Search
Search Results
-
Application of DNA markers for detection of scab resistant apple cultivars and selections
59-63.Views:104A DNA marker-based study was undertaken to identify the occurrence of major scab resistance genes in some apple cultivars and selections of importance for apple breeding. Unfortunately none of the RAPD-based markers previously reported to detect the Va, Vb, Vr and Vx genes produced unambiguous results. By contrast, the CAPS marker MI8 produced the expected three bands in all cultivars and selections already known or suspected to have the Vf gene, as well as in the Russian cultivar 'Antonovka Polotora Funtovaja' suspected to have Va resistance which however may be allelic to Vf. Vf-carrying selections and newly named cultivars 'Frida' and 'Fredrik' are grown successfully in Sweden without fungicides, suggesting that the Vr resistance breaking scab races 6 and 7 have not yet become a problem. The SCAR marker B12 detected the Vm gene in 'Prairifire', 'Rouville', clones 'OR45T132' and 'OR48T70', and selection '16-36-193'. The SSR locus 0102b10 detected one band at 118 by in 'Reka'. This is presumed to be identical to the Vr gene marker previously reported.
-
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.
-
Test of the utility of apple retrotransposon insertion patterns for molecular identification of 'Jonathan' somatic mutants
7-10.Views:171Up 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.
-
Development of microsatellite markers for Rhodiola rosea
37-42.Views:200Rhodiola 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.
-
High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells
99-102.Views:113We 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 recommend 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.
-
Genetic relatedness among Asian Cotoneaster species investigated with DNA marker analysis
43-46.Views:152The widespread genus Cotoneaster has its centre of diversity in the Himalayas and surrounding areas. Most taxa appear to be polyploid and apomictic, and many of them have become popular ornamentals due to their attractive foliage and berries. One of the taxonomically most critical groups is Section Alpigeni which contains several important ornamental plants. The number of species belonging to this section varies widely between different taxonomic treatises, depending on whether 'splitting' or 'lumping' of species is preferred. Using a rather narrow species definition, we have investigated 13 different species using RAPD analysis. A simple matching (SM) coefficient-based principle coordinate analysis (PCO) was calculated from the RAPD data. Some species were clearly more similar to each other than to other species in the analysis. The levels of similarity did, however, not correspond very well to the lumping together of several taxa under the same species name as performed e.g. in the recent Flora of China. Obviously, the complex hybridogenous origination and, in some cases, still ongoing recombination with sexual species or among the apomictic taxa themselves, produces a genetic variability structure that cannot be properly reflected in a hierarchical taxonomy.
-
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.
-
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.
-
RAPD analysis of grapevine hybrids and cultivars
63-66.Views:139Utilization 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.
-
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.
-
Aminoglycoside antibiotics affect the in vitro morphogenic response of chrysanthemum and tobacco
93-104.Views:109Broadly the success of genetic transformation of plants requires non-chimeric selection of transformed tissues and its subsequent regeneration. With rare exceptions, most plant transformation protocols still heavily utilize antibiotics for the selection of transgenic cells containing an antibiotic-degrading selectable marker gene. The morphogenic capacity of in vitro chrysanthemum and tobacco stem and leaf explants change with the addition of aminoglycoside antibiotics (AAs). Of 6 antibiotics tested, phytotoxicity occurred at 10-25 and 50-100 pgml-I in chrysanthemum and tobacco explants, respectively, depending on the size of the explant and the timing of application. The presence of light or darkness also had a significant effect. The use of transverse thin cell layers (tTCLs) in conjunction with high initial AA selection levels supported the greatest regeneration of transgenic material (adventitious shoots or callus) and the lowest number of escapes. Flow cytometric analyses demonstrate that regeneration can be predicted in both species, depending on the ploidy level of the callus. Endoreduplication was not observed in chrysanthemum, even at high AA levels, but occurred (8C or more) in tobacco callus, even at low AA concentrations (5-10 pgml-1). The higher the AA level, the greater the DNA degradation and the lower the 2C and 4C values.