Are <Emphasis Type="Italic">Melilotus albus</Emphasis> and <Emphasis Type="Italic">M. officinalis</Emphasis> conspecific?

The principal economic species of the genus Melilotus are white sweet-clover (Melilotus albus) and the extremely similar yellow sweet-clover (M. officinalis). Although they are widely recognized as distinct species, some influential references in North America reduce the former to a subspecific rank or even merely a conspecific synonym of the latter. Given their importance and the large numbers of germplasm collections, the doubt needs to be resolved. This review of relevant published evidence finds that in addition to the difference in floral colour, the traditional segregation of the two as distinct species is best supported by very strong reproductive barriers as well as divergent DNA sequences in three barcoding genes. Additional but weaker confirmation of separateness is provided by studies reporting differences in external morphology, biochemistry, seed protein profiles, karyotype and DNA microsatellites.     

Genetic mapping of the gene for brittle rachis in a <Emphasis Type="Italic">Triticum aestivum</Emphasis>—<Emphasis Type="Italic">Aegilops triuncialis</Emphasis> introgression line

Aegilops triuncialis L. (2n = 4x = 28, genome formula UUCC) has been used to obtain one of the “Arsenal collection”, the distant hybridization of Triticum aestivum L. The introgression line 124/00ⁱ, which was derived from the cross of T. aestivum ‘Rodina' with γ-irradiated pollen of an accession of Ae. triuncialis. However, undesirable genes can be incorporated into the cultivated varieties from Ae. triuncialis. The spikes of Ae. triuncialis disarticulate from stems at a single node at the bottom of the spikes. It was defined as a synaptospermic diaspore. However, the spike of 124/00ⁱ unexpectedly disarticulate below each spikelet showing wedge type of disarticulation. The brittle rachis gene Br ¹²⁴ (B rittle r achis of 124 /00 ⁱ ) of 124/00ⁱ was allelic to Br2 located on the short arm of chromosome 3A. From the microsatellite mapping, the Br ¹²⁴ gene was bracketed by Xbarc19 (20.3 cM) and Xgwm779.1(14.3 cM) on the short arm of chromosome 3A. The present study suggested that the dominant mutation from synaptospermic diaspore to wedge type disarticulation occurred in the gamete of Ae. triuncialis when Ae. triuncialis pollen was treated with γ-irradiation.     

<Emphasis Type="Italic">Samardala</Emphasis>: specificities and changes in the ethnobotanical knowledge about <Emphasis Type="Italic">Allium siculum</Emphasis> subsp<Emphasis Type="Italic">. dioscoridis</Emphasis> (Sm.) K. Richt. in Bulgaria

Allium siculum Ucria is a perennial bulbous plant that occurs in Bulgaria as A. siculum subsp. dioscoridis (Sm.) K. Richt. The plant is locally known as “samardala”. Its herbage is used traditionally by local communities in Bulgaria as edible green and for preparation of salty spice mixtures. The study was focused on the documentation of traditional knowledge about samardala, covering local collection and cultivation practices, processing methods and consumption preferences. The respondents (aged 30–86 years, 50% over 65) were local producers and small-scale salesmen of the herbage and samardala-containing spices. To assess the health features of samardala spices the total phenol and flavonoid content was measured in home-consumed or marketed flavoured salts. Overall appreciation of the plant and related food products was found to be very high; presenting a considerable engagement of the locals in plant cultivation and gathering, laborious processing and consumption. Traditional ecological knowledge about the biology, phenology and ecology of the samardala plant and methods for its processing is still preserved by elderly people who rely mostly on their own cultivated plants. Although the introduction of modern appliances has altered the method of production, its main stages and the recipes are preserved. Modernization of the processing method has not affected the quality of the products and faster processing could be a contributing factor for the higher content of biologically active substances. Phenolic content was about twice as low as the flavonoid content, following the same pattern for all tested flavoured salts. Measures for better management of natural populations and promotion of cultivation practices of A. siculum subsp. dioscoridis would ensure the sustainable quality of traditional products as well as conservation of the natural plant resources.     

<Emphasis Type="Italic">Arnica montana</Emphasis> subsp. <Emphasis Type="Italic">atlantica</Emphasis>: Really a subspecies?

In Arnica montana L. (Asteraceae) two subspecies are described, A. montana subsp. atlantica (AMA), present only on the Iberian Peninsula and A. montana subsp. montana (AMM) with a very wide distribution area. The morphological differences between the two subspecies are small and variable. Therefore, this concept is sometimes questioned. To establish the genetic background of the two subspecies, populations of AMA and AMM together with herbarium samples and DNA Bank material of AMM were tested with 12 microsatellite markers. A. montana propagates by seeds or by clonal propagation of its rhizome. In AMA, clonality was frequent while in AMM only one case of clonality could be identified. Therefore, further results were clone-corrected. Genetically, AMA separated very well from AMM with a G_ST between the subspecies of 0.81, genetically justifying the subspecies concept of A. montana. Genetic variability in AMA (H_exp?=?0.28) was lower than in the AMM populations (H_exp?=?0.70). A somewhat higher fixation index of AMA (F_ST?=?0.17, compared to an F_ST?=?0.08 for AMM) may indicate that geneflow in AMA is a bit more restricted than in alpine AMM. However, the fixation index of AMA is not deviating from Hardy–Weinberg equilibrium. No inbreeding was observed for AMA (F_IS?=?0.10) and AMM (F_IS?=?0.08).     

Recombination around the <Emphasis Type="Italic">P</Emphasis> locus for long glume phenotype in experimental introgression lines of <Emphasis Type="Italic">Triticum aestivum</Emphasis> – <Emphasis Type="Italic">Triticum polonicum</Emphasis>

A set of experimental introgression lines of Triticum aestivum L. cv. Novosibirskaya 67 (N67)—Triticum polonicum L. line IC 12196 was developed using a small-scale bulk breeding method. The linkage map in chromosome 7A was constructed using F₂ hybrids of N67/IC12196 and 34 microsatellite markers. The P gene was flanked by the centromeric markers, Xgwm890 (18.6 cM) and Xbarc108 (20.0 cM) on the long arm of chromosome 7A. Among 124 introgression lines, 118 lines were hexaploid (2n = 6x = 42), and 6 were tetraploid (2n = 4x = 28). Among hexaploid accessions, 68 were long-glumed, whereas 50 were normal-glumed. Thirty-four polymorphic microsatellite markers were scored for either the N67 alleles or IC 12196 alleles in 124 introgression lines derived from N67/IC 12196. The UPGMA dendrogram showed five clusters; Cluster 1 mainly contained hexaploid introgression lines with long glumes. Although the alleles around the P locus were recombined with IC1296 alleles, the distal end of the chromosome contained N67 alleles. Cluster 2 mainly contained normal glumed, hexaploid introgression lines. These predominantly had the N67 alleles on the long arm of chromosome 7A and the short arm proximal to the centromere. Cluster 3 contained long-glumed, hexaploid wheat lines with relatively high level of recombination. Cluster 4 contained non-parental alleles. Cluster 5 contained the group of tetraploid wheat lines. These tetraploid lines have IC12196 alleles on both arms of chromosome 7A. The frequency spectrum of parental alleles and chromosomal blocks among introgression lines suggested that T. aestivum – T. polonicum hybridization can rapidly give rise to a new landrace due to selective introgression of the P gene.     

Changes of gene expression in early generations of the synthetic allotetraploid <Emphasis Type="Italic">Cucumis</Emphasis> × <Emphasis Type="Italic">hytivus</Emphasis> Chen et Kirkbride

Changes of gene expression played an important role in the evolution of plant allopolyploids. Frequency, time and type of the changes of gene expression between the first two self-pollinated generations (S₁ and S₂) of a synthesized allotetraploid Cucumis × hytivus Chen et Kirkbride and in its diploid parents was analyzed with cDNA-AFLP and reverse-Northern blot technique. Sequences similarity of genes involved in changed expression were also analyzed with BLAST package. The results from cDNA-AFLP analysis showed that 36 (3.37%) genes showed silencing (27) or activation (9) in allotetraploids. These changes initiated in S₁ or S₂ generation. The silenced/activated genes included rRNA and protein-coding genes. Further reverse-Northern blot analysis validated the results obtained. Thus four types of changes of gene expression were observed, including silencing of genes from both parents, maternal parent, paternal parent, and genes novel expression. The results indicate rapid changes in gene expression in early generations of C. × hytivus, which contributed to the evolution of this synthetic allotetraploid.     

Taxonomic remarks on <Emphasis Type="Italic">Triticum</Emphasis> L. and ×<Emphasis Type="Italic">Triticosecale</Emphasis> Wittm.

The recent treatment of Triticum by Mac Key (2005) (broad species concept) is compared to the classical treatment by Dorofeev et al. (1979, English translation expected) (narrow species concept). A detailed infraspecific treatment was abandoned by Mac Key. Following his revision, names for many of the infraspecific taxa become obsolete. This is regarded to be a disadvantage for biodiversity and genetic resources studies. We propose maintaining ×Triticosecale Wittm. as a nothogenus, with ×T. rimpaui Wittm. for octoploid races, ×T. neoblaringhemii A. Camus for hexaploid races and ×T. semisecale (Mac Key) K. Hammer et A. Filat. (new combination) for tetraploid races.     

Inflorescence-visiting insects of a functionally dioecious wild grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> subsp. <Emphasis Type="Italic">sylvestris</Emphasis>)

The wild grapevine [Vitis vinifera L. subsp. sylvestris (Gmelin) Hegi] is the Eurasian dioecious ancestral form of mostly monoecious domesticated cultivars (Vitis vinifera subsp. vinifera). There are different hypotheses about grapevine pollination, varying from insect to wind pollination to spontaneous self-pollination. The aim of this paper is to update our knowledge on the biology of the wild grapevine by studying the insects visiting their inflorescences. During the fieldworks, floral visitors were observed, captured and identified. The most frequent insects visiting both sexes of wild grapevine were beetles (55%) followed by bees (45%). The present study shows that although in vineyards the wind may play a role in the cross-pollination, insects can contribute to the reproductive success of the wild grapevine. The knowledge of inflorescence-visiting insects, which potentially can act as pollinators may represent an important contribution to the conservation perspective of wild grapevine.     

Comparison of the efficiency of A–PAGE and SDS–PAGE,ISSRs and RAPDs in resolving genetic relationships among <Emphasis Type="Italic">Triticum</Emphasis> and <Emphasis Type="Italic">Aegilops</Emphasis> species

Species that belong to the genus Triticum L. and the genetically related Aegilops L. genus are important genetic and economic resources because they have an evolutionary relationship with the two main agricultural crops T. aestivum (bread wheat) and T. durum (durum wheat). Therefore, it is important to understand the genetic relationships among the cultivated wheat species and their wild relatives. The latter have a great role in the improvement of cultivated wheat. Molecular markers are the best choice and most reliable means to study these relationships accurately. In this study, we compared the efficiency of the biochemical methods A–PAGE and SDS–PAGE on seed storage proteins and the molecular methods RAPDs and ISSRs to explore the genetic relationships among seven species of Triticum and 20 Aegilops species. Three phylogenetic trees obtained in this study were compared with available classifications and phylogenetic trees constructed earlier for these species. It was noted that the tree based on ISSRs data was the most congruent with those classification and trees. This may be attributed to the fact that ISSRs is more specific, and therefore more reliable. This study is the first to study genetic relationships among all species studied here using biochemical and molecular techniques.     

Identification of unknown apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis>) cultivars demonstrates the impact of local breeding program on cultivar diversity

Apple (Malus?×?domestica Borkh.) trees, either abandoned or cared for, are common on the North American landscape. These trees can live for decades, and therefore represent a record of large- and small-scale agricultural practices through time. Here, we assessed the genetic diversity and identity of 330 unknown apple trees in northern Minnesota with 9 simple sequence repeat (SSR) markers. The unknown (not identified by cultivar name) trees were compared to >?1000 named cultivars in the U.S. Department of Agriculture Plant Genetic Resources Unit Malus collection and also to each other to identify repeated genotypes. Overall, the 330 unknown trees had high diversity (average H_e?=?0.75), and consisted of 264 unique genotypes. A total of 76 of the unknown trees were matched to 20 different named cultivars, and these cultivars were mainly derived from either the local breeding program at the University of Minnesota, or were Russian cultivars imported for horticulture in the northern US. This study demonstrates the importance of local breeding programs, and also the challenges associated with identifying clones in a genetically diverse crop like apple.     

Chromosome and genome composition of a <Emphasis Type="Italic">Triticum</Emphasis> × <Emphasis Type="Italic">Thinopyrum</Emphasis> hybrid by classical and molecular cytogenetic techniques

Triticum–Thinopyrum amphiploids arose from the need to obtain forage grasses highly resistant to pest, drought, soil salinity and frost and they can be used as efficient bridges to transfer desired genes from wheatgrass species to wheat. One of them is trigopiro SH16 INTA, it was introduced in Argentina in 1947 but its genomic composition was unknown. The aim of this work was to determine the chromosome number and genomic and chromosome composition of trigopiro SH16 INTA in order to use it in breeding programs. The simultaneous use of the in situ hybridization technique with different probes (genomic DNA of Th. ponticum (Podp.) Barkworth et D.R. Dewey, pSc119.2 and pAs1) allowed us to conclude that the chromosome number of trigopiro SH16 is 2n = 42 and the genome composition would be: 14 chromosomes of the B genome, the 2D and 4D chromosome pairs of wheat, 14 chromosomes of the J genome of Thinopyrum and the remaining chromosomes probably belong to the A genome of wheat.     

Comparative genetic diversity of <Emphasis Type="Italic">Triticum aestivum–Triticum polonicum</Emphasis> introgression lines with long glume and <Emphasis Type="Italic">Triticum petropavlovskyi</Emphasis> by AFLP-based assessment

Genetic diversity of a set of introgression lines of Triticum aestivum L./T. polonicum L. with long glume and T. petropavlovskyi Udacz. et Migusch. were analyzed by Amplified Fragments Length Polymorphism (AFLP). Small-scale bulk breeding method was applied throughout until F₆ generation to develop the introgression lines. Thirty-eight hexapolid F₇ plants with long glume phenotype and their parents were subjected to AFLP analysis by four primer combinations. A total of 47 polymorphic loci were detected between the parents, 15 of them were introgressed across the 38 lines. It was hypothesized that approximately 50% of A or B genomes associated polymorphic loci were introgressed. The variation of introgression lines was limited within the diversity between their parents, T. aestivum L. cv. Novosibirskaya 67 (N67) and T. polonicum L. cv. IC12196. N67 was closer to 38 introgression lines than that of IC12196. The UPGMA cluster and principal coordinate analysis (PCO) grouping showed 0.84 to 0.98 similarity values between N67 and the introgression lines. Eleven T. petropavlovskyi accessions were distinguished from introgression lines with UPGMA clusters and PCO groupings, and T. petropavlovskyi was located between the introgressions lines and IC12196. Several introgression lines resembled with T. petropavlovskyi for awning and glume length. The genetic variation among 38 introgression lines was much wider than that of T. petropavlovskyi. We concluded that T. petropavlovskyi was established by intensive selection of hybrid between T. aestivum/T. polonicum.     

Earthworm (<Emphasis Type="Italic">Metaphire guillelmi</Emphasis>) effects on rice photosynthates distribution in the plant–soil system

An experiment was conducted to investigate the effects of earthworm (Metaphire guillelmi) activities on rice photosynthates distribution in plant–soil system through ¹⁴C pulse-labelling method. Rice was planted in pots, and maize straw was mulched on the surface with or without earthworms. Rice plants at tillering stage or heading stage were labelled with ¹⁴CO₂. Plant and soil were sampled 15 days after labelling at the tillering or heading stage and at harvest. Rice growth was inhibited by earthworms (M. guillelmi) at early stage, but the inhibition disappeared at later stage. Earthworms significantly (P < 0.01) increased the ¹⁴C percentage in root at day 15 after tillering stage labelling, but the effect disappeared at harvest. Earthworms (M. guillelmi) significantly (P < 0.01) increased the ¹⁴C percentage in root at day 15 after heading stage labelling and increased ¹⁴C percentage in soil at harvest. Earthworms decreased the percentages of total organic ¹⁴C (TO¹⁴C) present as microbial biomass ¹⁴C (MB¹⁴C) and increased the percentages of total organic ¹⁴C present as dissolved organic ¹⁴C (DO¹⁴C) at all sampling times. It is suggested that earthworms might alter the transfer of plant photosynthates from the aboveground to the belowground, and thus, soil active C pool. However, these data should be also confirmed in the field.     

<Emphasis Type="Italic">Manihot fortalezensis</Emphasis> Nassar,Ribeiro, Bomfim et Gomes a new species of <Emphasis Type="Italic">Manihot</Emphasis> from Ceará, Brazil

A new species of Manihot has been collected from Fortaleza, Ceará state, Brazil. It was grown at the living collection in the experimental station, University of Brasilia since 1980 and left for natural reproduction. It bred true during all these years giving rise to identical typical plants of the ancestor one. The closest species to it is M. glaziovii. Apparently this new species was formed by sporadic hybridization between M. glaziovii and cassava itself in its natural habitat, Ceará. It possesses morphological marker gene of the latter one which is ribbed fruit that is found only in cassava. Interspecific hybrids produced experimentally between M. glaziovii and cassava showed similar morphological markers. Exam of its ovules revealed formation of multiple embryos, an indicator of apomixis. Grafting it with common cassava was compatible, giving increased root size up to seven times.     

Microsatellite genotyping of apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis> Borkh.) genetic resources in the Netherlands: application in collection management and variety identification

A highly informative set of 16 microsatellite markers was used to fingerprint 695 apple accessions from eight Dutch collections. Among the total sample, 475 different genotypes were distinguished based on multi-locus microsatellite variation, revealing a potential redundancy within the total sample of 32%. The majority of redundancies were found between collections, rather than within collections. No single collection covered the total observed diversity well, as each collection consisted of about 50% of unique accessions. These findings reflected the fact that most collection holders focus on common Dutch varieties, as well as on region-specific diversity. Based on the diversity patterns observed, maintenance of genetic resources by a network of co-operating collection holders, rather than by collecting the total diversity in a single collection appears to be an efficient approach. Comparison of microsatellite and passport data showed that for many accessions the marker data did not provide support for the registered variety names. Verification of accessions showed that discrepancies between passport and molecular data were largely due to documentation and phenotypic determination errors. With the help of the marker data the varietal names of 45 accessions could be corrected. Microsatellite genotyping of apple appears to be an efficient tool in the management of collections and in variety identification. The development of a marker database was considered relevant as a reference instrument in variety identification and as a source of information about thus far unexplored diversity that could be of interest in the development of new apple varieties.     

ITS sequence-based identification and utilization evaluation of <Emphasis Type="Italic">“</Emphasis>Nanjiang” <Emphasis Type="Italic">(Lonicera similis</Emphasis> Hemsl.), a local cultivar in Sichuan,China

The internal transcribed spacer (ITS) region was employed to analyze phylogeny of 20 populations, including the local cultivar “Nanjiang”, belonging to 7 species of Lonicera L. 20 ITS sequences that we obtained range from 614 to 618 bp, and the G+C content was higher than 60% with the average value 63.5%. The sequences of 5.8S nrDNA was highly conservative in both length and component, while ITS2 had more variation than ITS1 in sequence component and length. For the ITS complete sequence, the mutation and information sites were 139 and 117 with the content of 22.38 and 18.84%, respectively. The dendrogram analysed by neighbor-joining method showed that “Nanjiang” and Lonicera similis Hemsl. comprised a same cluster with a higher bootstrap support, indicating “Nanjiang” would be a variation or subspecies of L. similis Hemsl. The results of antibacterial activity test and active compounds identification showed that “Nanjiang” had the best antibacterial activity and the highest yield of chlorogenic acid, the active component of Flos Lonicerae, in the six species of Lonicera L.     

Regulation of macro,micro, and toxic element uptake by <Emphasis Type="Italic">Salix</Emphasis> × <Emphasis Type="Italic">smithiana</Emphasis> using liming of heavily contaminated soils

Purpose

Willow cultivation in soils heavily contaminated by risk elements is a challenging issue due to phytotoxic effects that restrict plant growth. Liming reduces the mobility of some risk elements in contaminated soils and therefore can be a suitable measure for contaminated soils but can also affect availability of nutrients for planted willows. We investigate how liming affects concentrations of macro, micro, and toxic elements in the organs of willows planted in contaminated soils.

Materials and methods

We established a 3-year pot experiment with Salix × smithiana planted in weakly acid and alkaline soils anthropogenically seriously contaminated by As, Cd, Pb, and Zn. Soils were both untreated and treated with two doses of lime and dolomite in the first year before planting. We determined biomass production, mortality, and the concentration of macro- and micronutrients and toxic elements in the willows’ aboveground organs.

Results and discussion

Lime application increased biomass production in both soils; dose of lime played an important role for its increase only in alkaline soil. Lime in a higher dose was incompatible with the vitality of just-planted willows in both soils. Doses of dolomite significantly affected the biomass production and mortality of willows, where lower doses caused a permanent decrease of biomass production and mortality in weakly acid soil. The toxicity of Cd and Zn in leaves was recorded in both untreated soils; the latent deficiency of P and deficiency of Fe in leaves was only recorded in weakly acid untreated soil.

Conclusions

Lime application irrespective of dose with foliar Fe application seemed to be the most suitable measure for increasing biomass production and decreasing toxic elements, especially Cd and Zn, without decreasing the macro- and micronutrients in the aboveground organs of willows in weakly acid soil. In alkaline soil, only higher doses of lime had a positive effect on the studied parameters. Dolomite application is not a suitable measure for planting willows in both contaminated soils. Dolomite in a lower dose impairs the growth of willows in weakly acid soil.

     

The introgression of 2× 1EBN <Emphasis Type="Italic">Solanum</Emphasis> species into the cultivated potato using <Emphasis Type="Italic">Solanum verrucosum</Emphasis> as a bridge

Wild diploid Solanum species with an endosperm balance number (EBN) of one are a rich source of disease resistance, pest resistance, and tuber quality traits. They are not sexually compatible with 4× 4EBN or 2× 2EBN forms of the cultivated potato. In this study, the wild 2× 2EBN Mexican species S. verrucosum (ver) was used as a bridge species to access 2× 1EBN germplasm. Hybrids (V1) were created between ver as a female parent and the 1EBN species S. cardiophyllum, S. chancayense, S. commersonii, and S. trifidum. Most of the V1 hybrids were successfully crossed as females to cultivated 2× 2EBN clones. However, due to stylar barriers, reciprocal crosses were unsuccessful. Additional studies are underway to determine the effectiveness of ver as a bridge to introgress specific traits from 2× 1EBN germplasm into the cultivated potato.     

Relationships in the <Emphasis Type="Italic">Diplotaxis–Erucastrum–Brassica</Emphasis> complex (Brassicaceae) evaluated from isoenzymatic profiles of the accessions as a whole. Applications for characterisation of phytogenetic resources preserved <Emphasis Type="Italic">ex situ</Emphasis>

Electrophoresis has become one of the most usual tools for plant studies. However, most analyses, using isoenzymes, have been carried out from a population geneticist’s point of view for the study of allelic frequencies. In present work, isoenzyme electrophoresis of bulked seeds was used as a rapid strategy for obtaining the electrophoretic profiles of accessions of a seed bank. The relationships amongst 30 taxa of the complex Diplotaxis-Erucastrum-Brassica (Brassicaceae family, tribe Brassicinae) were analysed, with the electrophoretic profiles of six isoenzyme systems. Each sample presented a stable and characteristic isoenzymatic pattern representative of the whole population. The enzyme markers provided 147 reliable bands which were scored as multistate traits (not with allelic value). UPGMA clustering method showed a dendrogram with three main groups and subgroups of samples that are in agreement with the recognised taxonomy of the taxa. This approach can be a rapid and efficient method to establish the specific enzyme patterns of conserved species populations of a germplasm bank, and the information could be included in the a database as part of documentation system.     

rDNA cytogenetics and some structural variability in an <Emphasis Type="Italic">Avena barbata</Emphasis> Pott ex Link × <Emphasis Type="Italic">A. sativa</Emphasis> subsp. <Emphasis Type="Italic">nuda</Emphasis> (L.) Gillet et Magne amphiploid after 5-azaC treatment

The paper reports on the study of DNA methylation patterns in chromosomes of an octoploid selection obtained from an amphiploid Avena barbata × Avena sativa ssp. nuda, their alteration and plant structural changes induced by 5-azacytidine (5-azaC) treatment. Subtelomere/telomere regions and some chromosomes remained heavily methylated after this practise. The increased concentration of 5-azaC strengthened the demethylation process. NOR rDNA loci on five and 5S rDNA doubled loci on three pairs of chromosomes were detected. Some 5S loci were colocalised with NORs. The number of active NORs was increased by one homologous pair after demethylation and this was related to minor loci. The NORs were mainly located on A/D genomes. Patterns of nucleoli fusion varied between main and lateral roots and demethylation increased the number of uninucleolar nuclei. Seed germination was inhibited and plant growth decreased under demethylation. 5-azaC also induced distinct anomalies in endosperm development. Extensive parts of the tissue were composed of defected cells or remained empty. Polyploidised clones of starchy or aleurone cells were observed, as were aleurone cells exhibiting new rare phenotypes.