Vol. 9 No. 4B December 2004

Volume 9 (2004) pp 795 - 804
Title THE CLONING OF SEQUENCES DIFFERENTIALLY TRANSCRIBED DURING THE INDUCTION OF SOMATIC EMBRYOGENESIS IN CUCUMBER (Cucumis sativus L.)
Authors Anna Linkiewicz1, Marcin Filipecki1*, Anna Tomczak1, Agnieszka Grabowska2 and Stefan Malepszy1
Abstract Somatic embryogenesis in cucumber cell suspension culture is a convenient tool to study differential gene expression, particularly during the early stages of this process. In this study, we used the cucumber somatic embryogenesis system to detect genes that were differentially transcribed during the induction of embryo development. We identified and cloned 120 candidate cDNA fragments from differential display gels. The selected cDNAs were confirmed by reverse northern, and 83 were sequenced. The obtained sequences represent 64 independent transcripts. The search for similarities in the databases gave a significant result in 16 cases. The potential involvement of these sequences in somatic embryogenesis is discussed.
Address and Contact Information 1Department of Plant Genetics, Breeding and Biotechnology, Warsaw Agricultural University, 02-787 Warsaw, Poland,
2Department of Biochemistry, Warsaw Agricultural University, 02-787 Warsaw, Poland
* Corresponding author, e-mail: filipecki@alpha.sggw.waw.pl
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Volume 9 (2004) pp 805 - 817
Title VERIFICATION OF STS MARKERS FOR LEAF RUST RESISTANCE GENES OF WHEAT BY SEVEN EUROPEAN LABORATORIES
Authors Lidia Błaszczyk1, Jerzy Chełkowski1*, Victor Korzun2, Jan Kraič3, Frank Ordon4, Jaroslava Ovesná5, Laszlo Purnhauser6, Melinda Tar6 and Gyula Vida7
Abstract A set of Thatcher near-isogenic lines and two breeding lines were used to examine sequence tagged site (STS) markers linked to leaf rust resistance genes Lr9, Lr10, Lr19, Lr24, Lr28, Lr29, Lr35, and a simple sequenced repeat (SSR) marker for Lr39. The selected STS markers for resistance genes Lr9, Lr10, Lr19, Lr24 and Lr28 were identified in seven accessions by seven European laboratories. Near-isogenic lines of the spring wheat Thatcher were used as positive controls. Markers for resistance genes Lr9, Lr10, Lr19, Lr24 were identified in all seven laboratories as amplification products of 1100 bp, 310 bp, 130 bp and 310 bp, respectively. The STS markers linked to resistance genes Lr9, Lr10, Lr19, Lr24, Lr29, Lr35 and the SSR marker for Lr39 were robust and highly specific for these genes and will be useful in marker-assisted selection in wheat. However, the amplification product of 378 bp that corresponded with resistance gene Lr28 was detected in all accessions including genotypes lacking this gene in all seven laboratories. This marker needs to be improved.
Address and Contact Information 1Institute of Plant Genetics Polish Academy of Sciences, Strzeszyłska 34, 60-479 Poznań Poland,
2Lochow-Petkus GmbH, PF 1197, D-29296 Bergen Germany,
3Division of Genetics and Plant Breeding, Research Institute of Plant Production, Bratislavská cesta 122, 92168 Pieštany, Slovakia,
4Institute of Epidemiology and Resistance, Federal Center for Breeding Research on Cultivated Plants, Theodor-Roemer-Weg 4, 06449 Aschersleben, Germany,
5Research Institute of Crop Production, Department of Molecular Biology, Drnovská 507,161 06 Praha 6 - Ruzyně, Czech Republic,
6Cereal Research Nonprofit Company, 6726 Szeged Also-Kikoto sor 9, Hungary,
7Hungarian Academy of Sciences Plant Protection Institute, Budapest, Hungary
* Corresponding author; e-mail: jche@igr.poznan.pl
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Volume 9 (2004) pp 819 - 827
Title COMBINED USE OF LINKED MARKERS FOR GENOTYPING THE Pm1 LOCUS IN COMMON WHEAT
Authors Łukasz Stępień1*, Jerzy Chełkowski1, Gerhard Wenzel2 and Volker Mohler2
Abstract Genotyping of 98 wheat cultivars/lines was carried out with molecular markers that are linked to the Pm1 locus: two bi-allelic (dominant) markers: the sequence-tagged site Xsts638-7A and the amplified fragment length polymorphism XE39M58-77-7A; and the multi-allelic simple sequence repeat marker Xgwm344-7A. Employing segregation data recorded in the population Chinese Spring x Virest (Pm1e), genetic mapping revealed that Xgwm344-7A and XE39M58-77-7A were distally linked to Pm1e in the repulsion phase with respective linkage distances of 0.9 cM and 4.8 cM, while Xsts638-7A was found to co-segregate with Pm1e in the coupling phase. The genotyping results of Xsts638-7A and XE39M58-77-7A confirmed disease scoring, except for the accessions of cultivars Omega, Remus and Weihenstephan Stamm M1N. The SSR marker Xgwm344 amplified 15 different fragments ranging from 102 bp to 147 bp, with 15 entries being null-allelic at the 7A and 7B homoeoloci. It was found that wheat lines having resistance alleles at the Pm1 locus mainly show the null allele at the Xgwm344-7A locus. Due to their fast-evolving nature, the use of multi-allelic SSRs for genotype determination may be complicated. However, the combined use of multiple linked marker alleles seems to be a promising approach for genotyping a broad range of plant materials.
Address and Contact Information 1Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyłska 34, 60-479 Poznań, Poland,
2Department of Agronomy and Plant Breeding, Center for Food and Life Sciences Weihenstephan, Technical University Munich, 85350 Freising, Germany
* Corresponding author; e-mail: lste@igr.poznan.pl
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Volume 9 (2004) pp 829-842
Title OZONE-INDUCED OXIDATIVE STRESS RESPONSE IN Arabidopsis: TRANSCRIPTION PROFILING BY MICROARRAY APPROACH
Authors Agnieszka Ludwikow1, Patrick Gallois2 and Jan Sadowski1, 3*
Abstract High ozone concentration generates oxidative stress in plants. To investigate the detailed transcriptional regulation of Arabidopsis thaliana genes encoding antioxidant enzymes upon ozone stress, we performed a microarray analysis using Affymetrix GeneChip technology. Our transcription profiling revealed a differential expression equal or greater than 2-fold change for 2385 genes (at confidence 99%) in response to 350 ppb ozone dose after 3 and 6 hours of treatment. Among these, we chose 38 genes to be oxidative stress related in ozone treatment: 29 of them were 2 times up-regulated and 9 were shown to be down-regulated in at least one of the time points. Our study revealed a new transcription pattern for catalase genes and showed the first detailed transcriptional analysis of phenylopropanoid-related genes in ozone stress conditions.
Address and Contact Information 1Department of Biotechnology, Adam Mickiewicz University of Poznań, Międzychodzka 5, 60-371 Poznań, Poland,
2School of Biological Science, University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester M139PT, UK,
3Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyłska 34, 61-479 Poznań, Poland
* Corresponding author; e-mail: jsad@amu.edu.pl
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Volume 9 (2004) pp 843 - 853
Title TRANSCRIPTIONAL REGULATION OF THE gluB PROMOTER DURING PLANT RESPONSE TO INFECTION
Authors Agnieszka Mac, Magdalena Krzymowska, Anna Barabasz and Jacek Hennig*
Abstract Several studies suggest that plant hydrolytic enzymes, such as 1,3-b- glucanases, may be components of a general defense system against pathogen invasion in several different plant species. We isolated and characterized a genomic sequence coding for a new acidic 1,3-b-glucanase (gluB) from Solanum tuberosum. The 5' flanking region of the gluB gene was also characterized. A chimeric gene composed of 2998 bp of the promoter sequence from the gluB gene was fused to the b-glucuronidase (GUS) coding region and used to transform potato and tobacco plants. Transcriptional activation of the gluB promoter was investigated in response to inoculation with Phytophthora infestans (Pi) or tobacco mosaic virus (TMV). In pathogen inoculated transgenic plants, GUS activity was strongly induced locally around necrotic lesions.
Address and Contact Information Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawiłskiego 5A, 02-106 Warsaw, Poland
*Corresponding author; tel: +48 22 8237186, e-mail: jacekh@ibb.waw.pl
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Volume 9 (2004) pp 855 - 867
Title GENETIC FACTORS ENCODING RESISTANCE TO LATE BLIGHT CAUSED BY Phytophthora infestans (MONT.) DE BARY ON THE POTATO GENETIC MAP
Authors Jadwiga Śliwka
Abstract Late blight, a potato disease caused by Phytophthora infestans (Mont.) de Bary, is of great economic significance, and has been the subject of numerous research projects aimed at both introducing resistance to the disease into potato cultivars, and at unravelling the mechanisms and genes underlying this resistance. This report is on publications about mapping the resistance to P. infestans encoded by major resistance genes or polygenes, introduced into the potato from different sources. Applied methods for resistance evaluation, methods for revealing DNA polymorphisms and for the construction of genetic maps are described and compared, as are results obtained by independent authors working in this field.
Address and Contact Information Plant Breeding and Acclimatization Institute, Młochów Research Centre, 05-831 Młochów, Poland
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Volume 9 (2004) pp 869 - 878
Title IDENTIFYING LEAF RUST RESISTANCE GENES AND MAPPING GENE Lr37 ON THE MICROSATELLITE MAP OF WHEAT
Authors Lidia Błaszczyk1, Henriette Goyeau2, Xiu-Qiang Huang3, Marion Röder4, Łukasz Stępień1 and Jerzy Chełkowski1*
Abstract Based on seedling resistance tests, five resistance genes (Lr10, Lr3, Lr13, Lr14a and Lr37) against leaf rust (Puccinia triticina) were identified in 16 cultivars of European winter wheat. STS and SCAR markers were used to verify the presence of the resistance genes Lr37 and Lr10 against leaf rust in cultivars, near-isogenic lines and segregating populations. The Lr37 gene is present in a small translocation from Triticum ventricosum Ces. (Aegilops ventricosa Tausch) and is tightly linked with resistance genes Yr17 and Sr38. The Lr37 gene was identified in the cultivars Kris, Clever, Slade, Apache, Caphorn, Lorraine, Balthasar, Renan and confirmed by two PCR markers. The F3 progenies of the crosses Kris (Lr37) x Nutka (Lr37 not present) were used for map construction. Two STS/SCAR markers specific for Lr37 were mapped in relation to nine polymorphic microsatellites on chromosome 2AS. The microsatellite marker Xgwm1176 mapped relatively close to the STS and SCAR markers for Lr37 with a linkage distance of 4.1 cM.
Address and Contact Information 1Institute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszyłska 34, 60-479 Poznań, Poland,
2INRA, Laboratory of Plant Pathology, 78 850 Thiverval Grignon, France,
3Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, R3T 2M9, Manitoba, Canada,
4Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany
*Corresponding author; e-mail: jche@igr.poznan.pl
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Volume 9 (2004) pp 879 - 889
Title DEVELOPMENT OF THE SINGLE NUCLEOTIDE POLYMORPHISM MARKER OF THE WHEAT Lr1 LEAF RUST RESISTANCE GENE
Authors Mirosław Tyrka1*, Lidia Błaszczyk2, Jerzy Chełkowski2, Volker Lind3, Ilona Kramer3, Marlis Weilepp3, Halina Wiśniewska2 snd Frank Ordon3
Abstract The range of publicly available data on plant nucleotide sequences opens a new possibility in the design of SNP assays. The purpose of this study was to identify point mutations in genomic sequences closely linked to the Lr1 leaf rust resistance gene, and to develop SNP markers based on primer extension (SNuPE) facilitating efficient marker-based selection procedures, e.g. the pyramiding of resistance genes. Studies were performed on the panel of 37 wheat cultivars, the set of 41 Thatcher near-isogenic lines of spring wheat and on the 21 individuals derived from doubled-haploid (DH) lines derived from ‘Henika’ (Lr1) x ‘IPG-SW-14’. A minisequencing reaction run with Lr1_98F primer detected four genotypes (T, C+T, C and “null”) in the set of all Triticum aestivum varieties tested. In this study, it turned out that the T allele is associated with the Lr1 gene in a wide genetic background.
Address and Contact Information 1Laboratory of Population Genetics, Polonia University, Pułaskiego 4/6, 42-200 Częstochowa, Poland,
2Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyłska 34, 60-479 Poznań, Poland,
3Federal Centre for Breeding Research on Cultivated Plants, Institute of Epidemiology and Resistance, Theodor- Roemer-Weg 4, 06449 Aschersleben, Germany
*Corresponding author; tel: +48 (034) 3684233, fax: +48 (034) 3249662, e-mail: mtyrka@ap.edu.pl
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Volume 9 (2004) pp 891 - 902
Title TRANSCRIPTIONAL EXPRESSION OF A Solanum sogarandinum pGT::Dhn10 GENE FUSION IN CUCUMBER, AND ITS CORRELATION WITH CHILLING TOLERANCE IN TRANSGENIC SEEDLINGS
Authors Zhimin Yin1, Izabela Pawłowicz1, Grzegorz Bartoszewski2, Robert Malinowski2, Stefan Malepszy2 and Tadeusz Rorat1,*
Abstract The expression pattern of a Solanum sogarandinum pGT::Dhn10 gene fusion encoding a dehydrin DHN10 protein and the potential role of that protein in cold tolerance in cucumber were analysed in three T1 transgenic lines. An accumulation of Dhn10 mRNA was detected in the leaves, cotyledons, hypocotyls and roots of the transgenic seedlings both under the control conditions and after a cold treatment at 6oC for 24 h. This was confirmed by RTPCR. However, no DHN10 protein was detected by the alkaline phosphataseconjugated antibody. The transgenic lines exhibited different levels of chilling tolerance. The TCC5/1 line showed a significant increase in its chilling tolerance compared to the non-transgenic line. No chilling injury was observed when the cold hardened (6oC, 24 h) TCC5/1 plants were subsequently exposed to a temperature of 2oC for 6 h. The other two transgenic lines, TCC2/1 and TCC3/2, exhibited a comparable level of chilling tolerance to that of the non-transgenic control. The transgenic lines showed similar or significantly decreased freezing tolerance compared to the non-transgenic control, as evaluated by an electrolyte leakage test. We concluded that the S. sogarandinum GT promoter is functional in the chilling sensitive species Cucumis sativus L., and that the pGT::Dhn10 gene fusion is expressed at the transcriptional level.
Address and Contact Information 1Institute of Plant Genetics, Polish Academy of Science, Strzeszyłska 34, 60-479 Poznań, Poland,
2Department of Plant Genetics, Breeding and Biotechnology, Faculty of Horticulture and Landscape Architecture, Warsaw Agricultural University, Nowoursynowska 166, 02-787, Warsaw, Poland
* Corresponding author; e-mail: tror@igr.poznan.pl
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Volume 9 (2004) pp 903 - 917
Title Agrobacterium-MEDIATED TRANSFORMATION OF POLYPLOID CEREALS. THE EFFICIENCY OF SELECTION AND TRANSGENE EXPRESSION IN WHEAT
Authors Anna Przetakiewicz, Agnieszka Karaś, Wacław Orczyk and Anna Nadolska-Orczyk
Abstract Three combinations of Agrobacterium tumefaciens strains and vectors were used in the transformation of selected Polish wheat cultivars. The combinations were: two hypervirulent strains, AGL1, containing the pDM805 binary plasmid, and EHA101, containing pGAH; and the common Agro strain LBA4404, harboring the super-binary pTOK233 vector. pDM805 contained bar under the control of Ubi1 promoter, pGAH had nptII under nos, and pTOK233 had hpt under 35S. Additionally, pDM805 and pTOK233 carried the gus reporter gene under the Act1 promoter or 35S promoter, respectively. The highest selection rate was 12.6% and was obtained with EHA101(pGAH) on a kanamycin-containing medium. Sixty-five of the plants grown on that medium were PCR positive. The second best combination was LBA4404(pTOK233) and kanamycin selection, which gave an average transformation rate of 2.3%. Phosphinothricin selection gave 1.0% transformation efficiency, while hygromycin, depending on the strain/vector used, gave from 0.2 to 0.4%. PCR tests in T1 revealed that 67% of the lines showed a 3:1 segregation ratio, and 11% a 15:1 ratio, while in 22%, segregation was non-Mendelian. The high number of T0 transgenic plants containing one copy of the transgene was confirmed via Southern blot analysis. Kanamycin resistance in the T1 generation was very low; in some lines, all the progeny were kanamycin sensitive. GUS expression, only tested in young T1 plants, was in agreement with Mendelian segregation in three out of the twelve tested. The factors influencing the efficiency of selection and transgene expression are discussed in this paper.
Address and Contact Information Plant Transformation and Cell Engineering Lab, Plant Breeding and Acclimatization Institute, Radzików, 05-870 Błonie, Poland
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Volume 9 (2004) pp 919 - 933
Title POLYMORPHOM OF SEXUALLY DIFFERENT CUCUMBER (Cucumis sativus L.) NIL LINES
Authors Zbigniew Przybecki, Magdalena Ewa Kowalczyk, Justyna Witkowicz, Marcin Filipecki and Ewa Siedlecka
Abstract Isolations of polymorphic sequences of two pairs of the NIL lines of cucumber (Cucumis sativus L.), which differ with respect to sex, were carried out using the subtraction hybridization methods of DSC (Differential Subtraction Chain) and GDDSC (Genetically Directed DSC). 266 DSC tags were isolated from the entire genome region, and 38 GDDSC tags were isolated from the region containing the sex genes. Based on the obtained results, the methods used may be considered highly effective. The attained sequences, like 11 AFLP clones obtained earlier [Witkowicz, J. et al. Cell. Mol. Biol. Lett. 8 (2003) 375- 381], were characterized by analyzing their hybridization with differential (dhaom) and subtractive cDNA libraries (cDNAsubtractom) from 1- to 2- mm floral buds of the same lines, and by the sequencing of 28 tags. A high average degree of homology was found to exist in the genpolom to dhom and cDNAsubtractom, particularly in the case of "dominant" (when the tester used was a line in which the sex of the plants was dependent upon the dominant allele). This indicates a significant share of coding sequences in the polymorphic genomic tags as well as their share in flower formation. Many of these sequences originate from the sex gene region. Analysis of the sequenced tags showed their interesting composition, including many organelle sequences which transferred into the nucleus, and coding sequences that may participate in flower development, including sex formation.
Address and Contact Information Department of Plant Genetics, Breeding and Biotechnology, Faculty of Horticulture, Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warsaw, Poland
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Volume 9 (2004) pp 935 - 946
Title GLUCOSYLTRANSFERASE: THE GENE ARRANGEMENT AND ENZYME FUNCTION
Authors Katarzyna Lorenc-Kukuła1, Alina Korobczak1, Anna Aksamit-Stachurska2, Kamil Kostyń1, Marcin Łukaszewicz2 and Jan Szopa1*
Abstract Glucosyltransferases were isolated and characterised from many plant sources. The enzymes show middle amino acid similarity and broad substrate specificity. The promoter of the potato 5-UGT gene reveals strong environmental induction. The activation of the gene expression by UV radiation, ABA and cold treatments was detected. Overexpression of 5-UGT resulted in the accumulation of the diglucoside derivative of petunidin in transgenic tubers; the latter is most probably the reason for plant resistance to pathogen infection. Overexpressing plants produced more tubers, and the overall yield was higher when compared to nontransformants.
Address and Contact Information 1Institute of Biochemistry and Molecular Biology,
2Institute of Genetics and Microbiology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland
* Corresponding author, e-mail: szopa@ibmb.uni.wroc.pl, tel: (4871) 3756202, fax: (4871) 3252930
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Volume 9 (2004) pp 947 - 961
Title DHN10 DEHYDRIN IS NOT EXPRESSED IN TRANSGENIC Solanum SPECIES PLANTS WHEN THE Dhn10 GENE IS FUSED TO A GLUCOSYL TRANSFERASE PROMOTER
Authors Izabela Pawłowicz, Wojciech J. Grygorowicz and Tadeusz Rorat*
Abstract A gene fusion system was used to study the expression pattern of the Dhn10 gene, encoding the DHN10 dehydrin protein in transgenic Solanum tuberosum plants carrying a combined GT-Dhn10 transgen in which the glucosyl transferase (GT) promoter region was fused to the coding sequence of the Dhn10 gene. Expression of the native Dhn10 gene and the GT-Dhn10 constructs was analysed in regenerated S. tuberosum transgenic plants, both at the transcript accumulation and protein levels. We showed that the expression of both the GT-Dhn10 transgen and the Dhn10 gene was regulated in the regenerated plants at the transcriptional level in an independent way, but only the protein product of the native Dhn10 expression was detected. The transcription product of the GT-Dhn10 transgen did not affect the expression of the Dhn10 gene either at the transcription level or at the protein level. The GT-Dhn10 plants did not show changes in freezing capacity compared to the control, non-transgenic ones.
Address and Contact Information Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyłska 34, 60-479 Poznań, Poland
*Corresponding author; e-mail: tror@igr.poznan.pl
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