AGRICULTURAL RESEARCH INSTITUTE OF THE HUNGARIAN ACADEMY OF SCIENCES
Martonvásár, H-2462, Brunsvik str. 2, Hungary.
Wheat breeding.
Z. Bedö, L. Szunics, L. Láng, Lu. Szunics, O. Veisz, I. Karsai, Gy. Vida, P. Szücs, A. Juhasz, M. Gal, Sz. Bencze, M. Megyeri, K. Puskás, and Cs. Horváth.
Wheat season. Due to the early appearance of rain in the autumn of 1998, only about two-thirds of the winter wheat growing area in Hungary were sown at the proper time. During the vegetative period, attacks by various pathogens further reduced the yield. In 1999, substantial local damage was caused in several parts of the country by Helminthosporium tan spot and Septoria leaf blotch. Leaf rust infection was observed extremely early in the southern counties of Hungary in late April, and on fields where susceptible varieties were grown or no chemical control was applied, considerable yield losses were incurred. In a nursery in Martonvásár that was not artificially inoculated or protected with fungicides, severe natural infection was detected during the later phases of the vegetative period, causing intense sporulation even on some varieties previously resistant to leaf rust.
Breeding. One medium-maturing, Martonvásár-bred, winter wheat variety was registered in 1999. optimal gluten strength. The water uptake of the flour is the highest among the three blending Martonvásár varieties (Mv Csárdás, Mv Emma, and Mv Magdalena), which is important to the bakery industry. The pharinograph value of this new variety was in between those of Mv Emma and Mv Magdalena. All other quality parameters are excellent or good. The yielding ability of Mv Csárdás is medium, but it had a 7.5 % higher yield level than Mv Emma, averaged over the last 2 years. The average yield of Mv Csárdás was close to 6 t/ha.
Along with this new registration, 24 Martonvásár-bred winter wheat varieties having a market share of over 50 % on the National List. As a result of the newly registered lines, seed distribution of the Martonvásár varieties increased further in the autumn of 1999. The greatest demand was for Mv Magdalena and Mv Magvas, but Mv Emma, Mv Pálma, and Mv Vilma also belong to the group of varieties grown in the largest area.
Winter durum wheat breeding. The new, winter durum wheat variety Martondur 3 was state registered in 1999. The frost resistance of Martondur 3 approaches that of moderately frost-resistant winter wheats and is much better than that of winter barleys. On the basis of phytotron freezing tests, the survival percentages averaged over 3 years were 80.6 % at -13°C and 69.1 % at -15°C, far exceeding that of the winter durum wheats currently cultivated. Martondur 3 has excellent vitreousness, protein and wet-gluten content, and its yellow-pigment content meets the standard criteria.
Examination of storage proteins with the SE-HPLC technique. The SE-HPLC technique is used widely for quantifying the storage proteins of wheat. Based on the results of this technique, not only the HMW-glutenin composition but also the distribution in size and quantity of the different polymers are indicated to have a significant effect on the dough properties. Determining the amounts of individual protein fractions and their ratios is a more precise tool for interpreting the good bread-making quality of the old Hungarian wheat variety Bánkúti 1201. In coöperation with CSIRO (Ferenc Békés, CSIRO Plant Industry, Australia), SE-HPLC was used to separate the storage proteins of the Bánkúti lines based on their size. Quantities of the monomeric proteins (gliadin and albumin + globulin fraction) and polymeric proteins (glutenins) and the insoluble polymeric protein fraction (UPP) were established. The ratios of fractions such as HMW to LMW and soluble polymer protein to UPP were calculated. The amount of single HMW-glutenin subunits in the lines was determined as well. Moderate levels of HMW glutenin ranging between 31.4 % and 46.3 % and different HMW:LMW proportions (0.46-0.86) were measured, which are in contrast to reports in the literature that high HMW:LMW ratios are characteristic of high quality wheat varieties. The unusually high level of UPP, ranging between 35.8 and 62.1 % of the total polymeric fraction, can be one explanation for the high-gluten quality of the Bánkúti 1201 wheat lines. The SE-HPLC data confirm our earlier electrophoretic results concerning the high genetic variability in the Bánkúti 1201 population. Identification of the different genotypes and their application as a germ plasm can be an important contribution to breeding programs.
Rheological properties of wheat varieties with different endosperm composition. Ten SRWW and 10 HRWW varieties were involved in a 3-year experiment (1997-99) to characterize their rheological properties (Brabender Fharinograph, 10-gram Mixograph, and Chopin Alveograph); gluten and protein contents (Inframatic 8611); and kernel hardness (Perten SKCS 4100). Twelve mixograph parameters were selected in order to maintain a good representation of dough properties. Tail value at the eighth minute (H) gave the best correlation to the farinographic quality number (ESZ) and to the planimetric area (PT). Water absorption had good correlation to all the mixograph values (0.65-0.85). From the alveograph parameters, S and W gave the best correlation with H, with the area under the curve (T), and with the ESZ. The protein content (F) had strong correlation to all the mixograph values, the P value, and hardness. Endosperm composition had significant effect on almost every parameter with the exception of G, L, PT, gluten spread, and kernel weight. Among the years, significant differences only occurred in wet-gluten content, protein content, PT, and kernel weight. The interaction of the two factors influenced the PT. Hard wheat varieties had significantly higher F, VF, S, P, W, H, and T in all 3 years than the soft wheats. In the case of G, L (in 1998 and 1999), gluten spread (in 1996 and 1999), and PT (in 1997 and 1999), lower values were measured in hard wheats.
Disease studies. In the artificially inoculated nursery, leaf rust appeared early on spreader plants, but due to a brief spell of dry, warm weather the development of secondary infections slowed. Nevertheless, the selection pressure was sufficient for the selection of resistant lines. The data indicate that the leaf rust resistance genes Lr9, Lr19, Lr24, Lr25, Lr27, Lr28, Lr29, Lr35, Lr38, and Lr44 provided satisfactory resistance to the Hungarian rust population in 1999. Within the framework of the Winter Wheat Nursery for Adult Plant Resistance to Leaf Rust set up as part of the COST 817 Action, four Martonvásár varieties (Martonvásári 17, Mv Magvas, Mv Pálma, and Mv Magdaléna) and one line (Mv 04-96) were tested in nine European countries.
Stem rust has not caused an epidemic in Hungary for several decades, chiefly because of the excellent resistance of the cultivated varieties. Because there is a potential danger for the breakdown of resistance, new sources are constantly tested and identified in an artificially inoculated nursery. The stem rust resistance genes Sr31 and Sr36, present in the majority of Hungarian winter wheat varieties, provide excellent resistance to the present rust population, and satisfactory, although not complete, protection also is provided by genes Sr7a, Sr9b, Sr11, Sr24, Sr27, and SrGT.
Powdery mildew appeared early in the fields, but sporulation was soon retarded, and an epidemic that might have caused severe economic losses did not develop. During virulence tests on greenhouse seedlings, 19 races were identified from 108 isolates using the test collection recommended by Nover. The dominant races and their frequencies were as follows: 90 (22.23 %), 51 (13.89 %), 72 (12.97 %), 59 (10.19 %), and 70 (9.26 %). Apart from the resistance gene Pm4a (Khapli), none of the known major resistance genes present in the experiments provided complete protection against the pathogen. Below-average virulence was found on differentials with Pm1+2+9 (22.22 %), Pm2+Mld (23.15 %), Pm4b+ (33.33 %), and Pm4a+ (37.04 %).
Abiotic stress resistance studies. One of the main components of yield reliability in winter cereals is winter hardiness. In winter hardiness tests made in the field over a period of nearly 20 years, the lack of snow cover has caused considerable frost damage in 14 of the years. In all the experimental years, a significant difference was observed in the overwintering survival of varieties with poor and good frost resistance grown in boxes placed on the ground, and differences were only found between varieties with moderate or better frost resistance in 10 of the years. The success of these field winter hardiness tests depended to a great extent on the weather.
The average overwintering survival of winter durum wheats in boxes placed on the ground was significantly less than that of the T. aestivum wheat variety Bánkúti 1201, which has moderate winter hardiness. However, because of breeding for winter hardiness, the majority of varieties and advanced lines introduced or bred in Martonvásár gave results equivalent to or better than those of Bánkúti 1201, even under extreme conditions. The winter hardiness of winter oat varieties was significantly less than that of Bánkúti 1201 at the 0.1 % level.
The results of phytotron experiments indicated that the genes or gene complexes responsible for frost resistance became active during different phases of hardening and improved the frost resistance of the recipient variety to different extents and at different rates. The functioning of these genes is influenced substantially by environmental factors. Genes on chromosomes 5A and 5D responded well to enhanced atmospheric CO2 concentration, significantly increasing the frost resistance of the recipient varieties. Frost resistance also was improved by these genes at normal CO2 concentrations. The frost resistance of the Chinese Spring/Cheyenne substitution lines had a very close, positive correlation with the fructose and glucose contents in plants grown at both normal (350 ppm) and double (700 ppm) atmospheric CO2 levels.
Frost tolerance studies in winter durum wheat. Changes in freezing resistance during the hardening of nine durum wheat cultivars were evaluated in a phytotron in Martonvásár. Hardening was at a constant temperature of +2°C for 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days, after which the plants from all the treatments were frozen at -13.5°C. Considerable varietal differences in the hardening kinetics were recorded for the durum genotypes investigated. The freezing resistance of the genotypes was negligible after 0-20 days of hardening. The maximum degree of freezing resistance was reached after 30-50 days of hardening. A considerable decrease in freezing resistance was observed in plants kept at +2°C for 60 days or more. Judging by the results of these and earlier experiments, T. turgidum subsp. durum genotypes achieve a lower level of hardening on average, which generally has a shorter duration than that observed in winter T. aestivum subsp. aestivum cultivars.
Dynamics of winter hardiness in Triticum turgidum subsp. durum genotypes. The winter hardiness of seven T. turgidum subsp. durum genotypes of different origin and with different degrees of frost resistance was evaluated under field conditions between 1995 and 1999. In order to trace changes in winter hardiness over time, the number of surviving plants was counted in early December, January, February, March, and April. With the exception of 1995-96, temperatures in the years examined were not critical for wheat. As a consequence, the T. turgidum subsp. durum varieties, which are more sensitive to frost, overwintered without any great loss. The results of experiments in the Martonvásár phytotron and the overwintering data for 1995-96 indicate that the mean level of hardiness of T. turgidum subsp. durum genotypes is generally lower and lasts for a shorter time than that of the winter T. aestivum subsp. aestivum varieties grown in Hungary. Of the four winters examined, a close positive correlation between field winter hardiness dynamics and the dynamics of frost resistance in the phytotron only was obtained under the meteorological conditions of 1995-96. These results confirm that if selection for frost resistance and winter hardiness is to be successful, the evaluation of frost resistance in T. turgidum subsp. durum wheat varieties and other genotypes should be made under controlled conditions in a phytotron.
Genetic diversity in durum wheat. A diversity analysis
was made using RFLP and RAPD markers to reveal differencesamong
T. turgidum subsp. durum genotypes. In RAPD analyses,
23 winter T. turgidum subsp. durum genotypes of
different origins were examined (using three T. aestivum
subsp. aestivum and three T. monococcum subsp. monococcum
varieties as reference genotypes). Five of these also were tested
using the RFLP method. Polymorphisms were demonstrated among the
five winter T. turgidum subsp. durum genotypes by
13 of the 47 RFLP probe-restriction endonuclease combinations
(28 %) and by eight of the 16 RAPD primers (50 %). In the course
of the divergence analysis of the 23 T. turgidum subsp. durum
genotypes using the RAPD method, 87.5 % of the 16 primers revealed
polymorphisms. In this case, the value of the JD coefficient was
between 0.03 and 0.74. The JD coefficients closely correlated
to the groupings suggested by the pedigrees. In the present experiments,
polymorphisms were demonstrated successfully among T. turgidum
subsp. durum genotypes with a relatively narrow genetic
basis using RFLP probes and RAPD primers. These results can be
utilized in the design of breeding programs, help in the development
of T. turgidum subsp. durum crosses aimed at mapping
DNA markers linked to various agronomic traits, and also draw
attention to the restricted genetic basis of the cultivated T.
turgidum subsp. durum genotypes.
B. Barnabás, E. Paldi, G. Kovács, I. Takács, T. Janda, G. Szalai, Z. Pónya, and I. Tímár.
Genetic transformation of egg cells and zygotes of wheat via microinjection. An expeditious and highly efficient technique of microinjection has been developed with the aim of introducing exogenous DNA into egg cells and zygotes of wheat. Using a mechanical-dissection method and a novel immobilization approach enabled us to microinject approximately 15 egg cells of wheat per hour. Exposing the protoplasts to a high-frequency, alternating-current field for immobilization, achieved a significantly higher transient expression rate of the injected genes (46 % and 52 % for egg cells and zygotes, respectively) than has been reported previously for plant protoplasts. We do not know if this high transformation efficiency is because of the high frequency electrical field applied for immobilizing the protoplasts. The transformation rate appeared to depend upon the time of egg-cell isolation. Observations of the ultrastructure seem to reflect a variation in competence of the egg cells during in situ development. In order to conduct studies directed towards establishing the optimal time window for DNA delivery into the fertilized egg cell, the time course of DNA dynamics during zygotic development has been quantified via quantitative microspectrofluorometry.
Morphological characterization of wheat egg-cell protoplasts. The morphological features and fine structure of wheat egg cell protoplasts isolated from premature (3 days prior to anthesis) and over-aged (12 days after anthesis) caryopses were compared. Except for shape, the young egg-cell protoplast showed the same morphological characteristics as the egg cell in plants at the time of anthesis. Young and adult egg-cell protoplasts were spherical in shape. Polarity could not be identified exactly with the methods used. During aging, the egg cell increased considerably in volume. The adult egg showed the typical features (membrane blebbing, autophagous vacuoles) of programmed cell death. After a long life-span (about 18 days), cells of the female gametophyte appear to undergo apoptosis.
Diploid pollen production in Triticum turgidum subsp. carthlicum. The well-known antimitogenic alkaloid colchicine has long been used for the reduplication of the genomes of animal and plant cells. In the present experiments, spikelet cultures of the wild wheat species T. turgidum subsp. carthlicum, which has relatively high in vitro fertility, were exposed to colchicine treatment at various stages of development. The colchicine treatment drastically reduced the number of fertile pollen grains and percentage seed set depending on the developmental stage of the cultures.
Cytological analyses on the offspring revealed a triploid karyotype (3n = 42), indicating the fusion of gametes with different chromosome numbers. The doubled gamete was very probably the sperm cell. Based on the preliminary results, the colchicine treatment of floret cultures would appear to be an efficient alternative method in polyploid research.
J. Sutka, G. Galiba, M. Molnár-Láng, G. Kocsy, G. Kovács, G. Linc, A. Vájújfalvi, E.D. Nagy, J. Jakab, and A. Bálint.
Genetic study of glutathione accumulation. The effect of cold hardening on the accumulation of glutathione (GSH) and its precursors was studied in the shoots and roots of wheat varieties Cheyenne (CNN, frost-tolerant) and Chinese Spring (CS, moderately frost-sensitive), in a T. aestivum subsp. spelta accession (TSPL, frost sensitive) and in chromosome substitution lines DS CS-CNN 5A and CS-TSPL 5A. Fast induction of total glutathione accumulation was detected during the first 3 days of hardening in the shoots, especially in the frost-tolerant CNN and DS CS-CNN 5A lines. This observation was corroborated by the study of de novo GSH synthesis using [35S]-sulphate. In CNN and the DS CS-CNN 5A, the total cysteine, g-glutamylcysteine (precursors of GSH), hydroxymethylglutathione, and GSH contents were greater during the whole 51-day treatment than in the sensitive genotypes. After 35 days of hardening, when the maximum frost tolerance was observed, a greater ratio of reduced and oxidized hydroxymethylglutathione and glutathione was detected in CNN and DS CS-CNN 5A compared to the sensitive genotypes. Correspondingly, a greater glutathione reductase (EC 1.6.4.2) activity also was found in CNN and DS CS-CNN 5A. Chromosome 5A of wheat is assumed to have an influence on GSH accumulation and the ratio of reduced to oxidized glutathione as part of a complex regulatory function during hardening. Consequently, GSH may contribute to the enhancement of frost tolerance in wheat.
Frost hardiness. The effect of cold hardening on the dynamics of frost tolerance and carbohydrate metabolism was studied in the frost-sensitive Chinese Spring and the frost-tolerant Cheyenne genotypes and in some of the chromosome substitution lines derived from crosses between Cheyenne and Chinese Spring. Total water-soluble carbohydrate, glucose, fructose, sucrose, and fructan contents were measured in the leaves.
Differences in the accumulation of carbohydrates associated with cold tolerance occurred early in response to low temperature. Total water-soluble carbohydrates and total fructan content increased continuously during the cold treatment in all genotypes, resulting in higher contents in tolerant genotypes than in sensitive ones. Their rate of accumulation correlated significantly with the frost tolerance after 19 days of cold treatment. During cold acclimation, the maximum accumulation of fructose preceeded that of sucrose. Significant correlation was detected between fructose and sucrose content and frost hardiness on the 43rd day of cold treatment. Fructose accumulated to a greater extent in the most tolerant genotypes with a sharp peak on the 35th day of cold hardening, followed by a decrease. In the chromosome substitution lines, considerable sucrose accumulation started after the 11th day with a maximum on the 43rd day of cold hardening, coinciding with the tolerance test.
T1B·1R translocations. An octoploid triticale developed between the common wheat line Martonvásári 9 kr1 and the rye cultivar Lovászpatonai was crossed with the T1B·1R wheat variety Matador. The frequency of chromosome associations involving the T1B·1R translocation was examined at metaphase I of meiosis in BC1 plants using GISH. Most of the chromosome bonds were rod bivalents (67 %) and trivalents (26.6 %); one quadrivalent also was observed (1.2 %). The 1RS arm of the translocation chromosome paired with that of Lovászpatonai rye in only 32 % of the PMCs, whereas the translocated 1BL arm exhibited much higher pairing (90 %) with its wheat homoeologues.
Winter wheat-winter barley hybrids. New winter wheat-winter barley hybrids were produced in the following combinations: Mv9 kr1/Igri, Mv9 kr1/Osnova, and Asakazekomugi/Manas. The hybrids were multiplied in tissue culture because of a high degree of sterility. The regenerated hybrids were pollinated with wheat to produce backcross progenies. The selection of disomic additions among the selfed progenies of the BC2F1 plants from the Mv9 kr1/Igri combination is now in progress.
A meiotic analysis was made using the Feulgen method and GISH on hybrids from the crosses Mv9 kr1/Igri and Asakazekomugi/Manas and in their in vitro regenerated progenies. The number of chiasmata was 1.59/cell in both of the initial hybrids. The number of bivalents increased after in vitro culture, and the number of chiasmata increased to 4.72 and 2.67, in the Mv9 kr1/Igri and Asakazekomugi/Manas regenerated hybrid combinations, respectively. According to the GISH analysis, wheat-barley pairing made up 3.6 % of the total pairing in the initial Mv9 kr1/Igri hybrid and 6.6 % and 16.5 % of the total pairing in in vitro regenerated progenies of the Asakazekomugi/Manas and Mv9 kr1/Igri hybrids, respectively. The demonstration by GISH of wheat-barley chromosome arm associations in the hybrids and especially in their in vitro regenerated progenies shows that recombinants could be between these two genera, and, thus, transfer useful characters from barley into wheat.
Phenotypic stability. The genetic properties of different
types of stability parameters for individual genotypes were investigated
using an eight-parent, half-diallel cross in a randomized complete
block design with three replications. Data pertaining to the parents
and F1s were subjected to Griffing and Hayman methods for the
genetic analysis of eight types of stability statistics. The results
of analysis of variance showed that genetic variation exists for
almost all the stability parameters. Moderate heritability estimates
were observed for coefficient of variation and superiority measure.
The estimates of variance components suggested that genes controlling
environmental variance, regression coefficient, ecovalence, and
superiority measure are predominantly additive, although nonadditive
gene action was predominant for coefficient of variation, stability
variance, and coefficient of determination. The results of combining
ability effects indicated that the best general combiners for
the improvement of adaptation were Chinese Spring and Shakha,
and the best stable specific combination was Shakha/Kobomugi.