Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.     

Characterization of an expansin gene and its ripening-specific promoter fragments from sour cherry (<Emphasis Type="Italic">Prunus cerasus</Emphasis> L.) cultivars

The presence of expansins was investigated in various developmental and ripening stages of cherry fruits by SDS-PAGE and immunoblotting. An expansin gene and three fragments (242, 607 and 929 bp) of its promoter region were cloned. The genomic clone of the expansin gene contained three introns, two exons spanning a 1.6 and a 1.0 kb upstream region. Semi-quantitative PCR analysis showed that this gene was ripening specific. Chimeric promoter—GUS constructs were made and truncated forms of the expansin promoter were introduced into tomatoes by agroinjection and fruits were analyzed for GUS expression by histochemical GUS staining and enzyme activity assays. The 0.60 kb expansin promoter efficiently induced GUS expression in transgenic tomatoes, whereas constructs with the 0.25 kb promoter did not display significant GUS staining. The highest GUS activity was detected in tomatoes containing the 1.0 kb promoter construct. Both large base pair promoter constructs drove the expression of the GUS gene at an equal or higher rate than the tomato E8 promoter.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Development and characterization of microsatellite markers for <Emphasis Type="Italic">Prunus verecunda</Emphasis> and <Emphasis Type="Italic">Prunus grayana</Emphasis> (Rosaceae)

Simple sequence repeat (SSR) markers were developed for Prunus verecunda and Prunus grayana to help understand the seed dispersal pattern of each species. We isolated and characterized nine microsatellite loci (four from P. verecunda and five from P. grayana). In P. verecunda, the number of alleles detected and the expected heterozygosity of five loci ranged from 11 to 24 and 0.59 to 0.92, respectively. In P. grayana, the number of alleles detected and the expected heterozygosity of five loci ranged from 4 to 14 and 0.62 to 0.86, respectively. These results show that the markers described here will be useful in studying the seed dispersal pattern of Prunus species.     

<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.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Pedogamy in <Emphasis Type="Italic">Neidium</Emphasis> (<Emphasis Type="Italic">Bacillariophyceae</Emphasis>)

Single (unpaired) vegetative cells of freshwater pennate diatom Neidium cf. ampliatum differentiated into gametangia and produced a single zygote (auxospore) via a pedogamic process. The gametic nuclei fused after auxospore expansion had begun. The auxospore expanded in parallel to the apical axis of the gametangium.     

Breakdown of distyly in a tetraploid variety of <Emphasis Type="Italic">Ophiorrhiza</Emphasis><Emphasis Type="Italic"> japonica</Emphasis> (Rubiaceae) and its phylogenetic analysis

We examined the floral morph of tetraploid Ophiorrhiza japonica Blume var. amamiana Hatus. and diploid O. japonica var. japonica to elucidate the association of distyly and ploidy levels. Chloroplast DNA phylogeny was reconstructed to determine the number of tetraploidization events and floral morph shifts in O. japonica. All individuals of O. japonica var. amamiana proved to be long-homostylous, whereas O. japonica var. japonica was distylous with typical long- and short-styled flowers. Distyly is related to the ploidy level. The bagging treatment of flowers indicated that O. japonica var. amamiana is self-compatible and potentially automatically self-pollinating. In cpDNA sequencing analysis, no haplotype was shared between the two varieties. The cpDNA haplotype network displayed the monophyly of O. japonica var. amamiana, suggesting a single origin of this variety. Hence, both tetraploidization and the breakdown of distyly to homostyly in O. japonica var. amamiana likely occurred just once. Because O. japonica var. amamiana having the morphological and cytological entity is recognized as a single lineage and clearly separated from O. japonica var. japonica, this variety can be considered to be a distinct species. We therefore propose to raise O. japonica var. amamiana to the rank of species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Genomic clues to the parental origin of the wild flowering cherry <Emphasis Type="Italic">Prunus yedoensis</Emphasis> var. <Emphasis Type="Italic">nudiflora</Emphasis> (Rosaceae)

Prunus yedoensis Matsumura is one of the popular ornamental flowering cherry trees native to northeastern Asia, and its wild populations have only been found on Jeju Island, Korea. Previous studies suggested that wild P. yedoensis (P. yedoensis var. nudiflora) is a hybrid species; however, there is no solid evidence on its exact parental origin and genomic organization. In this study, we developed a total of 38 nuclear gene-based DNA markers that can be universally amplifiable in the Prunus species using 586 Prunus Conserved Orthologous Gene Set (Prunus COS). Using the Prunus COS markers, we investigated the genetic structure of wild P. yedoensis populations and evaluated the putative parental species of wild P. yedoensis. Population structure and phylogenetic analysis of 73 wild P. yedoensis accessions and 54 accessions of other Prunus species revealed that the wild P. yedoensis on Jeju Island is a natural homoploid hybrid. Sequence-level comparison of Prunus COS markers between species suggested that wild P. yedoensis might originate from a cross between maternal P. pendula f. ascendens and paternal P. jamasakura. Moreover, approximately 81% of the wild P. yedoensis accessions examined were likely F1 hybrids, whereas the remaining 19% were backcross hybrids resulting from additional asymmetric introgression of parental genotypes. These findings suggest that complex hybridization of the Prunus species on Jeju Island can produce a range of variable hybrid offspring. Overall, this study makes a significant contribution to address issues of the origin, nomenclature, and genetic relationship of ornamental P. yedoensis.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

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.     

Identification of <Emphasis Type="Italic">S</Emphasis>-haplotype-specific F-box gene in Japanese plum (<Emphasis Type="Italic">Prunus salicina</Emphasis> Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, i.e., the S-RNase gene and the pollen-expressed SFB/SLF (S-haplotype-specific F-box/S-locus F-box) gene. However, the SFB gene in Japanese plum (Prunus salicina Lindl.) has not yet been identified. We determined eight novel sequences homologous to the SFB genes of other Prunus species and named these sequences PsSFB. The gene structure of the SFB genes and the characteristic domains in deduced amino acid sequences were conserved. Three sequences from 410 to 2,800 bp of the intergenic region between the PsSFB sequences and the S-RNase alleles were obtained. The eight identified PsSFB sequences showed S-haplotype-specific polymorphism, with 74–83% amino acid identity. These alleles were exclusively expressed in the pollen. These results suggest that the PsSFB alleles are the pollen S-determinants of GSI in Japanese plum. Nucleotide sequence data reported are available in the NCBI database under the accession numbers DQ849084–DQ849090 and DQ849118.     

Identification of <Emphasis Type="Italic">S</Emphasis> haplotypes in <Emphasis Type="Italic">Brassica</Emphasis> by dot-blot analysis of <Emphasis Type="Italic">SP11</Emphasis> alleles

A self-incompatibility system is used for F(1) hybrid breeding in Brassicaceae vegetables. The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen. Nucleotide sequences of SP11 alleles are more highly variable than those of SRK. We analyzed the S haplotype specificity of SP11 DNA by Southern-blot analysis and dot-blot analysis using 16 S haplotypes in Brassica oleracea, and found that DNA fragments of a mature protein region of SP11 cDNA, SP11(m), of eight S haplotypes can detect only the SP11 alleles of the same S haplotypes. This specificity makes these methods useful for S haplotype identification. Therefore, we developed two methods of dot-blot analysis for SP11. One is dot blotting of DNA samples, i.e. plant genomic DNA probed with labeled SP11(m), and the other is dot blotting of SP11(m) DNA fragments probed with labeled DNA samples, i.e. the SP11 coding region labeled by PCR using a template of plant genomic DNA. The former is useful for testing many plant materials. The latter is suitable, if there is no previous information on the S haplotypes of plant materials.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Genomewide analysis of <Emphasis Type="Italic">ABCB</Emphasis>s with a focus on <Emphasis Type="Italic">ABCB1</Emphasis> and <Emphasis Type="Italic">ABCB19</Emphasis> in <Emphasis Type="Italic">Malus domestica</Emphasis>

The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon–intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots of M9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple.     

<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.