We report the method for the establishment of rapidly growing callus cultures of Phaseolus vulgaris and the conditions required for efficient transformation using high velocity microprojectiles and high level of transient gene expression. Using hypocotyl explant and vertical culture on B5 medium with lmg/1 kinetin and 2 mg/1 2,4-D, we can recommen...d to get a rapidly growing callus from bean which is a good starting material to introduce foreign DNA into bean cells. The GeneBooster particle delivery system was used for the bombardment of bean callus and the Hgm resistance gene (Hgmr) was used as a selectable marker gene. 25mg/I hygromycin (Hgm) concentration was sufficient to kill the control callus. We used the standard physical factors, the appropriate pressure of N2 gas for the bombardment of the callus tissue, the shooting distance and the size of tungsten particles used as microprojectiles. Selective and nonselective tests were made by transferring the healthy green and white calluses, subcultured for 4 months on selective and nonselective medium. Several Hgm resistant calli had been obtained. Selective pressure was maintained over a period of 10 months.
Utilization 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.
Thirty-one old Hungarian grapevine (Vitis vinifera L.) cultivars were investigated on 7 microsatellite loci to characterize them, to separate the cultivars from synonym names, and to confirm parent-offspring connections. Conculta (group of cultivars or bud sports) members, such as `Goher' and Tajor' representatives, were studied to find a suitable... locus for the separation. Synonyms, conculta members, subcultivars and clones of Turmine, which was the most important cultivar of Tokaj, were also analyzed to separate the members of the different taxonomic levels. Pedigree of 'Kiralyleanyka' was examined to find the missing ancestor, because the parent-offspring connection between the natural hybrid and `Koverszolo' is questionable.
Cotyledonary segments of five different genotypes of watermelon were used to induce organogenesis. Five different hormone combinations were applied to enhance the induction of shoot formation on the surface of the segments. The phases of organogenesis were followed with light and scanning electron microscope. Shoots were obtained after four weeks,... then the shoots were transferred to hormone free medium for root induction.
This method of regeneration can be applied in transformation experiments. GUS histochemical assay was made to check the expected success of using Agrobacterium for the transformation.
Vitamin C (L-ascorbic acid) being essential for many living organisms, including man, became once more into the focus of interests because of its numerous physiological effects. Its anti-scurvy and anti-oxidant properties have already been recognised since long in the human body, but it turned out gradually that it has many other functions. In pla...nts, its primary importance is defense against the photo-oxidative stress.
The present review is intended to reveal some details of the artificial synthesis of vitamin C. Emphasis is put on the metabolism of L-ascorbic acid in higher plants. Biosynthetic processes, translocation and accumulation are discussed in detail on the basis of recent results published in the scientific literature.
The cluster thinning is a method of the yield regulation.With the removal one part of the clusters, the yield pro leaf area will be lower, hereby the grape and wine quality will be improved. The regulation of the yield can lead to further advantages: the ratio of the vegetative and generative performance of the vines will improve, the condition of... the plants will better, the number of the physiological diseases can be reduced and the growth of the shoots and roots can be promoted. The grape growers make the cluster thinning almost exclusive by creating one cluster shoots. Usually the upper clusters are removed, because the sugar content of these second or third clusters will be lower. The cluster thinning is an easy task, can be done without special skills. It is an effective method improving wine quality, but its use can lead to other problems. The grapes try to compensate the removed clusters. Therefore the clusters will be bigger and thicker, but more sensitive to bunch rot. Moreover the treatment is expensive, because it needs manual work. It is worth to get acquainted and try the new yield regulation methods, which can help to avoid the occurring problems. Our aim is to show the results of our experiment, which was carried out in Eger, examining the red grape cultivar Kékfrankos. During our 4 years long experiment we compared the effects of cluster thinning, cluster shredding, cluster tipping and defoliation at the flowering, on the vegetative and generative vine performance.
Common bacterial blight (CBB), caused by Xanthomonas campestris pv. phaseoli (Xcp). is a major disease problem of common bean (Phaseolus vulgaris L.). The inheritance of resistance in Xrl and Xr2 lines to two isolates of Xcp was studied in the F2 and F3 populations from the crosses between these lines and the Masay variety (susceptible to Xcp). ...Segregation patterns indicated that different single recessive genes presumably in coupling phase linkage determined the resistance to the HUN and EK-1 1 strains of Xcp in both lines. The presence of some minor, modifying genes beside the monogenic genetic background of resistance was also observed. Xrl and Xr2 lines represent valuable new monogenic genetic sources in resistance breeding to CBB.
RAPD 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.
The use of pathogen-free planting stock for new vineyard establishment is a key component in the maintenance and expansion of vine and quality table grape production. The success of the necessary changes in the structure of the grape industry is forced by the globalization process, the climate change, the rediscovery of autochton varieties as well... as breeding of new tolerant and resistant varieties. The renewal of vineyards largely depend on the availability of planting stocks. Serbia and Hungary found a common interest in establishing pathogen-free stock materials from newly breed resistant varieties and clonal selections of varieties which are traditional in the Serbian-Hungarian border area. During a cross-border cooperation program a complex system for the production of pathogen-free grapevine propagating material was established. Using heat therapy, in vitro shoot tip culture and traditional and molecular diagnostic techniques new pathogen-free stock materials were established from 26 varieties. They have been or will be tested for the presence of most important grapevine viruses, phytoplasmas, as well as bacterial and fungal pathogens. The complex system applying green grafting for indexing on grapevine indicators can shorten the duration of the procedure from 4 years to two-three years.
In 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 ma...rker. 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.
The 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 a...nd 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.
Viruses and viroids are submicroscopic infectious particles which can cause disease symptoms on grapevine. These parasites are depending completely on the energy metabolism of the plant cell. To enter the host cell plant viruses depend on injuries or on transmission via invertebrates (insects, nematodes, etc.). Viruses are classified by many chara...cters including particle morphology, host range and information content of the genome. At present about 70 viruses including 7 viroids infecting grapevine are known. In single or mixed infections they are potentially detrimental to the quality and quantity of grape production in any growing area of the world. Some viruses can cause severe economic damage in vineyards. In Hungary many important viruses and viroids have been detected in grape. This review summarises characteristics of viruses and the results of detection and characterization of virus and virus like diseases of grapevine in Hungary. The identification of the causal agent, its transmission, geographical distribution and the development of the diagnostic methods are also discussed.
Some 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 sele...ctive 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.
We 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.
Grapevine 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 ba...sed 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.
Cotyledonary 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 wit...h 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.
Tissue 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 napht...ylacetic 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.
About 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 turne...d 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.
Dry seeds from two cultivars of common bean (Phaseolus vulgaris L.) were germinated on sterile cotton and sterile deionized distilled water. Cotyledonary node tissue of seedlings were cultured on Murashige and Skoog(MS)-based media supplemented with different combination of N6-benzyl-aminopurine (BAP) and indole-3-acetic acid (IAA), and benzyladen...ine (BA) and a-naphthaleneacetic acid (NAA). The results revealed that the regeneration percent and the average number of buds and shoots per explant were influenced by the type of explants and exogeneously added hormones. Multiple shoot induction on dry bean cotyledonary node that contain 4-5 mm from cotyledons and hypocotyl on a medium containing full concentration of MS inorganic salts supplemented with 0.5mg/1 BA and 0.1mg/1 NAA was feasible and the method can be applied in transformation experiments.