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Changing of carbohydrates by inoculation of Pseudomonas savastanoi pv. phaseolicola oil bean lines with different resistance
82-85.Views:161The Pseudomonas savastanoi pv. phaseolicola (PS) is one of the most significant stressors of bean (Phaseolus vulgaris L.). Chemical and agrotechnical treatments have minor importance, so breeding has great part in the protection against this pathogen. Most of the cultivars are susceptible to PS. The genetic background of resistance in the plant is a complex system. Leaf resistance is a monogenic system, but there are some modifier genes. The pathogen species can be divided into different races.
To understand the functioning of this resistance gene, experiments were carried out using bean varieties with different genotypes and near isogenic lines of bean. Eight lines were tested. Our main objective was to test bean lines with PS with high virulence.
The experiment was made in greenhouse and on field. The virulent bacterium strain has been isolated in Hungary.
The changes of carbohydrates were tested after infection. In homeostasis the level of carbohydrates (especially glucose and fructose) were higher in susceptible lines. In case of artificial and natural infection the decrease of glucose were more significant in susceptible lines than in resistant lines. In the leaf samples from systemic chlorosis the level of this carbohydrate increased.
These changes are connected with the level of resistance, but more experiments are needed to verify this assumption.
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Molecular diversity of Hungarian melon varieties revealed by RAPD markers
11-13.Views:142RAPD markers were used to reveal genetic diversity between nine varieties of Cucumis melo L. and to identify the studied varieties. Of the 60 primers tested 12 primers produced polymorph patterns. A set of 4 primers was sufficient for distinction the nine investigated melon varieties.
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Production of transgenic carnation with antisense ACS (1-aminocyclopropane44-carboxy late synthase) gene
104-107.Views:177Dianthus chinensis and Dianthus caryophyllus varieties were tested for shoot regeneration from leaf and petal explants and transformed with Agrobacterium tuniefaciens strains (EHA 105 and LBA 4404) harbouring an apple derived ACS cDNA in antisense orientation in order to reduce ethylene production and influence the ethylene dependant traits in carnation. After transformation regenerating shoots were selected on MS medium containing 50-75-100-125-150 mg/1 kanamycin and supplemented with 1 mg/1 BA, 0.2 mg/1 NAA. Transgene integration was proved by PCR analysis with npt II spcific primers followed by Southern hybridisation of DNA isolated from green shoots on medium containing 150 mg/1 kanamycin. Several putative transformants were subjected to RT-PCR in order to examine the npt 11 expression at mRNA level. Both the transformant and the non-transformant plants were potted into glasshouse to observe the effect of changed ethylene production on flowering time, petal senescence and vase life.
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Molecular identification of old Hungarian apple varieties
37-42.Views:256Altogether 40, mainly old Hungarian apple varieties were screened with six previously described microsatellite markers. A total of 71 polymorphic alleles were detected (average 11.8 alleles/locus) and the heterozygosity of markers averaged very high (0.8). The genetic variability among the genotypes proved to be so remarkable that as few as three markers from the applied six were enough to distinguish between the 40 varieties. This was also confirmed by the cumulative probability of obtaining identical allele patterns for two randomly chosen apple genotypes for all loci, which value was quite low: 2.53 x 10-5. The molecular identification of these genetically very different old apple genotypes could be very useful in future breeding programs.
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RAPD analysis of grapevine hybrids and cultivars
63-66.Views:156Utilization of the Randomly Amplified Polymorphic DNA (RAPD) technique as a molecular marker was tested to investigate the relationships between some representative grapevine cultivars and hybrids established at the Department of Genetics and Plant Breeding (CUB), to distinguish clones as well as to characterize various hybrids between species or cultivars and their parents. Vitis vinifera cultivars were easily and successfully distinguished by the RAPD technique and they were grouped according to the traditional taxonomic classification. RAPD patterns of the examined Pinot gris clones proved to be completely identical. Number of generations was reflected by the value of genetic distance of the examined hybrids. Genetic identity of parents and their offsprings was influenced by the selection applied in the process of plant breeding. Parental phenotypic and morphologic characteristics showed high degree of segregation in hybrids, but RAPD analysis revealed that their genetic similarity is considerable. The three Vitis anntrensis clones were properly discriminated from every cultivar and hybrid of Vitis vinifera, i.e. hybrids are much closer to the cultivated grapevine than to V. anzurensis due to the phenotypic selection carried out during the life-cycle of one or two generations.
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Optimization of RNA isolation from stone fruits at different ripening stages
101-104.Views:202This study was conducted to select the most appropriate RNA isolation method that can be used successfully in case of stone fruits. The changing pattern of gene expression during the ripening process of stone fruits may elucidate the molecular background of several phenotypical or phytochemical alterations present among different genotypes. Our laboratory aims to study the expression of genes encoding for enzymes that catalyze crucial steps in the flavonoid biosynthesis pathway. RNA isolation from fruit mesocarp is a challanging task due to high levels of sugars and polyphenolics accumulating during fruit development. Therefore, at first, the optimal techniques eligible for RNA isolation from fruit tissues at different ripening stages must be selected. Our study compares three different RNA isolation protocols and describes their potential applicability according to different fruit species and ripening stages.
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Identification of ripening-related genes in strawberry fruit by cDNA-AFLP
33-41.Views:155An RNA fingerprinting study of strawberry receptacle and achene tissue was performed to identify candidate genes involved in fruit ripening. Quantitative cDNA-AFLP was used to detect differential gene expression in green, white, pink and red stages of fruit ripening. Based on hierarchical average linkage clustering the differentially expressed genes formed three major groups, genes expressed only in green receptacle, genes expressed mainly in white, pink and red receptacle, and in achene. 130 transcript-derived fragments (TDFs) were isolated and sequenced. Most TDFs did not show any homology to sequences with known functions, others were homologous to genes involved in oxidative stress response, signal transduction, regulation of development and cell-wall metabolism. Novel genes, so far not associated with strawberry ripening and ripening in general, were identified, such as genes encoding a bHLH protein, putative nitrilase-related protein, putative HD-zip protein. The differential pattern of gene expression draws the attention to the significance of ripening induced-or repressed promoters in strawberry fruit, whose isolation and characterization can be useful tool for functional genomics. For this purpose nine cDNA-AFLP fragments related either to ontogeny or senescing were completed with 5'UTR aiming at more precise annotation and future promoter isolation. Although tens of potentially important transcriptome changes were identified, the function of many ripening induced genes remain unknown.
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Development of microsatellite markers for Rhodiola rosea
37-42.Views:223Rhodiola rosea L. is an important adaptogen medicinal plant. In this study two new microsatellite markers were developed. The assessment of the genetic diversity of R. rosea has recently started with molecular markers, but only a few species-specific microsatellite markers have been published so far. However the small number of markers allows only a limited insight into the genetic variability of the species therefore the aim of our work was to develop new microsatellite markers for R. rosea with a microsatellite enrichment library technique. Genomic DNA was cleaved with an endonuclease enzyme followed by adaptor ligation and PCR amplification. DNA fragments that contained microsatellites were first isolated using a biotin-streptavidin linkage based magnetic selection and then cloned into plasmids. Out of forty-three sequenced clones three contained microsatellites, in these cases primers were designed for the amplification of the microsatellite repeats. The newly developed primer pairs were tested on individuals from distant R. rosea populations and the variability of the amplified fragments was estimated by fragment-length analysis. The locus RhpB14a was found to be monomorphic while RhpB14b and RhpB13 were polymorphic. As a result of the present study, two novel variable microsatellite loci were identified in the genome of R. rosea.
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Ripening related processes in strawberry, a nonclimacteric fruit: a short overview
105-109.Views:201Fruits are essential part of the human diet: they provide vitamins, minerals, antioxidants to the mankind. Physiologically they can be divided into two groups-climacteric and nonclimacteric - depending if they display any respiratory peak and dramatic increase in ethylene biosynthesis or do not. Ethylene is a gaseous hormone playing a very important role in several physiological processes in plants. While climacteric fruits, like apples, bananas, tomatoes, peaches, apricots show increased ethylene biosynthesis and dramatic respiratory peak during their ripening, nonclimacteric fruits, like strawberries, grapes, citrus do not.
The most widely used fruits for studying nonclimacteric ripening are strawberries: several papers are focusing on the identification and characterization of ripening related genes from this plant. Therefore here we attempt to summarize the most important advances in strawberry fruit development, and ripening.
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The use of SSR markers in family Rosaceae
29-32.Views:144The identification of plant species and study of their genetic relatedness is an important object of plant genetics. The Rosaceae family contains a lot of economically important fruit, ornamental, and wild plant species. The microsatellite markers have been proven to be an efficient tool for description of the genetic relatedness among varieties and species. Their evolutionary conserved regions enable them to differentiate among various accessions. This article intends to show proceeded identification and characterization projects on Rosaceae species by using SSR markers. The article presents sources of already published primer sequences. The use of already published primers can highly reduce the cost and duration of this kind of researches.
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Molecular analysis of strawberry cultivars using RAPD, AP-PCR and STS markers
24-28.Views:130Seventeen strawberry (Fragaria x ananassa Duch.) cultivars representing the national list of Hungary, were subjected to RAPD, AP—PCR and STS analysis. Of the 31 decamer and oligomer primers tested 26 primers produced polymorphic patterns. 45 polymorphic fragments were analysed, ranging between 200-2800 by in size. Based on the data, similarity coefficients (Jaccard index and Simple matching coefficient) were calculated, and dendrograms were constructed using the unweighted pair group method of arithmetic averages (UPGMA). The dendrograms only partly reflect the known pedigree data. Specific RAPD markers were identified for cultivars F5, Pocahontas and Rabunda.
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The Effects of Some Parameters on Agrobacterium-Mediated Transformation in Muskmelon
46-49.Views:181Some parameters involved in Agrobacterium-mediated transformation in muskmelon Hales best (HBS) were studied. Cotyledon explants excised from 3.5-day-old seedlings were co-cultivated with Agrobacterium tumefaciens harbouring binary vectors which contained GUS and BAR genes. After co-cultivation on a low pH medium, explants were transferred to selective medium, with higher pH, containing Claforan and Finale. The medium was changed every two weeks till shoots were induced. All shoots rooted on MS medium supplemented with 0.3 mg/L IBA. These parameters combined as a whole led to successful transformation. The expression of the introduced gene construct was confirmed by GUS staining of shoot segments.
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Preliminary evaluation of breeding perspectives of Ukrainian sweet cherry cultivars: nutraceutical properties and self-incompatibility
7-11.Views:336Some traditional sweet cherry cultivars of Ukrainian origin may represent perspective material for Hungarian cherry breeding. A total of eight cultivars analysed represent great diversity in several phenotypic traits including fruit ripening time or fruit flesh colour. Considerable differences in the anthocyanin content may result in different antioxidant capacity of fruits. In the present study, we used ferric reducing antioxidant power (FRAP) and total phenolic content (TPC) assays to characterize fruits’ nutraceutical properties. These values were compared with the respective values measured for eight commercial cultivars grown in Hungary. The average of FRAP and TPC values was higher for the Ukrainian cherries compared with commercial cultivars suggesting they might be included in functional breeding programs. Since, cherry is a self-incompatible species, the determination of S-genotype is required for both breeding and successful cultivar association in commercial orchards. Complete or partial S-genotypes were determined for 5 and 3 cultivars, respectively.
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Shoot induction and plant regeneration from cotyledon segments of the muskmelon variety "hógolyó"
61-64.Views:146Cotyledonary segments of the casaba type muskmelon variety "Hógolyó" were used to induce organogenesis. Fifty different hormone combinations were applied to enhance the induction of shoot formation on the edge of the segments. The phases of organogenesis were followed with light- and scanning electron microscope. Shoot induction was achieved with high frequency. The shoots were transferred to hormone free media for root induction. The rooted plantlets were planted out to soil.
NAA was feasible and the method can be applied in transformation experiments.
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Grape variety comparison of different stress tolerance based on the quantitative measurement of carbohydrates
37-40.Views:167The analyses of various host-pathogen relationships have established the response reaction roles of carbohydrates — especially monosaccharides — measurable in the vegetal parts of the host. Published results also provide information concerning the way various pathogens utilize carbohydrates and concerning the carbohydrates pathogens prefer out of the "selection" provided by the host plant. The role of carbohydrates in the response reactions to abiotic stress has been studied on several plant species as well — currently, too, it is an often discussed area of research. The above-mentioned results form the basis of our intention to study the connection between susceptibility to grey mould and the quantity of measurable carbohydrates in the leaves of grape varieties of various stress tolerance levels.
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Test of the utility of apple retrotransposon insertion patterns for molecular identification of 'Jonathan' somatic mutants
7-10.Views:202Up until today, apple sport mutants proved to be indistinguishable from each other and their progenitors at the molecular level using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) marker techniques. This is not surprising, since the genomes of these somatic mutants differ only in one or a few small regions that affect economically important characteristics, such as improved fruit colour, size, or flavour. In most cases, these genome differences are probably caused by retrotransposons which are able to convert their RNA transcripts to DNA with reverse transcriptase enzyme prior to reinsertion, but unable to leave the genome and infect other cells. Retrotransposon insertions can alter the expression of other genes and/or the structure of encoded proteins. The sequence-specific amplified polymorphism (S-SAP) technique is capable of revealing the genetic distribution of retrotransposable elements over the whole genome. The present study used this approach to try to characterize and distinguish 'Jonathan' somatic mutants via fingerprinting, which is an unsolved problem.
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Examinations of 600-year-old seeds by means of archaeobotanical and genetical methods
79-80.Views:143About 600-year-old plant seeds were discovered in a well of a mediaeval cellar in the course of an excavation in Budapest. After the archaeobotanic purification seed of 16 species were found in large quantities. Seeds preserved in the best state were selected from each group. The existence of endosperm was analysed in these subfossils, which turned to be successful mainly in the case of grapes (Vitis vinifera) and cornels (Cornus mas). Seeds of these two species contained the most endosperm and remains of the embryo. DNA was extracted with the help of DNEasy Plant Mini Kit and analysed by RAPD-PCR method. The amplification of DNA extracted from cornel seeds resulted in detecting a 1500 by fragment, which makes the comparison of these samples with present-day cornels possible.
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Characterization of quince (Cydonia oblonga Mill.) cultivars using SSR markers developed for apple
7-10.Views:357Quince (Cydonia oblongaMill.) is a minor fruit crop, which is primarily used for marmalade, jam and sauce.Very few quince cultivars are known all over the world and in many cases similar names are used for presumably different cultivars. The aim of the present study was to evaluate and characterize the genetic diversity of 36 quince cultivars and selections with SSR markers. Seven out of 8 SSR markers designed from apple sequences could successfully yield amplification also in quince cultivars. Number of alleles per locus ranged from 2 to 3 alleles. These allele numbers are quite low when compared to apple. It is supposed to be the consequence of a genetic bottleneck. In spite of the low allele number per locus, the 36 quince cultivars formed 30 different genotypes. The ratio of homozygosity was low, which might be coupled with the self-(in)compatibility phenotype of quinces. SSR markers proved unable to differentiate putatively closely related cultivars (e.g. ‘Bereczki’ and ‘Bereczki bôtermő’). In general, the level of polymorphism among the tested quince genotypes was much restricted due to the low allele number detected. However, it must be considered that the number of analysed SSR loci is not enough high to estimate the overall heterozygosity of the quince genome. Further experiments are needed and the SSR markers proved to be a reliable and useful tool for such analyses.
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Production of transgenic carnation with a heterologous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase bifunctional enzyme cDNA
75-79.Views:121Transgenic carnations were produced with a modified mammalian bifunctional enzyme cDNA coding 6-phosphofructo-2- kinaseffructose 2,6-bisphosphatase. Relative activity of this enzyme determines the fructose 2,6-bisphosphate (fru 2,6-P2) cytosolic concentration. This metabolite — as a signal molecule — is one of the carbohydrate metabolism regulators. The regenerated Dianthus chinensis and Dianthus caryophyllus shoots were selected on MS basal medium containing 150 mg/1 kanamycin. Transgene integration was proven by PCR analysis with cDNA specific primers followed by Southern hybridization of DNA isolated from selected green shoots, which survived on kanamycin containing medium, so 3 D. chinensis and 20 D. caryophyllus transgenic plants were produced. Transgene expression were examined by RT-PCR. Transformed and control plants were potted in glasshouse to evaluate the effect of modified fru 2,6-P2 on development, growth and carbohydrate metabolism.
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Co-transformation of bean callus using high-velocity microprojectiles- mediated DNA transfer
76-78.Views:128We have found that 50 mg/I kanamycin and 0.8 Mo1/1 mannitol concentration was sufficient to kill the control callus of bean (Phaseolus vulgaris L.) and differentiate transgenic from the non-transgenic cells. The GeneBooster particle delivery system was used for the bombardment of bean callus. The kanamycin resistance gene was used as a selectable marker. The test was made by transferring the healthy white callus, subcultured for three months on selective and non-selective medium. After selection on kanamycin containing media, several kanamycin resistant calli had been obtained, survived and grew. After selection on mannitol containing media no drought resistant calli had been obtained. Resistance of the selected calli were verified by their ability to grow repeatedly on selective medium containing 150 mg/I kanamycin. Selective pressure was maintained over a period of 8 months.
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Self-incompatibility alleles in Esatern European and Asian almond (Prunus dulcis) genotypes: a preliminary study
23-26.Views:200Almond [Prunus dulcis (Mill.) D. A. Webb.] as one of the oldest domesticated plants is thought to have originated in central Asia. Gametophytic self-incompatibility of almond is controlled by the highly polymorphic S-locus. The S-locus encodes for an S-ribonuclease (S-RNase) protein in the pistils, which degrades RNA in self-pollen tubes and hence stops their growing. This study was carried out to detect S-RNase allelic variants in Hungarian and Eastern European almond cultivars and Turkish wild growing seedlings, and characterize their S-allele pool. Five new alleles were identifi ed, S31H, S36-S39 in Eastern European local cultivars. The village Bademli and Akdamar island are two distinct places of almond natural occurrence in Turkey. Trees growing wild around Bademli city showed greater genetic diversity than those originated on Akdamar island. Many of the previously described 45 S-RNase alleles have been also detected in these regions. Homology searches revealed that Turkish almonds carried some P. webbii alleles indicating hybridization between the two cultivars and massive introgression events. Our results supply long-awaited information on almond S-allele diversity from regions between the main cultivation centres and the centre of origin of this species; and are discussed from the aspect of methodological developments and evolution of the cultivated almond.
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Somatic embriogenesis and regeneration of Vitis sp.
41-45.Views:154Somatic embriogenesis and regeneration of Vitis sp.
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Agrobacterium transformation of Rhodiola sp.: current status and limitations
37-42.Views:33The study of secondary metabolites has led to the discovery of new drugs for treating human diseases. However, consistent plant supply can be challenging, leading to the use of plant tissue culture techniques such as hairy root culture. Hairy roots have stable genetics, lateral branching, and can produce secondary metabolites, including alkaloids, flavonoids, and terpenoids. Research on hairy roots as a subject began in the late 19th century, and for the last four decades, hairy roots have been utilized for producing secondary metabolites and recombinant proteins. This article focuses on Rhodiola species - genus of perennial plants that belongs to the family Crassulaceae - and its potential as a source of secondary metabolites using hairy root culture techniques. Rhodiola sp. is widely distributed throughout the Arctic regions of the Northern Hemisphere, with several species having significant medicinal properties. The article discusses the possible use of hairy root cultures for the production of Rhodiola secondary metabolites, including salidroside and rosavins, which have demonstrated significant pharmacological activity in various studies. The use of elicitation and genetic engineering techniques to boost secondary metabolite production in Rhodiola hairy roots is also explored. Overall, the article highlights the potential of Rhodiola hairy root cultures as a valuable source of secondary metabolites with medicinal properties. However, despite some studies Rhodiola hairy root induction and culturing still remains highly unexplored.
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Nutritional and seasonal requirements for callus growth in Taxus baccata
111-114.Views:118Callus cultures derived from young stems of two varieties of Taxus baccata cv. aureovariegata (genotype I) and Taxus baccata L. (genotype II, III) were induced. Gamborg's B5 medium was supplemented with different concentrations of auxin (2,4-D) in combination with cytokinins (kinetin or topolin) and with a phenolic-binding compound (PVP) to prevent callus darkening and growth inhibition. Stem explants displayed different responses to in vitro culture depending on plant genotype and on the season. Genetic variability was observed in the growth rate of calli initiated from all three genotypes of the same Taxus species. We found the best growth of callus cultures originated from the genotype ill in defined media. After the first subculture the majority of the cream-coloured primary callus turned brown and ceased its growth. However, the long-term culture was initiated.
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Bean tissue culture and genetic transformation with Agrobacterium
32-35.Views:134In this paper we report the establishment methods of a rapidly growing callus culture of Phaseolus vulgaris bean as well as the conditions required for a high level of transient gene expression using Agrobacterium-mediated transformation. A vector is containing both the lindan-resistance gene as a selectable marker, and GUS gene as a screenable marker. By using hypocotyl explant and vertical culture on B5 medium supplemented with 1 mg/1 kinetin- and 2,4-D 2 mg/1 and subcultured every 3-4 weeks, we can recommend to get a good and much callus from bean. This will help in introducing foreign DNA into callus cells. One strain of Agrobacterium carrying plasmid as vector for introducing foreign DNA into plant cells was used. At different concentrations of lindan; 3, 4 and 4.5 mg/I, the transformed Maxidor callus survived and grew over a period of 6 month and subcultured every 3-4 weeks, but the control callus died. Callus were assayed for GUS activity to confirm the expression of the GUS gene using the histochemical assay test. The GUS gene was also correctly expressed in callus cultures grown on 4mg/I lindan-selected medium, the typical blue colour in the histochemical assay using the X-gluc as substrate. But the control, non-transformed callus was not able to grow in the presence of lindan, neither showed a positive reaction in the in vitro assays.