35-41.Vol 7No 12001
Symptoms resembling phytoplasma disease have been observed on peach trees in a seed-source plantation of stone fruits in south Hungary quite recently. In this publication we report on the results of woody indexing of symptomatic peach trees on GF 305 indicator in the field and under greenhouse conditions as well as on molecular studies. Phytoplasm...a infection detected on GF 305 indicators in greenhouse and field indexing was confirmed by PCR. Nested PCR was conducted using universal primer pairs followed by group and subgroup specific primers for the second amplification. RFLP analysis of nested PCR products was performed using Rsal restriction enzyme. Based on the results of molecular studies it can be concluded that phytoplasmas, belonging to the European stone fruit yellows subgroup (16SrX-B) were identified in peach trees. Further studies on symptomatic peach trees originating from different parts of Hungary are in progress.
Genotyping Hungarian apricot cultivars for self-(in)compatibility by isoelectric focusing and PCR analysis
69-72.Vol 11No 12005
Self-incompatibility (SI) in flowering plants is a widespread genetic system that promotes out-crossing. In Prunus species the SI is a gametophytic trait, which is controlled by a single multiallelic locus, termed S-locus. S-alleles codify stylar glycoproteins with ribonuclease activity (S-RNases). Our objective was to assess the S-genotype of som...e Hungarian apricot varieties by isoelectric focusing of stylar RNases as well as by PCR analysis using cherry consensus primers. Consensus primers amplified one or two bands of various sizes. Primers amplifying the 1st intron gained fragments the size of which ranged from 250 to 500 bp; while those amplifying the 2nd intron resulted in fragments of 800-2000 by length. Our data demonstrated that the first intron of the apricot S-RNase gene is shorter than the second one, which coincides with the structure of cherry S-RNase alleles. `Hargrand' (S1S2) and `Harcoe (S1S4) possessed one common S-RNase isoenzyme. Hungarian 'Orias' apricot cultivars showed different bands compared to the previous cultivars, but they shared completely identical patterns confirming that they possess the same S-genotype. 'Bergeron', `Harmat' and 'Korai zamatos' are characterised by an evidently distinct S-RNase pattern. The self-compatible cultivar (`Bergeron') had one allele, which suggests its correspondence to the Sc. Primers for the 2nd intron was unsuccessful in gaining fragments, which indicates that the 2nd intron in the Sc allele is too long to get any amplification. On the basis of our data, identities and differences were revealed in the S-allele constitution of some economically important Hungarian apricot cultivars at protein and DNA levels.
Primers designed for the detection of grapevine pathogens spreading with propagating material by quantitative real-time PCR
21-30.Vol 21No 1-22015
Several grapevine pathogens are disseminated by propagating material as systemic, but latent infections. Their detection and identification have a basic importance in the production and handling of propagating stocks. Thus several sensitive and reliable diagnostic protocols mostly based on molecular techniques have been developed. Of these methods... quantitative real-time PCR (q-PCR) has recently got an emerging importance. Here we collected primer data for the detection and identification of grapevine pathogens which are important in the production of propagating stocks by q-PCR. Additional novel techniques that use DNA amplification, hybridization and sequencing are also briefly reviewed.
43-47.Vol 9No 3-42003
Callus cultures were induced from hypocotyl of young bean seedlings. The B5 medium completed with 1 mg/1 KIN and 2mg/1 2,4-D proved the best. Callus developed and maintenaned on B5 medium supplemented with 1mg/1 kinetin and 2mg/I 2,4-D. The B5 medium supplemented with 1mg/1 KIN and 2mg/1 2,4-D induced much more callus than half strength MS medium ...supplemented with 0.5 or 0.75mg/1 BA and 0.1 mg/1 NAA. The results demonstrate that GeneboosterTM is convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus shot by GeneboosterTM can be transformed to get (kanamycin-resistant and stress mannitoltolerant) calli. The presence of mannitol-dehydrogenase gene (mt/) was verified by PCR, showing the integration of mt/ gene carried by two plasmids. Co-transformed calli were selected after bombardment on kanamycin, mannitol and (kanamycin+mannitop-containing media. Data of molecular analysis (PCR) confirmed the insertion of mtl gene in the genome of mannitol-tolerant callus lines.
37-42.Vol 21No 1-22015
Rhodiola 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.
Prediction infection risk on the basis of weather-related factors and Erwinia amylovora colonization in apple and pear flowers
39-54.Vol 10No 22004
Current infection risk prediction models utilize environmental parameters and field records, but do not take into account the estimated inoculum potential within the orchard. The object of this study was to survey the accuracy of three simple prediction methods under Hungarian climatic conditions, which could easily be used by the farmers. We also... tested whether the accuracy of infection risk predictions can be improved by taking into consideration the incidence and/or rate of flower colonization by Erwinia amylovora. After preliminary investigations in 1999-2001, data concerning the weather-related infection risk were recorded in 5 apple and 1 pear orchards in 2002, and in 12 apple and I pear orchards in 2003. The weather data were processed by the easy-to-use risk assessment models of the mean temperature prediction line (MTL), Smith's Cougarblight 98C and Billing's integrated system (BIS), and by the MaryblytTM 4.3 computer-assisted model for reference. The population size of E. amylovora in the flower samples was estimated within an order of magnitude by PCR. For all years and orchards tested, Maryblyt indicated 35 days on which there was an acute infection risk. The same days were indicated by all 3 methods in 23 cases (66%), 8 days were indicated by 2 methods (23%) and 4 days were indicated by 1 method only. A similarly good correlation was found for prediction of the date of the first massive infection risk: in 2003, for instance, there was a perfectly consistent prediction by all 4 models in 9 of the 13 participating orchards. A coincidental forecast was provided by 3 of the 4 models in the other 4 orchards. The results indicate that any of the risk assessment models could provide an increased accuracy of the actual infection risk prediction if combined with an estimation of the incidence of Erwinia amylovora colonization in the open flowers. We found no convincing differences in the size of the epiphytic population in flowers of cultivars possessing high or low susceptibility to Erwinia amylovora. We conclude that the easy-to-use methods tested could be used by the fanners to recognize weather-related risks, especially when coupled with an estimation of the proportion of the pathogen-infested flowers. This local prediction would provide rapid information (faster than the regional forecast systems) specifically for a given orchard.
Conventional PCR primers for the detection of grapevine pathogens disseminated by propagating material
69-80.Vol 20No 3-42014
Polymerase chain reaction driven by sequence specific primers has become the most widely used diagnostic method to detect and identify plant pathogens. The sensitive and cost-effective pathogen detection is exceptionally important in the production of propagating material. In this paper we have collected primer sequence data from the literature fo...r the detection of the most important grapevine pathogens disseminated by propagating stocks by conventional polymerase chain reaction. Basic protocols to obtain template nucleic acids have also been briefly rewieved.
Determination of (in)compatibility genotypes of Hungarian sweet cherry (Prunus avium L.) accessions by PCR based methods
37-42.Vol 9No 3-42003
Sweet cherries (Prunus avium L.) are generally self-incompatible and pollinator cultivars are needed in orchards for reliable yield. In Hungary, choosing the appropriate cross-compatible cultivar pairs has so far been based on traditional test-crosses in the field. In recent years PCR-based methods that allow the identification of the S-alleles re...sponsible for compatibility have been elaborated. We have determined the S-allele constitution of 24 cultivars and four selections important to Hungarian growers and breeders using PCR-based methods developed at Horticulture Research International, East Malling. The 28 accessions had various pairs of 9 alleles including one new allele, Sr. They could be assigned to 12 of the existing incompatibility groups or to a new group (S4S12) for which the designation 'Group XXVII' is proposed. The cultivars `Krupnoplodnaja' and 'Rita' had novel genotypes, S5S9 and S5Sx, respectively and can be placed into group 0 that holds universal pollen donors. The genotype of the cultivar ‘Hedelfingeni óriás' grown in Hungary was found to be S3S4 and therefore different from the cultivar `Hedelfingen' that is widespread in Western Europe.
Genetic engineering of apple (Malus domestica Borkh.) for resistance to fungal diseases using g2ps1 gene from Gerbera hybrida (Asteraceae)
15-12.Vol 20No 1-22014
In the present study, g2ps1 gene from Gerbera hybrida coding for 2-pyrone synthase which contribute for fungal and insect resistance was used. The aim was to work out an efficient approach of genetic transformation for apple cvs. ‘Golden Delicious’, ‘Royal Gala’ and ‘MM111’, ‘M26’ rootstocks for improving their fungal resistance us...ing genetic engineering techniques. Adventitious shoot formation from leaf pieces of apples studied was achieved using middle leaf segments taken from the youngest leaves from in vitro-grown plants.Optimum conditions for ‚direct’ shoot organogenesis resulted in high regeneration efficiency of 0%, 95%, 92%, 94% in the studied apples respectively. Putative transgenic shoots could be obtained on MS media with B5 Vitamins, 5.0 mg l-1 BAP, or 2.0 mg l-1 TDZ with 0.2 mg l-1 NAA in the presence of the selection agent “PPT” at 3.0-5.0 mgl-1. Shoot multiplication of transgenic shoots was achieved on: MS + B5 vitamins + 1.0 mg l-1 BAP + 0.3 mg l-1 IBA, 0.2 mg l-1 GA3+1.0 g/l MES+ 30 g/l sucrose + 7.0 g/l Agar, with the selection agent PPT at 5.0 mg l-1 and were subcultured every 4 weeks in order to get sufficient material to confirm transformation of the putative shoots obtained. Six, seven, one and six transgenic clones of the apples studied respectively have been obtained and confirmed by selection on the media containing the selection agent “PPT” and by PCR analysis using the suitable primers in all clones obtained for the presence of the selection” bar gene (447 bp) and the gene-of- interest “g2PS1” (1244 bp), with transformation efficiency of 0.4%, 0.6%, 0.1% and 0.3% respectively. These transgenic clones were multiplied further in vitro in the presence of the selection agent ‘PPT’ and rooted in vitro. Rooted transgenic plantlets were successfully acclimatized and are being kept under-containment conditions according to the biosafety by-law in Syria to evaluate their performance for fungal resistance .
Transformation of tobacco plants with virEl gene derived from Agrobacterium tumefaciens pTiA6 and its effect on crown gall tumor formation
53-56.Vol 7No 3-42001
The 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 transfor...mation. 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.
Production of transgenic carnation with a heterologous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase bifunctional enzyme cDNA
75-79.Vol 12No 42006
Transgenic 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 regul...ators. 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.
21-24.Vol 12No 42006
This study aimed to establish a new protocol for DNA isolation from the Pyrus genus to get high quality DNA that is suitable for the generation of molecular markers, such as RAPD and AFLP. This method is based on modified CTAB extraction procedure (Aldrich & Cullis, 1993). For isolation of high-quality DNA we used copper (II) acetate treatment... that enabled fixation and removing of tannins, present in abundance in Pyrus. DNA yield from this procedure is high. DNA is completely digestible with restriction endonucleases and amplifiable in the PCR, indicating freedom from common contaminating polyphenolic compounds.
Production of transgenic carnation with antisense ACS (1-aminocyclopropane44-carboxy late synthase) gene
104-107.Vol 6No 42000
Dianthus 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 car...nation. 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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