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  • Preliminary evaluation of breeding perspectives of Ukrainian sweet cherry cultivars: nutraceutical properties and self-incompatibility

    Some 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.

  • Identification of ripening-related genes in strawberry fruit by cDNA-AFLP

    An 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.

  • Changing of carbohydrates by inoculation of Pseudomonas savastanoi pv. phaseolicola oil bean lines with different resistance

    The 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.

  • Impact of foliar fungi on dogroses

    Wild roses of the section Caninae, commonly known as dogroses, have been described as more disease tolerant than ornamental roses and could therefore become valuable for breeding improved rose cultivars. Two fields with dogroses, one with plants obtained by open pollination in wild populations, and one with plants obtained from intra- and interspecific crosses, were evaluated for blackspot, powdery mildew, rust and leafspots in the autumn of 2005. Symptoms of the different fungi on different dogrose species were carefully evaluated in a microscope and documented by photography. Interestingly, almost no symptoms of powdery mildew were found in either field, although the fungus infected wild roses of a different section in a field closeby. Surprisingly few symptoms were found also of blackspot, and they differed considerably from those found on ornamental cultivars, indicating a lower susceptibility in dogroses. The most important fungal disease in 2005 was rust, followed by leafspot symptoms. The latter were apparently caused by Sphaceloma rosarum and Septoria rosae which can be properly discriminated only in a microscope. The investigated dogrose species and their progeny groups varied significantly in disease susceptibility and in the appearance of encountered symptoms but there was no evidence of major resistance genes, except possibly in Rosa rubiginosa which did not show any symptoms of Septoria. In 2006, a subset of the plant material in Field 1 was evaluated to check for consistency between the years. Leafspots had overtaken rust as the most important disease but results were otherwise very similar to those of 2005.

  • Resistance Gene Analogs (RGA) as a tool in fruit tree's breeding

    Breeding 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.

  • RAPD analysis of grapevine hybrids and cultivars

    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.

  • Molecular diversity of Hungarian melon varieties revealed by RAPD markers

    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.

  • Production of transgenic carnation with antisense ACS (1-aminocyclopropane44-carboxy late synthase) gene

    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 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.


  • Production of transgenic carnation with a heterologous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase bifunctional enzyme cDNA

    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 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.

  • Down-regulation of ethylene production in carnation (Dianthus Caryphyllus L.) by an apple derived ACC-cDNA

    Transgenic 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.

  • Optimization of RNA isolation from stone fruits at different ripening stages

    This 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.

  • The Effects of Some Parameters on Agrobacterium-Mediated Transformation in Muskmelon

    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 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.


  • Goals and results in improvement of biological background of medicinal plant production

    The choice of varieties among medicinal plant species is relatively small, compared to other horticultural crops. In Hungary, only poppy (Papaver somniferum) and mustard (Sinapis alba) have several cultivars. Recognising the problem, in the recent years breeding activity has been intensified all over the world, in spite of financial, technical and legal difficulties. The article reports on the results of breeding at the Department of Medicinal and Aromatic Plants of the Szent István University, 11 varieties of which has been officially registered till 2000. Main goals of genetic improvement are: increasing of the production capacity of utilised plant organs, enhancement of active material accumulation capacity, improvement of sensory quality and technological properties. The most often applied methods are selection, and recently, cross breeding, the results of which can be measured on new materials of caraway (Carum carvi), hyssop (Hyssopus officinalis), marjoram (Majorana hortensis), poppy (Papaver somniferum), etc. Efficacy of breeding work is established by additional, regular research on the genetics, physiology, floral and reproductive biology, chemosyndromes of medicinal plant species.


  • The effects of ACS (1-aminocyclopropane-l-carboxylate synthase) gene down regulation on ethylene production and fruit softening in transgenic apple

    A 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.

  • Development of microsatellite markers for Rhodiola rosea

    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.

  • Transmethylation and the general defense reaction of plants

    Plant 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.

  • Reaction of different Capsicum genotypes to four viruses

    The objective of this study was to examine the reaction of 44 Capsicum genotypes to common strain of Tobacco mosaic virus (TMV-C/U1), Obuda pepper virus (ObPV), NTN strain of Potato virus Y (PVYNTN) and legume strain of Cucumber mosaic virus (CMV­U/246). Reaction (extreme resistance, hypersensitive reaction, latent susceptibility, susceptibility) of the tested Capsicum species/hybrids and breeding lines seemed to be greatly depending on hosts and viruses. Out of the breeding materials 4/99 F2 and IX-8 in to CMV-U/246, while 32.Bogyisz. type, VI-57 ii. 57/83 and V-12=19/98 to TMV-C/Ui showed extreme resistance. Two lines (V-25 F1=32/98 F1 and V-27 in F4=35/98 F4) showed hypersensitive reaction to ObPV. Latent susceptibility to PVYNTN was observed in case of all eleven tested Capsicum genotypes and in case of several lines to TMV-C/U1, ObPV and CMV-U/246. Other breeding materials proved susceptible to the mentioned viruses. Pepper genotypes showing extreme resistance and hypersensitivity could be used for resistance breeding to viruses.

  • Shoot induction and plant regeneration from cotyledon segments of the muskmelon variety "hógolyó"

    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 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.


  • Malus taxa and the progenies of Malus floribunda selected in Hungary, as gene sources of resistance breeding

    One of our objectives in the apple breeding program of the Department of Fruit Science, beside creating new varieties, is to search for new gene sources. After evaluating the disease resistance of Malus taxa available in Hungary, we put the promising selected individuals through further examinations. Their habit and growth vigor was observed, as well as productivity and fruit characteristics were evaluated. Malus fusca seems to be a new, promising gene source in Hungary among the examined taxa. Beside its optimal canopy, growth vigor and fruit characteristics, it has the advantage to be in distant relationship to Malus x domestica, therefore its resistance is based on a different genetic basis. SBG 1 selection of Malus spectabilis can be recommended as a new gene source for resistance breeding. The range of gene sources can further be widened by selected clones of Malus floriblunda (BA I), Malus x zumi (BA) and Malus baccata (SBG 6). An additional value of Malus taxa chosen by us is that they show resistance not only to apple scab, but to powdery mildew as well, and according to our results, they have optimal habit, acceptable productivity and good fruit quality. Nine hybrids were selected from first Malus progenies which proved to be resistant to apple scab and powdery mildew. These are valuable not only as gene sources of breeding, but as pollenizers or ornamental trees, while their decorative fruits can be used in inside decorations and flower arrangements.

  • Grape variety comparison of different stress tolerance based on the quantitative measurement of carbohydrates

    The 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.

  • Molecular analysis of strawberry cultivars using RAPD, AP-PCR and STS markers

    Seventeen 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.

  • The effect of modified bacterial virulence to host-pathogen relationship (Phaseolus vulgaris L. Pseudomonas savastanoi pv. phaseolicola)

    The Pseudomonas savastanoi pv. phaseolicola is one of the most expressive biogen stressors of the bean (Phaseolus vulgaris L.) in Hungary. The chemical and agrotechnological defence is inefficient, so breeding is the only workable way. The conventional cultivars are susceptible to PS while most of the new industrial varieties have genetic resistance to the pathogen. The genetic background of resistance is, however, a complex system in the bean. Leaf resistance is a monogenic system, but this gene is not expressed in juvenile stage of the host. The pathogen species can be divided into different races. After inoculation with virulent strains, typical symptoms appeared on the leaves. To understand the details of host-pathogen relationships, there were carried out experiments using bacterial strains with altered virulence. Six transposon mutants of the PS were tested. Our main objective was to test these modified bacterial strains on bean cultivars of known genetic background. First we analysed the symptoms, and then the correlation between the symptoms and the multiplication of mutant bacteria. Three cultivars (Cherokee, Inka and Főnix) were tested.

    The infection by the virulent PS isolate produced typical symptoms on the three cultivars tested. Mutant bacteria (except strain 756) did not cause any significant symptoms on the hosts. The mutant 756 induced visible symptoms on the cultivars Cherokee and Inka. On Cherokee there were small watersoaked lesions, and HR (hypersensitivity reaction) was detected on Inka, but this was restricted to some cells only (mikro HR). The rate of multiplication of the wild type strain was much higher than the multiplication of the mutants. Bacteria were detected in the cotyledons and primordial leaf, but there is not any substantial number of bacteria in leaves, except for strains 757, 1212 and 1213. The rate of multiplication of strain 756 was intermediate. These, and other experiments can help to understand the genetic background of resistance and the host-pathogen relationship in the Pseudomonas-bean pathosystem.


  • Severely pollen-limited fruit set in a pear (Pyrus communis) orchard revealed by yield assessments and DNA-based paternity assignment of seedlings

    In commercial fruit tree orchards, consistently high yields are necessary for a durable economy. The Swedish pear cultivar 'Carola' has been noted for low setting in some orchards, possibly due to insufficient pollination. In this study, fruit set was evaluated in a research orchard where `Carola' had been planted together with four potential pollinators. Total yield and number of fruits was noted during three and four years, respectively. In 2003, seeds were germinated from the harvested `Carola' fruits, and the paternity of three seedlings from 50 trees was determined with RAPD analysis. 'Clapp's Favourite' had sired 39.6% of the seedlings, closely followed by `Seigneur d'Esperen' (30.7%) and 'Clara Frijs' (26.7%) whereas 'Skanskt sockerparon' only sired 1.1% of the seedlings. The remaining 2.3% appeared to have been derived by selfing. Pollen-limited seed set was indicated at surprisingly short distances; accumulated number of fruits on the `Carola' trees was significantly higher when separated by only 2 m from one of the two most efficient pollinators, 'Clapp's Favourite' or 'Seigneur d'Esperen‘, compared to trees 4—l0 in away in the same row. Number of viable seeds per fruit was also higher in fruits from trees immediately adjacent to the pollinators, suggesting an effect of improved pollination success. The importance of very short inter-cultivar distances for efficient pollen transfer became even more clear when comparisons involved the true pollination distances as determined by RAPD; the accumulated yields decreased linearly from 55 kg at a 2 in distance to only 17 kg at 13 m.

  • Genotyping Hungarian apricot cultivars for self-(in)compatibility by isoelectric focusing and PCR analysis

    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 some 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.

  • High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells

    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 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.