Comparison of Defense Body Movements of <Emphasis Type="Italic">Apis laboriosa</Emphasis>, <Emphasis Type="Italic">Apis dorsata dorsata</Emphasis> and <Emphasis Type="Italic">Apis dorsata breviligula</Emphasis> Honey Bees

Defense behavior of three, free living giant (Megapis) honey bee subspecies, Apis laboriosa, A. dorsata dorsata and A. dorsata breviligula, was compared. Disturbed worker bees responded with characteristic dorso-ventral defense body twisting (DBT). Workers of A. laboriosa twisted the thorax by 55°, and the two other A. dorsata subspecies by about 10° more. A. laboriosa workers raised the tip of the abdomen by 90° and workers of the two other bee subspecies by about 20° higher. Differences in those traits were highly significant between A. laboriosa and both A. dorsata subspecies, but were not significant between those two subspecies. The whole cycle of DBT was the most vigorous in A. d. breviligula (0.11 s), and it was twice as vigorous as in A. d. dorsata (0.26 s) and trice as in A. laboriosa (0.32 s). A. laboriosa twisted the body together with wings folded over the abdomen, while the two A. dorsata subspecies raised the abdomen between spread wings. This supports the opinion to treat A. laboriosa as a separate species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ERIC-PCR Genotyping of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> in Southern Italian Honey and Brood Combs

Given the considerable economic loss to beekeepers worldwide and the possible public health implications related to the presence of antibiotics in honey, an American Foulbrood (AFB) monitoring/prevention program for Paenibacillus larvae is regarded as essential. This study investigates the occurrence and distribution of P. larvae genotypes in honey and brood combs from Apulia (Italy). Genotyping of P. larvae isolates using ERIC-PCR generated a total of four different ERIC banding patterns (ERIC-A, ERIC-B, ERIC-C, ERIC-D), including fragments ranging from 200 to 3000 bp. Considering that the genotype has an influence on P. larvae infections and multi-genotype infections of colonies or apiaries may increase the complexity of P. larvae infections by influencing the type and speed of the development of clinical symptoms, the findings of the present study could be helpful for training veterinarians, bee inspector’s extension staff, and beekeepers, thus improving the detection of AFB infections in the field.     

Antimicrobial factor from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> inhibits <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>, the causative agent of American foulbrood

Bacillus amyloliquefaciens LBM 5006 produces an antimicrobial factor active against Paenibacillus larvae, a major honeybee pathogen. The antagonistic effect and the mode of action of the antimicrobial factor were investigated. The antibacterial activity was produced starting at mid-logarithmic growth phase, reaching its maximum during the stationary phase. Exposure of cell suspensions of P. larvae to this antimicrobial resulted in loss of cell viability and reduction in optical density associated with cell lysis. Scanning electron microscopy showed damaged cell envelope and loss of protoplasmic material. The antimicrobial factor was stable for up to 80°C, but it was sensitive to proteinase K and trypsin. Mass spectrometry analysis indicates that the antimicrobial activity is associated with iturin-like peptides. The antimicrobial factor from B. amyloliquefaciens LBM 5006 showed a bactericidal effect against P. larvae cells and spores. This is the first report on iturin activity against P. larvae. This antimicrobial presents potential for use in the control of American foulbrood disease.     

Polymorphism analysis of <Emphasis Type="Italic">csd</Emphasis> gene in six <Emphasis Type="Italic">Apis mellifera</Emphasis> subspecies

The complementary sex determination (csd) gene is the primary gene determining the gender of honey bees (Apis spp). In this study we analyzed the polymorphism of csd gene in six Apis mellifera subspecies. The genomic region 3 of csd gene in these six A. mellifera was cloned, and identified. A total of 79 haplotypes were obtained from these six subspecies. Analysis showed that region 3 of csd gene has a high level of polymorphism in all the six A. mellifera subspecies. The A. m. anatolica subspecies has a slightly higher nucleotide diversity (π) than other subspecies, while the π values showed no significant difference among the other five subspecies. The phylogenetic tree showed that all the csd haplotypes from different A. mellifera subspecies are scattered throughout the tree, without forming six different clades. Population differentiation analysis showed that there are significant genetic differentiations among some of the subspecies. The NJ phylogenetic tree showed that the A. m. caucasica and A. m. carnica have the closest relationship, followed by A. m. ssp, A. m. ligustica, A. m. carpatica and A. m. anatolica that were gathered in the tree in turn.     

Honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis> L., confinement behavior toward beetle invaders

We performed two experiments to study the hiding behavior of various beetles introduced into colonies of European honey bees, Apis mellifera L. In the first experiment, we studied the spatial distribution within confinement sites of six beetle species at eight time intervals following their introduction into honey bee observation hives. For each beetle species, we also determined whether the beetle’s level of integration into honey bee colonies correlated with its ability to hide at confinement sites within colonies. In experiment 1, we used five species of nitidulid beetles and one species of tenebrionid beetle, collectively representing three differing levels of integration into honey bee colonies. These species (and their level of integration) included Aethina tumida (highly integrated), Lobiopa insularis, and Epuraea luteola (accidentals), and Carpophilus humeralis, C. hemipterus, and Tribolium castaneum (non-integrated). There were always more A. tumida found in confinement sites than beetles of the other species. This difference became more pronounced over the 24-h observation period. In experiment 2, we determined whether previous A. tumida occupation of confinement sites predisposed those sites to hosting invading A. tumida never before exposed to honey bee colonies. The results from this study indicate that invading A. tumida find hiding sites within honey bee colonies more rapidly if other A. tumida previously occupied the confinement sites. Collectively, these studies suggest that A. tumida is unique among beetle invaders with respect to its ability to seek out and occupy confinement sites inside honey bee colonies.     

Genetic structure of <Emphasis Type="Italic">Varroa destructor</Emphasis> populations infesting <Emphasis Type="Italic">Apis mellifera</Emphasis> colonies in Argentina

Although mitochondrial DNA mapping of Varroa destructor revealed the presence of several haplotypes, only two of them (Korean and Japanese haplotypes) were capable to infest Apis mellifera populations. Even though the Korean haplotype is the only one that has been reported in Argentina, these conclusions were based on mites sampled in apiaries from a specific geographical place (Buenos Aires province). To study mites from several sites of Argentina could reveal the presence of the Japanese genotype, especially considering sites near to Brazil, where Japanese haplotype was already detected. The aim of this work was to study the genetic structure of V. destructor populations from apiaries located in various provinces of Argentina, in order to determine the presence of different haplotypes. The study was carried out between January 2006 and December 2009. Phoretic adult Varroa mites were collected from honey bee workers sampled from colonies of A. mellifera located in Entre Ríos, Buenos Aires, Corrientes, Río Negro, Santa Cruz and Neuquén provinces. Twenty female mites from each sampling site were used to carry out the genetic analysis. For DNA extraction a nondestructive method was used. DNA sequences were compared to Korean haplotype (AF106899) and Japanese haplotype (AF106897). All DNA sequences obtained from mite populations sampled in Argentina, share 98% of similitude with Korean Haplotype (AF106899). Taking into account these results, we are able to conclude that Korean haplotype is cosmopolite in Argentina.     

The pheromones of laying workers in two honeybee sister species: <Emphasis Type="Italic">Apis cerana</Emphasis> and <Emphasis Type="Italic">Apis mellifera</Emphasis>

When a honeybee colony loses its queen, workers activate their ovaries and begin to lay eggs. This is accompanied by a shift in their pheromonal bouquet, which becomes more queen like. Workers of the Asian hive bee Apis cerana show unusually high levels of ovary activation and this can be interpreted as evidence for a recent evolutionary arms race between queens and workers over worker reproduction in this species. To further explore this, we compared the rate of pheromonal bouquet change between two honeybee sister species of Apis cerana and Apis mellifera under queenright and queenless conditions. We show that in both species, the pheromonal components HOB, 9-ODA, HVA, 9-HDA, 10-HDAA and 10-HDA have significantly higher amounts in laying workers than in non-laying workers. In the queenright colonies of A. mellifera and A. cerana, the ratios (9-ODA)/(9-ODA + 9-HDA + 10-HDAA + 10-HDA) are not significantly different between the two species, but in queenless A. cerana colonies the ratio is significant higher than in A. mellifera, suggesting that in A. cerana, the workers’ pheromonal bouquet is dominated by the queen compound, 9-ODA. The amount of 9-ODA in laying A. cerana workers increased by over 585% compared with the non-laying workers, that is 6.75 times higher than in A. mellifera where laying workers only had 86% more 9-ODA compared with non-laying workers.     

Synergistic effect of surfactin from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C4 and <Emphasis Type="Italic">Achyrocline satureioides</Emphasis> extracts on the viability of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>

The aim of this work was to determine the in vitro effect of the mixture between the lipopeptide surfactin, synthesized by Bacillus subtilis C4 (strain isolated from honey) and the most active vegetal extract from Achyrocline satureioides, a traditional medicinal plant, on local strains of Paenibacillus larvae, the agent of American Foulbrood in honeybees. Five P. larvae strains isolated in Córdoba, Argentina, were phenotypically characterized. These and 12 other P. larvae strains from different regions of Argentina were analysed. The antimicrobial activities of the essential oil, hexane (HE) and benzene extracts from A. satureioides were assessed against P. larvae and the HE showed the highest anti-P. larvae activity. A combination of the biosurfactant surfactin, produced by B. subtilis C4, and the HE of A. satureioides revealed a synergistic action on P. larvae. The effective surfactin concentration in the mixture decreased from 32 to 1 μg ml⁻¹ and the HE concentration from 32 to 4 μg ml⁻¹, values similar or equal to minimal inhibitory concentrations observed for oxytetracycline. The fractional inhibitory concentration index confirmed synergism in 4 strains and partial synergism in one strain. The combination of surfactin synthesized by B. subtilis C4 and the HE from A. satureioides could be a natural alternative to help beekeepers to combat the American foulbrood agent P. larvae.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Reproductive biology of <Emphasis Type="Italic">Varroa destructor</Emphasis> in Africanized honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Since its first contact with Apis mellifera, the population dynamics of the parasitic mite Varroa destructor varies from one region to another. In many regions of the world, apiculture has come to depend on the use of acaricides, because of the extensive damage caused by varroa to bee colonies. At present, the mite is considered to contribute to the recent decline of honey bee colonies in North America and Europe. Because in tropical climates worker brood rearing and varroa reproduction occurs all year round, it could be expected that here the impact of the parasite will be even more devastating. Yet, this has not been the case in tropical areas of South America. In Brazil, varroa was introduced more than 30 years ago and got established at low levels of infestation, without causing apparent damage to apiculture with Africanized honey bees (AHB). The tolerance of AHB to varroa is apparently attributable, at least in part, to resistance in the bees. The low fertility of this parasite in Africanized worker brood and the grooming and hygienic behavior of the bees are referred as important factors in keeping mite infestation low in the colonies. It has also been suggested that the type of mite influences the level of tolerance in a honey bee population. The Korea haplotype is predominant in unbalanced host-parasite systems, as exist in Europe, whereas in stable systems, as in Brazil, the Japan haplotype used to predominate. However, the patterns of varroa genetic variation have changed in Brazil. All recently sampled mites were of the Korea haplotype, regardless whether the mites had reproduced or not. The fertile mites on AHB in Brazil significantly increased from 56% in the 1980s to 86% in recent years. Nevertheless, despite the increased fertility, no increase in mite infestation rates in the colonies has been detected so far. A comprehensive literature review of varroa reproduction data, focusing on fertility and production of viable female mites, was conducted to provide insight into the Africanized bee host-parasite relationship.     

Expression of <Emphasis Type="Italic">Pax group III</Emphasis> genes in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior–posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.     

New approach to the mitotype classification in black honeybee <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> and Iberian honeybee <Emphasis Type="Italic">Apis mellifera iberiensis</Emphasis>

The black honeybee Apis mellifera mellifera L. is today the only subspecies of honeybee which is suitable for commercial breeding in the climatic conditions of Northern Europe with long cold winters. The main problem of the black honeybee in Russia and European countries is the preservation of the indigenous gene pool purity, which is lost as a result of hybridization with subspecies, A. m. caucasica, A. m. carnica, A. m. carpatica, and A. m. armeniaca, introduced from southern regions. Genetic identification of the subspecies will reduce the extent of hybridization and provide the gene pool conservation of the black honeybee. Modern classification of the honeybee mitotypes is mainly based on the combined use of the DraI restriction endonuclease recognition site polymorphism and sequence polymorphism of the mtDNA COI–COII region. We performed a comparative analysis of the mtDNA COI–COII region sequence polymorphism in the honeybees of the evolutionary lineage M from Ural and West European populations of black honeybee A. m. mellifera and Spanish bee A. m. iberiensis. A new approach to the classification of the honeybee M mitotypes was suggested. Using this approach and on the basis of the seven most informative SNPs of the mtDNA COI–COII region, eight honeybee mitotype groups were identified. In addition, it is suggested that this approach will simplify the previously proposed complicated mitotype classification and will make it possible to assess the level of the mitotype diversity and to identify the mitotypes that are the most valuable for the honeybee breeding and rearing.     

Nestmate Recognition Differences between Honeybee Colonies of <Emphasis Type="BoldItalic">Apis cerana</Emphasis> and <Emphasis Type="BoldItalic">Apis mellifera</Emphasis>

Nestmate recognition in Apis cerana and Apis mellifera was studied by introducing sealed queen cells heterospecifically between queenless colonies. No A. cerana queens were accepted by queenless A. mellifera; but A. mellifera queens were accepted in queenless A. cerana colonies. A. mellifera queens oviposited in queenless A. cerana colonies, but A. cerana workers removed most eggs. In time, egg removals declined, and some A. mellifera larvae that hatched from these eggs reached adulthood, and eventually about half of the workers were newly emerged A. mellifera. Eventually, the colonies consisted only of A. mellifera after A. cerana workers died by attrition. A. mellifera workers are more sensitive to nestmate recognition and killed the A. cerana virgin queens. In mixed-species colonies, after newly emerged A. mellifera workers matured, they removed eggs laid by the A. cerana queens until there were no workers to replace the old ones.     

Deformed wing virus associated with <Emphasis Type="Italic">Tropilaelaps mercedesae</Emphasis> infesting European honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Mites in the genus Tropilaelaps (Acari: Laelapidae) are ectoparasites of the brood of honey bees (Apis spp.). Different Tropilaelaps subspecies were originally described from Apis dorsata, but a host switch occurred to the Western honey bee, Apis mellifera, for which infestations can rapidly lead to colony death. Tropilaelaps is hence considered more dangerous to A. mellifera than the parasitic mite Varroa destructor. Honey bees are also infected by many different viruses, some of them associated with and vectored by V. destructor. In recent years, deformed wing virus (DWV) has become the most prevalent virus infection in honey bees associated with V. destructor. DWV is distributed world-wide, and found wherever the Varroa mite is found, although low levels of the virus can also be found in Varroa free colonies. The Varroa mite transmits viral particles when feeding on the haemolymph of pupae or adult bees. Both the Tropilaelaps mite and the Varroa mite feed on honey bee brood, but no observations of DWV in Tropilaelaps have so far been reported. In this study, quantitative real-time RT-PCR was used to show the presence of DWV in infested brood and Tropilaelaps mercedesae mites collected in China, and to demonstrate a close quantitative association between mite-infested pupae of A. mellifera and DWV infections. Phylogenetic analysis of the DWV sequences recovered from matching pupae and mites revealed considerable DWV sequence heterogeneity and polymorphism. These polymorphisms appeared to be associated with the individual brood cell, rather than with a particular host.     

<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.     

Vitellogenin transcytosis in follicular cells of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis> and the wasp <Emphasis Type="Italic">Polistes simillimus</Emphasis>

Vitellogenin receptor (VgR) is a low-density lipoprotein receptor responsible for the mediated endocytosis of vitellogenin (Vg) during egg formation in insects. The maturing oocyte is enveloped by a follicular epithelium, which has large intercellular spaces during Vg accumulation (patency). However, Vg has been reported in the cytoplasm of follicular cells, indicating that there may be a transcellular route for its transport. This study verified the presence of VgR in the follicular cells of the ovaries of the honeybee Apis mellifera and the wasp Polistes simillimus in order to evaluate if Vg is transported via transcytosis in these insects. Antibodies specific for vitellogenin receptor (anti-VgR), vitellogenin (anti-Vg), and clathrin (anti-Clt) were used for immunolocalization. The results showed the presence of VgR on the apical and basal plasma membranes of follicular cells of the vitellogenic follicles in both species, indicating that VgR may have been transported from the basal to the apical cell domain, followed by its release into the perivitelline space, evidenced by the presence of apical plasma membrane projections containing VgR. Co-localization proved that Vg bind to VgR and that the transport of this protein is mediated by clathrin. These data suggest that, in these social insects, Vg is transported via clathrin-mediated VgR transcytosis in follicular cells.     

Interracial Differences in Expression of Genes of Antibacterial Peptides,Abaecin, Hymenoptaecin,and Defensin,in Bees <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> and <Emphasis Type="Italic">Apis mellifera caucasica</Emphasis>

Results of experiments are presented which have revealed differences in expression of genes of antibacterial peptides (ABP) after administration of pathogen (bacterial preparation, bitoxybacillin) between honeybees of the middle-Russian and Caucasian races as well as their hybrids. Interracial variations of the level of ABP gene expression were studied in the bees at the early stage of protective reactions. It was shown that the reaction was realized more rapidly in bees after their immunization and that purebred bees had some advantages over hybrids in development of the protective response with participation of ABP.     

The parasitic mite <Emphasis Type="Italic">Varroa destructor</Emphasis> affects non-associative learning in honey bee foragers, <Emphasis Type="Italic">Apis mellifera</Emphasis> L.

The parasitic mite Varroa destructor influences flight behavior, orientation and returning success of forager honeybees (Apis mellifera) infested as adults. As impaired orientation toward the nest entrance might be due to deficiency in recognition and responsiveness to stimuli in the environment, we examined effects of V. destructor on sensory responsiveness, non-associative and associative learning of honey bee foragers by using proboscis extension reaction paradigm (PER). Although infested and uninfested workers were initially equally responsive to different concentrations of sugar water, we found differences in non-associative learning. In habituation, PER to repeated sugar stimulation of the antennae occurred faster in infested foragers compared to uninfested foragers. In sensitization, infested foragers showed a lower response to an odor stimulus following sugar stimulation than non-infested foragers. Differences in non-associative paradigms were more pronounced in bees with lower responsiveness to sucrose. In conditioning learning experiments, a significant reduction in proboscis extension response was found 1 min but not 12 min after a single conditioning trial indicating that V. destructor predominantly affects the non-associative components of learning and its underlying neural and molecular processes. Jasna Kralj and Axel Brockmann have contributed equally to this study.     

The tiny Hairless protein from <Emphasis Type="Italic">Apis mellifera</Emphasis>: a potent antagonist of Notch signaling in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

The Notch signaling pathway is fundamental to the regulation of many cell fate decisions in eumetazoans. Not surprisingly, members of this pathway are highly conserved even between vertebrates and invertebrates. There is one notable exception, Hairless, which acts as a general Notch antagonist in Drosophila. Hairless silences Notch target genes by assembling a repressor complex together with Suppressor of Hairless [Su(H)] and the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). Now with the availability of genomic databases, presumptive Hairless homologues are predicted, however only in insect species. To further our understanding of Hairless structure and function, we have cloned the Hairless gene from Apis mellifera (A.m.H) and characterized its functional conservation in Drosophila.     

<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.