Evolutionary Divergence of TNL Disease-Resistant Proteins in Soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) and Common Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>)

Disease-resistant genes (R genes) encode proteins that are involved in protecting plants from their pathogens and pests. Availability of complete genome sequences from soybean and common bean allowed us to perform a genome-wide identification and analysis of the Toll interleukin-1 receptor-like nucleotide-binding site leucine-rich repeat (TNL) proteins. Hidden Markov model (HMM) profiling of all protein sequences resulted in the identification of 117 and 77 regular TNL genes in soybean and common bean, respectively. We also identified TNL gene homologs with unique domains, and signal peptides as well as nuclear localization signals. The TNL genes in soybean formed 28 clusters located on 10 of the 20 chromosomes, with the majority found on chromosome 3, 6 and 16. Similarly, the TNL genes in common bean formed 14 clusters located on five of the 11 chromosomes, with the majority found on chromosome 10. Phylogenetic analyses of the TNL genes from Arabidopsis, soybean and common bean revealed less divergence within legumes relative to the divergence between legumes and Arabidopsis. Syntenic blocks were found between chromosomes Pv10 and Gm03, Pv07 and Gm10, as well as Pv01 and Gm14. The gene expression data revealed basal level expression and tissue specificity, while analysis of available microRNA data showed 37 predicted microRNA families involved in targeting the identified TNL genes in soybean and common bean.     

Methane Yield and Feed Quality Parameters of Mixed Silages from Maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) and Common Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

European agricultural policy increasingly focuses on environmental friendly cropping systems. Intercropping of maize (Zea mays L.) and common beans (Phaseolus vulgaris L.) has been suggested as an alternative cropping system with environmental benefits. The aim of this study was to assess methane yield potential of mixed silages. Based on material from two field experiments at three sites in Germany, mixed silages were produced with proportions of individual components varying from 0 to 100 % of fresh matter in increments of 12.5 %. Chemical parameters (neutral detergent fiber (NDF), acid detergent fiber (ADF), crude protein (XP), starch, sugar, and crude fat) were determined, and batch tests were performed to measure methane yield potential from silages. With increasing bean proportion, concentrations of XP increased while NDF, methane yield, and methane content decreased. While methane yield showed a negative relationship with XP content (R ² = 0.56***), a positive relation was found with NDF (R ² = 0.55***). The reduction of methane yield of circa 1 L of methane per each additional bean percentage in the silages could not be explained by the chemical parameters of the silages. It is hypothesized that other chemical compounds, such as lectins, which were not determined in the present study, may have influenced methane production.     

Exploring the quantitative resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">phaseolicola</Emphasis> in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Pseudomonas syringae pv. phaseolicola is an important disease that causes halo blight in common bean. The genetic mechanisms underlying quantitative halo blight resistance are poorly understood in this species, as most disease studies have focused on qualitative resistance. The present work examines the genetic basis of quantitative resistance to the nine halo blight races in different organs (primary and trifoliate leaf, stem and pod) of an Andean recombinant inbred line (RIL) progeny. Using a multi-environment quantitative trait locus (QTL) mapping approach, 76 and 101 main-effect and epistatic QTLs were identified, respectively. Most of the epistatic interactions detected were due to loci without detectable QTL additive main effects. Main and epistatic QTLs detected were mainly consistent across the environment conditions. The homologous genomic regions corresponding to 26 of the 76 main-effect detected QTLs were positive for the presence of resistance-associated gene cluster encoding nucleotide-binding and leucine-rich repeat (NL) proteins and known defence genes. Main-effect QTLs for resistance to races 3, 4 and 5 in leaf, stem and pod were located on chromosome 2 within a 3.01-Mb region, where a cluster of nine NL genes was detected. The NL gene Phvul.002G323300 is located in this region, which can be considered an important putative candidate gene for the non-organ-specific QTL identified here. The present research provides essential information not only for the better understanding of the plant-pathogen interaction but also for the application of genomic assisted breeding for halo blight resistance in common bean.     

The Novel Use of a Combination of Sonication and Vacuum Infiltration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated Transformation of Kidney Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) with <Emphasis Type="Italic">lea</Emphasis> Gene

An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation.     

Embryo abortion and histological features in the interspecific cross between <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L. and <Emphasis Type="Italic">P. coccineus</Emphasis> L

In this paper, the causes of early embryo abortion in the reciprocal crosses between Phaseolus vulgaris L. (a cultivar) and Phaseolus coccineus L. (a wild form) were studied. Methacrylate resin sections, 3–5 μm thick, of 3 to 14 day-old seeds were used to examine the embryo developmental stages and the state of seed tissue. It was observed that, embryos aborted at different developmental stages (globular to early cotyledon) depending on the maternal parent. The use of P. coccineus cytoplasm resulted in a higher number of abortion than in reciprocal crosses. Many of them took place between 5 and 6 days after pollination (DAP). Histological analyses permitted to observe that the embryo development was slower in the cross between P. coccineus and P. vulgaris, compared to parental seeds. It would be related to a deficient endosperm development in reciprocal crosses and, in some extent, hypertrophy of the suspensor might be the main cause of early embryo abortion. Then, it would be practical to overcome this incompatibility by rescuing the embryo at the globular stage of development.     

Phosphoproteomic Analysis in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Roots Treated with <Emphasis Type="Italic">Rhizobium etli</Emphasis> Nodulation Factors

Legumes are unique in their ability to establish symbiotic interactions with rhizobacteria, providing a source of assimilable nitrogen; this symbiosis is regulated by complex signaling process between the plant and the bacteria. The participation of specific protein kinases during the initial steps of the nodulation process has been established. However, their substrates or the signaling networks implicated are not fully understood. Herein, a phosphoproteomic analysis of Phaseolus vulgaris roots treated for 24 h with specific Nod factors was performed using an immobilized metal ion affinity chromatography enrichment and two-dimensional gel electrophoresis approach with mass spectrometry identification. A total of 33 protein spots showing more than 1.5-fold shift were identified (17 protein spots in which the relative abundance increased and 16 that decreased). The majority of the identified root phosphoproteins displaying an increased relative abundance are presumed to have functions related to the biosynthesis and folding of proteins, energy metabolism, or cytoskeleton rearrangements, which reflect the metabolic status of the roots as being part of the developmental processes leading to nodule initiation and the importance of cytoskeleton rearrangement in the P. vulgaris–rhizobia symbiosis. The proteins in which relative abundance decreased are associated with defense and oxido-reduction processes, which could indicate a suppression of plant defense responses during the establishment of the rhizobia–legume interaction and an increase of reactive oxygen species production.     

Movement of Abscisic Acid in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Plants

THE movement of abscisic acid (ABA) in plants seems to have been studied only in isolated segments of tissue^1–4. We have used ¹⁴C-labelled ABA of relatively high specific activity to investigate its movement in a number of plant species, in both isolated tissue segments and whole plants. The movement of 2-¹⁴C-ABA in intact seedlings of Phaseolus vulgaris is described here.     

SNP marker diversity in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Single nucleotide polymorphism (SNP) markers have become a genetic technology of choice because of their automation and high precision of allele calls. In this study, our goal was to develop 94 SNPs and test them across well-chosen common bean (Phaseolus vulgaris L.) germplasm. We validated and accessed SNP diversity at 84 gene-based and 10 non-genic loci using KASPar technology in a panel of 70 genotypes that have been used as parents of mapping populations and have been previously evaluated for SSRs. SNPs exhibited high levels of genetic diversity, an excess of middle frequency polymorphism, and a within-genepool mismatch distribution as expected for populations affected by sudden demographic expansions after domestication bottlenecks. This set of markers was useful for distinguishing Andean and Mesoamerican genotypes but less useful for distinguishing within each gene pool. In summary, slightly greater polymorphism and race structure was found within the Andean gene pool than within the Mesoamerican gene pool but polymorphism rate between genotypes was consistent with genepool and race identity. Our survey results represent a baseline for the choice of SNP markers for future applications because gene-associated SNPs could themselves be causative SNPs for traits. Finally, we discuss that the ideal genetic marker combination with which to carry out diversity, mapping and association studies in common bean should consider a mix of both SNP and SSR markers.     

<Emphasis Type="Italic">Rhizobium cellulosilyticum</Emphasis> as a co-inoculant enhances <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> grain yield under greenhouse conditions

The Rhizobium-legume symbiosis is a complex partnership with many factors, with initial bacterial colonization of the plant root surface and primary infection as key early stages. Two molecules are strongly involved in these processes: the structural carbohydrate cellulose and the enzyme cellulase, which breaks down the former and allows rhizobia to infect the roots. Here, we report the effect on common bean (Phaseolus vulgaris L.) after co-inoculation of the non-nodulating, cellulase-overproducing strain Rhizobium cellulosilyticum ALA10B2^T and the P. vulgaris-nodulating R. leguminosarum strain TPV08. In order to elucidate the effect of combined inoculation with both strains, we designed greenhouse assays, including single inoculation with strain TPV08, co-inoculation with both strains and an uninoculated treatment in non-sterile peat. Chemical fertilizers were not added. Chlorophyll content in the leaves was measured after the flowering stage by spectrophotometry and was considered to be indicative of the nutrient status of the plants. Nodule formation was observed on roots of the inoculated plants, while no nodulation was observed on roots of the uninoculated plants. The results indicate a synergistic effect between the two Rhizobium strains. Co-inoculated plants exhibited significant increases in seed yield and nitrogen content in comparison with the uninoculated control plants and with plants inoculated with a single strain. It is suggested that co-inoculation with strain ALA10B2^T greatly increased the efficiency of N fixation by strain TPV08.     

Rapid regeneration of <Emphasis Type="Italic">Phaseolus angustissimus</Emphasis> and <Emphasis Type="Italic">P. vulgaris</Emphasis> from very young zygotic embryos

A rapid regeneration protocol for proembryos of Phaseolus angustissimus as young as 1 day after pollination (DAP) involving pod culture for 1 week followed by embryo culture for 2 weeks and embryo germination for 1 or 2 weeks is provided. Optimization of the media was conducted with pods collected 3 DAP. The best pod culture medium was composed of basal medium [(Phillips and Collins 1979) salts with (Geerts et al. 2001) vitamins], 1000 mg l⁻¹ glutamine, 1000 mg l⁻¹ casein hydrolysate, 3% sucrose and 0.5% agar. Embryo culture medium consisted of basal medium with 500 mg l⁻¹ glutamine, 250 mg l⁻¹ casein hydrolysate, 1.9 μM ABA, 3% sucrose and 0.5% bacto-agar. Embryos developed into plantlets on germination medium containing basal medium with 0.25 μM BA, 3% sucrose and 0.7% bacto-agar. Fertile, normal plants were recovered from direct embryogenesis and from micrografted embryo-derived shoots. Embryos obtained from pods collected 3 DAP regenerated plantlets at a rate of 29.3%, while embryos from pods collected 2 DAP and 1 DAP regenerated at rates of 20.2 and 4%, respectively. A second accession of P. angustissimusregenerated at a rate of 26.2%. Using this 5-week protocol for P. vulgaris resulted in a plantlet regeneration rate of 12.5%.     

<Emphasis Type="Italic">dw2</Emphasis>, a New Mutation of Beet <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

Twelve dwarf plants were found in the second hybrid generation of beet. The average height of mutant plants was 21.8 cm, their leaf blades and flowers were significantly smaller than normal, and the plants exhibited male and female sterility. This dwarfism was shown to be caused by a mutation differing from that previously described in beet, which is named dwarf2 (dw2). The experimental evidence suggests that this mutation appeared in one of the first-generation plants. Based on plant phenotype in the first hybrid generation and the number of mutant plants in the second one, this mutation is suggested to be under recessive monogenic control of the dw2 gene. The genotypic class segregation in the second hybrid generation indicates that the dw2 gene is inherited independently of genes m, a1, and ap that control choricarpousness, gene male sterility, and pollen grain aggregation into tetrads.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 657–660.Original Russian Text Copyright © 2005 by Mglinets, Osipova.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Nucleotide diversity of a genomic sequence similar to <Emphasis Type="Italic">SHATTERPROOF</Emphasis> (<Emphasis Type="Italic">PvSHP1</Emphasis>) in domesticated and wild common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Evolutionary studies in plant and animal breeding are aimed at understanding the structure and organization of genetic variations of species. We have identified and characterized a genomic sequence in Phaseolus vulgaris of 1,200 bp (PvSHP1) that is homologous to SHATTERPROOF-1 (SHP1), a gene involved in control of fruit shattering in Arabidopsis thaliana. The PvSHP1 fragment was mapped to chromosome Pv06 in P. vulgaris and is linked to the flower and seed color gene V. Amplification of the PvSHP1 sequence from the most agronomically important legume species showed a high degree of interspecies diversity in the introns within the Phaseoleae, while the coding region was conserved across distant taxa. Sequencing of the PvSHP1 sequence in a sample of 91 wild and domesticated genotypes that span the geographic distribution of this species in the centers of origin showed that PvSHP1 is highly polymorphic and, therefore, particularly useful to further investigate the origin and domestication history of P. vulgaris. Our data confirm the gene pool structure seen in P. vulgaris along with independent domestication processes in the Andes and Mesoamerica; they provide additional evidence for a single domestication event in Mesoamerica. Moreover, our results support the Mesoamerican origin of this species. Finally, we have developed three indel-spanning markers that will be very useful for bean germplasm characterization, and particularly to trace the distribution of the domesticated Andean and Mesoamerican gene pools.     

Characterization of AT-rich microsatellites in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Polymorphism of microsatellite markers is often associated with the simple sequence repeat motif targeted. AT-rich microsatellites tend to be highly variable and this appears to be notable, especially in legume genomes. To analyze the value of AT-rich microsatellites for common bean (Phaseolus vulgaris L.), we developed a total of 85 new microsatellite markers, 74 of which targeted ATA or other AT-rich motif loci and 11 of which were made for GA, CA or CAC motif loci. We evaluated the loci for the level of allelic diversity in comparison to previously characterized microsatellites using a panel of 18 standard genotypes and genetically mapped any loci polymorphic in the DOR364 × G19833 population. The majority of the microsatellites produced single bands and detected single loci, however, 15 of the AT-rich microsatellites produced multiple or double banding patterns; while only one of the GA or CA-rich microsatellites did. The polymorphism information content (PIC) values averaged 0.892 and 0.600 for the AT and ATA motif microsatellites, respectively, but only 0.140 for the CA-rich microsatellites. GA microsatellites, which had a large average number of repeats, had high to intermediate PIC, averaging 0.706. A total of 45 loci could be genetically mapped and distribution of the loci across the genome was skewed towards non-distal locations with a greater prevalence of loci on linkage groups b02, b09 and b11. AT-rich microsatellites were found to be a useful source of polymorphic markers for mapping and diversity assessment in common bean that appears to uncover higher diversity than other types of simple sequence repeat markers.     

Lead uptake,toxicity and accumulation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> plants

The effects of lead were investigated in bean plants (Phaseolus vulgaris L. cv. Zlota Saxa) grown hydroponically in nutrient solution and exposed to Pb(NO₃)₂ (0.1, 0.5, 1 mM) with or without equimolar concentrations of chelator ethylenediaminetetraacetic acid (EDTA). The roots treated only with Pb(NO₃)₂ accumulated up to 25 g(Pb) kg⁻¹(d.m.), during 4-d exposure. However, in bean plants exposed to 0.5 mM Pb + 0.5 mM EDTA or 1 mM Pb + 1 mM EDTA 2.5 times less Pb was determined. In bean plants treated only with Pb, less than 6 % of total lead accumulated was transported to the aboveground parts, while in the case of plants grown with Pb + EDTA, around 50 % of total Pb was transported to the shoots.     

Plasmid content of salt stress-tolerant <Emphasis Type="Italic">Rhizobium</Emphasis> strains from Egyptian soils nodulating common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Plasmid profile analysis is useful to characterize Rhizobium strains within the same species. Among the 16 Rhizobium strains examined, 14 had distinct plasmid profiles. The size of plasmids ranged from 40 to 650 kb, and three plasmids of 650, 510 and 390 kb were common to several strains. Plasmid analysis revealed that Rhizobium etli contained a mega-plasmid, similar in size to Rhizobium tropici. All the salt-tolerant strains examined had a plasmid of 250 kb, except for strain EBRI 29. This suggests that this plasmid may play an important adaptive role under salt stress conditions.     

Induced cytomictic variations and syncyte formation during microsporogenesis in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.

The intercellular translocation of chromatin material along with other cytoplasmic contents among the proximate meiocytes lying in close contact with each other commonly referred as cytomixis was reported during microsporogenesis in Phaseolus vulgaris L., a member of the family Fabaceae. The phenomenon of cytomixis was observed at three administered doses of gamma rays viz. 100, 200, and 300 Gy respectively in the diploid plants of Phaseolus vulgaris L. The gamma rays irradiated plants showed the characteristic feature of inter-meiocyte chromatin/chromosomes transmigration through various means such as channel formation, beak formation or by direct adhesion between the PMC’s (Pollen mother cells). The present study also reports the first instance of syncyte formation induced via cytomictic transmigration in Phaseolus vulgaris L. Though the frequency of syncyte formation was rather low yet these could play a significant role in plant evolution. It is speculated that syncyte enhances the ploidy level of plants by forming 2n gametes and may lead to the production of polyploid plants. The phenomenon of cytomixis shows a gradual inclination along with the increasing treatment doses of gamma rays. The preponderance of cytomixis was more frequent during meiosis I as compared to meiosis II. An interesting feature noticed during the present study was the channel formation among the microspores and fusion among the tetrads due to cell wall dissolution. The impact of this phenomenon is also visible on the development of post-meiotic products. The formation of heterosized pollen grains; a deviation from the normal pollen grains has also been reported. The production of gametes with unbalanced chromosomes is of utmost importance and should be given more attention in future studies as they possess the capability of inducing variations at the genomic level and can be further utilized in the improvement of germplasm.     

Integration of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) linkage and chromosomal maps

Fluorescent in situ hybridisation of pooled, closely linked RFLP markers was used to integrate the genetic linkage map and the mitotic chromosome map of the common bean. Pooled RFLP probes showed clear and reproducible signals and allowed the assignment of all linkage groups to the chromosomes of two Phaseolus vulgaris cultivars, Saxa and Calima. Low extension values for signals originating from clustered RFLPs suggest that these clones are physically close to each other and that clusters in the genetic map are not a result of suppression of recombination due to the occurrence of chromosome rearrangements. For linkage group K, clustering of markers could be associated with proximity to centromeres. High variation in the number of 45S rDNA loci was observed among cultivars, suggesting that these terminal sites are highly recombinogenic in common bean.     

<Emphasis Type="Italic">In vitro</Emphasis> plantlet production of the endangered <Emphasis Type="Italic">Pinguicula vulgaris</Emphasis>

This study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free ¼MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.