H. Niedermeierová

Cloning and characterisation of chs-specific DNA and cDNA sequences from hop (Humulus lupulus L.)

A complete sequence of chalcone synthase (CHS) gene from hop was cloned. The gene designated chs _H1 consists of two exons and one 187 bp intron. CHS protein predicted from chs_H1 cDNA has 42.5 kDa and retains conserved domains and residues including 26 amino acids at positions identical to those identified by crystallography as characteristic for catalytic domains of alfalfa CHS (EC 2.3.1.74). Cloned CHS_H1 protein shows specific CHS activity with 4-coumaroyl-CoA. Structure modelling revealed clear differences between CHS_H1 and phlorisovalerophenone synthase, the only published CHS-like homologue from hop. Conserved motifs like H, and G boxes characteristic for the light regulated and stress inducible genes were identified within promoter region of chs _H1 gene. Highly specific expression of chs _H1 mRNA was detected by quantitative RT PCR in glandular trichomes during cone maturation. Much lower, but significant levels of chs _H1 mRNA were detected at the stage of hop flowering in petioles (100%), developed flowers (96%), and in stem apexes (78%), while the lowest levels of mRNA were found in the roots (31%) and leaf blades (9%). Southern blot analyses predicted at least five additional chs -like genes related to chs _H1. A genomic arrangement different from phlorisovalerophenone synthase sequences was found for these genes. RFLP analyses using DNA from 15 genotypes revealed several distinct dendrogram clusters, suggesting specific re-arrangements of hop chs -like genes during evolution and/or during the breeding and selection processes. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Use of phosphomannose isomerase-based selection system for Agrobacterium -mediated transformation of tomato and potato

Two selection systems for Agrobacterium tumefaciens mediated transformation of tomato and potato were compared. In the tomato (Lycopersicon esculentum cv. Moneymaker), the highest transformation rate, 4.2 %, of cotyledon explants on mannose-selection medium was obtained when mannose/sucrose concentration in the regeneration medium was 5/15 g dm−3. The best transformation efficacy with the commonly used concentration of 100 mg dm−3 kanamycin as a selection agent was 9 %. In the potato (Solanum tuberosum cv. Bintje), the highest transformation frequency was 53.3 % when mannose concentration in the regeneration medium was 5 g dm−3 during the first 3 weeks after transformation and 10 g dm−3 afterwards. The optimum concentration of sucrose was 20 g dm−3. The transformation efficiency using kanamycin as a selection agent at a concentration 100 mg dm−3 was 33.3 % with potato. Our results demonstrate that the transformation efficiency using mannose selection is 1.6-fold higher for potato and about 2 times lower for tomato comparing with the ordinary protocol using kanamycin.

Transformation of Rhododendron spp. using Agrobacterium tumefaciens with a GUS-intron chimeric gene

Abstract The five Rhododendron cultivars, ‘America’, ‘Catawbiense grandiflorum roseum’, ‘Madame Carvalho’, ‘Mars’ and ‘Nova Zembla’ were used for transformation by Agrobacterium tumefaciens carrying T-DNA with the gusA gene encoding β-glucuronidase (GUS) gene and the neomycin phosphotransferase II gene as a selectable marker gene. The GUS reporter gene was successfully transferred into all five cultivars as indicated by fluorimetric staining, polymerase chain reaction (PCR) and Southern blot analysis. Some primary transformants appeared to be chimeric as both GUS expression and GUS nucleotide sequences were lost during vegetative propagation.

Production of human papillomavirus type 16 E7 oncoprotein fused with β-glucuronidase in transgenic tomato and potato plants

The human papillomavirus type 16 (HPV 16) oncogene E7 fused with the gene for β-glucuronidase (gus) was used in plant transformation experiments. The E7 gene modified for lower cancerogenicity and fused with the 5′ end of the gus in cassettes with cauliflower mosaic virus 35S promoter and transcription terminator produced high contents of fusion proteins in potato protoplasts. Expression vectors harbouring E7 fusion cassettes were used for Agrobacterium tumefaciens LBA4404 mediated transformation of either potato (Solanum tuberosum L. cv. Bintje) or tomato (Lycopersicon esculentum Mill. cv. Moneymaker). A fusion gene was found in all rooted regenerants using polymerase chain reaction with primers providing amplified fragments from E7 and gus genes. GUS activity was revealed in all regenerants obtained. Nevertheless, the level of GUS expression in different constructs varied much more than in transient expression experiments with potato protoplasts. Especially, expression level in plants carrying vectors with the whole E7 gene fused with gus was lowered by 2–3 orders of magnitude comparing with fusion of the first 41 codons of E7 and gus. Southern hybridisation of 18 tomato and 23 potato regenerants revealed mostly multiple tandem integration of T-DNA into the plant genome and Western blot proved the presence of the fusion protein in 9 tomato and 11 potato plants out of 41 tested individuals.

Germinal Excision and Reinsertion Frequencies of the Mobile Element Ds Transposed from Two Unlinked T-DNA Loci in Tomato

Acceptor sites of unlinked transposed Ds element from two T-DNA loci in tomato were mapped. Experimental data obtained from TC1 progeny testing were employed for estimation of germinal excision frequency (GEF) of Ds element and frequency of its reinsertion (FR). The donor T-DNAs 1481J and 1601D, containing a 35S:NPT transformation marker, a 35S:BAR or nos:BAR excision marker conferring phosphinothricine resistance and a Ds element in the 5′ untranslated leader of the nos (or 35S): BAR gene, were located on chromosome 7 and 8, respectively. Ds transposition was induced by 105121 T-DNA carrying stabilized Ac (sAc) which provides a source of transposase and 2′:GUS marker conferring β-glucuronidase activity. Tomato plants harbouring the Ds in 1481J or 1601D locus and sAc were crossed and F1D, were crossed individually as seed parents to wild-type plants to generate TC1 progenies. TC1 seed was germinated on phosphinothricine (Basta)-containing medium, and individual seedlings carrying a transposed Ds and lacking sAc were identified by PCR (to detect the Ds) on phosphinothricine resistant individuals that lacked β-glucuronidase activity. From segregation ratio in TC1 the germinal excision and reinsertion frequencies of the Ds element were estimated for individual F1 plants. A total of 14560 TC1 seedlings of 1481J and 16195 TC1 seedlings of 1601D was analyzed. We observed high variation between individual plants as regards both GEF and FR despite of donor locus (1481J or 1601D), however, the average germinal excision frequencies as well as average frequencies of reinsertion were very similar for both donor loci: GEF1481J = 24 %, GEF1501D = 25 %, FR1481J = 42 %, FR1601D = 46 %.

Transposition patterns of unlinked transposed Ds elements from two T-DNA loci on tomato chromosomes 7 and 8.

Abstract. We have previously reported that unlinked transposed Ds elements originating from chromosome 4 of tomato preferentially inserted in chromosome 2. This observation, together with data from other studies, suggested that there may be absolute preferences for transposition, irrespective of the chromosomal location of the donor site. The aim of the present work was to verify whether the distribution of transposed Ds elements on chromosome 2 was non-random and thus whether, unlike the case in maize, unlinked transpositions in tomato are not distributed randomly. To do this, unlinked acceptor sites of Ds elements originating from two donor T-DNA loci lying on chromosomes 7 and 8 were mapped. Receptor sites for trDs elements transposed from the 1601D locus on chromosome 8 exhibited a non-random distribution (P<0.01). Eleven out of 46 independent transpositions mapped to chromosome 2 and, as this was statistically significant (P<0.01), proves that receptor sites for this element are not randomly distribution on the chromosomes. In addition, deviation of the observed number from the expected number of trDss was close to being significant for chromosome 4 (P=0.05–0.1). In contrast, the distribution of unlinked receptor sites for trDss derived from the 1481J locus on chromosome 7 was random. χ2 tests were performed for each chromosome, and for chromosome 4 the difference between the observed and the expected number of trDss was very high but statistically non-significant (P=0.05–0.1). For chromosome 2 the difference was statistically negligible. Therefore, we conclude that chromosome 2 does not serve as a preferential receptor for the transposition of Ds elements independently of the location of the donor site.

Transformation of Tobacco cpDNA with Fusion E7GGG/GUS Gene and Homologous Recombination Mediated Elimination of the Marker Gene

ABSTRACT A transformation vector harboring the marker gene aadA conferring spectinomycin resistance and the fusion E7GGG/gus gene of interest was employed for the transformation of tobacco cpDNA via biolistics. The gene of interest consisted of the oncogenic E7GGG gene from human papillomavirus strain 16 fused with the reporter gus gene. Both transgenes were equipped with the same promoter and termination sequences arranged as direct repeats. Biolistics with circular vector yielded 20 shoots rooted in spectinomycin containing medium; with linear vector no rooted plants were obtained. After 4 months of in vitro selection the plants were burst into flower in a greenhouse and pollinated with non-transgenic plant pollen. Seeds were harvested individually from each plant capsule and planted onto selection medium. Rare white seedlings were recovered by transfer onto medium without spectinomycin and self-pollinated. GUS activity of 15 seedlings from each self-pollinated plant was measured and 26 plants with higher activity (and one without any GUS activity) were PCR analyzed for the presence of both the marker gene and the gene of interest. Finally, 8 plants were analyzed in detail by Southern hybridization, RT-PCR, and Western blot. We identified 6 plants harboring the E7GGG/gus gene only, where the marker gene was eliminated by homologous recombination; one plant with both transgenes; and one with the aadA gene only. RT-PCR showed the presence of the respective mRNAs in all 8 plants analyzed but Western blot proved that only the GUS part of the fusion E7GGG/GUS protein was present in all plants harboring the fusion gene. We discuss why E7GGG protein could not be detected.

GFLV coat protein constructs based on local isolates from the Czech Republic

Coat protein genes of grapevine fanleaf virus local strains isolated in South-Moravia, Czech Republic were sequenced, and artificial coat protein gene was designed and synthesized. It retains typical sequence features of local strains but is free of mRNA destabilizing sequences. Three variants of the synthetic gene were cloned into Agrobacterium plant expression vector and their function was tested after potato protoplasts transformation, assaying transient mRNA and coat protein production.