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Co-transformation of bean callus using high-velocity microprojectiles- mediated DNA transfer
76-78.Views:122We 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:184Almond [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:147Somatic embriogenesis and regeneration of Vitis sp.
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Nutritional and seasonal requirements for callus growth in Taxus baccata
111-114.Views:114Callus 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:117In 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.
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Molecular characterization of apricot (Prunus armeniaca L.) cultivars using cross species SSR amplification with peach primers
53-57.Views:200Apricot takes an important place in Hungarian fruit production. Considering morphological characteristics of apricots it was concluded that the genetics background of European cultivars is very limited. Molecular markers and their use for genotyping have revolutionized the identification of cultivars. In a classic apricot breeding program, it is important to be able to establish unique DNA profiles of selections to identify them unambiguously and to determine their genetic relationship. Presently SSR is far the most frequently performed technique for genetic diversity studies. In this study there were used peach and apricot primer pairs from four different sources in order to examine microsatellite polymorphism among cultivars and investigate relationships among them. The possibility of cross species amplification among different Prunus species using SSR primers allowed us to use primers developed in peach to study genetic diversity in apricot. In this work, 90% of the primers used were able to amplify SSRs in apricot and more than half of them were polymorphic. With the 10 primer pairs utilized were proven to be sufficient to set unique fingerprint for several cultivars studied. The obtained dendrogram classified of the 45 cultivars included in this study into two major groups and several subgroups.
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Callus induction on standard type Cymbidium cultivars
108-110.Views:119Tissue cultured Cymbidium PLBs (protocormlike body) were used as starting material to induce embryogenic callus which could serve as objects of genetic transformation. We obtained callus using two methods. The first method was culturing the PLB segments for one month in liquid MS medium in the presence of 0.5 mg/1 benzyladenine and 0.05 mg/1 naphtylacetic acid followed by cultivation on the same composition solid medium with 0.5 g/l activated charcoal for an additional month. Callus formation was observed on 30% of the explants. The second way was to propagate the PLB segments on solid MS medium supplemented with 1 mg/1 thidiazuron. In these cultures we also observed callus formation on 20% of the explants.
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Down-regulation of ethylene production in carnation (Dianthus Caryphyllus L.) by an apple derived ACC-cDNA
101-104.Views:128Transgenic carnations were produced with an apple derived antisense ACC-synthase cDNA. Transgenic carnation regenerants were potted in glasshouse. All transformed plants showed normal growth and were true-to-type. Ethylene production — measured at full opening stage — lowered by 30-60 %, no plant with 100 % decrease was identified. The vase-life has been observed for 5 years. 38 % of the transformant carnations showed a higher a relative value in days by more than 2 days to 6 days. Twenty six plants were found exhibiting the most marked alterations in the tested trait. In these plants ethylene production decreased by 37-67 %, they have longer vase-life (by 4 days or more). Since the fragrance variety 'Bíbor' was the plant material for genetic modification of vase-life, this trait has been conserved after transformation in spite of the fact that the position of transgene integration cannot be directed.
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Preliminary characterization of the self-incompatibility genotypes of European plum (Prunus domestica L.) cultivars
23-26.Views:211European plum is an important fruit crop with complex, hexaploid genome of unknown origin. The characterization of the selfincompatibility (S) locus of 16 European plum cultivars was carried out using the PaConsI-F primer in combination with the EM-PC1consRD primer for the first intron and the EM-PC2consFD and EM-PC3consRD primers for the second intron amplification. Altogether, 18 different alleles were scored indicating high genetic diversity. These alleles were labelled using alphabetical codes from SA to SS. We identified 5 different alleles in 9 cultivars, 4 alleles in 5 cultivars, while 3 alleles were shown in two of the assayed cultivars. A total of 16 different S-genotypes were assigned, and discrimination of all plum cultivars was successful based on their unique S-genotypes. However, further research is required to reliably identify the S-alleles based on their DNA sequence and clarify complete S-genotypes.
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Microsatellite based identification of grapevine cultivars traditional in Hungary and in the Carpathian Basin
71-73.Views:137Grapevine cultivars and clones traditional in Hungary and in the Carpathian Basin are maintained in the genetic collection of the Corvinus University of Budapest. Mostly ancient varieties and clones were genotyped using microsatellite loci. The investigated 6 loci were sufficient to distinguish all cultivars. Some clones could also be separated based mostly on the variable VVS2 loci. For 17 out of the 31 investigated cultivars this is the first report on characterization of the polymorphism of the allele lengths by microsatellite markers on loci: VVS2, VVMD7, VVMD27, vrZAG62.
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Resistance Gene Analogs (RGA) as a tool in fruit tree's breeding
7-15.Views:175Breeding for pest and disease resistance comes as a major objective behind the fruit traits. To increase the effectiveness of fruit resistance breeding application of the Marker Assisted Selection ( MAS) is advantageous. For generating molecular markers which enable the following of interesting traits basically two methods are available: targeted marker design based on conservative region of already known Resistance ( R) gene sequences or randomly generated markers. The creation and the application of these homology based markers are the object of this review in the main temperate zone fruit species.
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The effects of ACS (1-aminocyclopropane-l-carboxylate synthase) gene down regulation on ethylene production and fruit softening in transgenic apple
65-70.Views:113A detailed examination of the production of ethylene and other ripening parameters during storage period has been undertaken in transgenic apple fruits, where the ethylene biosynthesis was inhibited by antisense ACS (l-aminocyclopropane-l-carboxylate synthase) gene. Data indicate down regulation of ethylene production, softening and spoilage in some transgenic lines. In some cases ethylene production was inhibited for over 90 percent, considerable reduction of softening and spoilage was observed probably due to the reduced activity of cell wall degradable enzymes. ACS activity was also monitored during ripening. The fruits of the best transgenic lines could be stored for minimum 4-5 months longer under 5 °C cold room storage conditions and one month longer at normal room temperature. This molecular approach can provide an alternative way to replace the commonly used and costly atmospheric storage of fruits.
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Analyses of Hungarian sour cherry germplasm with simple sequence repeat markers
27-31.Views:198Twenty-four sour cherry cultivars (genotypes), belonging to four cultivar groups were fingerprinted using microsatellite markers. All genotypes have been arisen from the Carpathian basin, which could be secondary gene centre of sour cherry, since its progenitor species, ground cherry and sweet cherry overlap here. Five SSR primer pairs, earlier used for fingerprinting Turkish sour cherry germplasm were tested. None of the five primer pairs showed any polymorphism within the cultivar groups. The primer pairs were able to distinguish between the cultivar groups. The Oblacsinszka and the Cigánymeggy cultivar groups were the most difficult to separate, while the Pándy cultivar group was the most distinguishable.
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Luminescence variations in cucumber (Cucumis sativus L.) leaves derived from different regeneration systems
50-52.Views:154Plants obtained from in vitro culture can show increased susceptibility to environmental stress conditions. In the process of their adaptation to natural conditions it requires monitoring of their physiological state. The methods used to check this phenomenon should estimate quickly and exactly the tolerance to suboptimal environmental factors. Such requirements are satisfied by the methods of measuring chlorophyll luminescence in vivo, e.g. fluorescence induction and delayed luminescence. The objects of our studies were cucumber plants regenerated from cultures of callus and embryogenic cell suspension, as well as the plants obtained from seeds. The plants derived from in vitro cultures displayed a poor physiological condition at the early phase of adaptation characterised by higher susceptibility both to stress caused by increased density of the light flux and low temperature (4 °C) in comparison with the plants obtained from seeds.
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Determination of the cold tolerance off sour cherry cultivars with frost treatments in climatic chamber
49-54.Views:201Nowadays, sour cherry buds can be seriously damaged by spring and winter frosts. Unlike other fruit species threatened by high frost damage, sour cherry cultivars have not been assessed for frost tolerance. The aim of .our survey was to establish the relative cold tolerance of the Hungarian cultivars after treatment in a climatic chamber, and to optimize the methodology formerly elaborated for the frost treatment of apricot. Fourteen cultivars of Hungarian sour cherry (Prunus cerasus) were used in the experiments, which spanned the winters of 2005/2006 and 2006/2007. Our data were used to rank cultivars in two groups according to their levels of cold resistance. We also recommend critical temperatures and treatment times for the testing of sour cherry cultivar resistance to cold in climatic chambers.
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General defense system in the plant kingdom III.
45-54.Views:153Our observations regarding the symptoms not fitting into, significantly differing from the hypersensitive defense system, which we noticed during the judgment of several plant species, symptoms provoked on several million plants have constituted a unified entity. They have provided evidence for the existence of a different plant defense system. We called this so far unknown basic response of plants to biotic effects as general defense system. This system defends them from the attack of numerous microbe species in the environment.
The evolutionary intermediate phase between the general and the specific, the two defense systems is the susceptible host—pathogen relation. The vertical resistance system of plants escaping from the susceptible host—pathogen relation, based on specific hypersensitive reaction also suggested the existence of a more original, general defense system and the susceptible host—pathogen relation developed as a result of the collapse of that system.
The evolutionary relation of the two defense systems is proved by the only recessive inheritance of the older general defense system and in the majority of cases dominant hereditary course of the specific defense system. In our experiences, the modifying genes of the recessive general defense system, in most cases, are behind the specific defense systems, which are known to have monogenic dominant hereditary course and react with hypersensitive tissue destruction. This seemingly striking genetic fact is explained by the following: the general defense system less dependent on environmental effects regulates much faster pathophysiological reaction than the specific resistance genes strongly dependant on environmental effects coding dominant hypersensitive reaction.
The general and specific defense reactions, the processes excluding the microbes attacking plants with compacting of cell growth and tissue destruction, which mean two opposite strategies, building on and regulating each other constitute the entity of resistance to plant disease.
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Review of the molecular background of self-incompatibility in rosaceous fruit trees
7-18.Views:193This review gives a presentation of the gametophytic self-incompatibility system in the roscaeous fruit trees. Studies to discover the pistil (S-ribonucleases) and pollen-part components (F-box molecules) are summarized and models for the self-incompatibility reactions as well as their molecular background are discussed. We describe how mutations within the S-RNase or F-box genes can contribute to the transition from self-incompatibility to the self-compatible phenotype in many fruit tree crops. The current state of the arts is compared to the information obtained in other plant species possessing similar incompatibility system.
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Evaluation of different sources of resistance for breeding powdery mildew resistant grapevine varieties
35-40.Views:163The appearance of resistance to powdery mildew was investigated on seedling progenies of interspecific crossings. Hybrids of American and East-Asian wild species and one Vitis vinifera variety (Janjal kara) were used as source of resistance in the crossings. The resistance of foliage and of berries were tested in the field at the time of vintage. The degree of resistance was ranked into six classes.
In American and Asian hybrid derivatives there were few highly resistant progenies, but in the hybrids of Janjal kara variety more than 90 % of the progenies proved to be highly resistant to powdery mildew. According to the distribution of classes of resistance in the populations the resistance of American and Asian sources are mostly influenced by additive gene affects. The resistance of Janjal kara seemed to be mono or digenic.
No close correlation was found between the powdery mildew resistance of the foliage and that of the berries.
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Transmethylation and the general defense reaction of plants
35-40.Views:165Plant breeding for resistance, namely building specific resistance genes into cultivated plants to ensure resistance against certain pathogen species, is a several-decade-long practice. While looking for purposes of failures appearing during the cultivation of varieties created in this way, a plant feature that ensures non-specific reactions against effects which evoke biotic stress attracted our attention. We named this plant defense form the general defense reaction. The general defense reaction is a fundamental attribute of the plant kingdom, fulfils the role of plant immune system and manifests itself in cell enlargement and cell division. Plants with a high level general defense reaction endure abiotic stresses as well.
In studying the biochemical background of the interaction of the general defense reaction and transmethylation, we found that transmethylation has important role in warding off both biotic and abiotic stresses. According to our observations, plants possessing high level general defense system are suitable for thorough examination of the process and plant physiological role of transmethylation. Biochemical studies also strengthened our observation, which has been taken on the basis of phenotype, that the general defense system can not be ignored during future plant breeding.
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In vitro multiplication and hardening of grapevine plants in aeriated media
15-18.Views:207In vitro cultures have widely been used in horticulture for rapid multiplication of new varieties and clones as well as to produce pathogen-free stock material. To improve efficient hardening and transfer in vitro grown grapevine plants were multiplied by cutting them into single-node internodes with the whole leaf. Microcuttings including the shoot tips were rooted in granulated perlite moisted with tapwater under sterile conditions. After 2-3 weeks the rooted microcuttings were supplied by nutrients and hardened by gradual opening and finally by complete removal of the lids of jars or plastic boxes used for growth. Using this method microcuttings of Vitis vinifera cvs. „Chardonnay", „Cabernet franc", „Riesling" and „Sauvignon blanc" and the rootstock varieties Vitis riparia x Vitis cinerea cv. „Barrier" and Vitis berlandieri x Vitis rupestris cv. „Richter 110" formed new roots and shoots and 100% of the tested plants survived the acclimatization procedure. Similar results were obtained when perlite was replaced with rockwool-, or pit-pot blocks. This method may highly increase the efficiency of producing pathogen-free propagating material and new transgenic lines.
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Genetic transformation of bean callus via Agrobacterium- mediated DNA transfer
49-53.Views:120Callus cultures were induced from hypocotyl of young bean seedlings. Callus developed and maintenaned on B5 medium supplemented with 2mg/1 2,4-D and 1 mg/1 kinetin. The results demonstrate that A. tumefacins-mediated transformation is a convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus co-cultivated with A. tumefaciens can be transformed to get heibicide Finale (glufosinate-ammonium) resistant GUS positive tissues. Southern blot analysis of transformed calli showed integration of gusA marker gene carried by a binary vector. Transformed calli were selected on herbicide containing media. Data of molecular analysis (Southern blotting) confirmed the insertion of gusA gene in the genome of herbicide resistant calli with bar gene. There are three evidences that calli are stable transformants: (1) herbicide resistance, (2) GUS activity which is indicative since the coding region containing an intron, (3) the results of Southern hybridization technique.
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Evaluation of some Achillea L. accessions based on morphological, cytological and chemical characteristics
69-74.Views:152Taxonomical evaluation of six taxa of the Achillea aggregate was carried out to determine their potential for breeding and cultivation. We used complex morphological, cytological and chemical aspects of characterisation. Three taxa of tetraploid genom and high chamazulene contents (33-40% ess.oil) proved to be A. collina Becker. A strain of wild origin had also high -bisabolol content and high oil level, comparable with the selected cultivars. A population was identified as new chemotype of A. pannonica Scheele with typical morphological and cytological traits, a wide sesquiterpene spectrum but lacking of 1.8 cineole and -pinene. We found a mixed population which consisted of both A. collina and A. pannonica plants. A tetraploid, azuleneless taxon could be concluded as A. pratensis Saukel and Langer not described before in Hungary. The diverse performance of the populations calls the attention to the significance of controlled plant raw in phytopharmaceutical products.
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Self-incompatibility in plums (Prunus salicina Lindl., Prunus cerasifera Ehrh. and Prunus domestica L.). A minireview
137-140.Views:424Japanese plums (P salicina) and cherry plums (P cerasifera) are diploid species, while European plum (P. domestica) cultivars are hexaploids. Most diploid species are self-incompatible while fertility relations of the hexaploid European plums are variable between self-incompatibility and self-compatibility. About twenty S-alleles and six inter-incompatibility groups and one S-haplotype responsible for the self-fruitful phenotype were described in Japanese plum cultivars, but studies on cherry plums and even on the European plum cultivars are severely restricted. This review is focused on the available information obtained from myrobalans and European plums; and discusses recent hypotheses regarding the putative origin of the hexaploid plums, and thereby indicates the possibility of allele flow between different plum species.
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Transformation of tobacco plants with virEl gene derived from Agrobacterium tumefaciens pTiA6 and its effect on crown gall tumor formation
53-56.Views:137The VirEl protein plays a key role in the transport of VirE2 protein from the bacterium to the plant cell during crown gall tumor induction by Agrobacterium. The virEl gene of A. tutnefaciens pTiA6 was cloned into the plant transformation vector pTd33 yielding pTd93virEl that was introduced into A. tuniefaciens EHA101 and used for tobacco transformation. The presence of the foreign DNA in the putative transgenic plants was confirmed by PCR analysis. Nine of the 41 transformed plants formed only small tumors following infection with the wild-type A. vitis octopine strain AB3. This property was inherited into the T1 generation. The expression of virEl gene in TI plants was demonstrated by Northern blot analysis.
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S-locus genotyping on stone fruits in Hungary: a review of the most recent achievements
39-43.Views:199Central Europe can be taken as a geographical and historical connection zone between the western growing countries and Asian gene centres of Prunus tree fruits. The determination of the S-genotype of stone fruit (mainly almond, plum, cherries and apricot) cultivars and landraces has both practical and theoretical significance. Our group has allocated complete S-genotypes for more than 200 cultivars and selections of almond, Japanese plum, sweet cherry and apricot. Among Eastern European almond cultivars, two novel cross-incompatibility groups (CIGs) were identified. S-alleles of a related species were also shown in P. dulcis accessions; a fact seems to be indicative of introgressive hybridization. Our results with Japanese plum clarified and harmonized two different allele nomenclatures and formed a basis for intensive international studies. In apricot, a total of 13 new S-alleles were identified from Eastern European and Asian accessions. Many Turkish and North African cultivars were classified into new CIGs, III–XVII. Results suggest that the mutation rendering apricot self-compatible might have occurred somewhere in south-east of Turkey and we were successful to confirm the presumed Irano-Caucasian origin of North African apricots based on the geographical distribution of S-alleles. In sweet cherry, new alleles have been identified and characterized from Turkish cultivars and selections. In addition, wild sweet cherry and sour cherry S-alleles were also shown indicating a a broader gene pool in Turkey as compared with international cultivars. We also used S-genotype information of Ukrainian sweet cherry cultivars to design crosses in a functional breeding program. Our results exhibit an increased number of S-alleles in tree fruit accessions native to the regions from Eastern Europe to Central Asia, which can be used to develop S-genotyping methods, to assist cultivation and draw inferences for crop evolution.