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu⁺ transformants (μg pRPLeu10 DNA)^-1 (1×10⁶ viable protoplasts)^-1. Blot analysis of DNA from the transformants showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus. Received: 2 October 1995/Received last revision: 5 December 1995/Accepted: 11 December 1995     

DNA-mediated transformation system in a bipolar basidiomycete, <Emphasis Type="Italic">Pholiota microspora</Emphasis> (<Emphasis Type="Italic">P. nameko</Emphasis>)

We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was about 88.8 transformants/μg pMBsip2 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation was about 122.4 transformants/μg pMBhph1 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.     

A simple method for isolation,liquid culture,transformation and regeneration of Arabidopsis thaliana protoplasts

Summary An efficient technique was developed for the isolation, culture, transformation and regeneration of protoplasts derived from auxin conditioned Arabidopsis root cultures. On an average 30% of root protoplasts underwent cell division in liquid culture and formed somatic embryolike structures which regenerated to plants without embedding in Ca²⁺-alginate. The protoplast protocol was applicable to different landraces of Arabidopsis thaliana (L.) Heynh., such as RLD, Columbia or C24. PEG-mediated DNA uptake into protoplasts using different uidA reporter gene constructs yielded transient gene expression in over 25% of treated cells indicating that root-derived protoplasts are suitable recipients for transformation.Abbreviations BA 6-benzylaminopurine - 2,4D 2,4dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ISA indole-3buryric acid - IPAR 6-(,-dimethylallylamino)purine riboside - NAA naphthaleneacetic acid - uidA ß-glucuronidase gene - GUS ß-glucuronidase enzyme - CaMV Cauliflower Mosaic Virus - nos nopaline synthase - MES 2[N-morpholino]ethane-sulfonicacid - PEG polyethylene glycol - X-gluc 5bromo-4-chloro-3-indolyl glucuronide - MUG 4-methyl umbelliferyl glucuronide - MU 4-methylumbelliferone     

Genetic introduction of foreign genes to <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> by restriction enzyme-mediated integration

Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing 1 μg of the HindIII-digested plasmid DNA and 10⁶ mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10–40 hygromycin-resistant transformants. Successful transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants. To date, this is the first report on the transformation of P. eryngii by REMI technique.     

Transfer of hygromycin resistance into Brassica napus using total DNA of a transgenic B. nigra line

The successful transfer of a marker gene (hpt gene) from Brassica nigra into B. napus via direct gene transfer was demonstrated. Total DNA was isolated from a hygromycin-resistant callus line, which contained three to five copies of the hpt gene. This line had been produced via direct gene transfer with the hygromycin resistance-conferring plasmid pGL2. The treatment of B. napus protoplasts with genomic DNA of B. nigra (Hyg^R) resulted in relative transformation frequencies of 0.1–0.4%. Similar transformation rates were obtained in direct gene transfer experiments using B. napus protoplasts and plasmid pGL2.     

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     

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     

Hybrid genes in the analysis of transformation conditions

Direct gene transfer into plant protoplasts has been recently developed, and conditions for high frequency transformation of SR₁ tobacco protoplasts established. In this paper we analyse numerous transformation parameters in a comparative study on SR₁ Nicotiana tabacum and N. plumbaginifolia, and report on a simple chemical technique for very efficient protoplast transformation. It is based on the synergistic interaction of MgCl₂ and PEG. The technique yielded up to 1400 transformants per 3×10⁵ treated N. tabacum protoplasts (up to 4.8% of the survivors, late selected clones). Using N. plumbaginifolia, the frequencies were 10-fold lower, indicating that the competence for transformation has a species-specific component.Abbreviations PEG polyethyleneglycol - RTF relative transformation frequency - ATF absolute transformation frequency     

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.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Avena sativa L. and of mesophyll and guard cell protoplasts of Vicia faba L. were found to be 1.9±0.9·10^-6, 3.2±2.2·10^-6, and 0.8±0.7·10^-6 cm·bar^-1·s^-1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe.Abbreviations GCP guard cell protoplast - Lp hydraulic conductivity - MCP mesophyll cell protoplast     

Transgenic plants of Agrostis alba obtained by electroporation-mediated direct gene transfer into protoplasts

A system for genetic transformation of Agrostis alba plants by electroporation-mediated DNA transfer to protoplasts is described. The npt II gene was used as a selectable marker. Selection with 20 mg/1 G418 (geneticin) yielded a total of over 50 resistant cell colonies from three independent experiments. Overall frequency of resistant colony formation was 1–3 × 10^–6 based on the number of protoplasts plated and 1–2 × 10^–5 based on the number of cell colonies recovered. Subsequent subcultures led to the development of plants with an apparently normal morphology. DNA analysis (PCR and Southern hybridization) and enzymatic analysis showed that the G418 resistant plants carried the transgene and expressed it. This is the first successful genetic transformation of an economically important temperate grass, Agrostis.     

Identification of the aph IV gene from protoplast-derived maize plants by a simple nonradioactive DNA hybridization method

Summary Protoplast-derived, transformed maize plants were evaluated by Southern analysis for the presence of the aph IV gene which codes for resistance to the antibiotic, hygromycin B. This gene was used as a selectable marker for the transformation of maize protoplasts. Southern analysis was performed with fluorescein-labeled probe DNA. A new method for labeling molecular weight markers with fluorescein-N⁶ is presented. The nonradioactive Southern analysis method is compared to the radioactive method and the results show that the nonradioactive method is as sensitive as the radioactive method.     

Transfection of protoplasts from Streptomyces lividans 66 with actinophage SH10 DNA

Summary The slightly modified procedure for the transformation of protoplasts of S. coelicolor A3 (2) with SCP2 plasmid DNA and polyethylenglycol (PEG) (Bibb et al., 1978) was extented to infection of protoplasts of S. lividans 66 with actinophage SH10 DNA (Klaus et al., 1979).Maximal yield of transfected protoplasts was obtained at 20% PEG, 3 mM sodium-citrate and 150 mM NaCl final concentrations. The efficiency of transfection was determined to be about 2×10^-8 to 2×10^-7. The average value of competent protoplasts was about 1–2×10^-4 of regenerating protoplasts.In comparison with outgrowing spores infected with phage particles the average burst size of transfected protoplasts was reduced from 100 to 10 pfu/infected cell, the latent period prolonged from 45 min to 120 min and the rise period was not affected.     

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.     

Development of Chainia as a host for xylanase gene cloning : Evidence for occurrence of a restriction-modification system

Summary Conditions for genetic transformation of the xylanase-negative (X^–) strain of Chainia with pIJ 702 were optimized. The growth of Chainia at 30°C for 36 – 40 h and addition of geletin (1%) to the medium resulted in the maximum yield of protoplasts and regeneration efficiency. Poor transformation efficiency of Chainia (X^–) protoplasts by native pIJ 702 versus improved efficiency (16 transformants ug^–1 of plasmid DNA) by prior heating of protoplasts at 42°C for 10 min suggests the occurrence of a restriction system in Chainia. Increased transformation efficiency by passage of the plasmid through Chainia together with the altered methylation status of the transformant plasmid presents evidence for the existence of an operative modification system in Chainia. Development of thiostrepton resistance and formation of me1amin pigment in Chainia (X^–) by transformation with pIJ 702 reveal that genes from Streptomyces can be functionally expressed by Chainia (X^–).(NCL Communication No. 6207)     

Efficient transformation ofMicromonospora melanosporea protoplasts byStreptomyces plasmid

An efficient and relatively simple procedure forMicromonospora melanosporea protoplast preparation and transformation is described. Transformation ofM. melanosporea protoplast by theStreptomyces plasmid pIJ702 was optimized by altering parameters affecting the formation, regeneration, and transformation of protoplasts. Improvement of regeneration medium resulted in relatively quick growth of transformants (only 7 days). As a result of these experiments we describe a new transformation method that has routinely yielded 10⁶ transformants/µg plasmid DNA.     

Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake

Summary Efficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.Abbreviations bar/PAT bialaphos resistance gene/phosphinotricin acetyltransferase - 2,4-D 2,4-di-chlorophenoxyacetic acid - dhfr/DHPR dihydrofolate reductase gene/enzyme - gus/GUS -glucuronidase gene/enzyme - hpt/HPT hygromycin phosphotransferase gene/enzyme - Kn kinetin - PEG polyethylene glycol - RH relative humidity     

Study of the phenomenon of marker rescue in plasmid transformation and transduction of intact cells, protoplasts and different rec mutants of Bacillus subtilis

Summary Marker rescue in plasmid transformation of competent cells of different rec mutants of B. subtilis was studied. In most cases the value of marker rescue decreased proportionally to reduction of plasmid transformation efficiency (although there were certain exceptions). Marker rescue was not observed either in plasmid transformation of protoplasts or in plasmid transduction of intact cells.Abbreviations Km ^R Kanamycin-resistant - Cm ^R Chloramphenicol-resistant