Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the <Emphasis Type="Italic">gpd</Emphasis> promoter for transformation in <Emphasis Type="Italic">Flammulina velutipes</Emphasis>

The glyceraldehyde-3-phosphate dehydrogenase gene of Flammulina velutipes was isolated. The complete gpd sequence (from ATG to TAA) was 1,489 bp in length and contained nine introns. The locations of these nine introns were similar to those of other basidiomycetes, which might reflect the evolutionary divergence of these mushrooms. The F. velutipes gpd gene was found to encode a protein of 339 amino acids and its putative amino acid sequence revealed a high similarity to an analogous protein deriving from other basidiomycetes. Results of Southern blot analysis suggested that there existed only one copy of the gpd gene in the genome of F. velutipes and that there was one typical TATA box and two CAAT boxes located in the 5 flanking region. The F. velutipes gpd promoter was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selection marker. Using the resulting construction, hph was efficiently transformed into F. velutipes by basidiospore electroporation. No false-positive antibiotic-resistant cultures were detected by PCR amplification and the hygromycin resistance trait was maintained stably during mitotic cell division for 3 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. This rapid and convenient electroporation procedure offers new prospects for the genetic manipulation and a tool for tagging genes of this important edible mushroom species. Sequence data will appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AF515622.     

Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells

Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3 were established within 6–8 weeks from 3–4 week old calli derived from mature seeds. Transgenic rice plants were obtained by introducing a plasmid carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV 35S promoter, via particle bombardment of embryogenic suspensions. The effect of osmotic conditioning on transformation was evaluated. Regenerated plants were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth of mother plants (R₀) was normal and seeds were produced. Southern blot analysis of R₀ and R₁ plants showed that hygromycin resistant plants contained intact hph genes that were inherited in a Mendelian fashion. A protocol for a simple, efficient, repeatable, genotype- and environment-independent Indica rice transformation system is described.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - kb kilobase - GUS ß-glucuronidase - hph hygromycin B phosphotransferase     

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa based on ligninolytic enzymes

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa using ligninolytic enzymes as markers was evaluated with the detection and analysis of activities and composition of 15 edible fungi. The results showed that the activity and composition of ligninolytic enzyme system varied in response to changes of fungal strains. By analyzing the growth rate of mycelia and their ability to produce ligninolytic enzymes, H. marmoreus and P. geesteranus, G. frondosa and P. sajor-caju were screened for further study. Three colonies of 26 regenerated colonies of H. marmoreus and P. geesteranus protoplast fusion and one colony of 48 regenerated colonies of G. frondosa and P. sajor-caju were selected respectively. At the same time, these four strains were identified using RAPD and ISSR molecular markers. The results showed that the strains HM5G1and PS7F1are new strains and have low similarity to parental strains H. marmoreus and G. frondosa. These results are supported by the results of antagonism tests. These two fusants were significantly higher in their ligninolytic enzyme activity than H. marmoreus and G. frondosa. The growth rates of strains HM5G1and PS7F1 were also noticeably higher than those of H. marmoreus and G. frondosa, by 1.36 and 1.5 times respectively. The biological efficiency of the strain HM5G1 was 11.5 % higher than that of the parental strain H. marmoreus. This work suggests that it is an efficient way of breeding new strains to use the decolorization of ligninolytic enzymes as a preliminary screening marker.     

Development of a Gene Transfer System for the Mycelia of Flammulina velutipes Fv-1 Strain

To develop a gene transformation method for Flammulina velutipes, we constructed a vector with hph gene under control of the trp1 gene promoter. The vector was integrated into protoplast derived from mycelia by the calcium-polyethylene glycol method, as it has not been reported for F. velutipes. Transformation efficiency was much improved when transformation was performed by the restriction enzyme mediated integration method.     

Genetic transformation of Monascus purpureus DSM1379

Monascus purpureus was transformed into hygromycin B resistance with hygromycin B phosphotransferase (hph) fused to Aspergillus nidulans trpC or a putative Monascus purpureus gpd1 promoter by electroporation. Among five strains, only M. purpureus DSM1397 was a competent recipient. Normal growth and sporulation on media containing up to 500 mg hygromycin B l^–1 occurred up to five generations. Upon transformation of the strain with the green fluorescent protein gene (sgfp) as a model gene and hph as a selection marker, characteristic green fluorescence was observed under fluoromicroscopy indicating successful transformation.     

DNA mediated transformation of the filamentous fungus Curvularia lunata using a dominant selectable marker

Summary A DNA mediated transformation system was developed for the filamentous fungus Curvularia lunata. Incubation of protoplasts with a plasmid (pAN7-1) which contains the Escherichia coli hygromycin B phosphotransferase gene (hph) fused to an Aspergillus nidulans promoter resulted in the formation of hygromycin B resistant transformants. DNA-DNA hybridization experiments revealed that plasmid pAN7-1 became integrated into the fungal genome. All transformants analyzed so far proved to be stable even after vegetative growth for three months without selective pressure.Dedicated to Professor Dr. Dr. h. c. Esser on the occasion of his 65th birthday     

Hygromycin resistance gene cassettes for vector construction and selection of transformed rice protoplasts

Hygromycin resistance gene cassettes were designed to facilitate vector construction for plant transformation. Unique EcoRI, Pstl, and Sacll sites in the coding sequence of a hygromycin B phosphotransferase gene (hph) from Escherichia coli were eliminated. The mutated hph genes were used to form gene cassettes flanked by EcoRI-Sacll-Kpnl-Hindlll sites. Hygromycin resistance of wild-type and mutated hph genes was indistinguishable in E. coli and rice protoplast growth assay.     

Trans-activation and stable integration of the maize transposable element Ds cotransfected with the Ac transposase gene in transgenic rice plants

To develop an efficient gene tagging system in rice, a plasmid was constructed carrying a non-autonomous maize Ds element in the untranslated leader sequence of a hygromycin B resistance gene fused with the 35S promoter of cauliflower mosaic virus. This plasmid was cotransfected by electroporation into rice protoplasts together with a plasmid containing the maize Ac transposase gene transcribed from the 35S promoter. Five lines of evidence obtained from the analyses of hygromycin B-resistant calli, regenerated plants and their progeny showed that the introduced Ds was trans-activated by the Ac transposase gene in rice. (1) Cotransfection of the two plasmids is necessary for generation of hygromycin B resistant transformants. (2) Ds excision sites are detected by Southern blot hybridization. (3) Characteristic sequence alterations are found at Ds excision sites. (4) Newly integrated Ds is detected in the rice genome. (5) Generation of 8 by target duplications is observed at the Ds integration sites on the rice chromosomes. Our results also show that Ds can be trans-activated by the transiently expressed Ac transposase at early stages of protoplast culture and integrated stably into the rice genome, while the cotransfected Ac transposase gene is not integrated. Segregation data from such a transgenic rice plant carrying no Ac transposase gene showed that four Ds copies were stably integrated into three different chromosomes, one of which also contained the functional hph gene restored by Ds excision. The results indicate that a dispersed distribution of Ds throughout genomes not bearing the active Ac transposase gene can be achieved by simultaneous transfection with Ds and the Ac transposase gene.     

Agrobacterium tumefaciens-mediated genetic transformation of the white root rot ascomycete Rosellinia necatrix

Hygromycin B resistance was conferred to the mycelium of the white root rot fungus Rosellinia necatrix by transformation with the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. In all three transformants, the presence of hph and single-copy integrations of the marker gene were demonstrated by Southern analysis. This is the first report describing A. tumefaciens-mediated transformation of R. necatrix     

Transgenic Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>) plants obtained by biolistic transformation of embryogenic suspension cells

Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates. We are interested in developing biotechnological methods to improve this monocot forage species. Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric -glucuronidase (gusA) gene was co-transformed with hph. Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin. Plants were regenerated from 45% of the hygromycin resistant calli. Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed. Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues. Fertile transgenic plants were obtained after two winters of vernalization under field conditions. This is the first report on the generation of transgenic plants in Russian wildrye.     

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.     

Production of fertile transgenic barley (Hordeum vulgare L.) plant using the hygromycin-resistance marker

Fertile transgenic barley (Hordeum vulgare L.) plants were obtained by high velocity particle bombardment. The plasmid pBCl was used to deliver the selectable hph gene and reporter Gus gene into immature embryo. After the selection culture 18 hygromycin resistant plants were obtained. Samples for Southern hybridization and enzymatic Gus assay were obtained from 11 plants. Southern hybridization confirmed the presence of the hph gene in the 11 hygromycin resistant plants(T₀). Enzymatic assay indicated that all the t₀ plants that showed hph positive in Southern analysis possessed detectable amount of Gus activity. To date all the 11 t₀ plants reached maturity and mature seeds were obtained Transmission of the hph gene to progeny(T₁) of two independent t₀ plants was confirmed by Southern hybridization.Abbreviations Adh Alcohol Dehydrogenase - BA 6-Benzylaminopurine - cv cultivar - 2,4-D 2,4-Dichlorophenoxyacetic Acid - Gus -Glucuronidase - hph Hygromycin Phosphotransferase - 4MU 4-Methyl-umbelliferone     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the plant pathogenic fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.     

Agrobacterium tumefaciens-Mediated Transformation of the Vegetative Dikaryotic Mycelium of the Cultivated Mushroom Flammulina velutipes

Agrobacterium tumefaciens was used to transform the vegetative dikaryotic mycelium of Flammulina velutipes using a hygromycin B resistance gene as selectable marker. The gene coding for urogen III methyltransferase (cob) was introduced into F. velutipes dikaryotic cells. The resulting transformant cells generated a bright red fluorescence, indicating that cob is promising as a reporter gene in F. velutipes.     

Agrobacterium rhizogenes-mediated transformation of a high oil-producing fil a m en to us fungusUmbelopsis isabellina

TheAgrobacterium rhizogenes-mediated transformation procedure was developed by using the hygromycin B phosphotransferase gene (hph) as a selective marker for the oil-producing fungusUmbelopsis isabellina. Different conditions were combined to increase the transformation efficiency. The highest efficiency was obtained by usingA. rhizogenes strain R105 and a vector with zygomycete promoter. Southern blot analysis demonstrated that 71 % of transformants contained random integrations of T-DNA sequences under optimal conditions. We randomly selected 115 positive transformants resistant to hygromycin to analyze the amount of total fatty acid and gamma-linolenic acid (GLA). Six transformants produced a higher amount of total fatty acids than the wild strain, and one transformant also produced a higher level of GLA than the wild strain in gas chromatography analysis. This is the first report about usingA. rhizogenes strain R105 and germinated conidia to transform successfully the recalcitrant zygomycetes and to obtain transformants with a stable phenotype.     

Transgenic Japanese lawngrass (Zoysia japonica Steud.) plants regenerated from protoplasts

Transgenic Japanese lawngrass (Zoysia japonica Steud.) plants were generated by means of polyethylene glycol (PEG)-mediated direct gene transfer into protoplasts. The plasmid pBC1 was used to deliver the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes into protoplasts. Selection with a high concentration (400 mg/l) of hygromycin yielded a number of resistant calli and about 400 plants were generated. Polymerase chain reaction (PCR) and Southern hybridization analyses revealed that all of then plants tested contained introduced genes. The gus gene regulated by the maize alcohol dehydrogenase-1 (Adh 1) promoter was expressed in the leaves and roots of transgenic Japanese lawngrass plants. Received: 13 December 1996 / Revision received: 9 June 1997 / Accepted: 2 September 1997