Microprotoplast-mediated transfer of single specific chromosomes between sexually incompatible plants.

Microprotoplast-mediated chromosome transfer (MMCT) through fusion of small (subdiploid) microprotoplasts of a transgenic triploid potato (Solanum tuberosum) cell line with leaf protoplasts of tobacco (Nicotiana tabacum) and the wild tomato species Lycopersicon peruvianum is reported. The microprotoplasts contained one or a few chromosomes. Monosomic addition plants were produced from the fusion products. We employed mass-scale induction of micronuclei in donor suspension cells of potato using the microtubule inhibitor Cremart. Protoplasts were isolated from micronucleated cells after incubation in a cell wall digesting enzyme mixture. The microprotoplasts were isolated from the micronucleated protoplasts by high-speed centrifugation. By using sequential filtration, small microprotoplasts containing one or few chromosomes were separated from the bigger subdiploid microprotoplasts. These small microprotoplasts were fused with recipient protoplasts of tobacco or tomato using polyethylene glycol. The selectable marker kanamycin resistance (KanR) and the reporter gene β-glucuronidase (gus), carried by the donor potato chromosome, were used for the selection of fusion products and the isolation of hybrid calli. Several monosomic addition plants were obtained within the short period of 3-4 months after fusion. These contained one potato chromosome carrying a single copy of gus and one or two copies of the neomycin phosphotransferase (nptII) gene conferring KanR, and the complete set of chromosomes of tobacco or tomato, as revealed by genomic in situ hybridization and Southern blot hybridization. The alien genes, gus and nptII, were stably expressed in both the tobacco and tomato backgrounds. They were transmitted to the progeny after backcrossing to tomato. Monosomic and disomic additions, and some introgression plants showing integration of gus and nptII in the tomato genome, were recovered in the first backcross progeny. The potential value of MMCT for the transfer of economically important traits, genome analysis, and gene expression is discussed. Key words : chromosome transfer, microprotoplast fusion, monosomic-disomic additions, sexual transmission, DNA integration, alien gene expression.

Activity of constitutive promoters in various species from the Liliaceae.

In this paper we first review literature on the performance of various promoters in monocotyledonous species. In general, promoters isolated from monocots show a higher activity in monocot species. Moreover, the presence of an intron between the promoter and reporter gene increases transcription levels. We used the same approach to study gene expression in Liliaceae. The activities of the CaMV 35S, maize Adh1-based pEmu, rice Act1 and maize Ubi promoters, coupled to the beta-glucuronidase (gus) reporter gene, were evaluated for transient gene expression upon particle bombardment of tissues of tobacco, rice, tulip, lily and leek. Although monocot promoters performed very well in rice tissues, the results of this study show that this cannot be generalized for other monocot species. The transcription inducing effects of monocot promoters were less pronounced or even absent in tissues of Liliaceae, while the presence of an intron between promoter and gus gene reduced promoter activity.

Expression of the GUS-gene in the monocot tulip after introduction by particle bombardment and Agrobacterium.

Gene transfer to the monocotyledon tulip (Tulipa sp. L.) was obtained both by particle bombardment and Agrobacterium transformation. Using a Particle Delivery System, transient expression of the reporter gene for ßglucuronidase was demonstrated. It was shown that the CAMV 35S as well as the TR2' promoter were active in flower stem expiants. Various wildtype and disarmed Agrobacterium strains, harbouring the 35S GUSintron gene on a binary plasmid, were used for infection of flower stem expiants of 7 cultivars and 7 botanical Tulipa species. In nine genotypes the GUSintron gene was expressed, despite the fact that tulip tissue did not produce detectable amounts of virulence-inducing substances. Agrobacterium rhizogenes appeared to be most effective in gene transfer to tulip tissue.

Effects of Alternaria alternata f.sp. lycopersici toxins on pollen.

Effects of the phytotoxic compounds (AAL-toxins) isolated from cell-free culture filtrates of Alternaria alternata f.sp. lycopersici on in vitro pollen development were studied. AAL-toxins inhibited both germination and tube growth of pollen from several Lycopersicon genotypes. Pollen from susceptible genotypes, however, was more sensitive for AAL-toxins than pollen from resistant plants, while pollen of species not belonging to the host range of the fungus was not significantly affected by the tested toxin concentrations. AAL-toxins elicit symptoms in detached leaf bioassays indistinguishable from those observed on leaves of fungal infected tomato plants, and toxins play a major role in the pathogenesis. Apparently, pathogenesis-related processes and mechanisms involved in disease resistance are expressed in both vegetative and generative tissues. This overlap in gene expression between the sporophytic and gametophytic level of a plant may be advantageously utilized in plant breeding programmes. Pollen may be used to distinguish susceptible and resistant plants and to select for resistances and tolerances against phytotoxins and other selective agents.

Sequence analysis of a split gene involved in fruiting from the fungus Schizophyllum commune.

The sequence of a gene and its mRNA, which is abundantly expressed during fruiting body initiation in the Basidiomycete Schizophyllum commune, is described. This gene (1G2), the first to be analyzed in this group of fungi, contains an open reading frame coding for a polypeptide of 94 amino acids and a mol. wt. of 9842. A possible signal peptide of 20 residues and one glycosylation site were found. The sequence analysis was hampered by a sequence rearrangement in one of the cDNA clones, probably due to base pairing between short complementary sequences present at the 5' and 3' ends of the mRNA. The 5' untranslated leader sequence is 57 bp long and harbors a possible ribosome binding site close to the AUG start codon. A TATA box is found at position -31 upstream of transcription initiation. The 3' untranslated sequence is 200 bp long and contains the sequence -TATATAAT-, which most likely represents the polyadenylation signal. Some heterogeneity as to the site of addition of the poly(A) tail was observed. The coding region of the gene is interrupted by three very small introns of 53, 49 and 49 bp, respectively. The 5' and 3' splice junctions are conserved: GTGAGT- and -AG-, respectively. Each intron contains a sequence complementary to the 5' end of the intron. These sequences are compared with internal conserved sequences in yeast and filamentous fungi with regard to their possible role in splicing.

Molecular cloning of a gene abundantly expressed during fruiting body initiation in Schizophyllum commune.

Complementary DNA was synthesized on polyadenylated RNA from a dikaryotic mycelium of the basidiomycete Schizophyllum commune bearing fruiting body initials. The complementary DNA was cloned into the PstI site of pBR327 by the deoxyguanidylate-deoxycytidylate tailing approach. After transformation into Escherichia coli cells, a differential screening was performed by colony hybridization with complementary [32P]DNA made on the RNAs of the monokaryon and dikaryon strains. Two clones were selected for further analysis by Northern blotting and hybrid release translation. Clone 1D10 hybridized with an mRNA of 775 nucleotides, coding for a polypeptide with an Mr of 15,000. Although this RNA was present in both monokaryotic and dikaryotic mycelia, its concentration appeared to change considerably over time and with different cultivation conditions. This mRNA is probably the most abundantly expressed sequence in S. commune. Clone 1G2 and its homologs hybridized with an mRNA of 650 nucleotides, coding for a polypeptide with an Mr of 13,000. This gene was exclusively expressed in the dikaryon strain. In liquid-grown cultures, the concentration of this mRNA was low but increased ca. 20-fold during the establishment of fruiting body primordia. A chromosomal fragment of 9 kilobase pairs which contained the 1G2 gene was cloned into pBR327 and used as a probe in Northern blot hybridization. It was found that surrounding sequences were not expressed at the same time or to the same extent as the 1G2 gene.

Sequence organization of the nuclear DNA of Schizophyllum commune.

Several methods were used to characterize the organization of repetitive DNA in the fungus Schizophyllum commune. They all failed to show interspersion of repetitive sequences among single copy sequences. Saturation hybridization showed that 2.2% of the double-stranded nuclear DNA coded for rRNA. The size of the ribosomal cistron (11.9.10(6) daltons) was determined by restriction enzyme analysis. From these values it was calculated that about 6% of the nuclear DNA consisted of ribosomal cistrons, which approx. equals the amount of repetitive DNA present. Thus, this simple sequence organization in Schizophyllum commune is fundamentally different from organization patterns in higher eukaryotes.

Characterization of the genome of the basidiomycete Schizophyllum commune.

DNA of Schizophyllum commune was isolated both from mycelial cells and from protoplasts. Nuclear DNA was isolated after solubilization of the mitochondria with the detergent Nonidet. The G + C content of the nuclear DNA was 57%, calculated from its buoyant density (1.7165 g/ml) and from the Tm (77.4 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). The buoyant density of the ribosomal cistrons was 1.707 g/ml. DNA isolated from purified mitochondria had a very low G + C content: 22% (rho = 1.6845 g/ml, Tm = 61.8 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). Analysis of CsCl profiles and melting patterns suggested that mitochondrial DNA contains interspersed (A + T)-rich sequences. From reassociation analysis of sheared nuclear DNA the genome size of S. commune was determined to be 22.8 . 10(9) daltons. A small amount of DNA (0.5 . 10(9) daltons) bound to hydroxyapatite at zero time Cot. 7% of the genome (1.6 . 10(9) daltons) represented repetitive DNA.

On the isolation of TI-plasmid from Agrobacterium tumefaciens.

An efficient lysis method for Agrobacterium cells was developed, which allows a reproducible isolation of the tumor inducing (TI)-plasmid. The lysis method is based on the sensitivity of this bacterium to incubation with lysozyme, n-dodecylamine,EDTA, followed by Sarkosyl, after growth in the presence of carbenicillin. We also present a procedure for the isolation of the TI-plasmid on a large scale, that might be used for the mass isolation of other large plasmids which like the TI-plasmid, can not be cleared with earlier described procedures. The purity of the plasmid preparations was determined with DNA renaturation kinetics, which method has the advantage that the plasmid need not to be in the supercoiled or open circular form.