Table S5. The unigenes differentially expressed between the control and WW-transgenic plants of hop. (XLS 296 kb)

BACKGROUND:The hop plant (Humulus lupulus L.) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils. These compounds are widely implicated in the beer brewing industry and are having potential biomedical applications. Several independent breeding programs around the world have been initiated to develop new cultivars with enriched lupulin and secondary metabolite contents but met with limited success due to several constraints. In the present work, a pioneering attempt has been made to overexpress master regulator binary transcription factor complex formed by HlWRKY1 and HlWDR1 using a plant expression vector to enhance the level of prenylflavonoid and bitter acid content in the hop. Subsequently, we performed transcriptional profiling using high-throughput RNA-Seq technology in leaves of resultant transformants and wild-type hop to gain in-depth information about the genome-wide functional changes induced by HlWRKY1 and HlWDR1 overexpression.RESULTS:The transgenic WW-lines exhibited an elevated expression of structural and regulatory genes involved in prenylflavonoid and bitter acid biosynthesis pathways. In addition, the comparative transcriptome analysis revealed a total of 522 transcripts involved in 30 pathways, including lipids and amino acids biosynthesis, primary carbon metabolism, phytohormone signaling and stress responses were differentially expressed in WW-transformants. It was apparent from the whole transcriptome sequencing that modulation of primary carbon metabolism and other pathways by HlWRKY1 and HlWDR1 overexpression resulted in enhanced substrate flux towards secondary metabolites pathway. The detailed analyses suggested that none of the pathways or genes, which have a detrimental effect on physiology, growth and development processes, were induced on a genome-wide scale in WW-transgenic lines.CONCLUSIONS:Taken together, our results suggest that HlWRKY1 and HlWDR1 simultaneous overexpression positively regulates the prenylflavonoid and bitter acid biosynthesis pathways in the hop and thus these transgenes are presented as prospective candidates for achieving enhanced secondary metabolite content in the hop.

Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.

Modified versions of the Cry3A gene of Bacillus thuringiensis (Bt) were transferred into Norway spruce (Picea abies). Both the biolistic approach and Agrobacterium tumefaciens mediated procedure were employed for transformation of embryogenic tissue (ET) cultures. The latter method proved to be more efficient yielding 70 transgenic embryogenic tissue lines compared with 18 lines obtained by biolistics. The modified Cry3A genes were driven by a 35S promoter and the nptII screenable selection marker gene was used in all vectors. The transgenic ETs were molecularly characterized and converted into mature somatic embryos. Germinating embryos formed plantlets which were finally planted into perlite and their Cry3A gene transcription activities were demonstrated by RT-PCR.

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 Biotechnol. & Biotechnol. Eq. 2013, 27(2), 3644-3648

Bacillus thuringiensis Cry3A gene was redesigned for high expression in Norwegian spruce and the sequence was slightly modified to allow for simple N- and C- terminal deletions and domain II loop 1 exchange for synthetic oligos. Modified Cry3A toxins from 13 variants of the synthetic gene were expressed in Escherichia coli BL21 and their toxicity on spruce bark beetle larvae was tested using spruce bark sandwiches. Mutant toxins with N-terminal deletion and loop 1 duplication showed increased toxicity. Key words : Bacillus thuringiensis , Ips typographus, Picea abies, resistance.

In conifers and other plants with long reproductive cycles, transformed embryogenic tissues can serve as a convenient source of plant material for the testing of insecticidal or fungicidal transgene efficiency. In this report, transgenic embryogenic tissue was obtained after the transformation of somatic embryos of Norway spruce (Picea abies (L.) Karst.) by Agrobacterium tumefaciens with the gus-intron chimeric gene. The stable integration of transgenes was confirmed by PCR and Southern hybridization. The transformation was successful only in a suitable embryogenic cell line sensitive to Agrobacterium. Out of the nine embryogenic lines tested only one gave transgenic callus.

A positive selection system using phosphomannose isomerase was employed for Agrobacterium tumefaciens mediated transformation of lettuce (Lactuca sativa L. var. 'Achát'). It was shown that the mannose-based selection system works very well with the lettuce genotype used, reaching up to 25% transformation efficiency on the medium with 20 g/L mannose and 20 g/L sucrose. The best transformation efficacy with the commonly-used kanamycin at 100 mg/L as a selection agent was 21%. Southern blot analyses of thirteen chosen mannose-resistant regenerants revealed that some of them have clonal origin, about one-half harbour a single T-DNA copy and one plant contains an incomplete T-DNA segment with only the left part of T-DNA with the pmi gene present in the genomic DNA. The following Northern analysis showed transcriptional activity of the introduced pmi gene in all plants analysed with very high differences in the level of pmi specific mRNA. The results demonstrate that both mannose and kanamycin provide comparable transformation efficiencies in our lettuce genotype. An alternative selection method with mannose as a selection agent is now available for lettuce transgenosis.

Somatic embryogenesis is conveniently utilized for the preparation of Norway spruce (Picea abies [L.] Karst.) transgenic clones by means of Agrobacterium. The establishment of successful transformation protocol requires to determine the tolerance of growing embryogenic tissue to antibiotics in culture and selective media. In 5 Norway spruce lines (genotypes) differences in the tolerance of embryogenic tissues to penicillin antibiotics (amoxicillin, carbenicillin, and ticarcillin), carbapenem antibiotic (meropenem) used for the Agrobacterium growth prevention, and aminoglycoside antibiotic (kanamycin) used in selective media were determined. Of the penicillin derivatives, amoxicillin was optimally tolerated in all lines and, in addition, its highest concentration accelerated growth in more rapidly growing lines. Ticarcillin was similarly tolerated but no growth acceleration was observed in any line. As regards carbenicillin, only the lowest concentration was observed to be well tolerated by all lines whereas all concentrations of meropenem were well tolerated in all lines except for slowly growing line 28, the growth of which was retarded by the concentration of 20 mg/l. The aminoglycoside antibiotic kanamycin was well tolerated by the embryonic tissue of all lines in the concentration of 10 mg/l and less in the concentration of 25 mg/l. The concentrations of 50 mg/l and 100 mg/l appeared as intolerable in all lines. Toxicity of kanamycin manifested at first in the browning and later in the growth cessation of embryogenic tissue.

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.

Biologia plantarum, an international journal for experimental botany founded in 1959 by Professor Bohumil Němec. Covers all branches of experimental botany ranging from molecular biology and biotechnology to whole-plant and stand functioning.

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.

Valerophenone synthase homologue of chalcone synthase (CHS) is the first key enzyme described to be involved in the biosynthesis of bitter acids, the compounds produced in hop lupulin glands valuable for the taste of beer. The complete sequence of a novel homologue of CHS chs 4 was isolated from hop. Protein predicted from chs 4 cDNA has 43.45 kDa and length 395 amino acids. It was found by the analysis of chs 4 flanking sequences that this gene is in the cluster with other CHS homologues — chs 3 and vps. The intron identified in chs 4 has been found to be homological to vps and chs 3 introns. Expression of chs 4 was partially characterized using reverse transcription polymerase chain reaction and it was found that chs 4 is specifically expressed in the glandular tissue of hop cones likewise vps. The predicted protein sequence CHS 4 was compared with other CHS-like proteins.

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.

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.

The complete sequence hop gene, which corresponds to true chalcone synthase (EC 2.3.1.74), was amplified using a combination of PCR, RT PCR and inverse PCR methods and cloned from Czech Osvald's clone 72. The gene designated chs_H1 was found to be specifically expressed on glandular trichomes, whereas negligible level of specific mRNA was found in leaves. Thus, chs_H1 may co-determine biosynthesis of prenylated chalcones, compounds valuable as anticancer and antiproliferative components of lupulin. It was shown by the comparative analyses and by the structure modelling that the true hop chalcone synthase differs from previously described CHS-like protein, phlorisovalerophenone synthase, which is involved in biosynthesis of bitter acids. Several hop cultivars were analysed for the presence of genes homologous to chs_H1 using chs_H1 cDNA as probe. 2-4 HindIII specific genomic fragments were detected by Southern blots, depending on cultivar. More detailed analysis revealed at least six homologous genes in Osvald's 72 hop, suggesting a great potential of this hop also as a genetic source for modern biotechnology. High level of xanthohumol in some Osvald's 72-derived hybrid cultivars like cv. Sladek, as well as a comparatively high level of prenylated flavonoids in some comprehensive hops, suggest also the presence of additional, regulatory genes co-determining levels of prenylated flavonoids valuable for medicinal hops.

The timing of excision of maize transposable element Ac was studied using visual histochemical assay based on Ac excision restoring activity of β-glucuronidase (GUS). The Solanum tuberosum L. cv. Bintje was used for Agrobacterium-mediated transformation with pTT230 plasmid harbouring Ac-interrupted gus A gene and npt II gene as a selectable marker gene. Twenty-eight out of 72 kanamycin resistant calli did not express any GUS activity, 31 calli showed partial GUS expression and 13 out of assayed calli revealed strong expression of gus A gene. Plants were regenerated from calli without and/or with partial expression of gus A gene. The regenerated transformants which did not express GUS during the callus phase often contained many small GUS expressing spots on leaves. A phenotypic selection assay for excision of Ac has been also used. This non-detectable excision of Ac in callus tissue could be followed by a "late" timing excision during leaf development. After transformation with pTT224 plasmid harbouring Ac-interrupted hpt II gene and npt II gene transgenic calli containing Ac within the hygromycin resistance gene were derived and hygromycin sensitive plants were regenerated from them. Protoplasts isolated from leaves of transgenic regenerated plants were selected on hygromycin. Hygromycin resistant minicalli showed to harbour multiple copies of Ac and mark out low uniqueness of integration sites.

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 %.

Twelve Rhododendron cultivars (Marcel Menard, Blutopia, Madame, Old Port, Lugano, Omega, Album Novum, Franceska, Polar Nacht, Lumina, Erato and Blurettia) were tested for in vitro propagation ability from flower buds. The flower buds were withdrawn from the outdoor stock plants during the dormand period. Outer resinous bud scales were removed, buds were surface disinfected in 10% SAVO, a commercial bleach (5% NaClO), with 0.1% Tween 20 and rinsed thrice with sterile distilled water. Florets were excised from the buds and cultivated on agar Anderson's medium supplemented with indole-3-acetic acid (IAA) and 6-(dimethylallylamino)-purine (2iP). The concentrations of IAA/2iP of 1/5 and 4/15 mg/l stimulated to growth of granular callus, while the concentrations of IAA/2iP of 1/8 and 4/15 mg/l stimulated shoot proliferation. Nine out of twelve Rhododendron cultivars tested were found to be able to propagate from flower buds.