Éva Szakács

Substitution analysis of callus induction and plant regeneration from anther culture in wheat (Triticum aestivum L.).

The genetic determination of callus induction, total plant regeneration and green plant regeneration from anther culture were studied using a “Chinese Spring”/“Cheyenne” substitution series. All the three characteristics were found to be polygenically determined, but their inheritance was independent from one another. The 7A and 18 chromosomes had a considerable effect on callus induction. In the case of total plant regeneration the most influential chromosome “as the 3A while the 2D chromosome showed a definite influence on green plant regeneration. The interaction between the genetic background of the recipient plant and the substituted chromosome plays an important role in the manifestation of the studied features.

In Vitro Androgenesis of Wheat from Fundamentals to Practical Application

Wheat anther cultures have a history of almost 30 years and are nowemployed efficiently in many countries of the world for the developmentof doubled haploid lines for breeding. The present paper discusses keyquestions of the elaboration and perfection of the method: cytologicalaspects of in vitro androgenesis, the conditions required for theembryogenic development of microspores and the applicability of anthercultures in the Martonvásár wheat breeding programme.

Molecular cytogenetic evaluation of chromosome instability in Triticum aestivum-Secale cereale disomic addition lines

The genetic stability of wheat/rye (‘Chinese Spring’/‘Imperial’) disomic addition lines was checked using the Feulgen method and fluorescent in situ hybridization (FISH). Feulgen staining detected varying proportions of disomic, monosomic, and telosomic plants among the progenies of the disomic addition lines. The greatest stability was observed for the 7R addition line, while the most unstable lines were those with 2R and 4R additions. Chromosome rearrangements were also detected using FISH. Based on the specific hybridization patterns of repetitive DNA probes pSc119.2 and (AAC)5, as well as ribosomal DNA probes (5S and 45S), isochromosomes were identified in the progenies of 1R and 4R addition lines. The results draw attention to the importance of continuous cytological checks on basic genetic materials by using FISH, because this method reveals chromosome rearrangements that could not be detected either with the conventional Feulgen staining technique or with molecular markers.

The effect of colchicine treatment on microspore division and microspore-derived embryo differentiation in wheat (Triticum aestivum L.) anther culture

SummaryThe relationship between division symmetry andin vitro microspore embryo genesis was studied using two winter wheat varieties of high embryogenic capacity. Anther cultures were treated with colchicine added to the induction media at concentrations of 0.01%, 0.02% and 0.04%. As a result of the colchicine treatment, the rate of symmetrical divisions increased significantly which was followed by a significant increase in the microspore-derived embryo frequency. The effect of colchicine was not dependent on the concentrations used. On the basis of this it can be supposed that there is a clear relationship between symmetric divisions and microspore-derived embryo differentiation.

Cytological aspects of in vitro androgenesis in wheat (Triticum aestivum L.) using fluorescent microscopy

SummaryTwo winter wheat genotypes (‘Diószegi 200’ and ‘Mv 15’) were compared for their in vitro androgenic capacity. On average, the induction frequency of embryogenic structures was 71.7% in ‘Diószegi 200’ and only 4.3% in ‘Mv 15’. The haploid induction ability of the two genotypes differed considerably, with ‘Diószegi 200’ being much higher. The difference in the in vitro inductability of the microspores may result from genetic differences which are manifested in the survival rate of the microspores during the culture period and their adaptability to in vitro conditions. Special DNA fluorochrornes were suitable for studying the different pathways of in vitro androgenesis. Our data indicate that the repeated equal divisions of the microspore nucleus might lead to pollen embryo formation, and subsequent divisions of the vegetative portion of the pollen grain after the first asymmetric microspore mitosis can result in pollen callus formation.

Characterization of a 5HS-7DS.7DL wheat-barley translocation line and physical mapping of the 7D chromosome using SSR markers

A spontaneous wheat-barley translocation line was previously detected in the progenies of the Mv9kr1 × ‘Igri’ wheat-barley hybrid and the translocation was identified as 5HS-7DS.7DL. Multicolor genomic in situ hybridization (mcGISH) with D and H genomic DNA probes and three-color fluorescence in situ hybridization (FISH) with repetitive DNA probes (Afa-family, pSc119.2, and pTa71) were performed to characterize the rearranged chromosome. The effect of 5HS and the deleted 7DS fragment on the morphological traits (plant height, fertility, yield, and spike characteristics) of wheat was assessed. Despite the non-compensating nature of the translocation, the plants showed good viability. The aim of the study was to physically localize SSR markers to the telomeric and subtelomeric regions of the 7DS chromosome arm. Of the 45 microsatellite markers analyzed, ten (Xbarc0184, Xwmc0506, Xgdm0130, Xgwm0735, Xgwm1258, Xgwm1123, Xgwm1250, Xgwm1055, Xgwm1220, and Xgwm0635) failed to amplify any 7DS-specific fragments, signaling the elimination of a short chromosome segment in the telomeric region. The breakpoint of the 5HS-7DS.7DL translocation appeared to be more distal than that of reported deletion lines, which provides a new physical landmark for future deletion mapping studies.

Production and Molecular Cytogenetic Identification of Wheat-Alien Hybrids and Introgression Lines

Barley, rye, Aegilops and Thinopyrum (syn. Agropyron) species belonging to the Triticeae tribe have large genetic diversity and serve as a valuable genetic reservoir for wheat improvement. Many of these species have been used for more than a century for the production of wheat × alien hybrids and introgression lines. The most up-to-date molecular cytogenetic techniques make it possible to detect and identify alien chromosomes in the wheat genome. The first methods used to identify rye, barley, Aegilops and Thinopyrum chromosomes in the wheat genome were C- and N-banding. Genomic in situ hybridization (GISH) is the most accurate way of detecting the translocation breakpoint in introgression lines. Alien chromosomes can be identified in the wheat genome using fluorescence in situ hybridization (FISH) with the help of repetitive DNA probes.Multicolor GISH (mcGISH) was developed to demonstrate the various genomes in polyploid plant species and in interspecific and intergeneric hybrids, amphiploids and derivatives. Sequential GISH and FISH are useful methods for identifying alien translocations in the wheat genome.

Development and identification of a 4HL.5DL wheat/barley centric fusion using GISH, FISH and SSR markers

The 4H(4D) wheat/barley substitution line was crossed with the ‘Chinese Spring’ ph1b mutant genotype in order to induce wheat-barley homoeologous recombinations. F3 and F4 seeds of the 4H(4D) × ‘Chinese Spring’ ph1b mutant cross were analysed using genomic in situ hybridization, and a Robertsonian translocation was detected in monosomic form. Disomic centric fusions were selected among the self-fertilized progenies. The presence of the long arm of 4H was confirmed with SSR markers. The long arm of the 5D wheat chromosome in the Robertsonian translocation was identified using fluorescent in situ hybridization with the help of three DNA probes: pSc119.2, Afa family and pTa71. The wheat/barley centric fusion was identified as a 4HL.5DL translocation. This line exhibited supernumerary spikelet character, but the number of seeds/plant did not increase. The 4HL.5DL centric fusion line is suitable genetic material to study the expression of genes located on 4HL in a wheat genetic background.

Induction of chromosome rearrangements in a 4H(4D) wheat-barley substitution using a wheat line containing a Ph suppressor gene

The aim of the experiments was to develop translocation lines by inducing homoeologous chromosome pairing in a 4H(4D) wheat-barley substitution line previously developed in Martonvasar. It was hoped to incorporate various segments of the barley 4H chromosome from the 4H(4D) substitution into wheat. Observations were made on the frequency with which wheat-barley translocations appeared in the F 2 progeny grains from a cross between the line CO4-1, which carries the Ph suppressor gene from Aegilops speltoides and thus induces a high level of homoeologous chromosome pairing, and the 4H(4D) wheat-barley substitution line, and on which chromosome segments were involved in the translocations. The translocations were identified by means of genomic in situ hybridisation. Of the 117 plants examined, three (2.4 %) were found to contain translocations. A total of four translocations were observed, as one plant contained two different translocations. The translocations consisted of one centric fusion, two dicentric t...

Analysis of chromosomal polymorphism in barley (Hordeum vulgare L. ssp. vulgare) and between H. vulgare and H. chilense using three-color fluorescence in situ hybridization (FISH)

The aim of the present work was to study chromosomal polymorphism within cultivated barley (Hordeum vulgare ssp. vulgare) using three-color fluorescence in situ hybridization (FISH). The physical distribution of the most frequently used, highly repetitive DNA sequences (GAA)7 specific for pericentromeric heterochromatic regions, the ribosomal DNA clone pTa71, specific for the 45S rDNA, and the barley-specific telomere-associated sequence HvT01, was investigated to reveal genetic diversity in metaphase spreads of ten barley genotypes with diverse geographical origin, growth habit and row number. A wild relative of barley, Hordeum chilense was also studied in order to compare the polymorphism between and within Hordeum species. Significant differences in the hybridization patterns of all three DNA probes could be detected between the two related species, but only probes pTa71 and HvT01 showed variation in the intensity and/or position of hybridization sites among genotypes of H. vulgare ssp. vulgare. The extent of polymorphism was less than that earlier reported for molecular markers and was restricted to the long chromosome arms, with differences between the chromosomes. 1H and 3H proved to be the most variable chromosomes and 4H and 6H the most conserved.