ITEMS FROM HUNGARY

Agricultural Research Institute of the Hungarian Academy of Sciences

Martonvasar 2462, Hungary.

Z. Bedoe, L. Lang, L. Szunics, L. Balla, O. Veisz, I. Karsai, Lu. Szunics, and Gy. Vida.

The precipitation in autumn 1993 and the mild weather in winter and early spring had a beneficial effect on cereal development, but the microclimate in the strong, dense, and, in places, lush plant stands favoured intense attacks by certain pathogens. Thus, in 1994, susceptible wheat varieties were afflicted by a very severe nationwide infection of leaf rust (Puccinia recondita). There also was a severe epidemic of powdery mildew (Erysiphe graminis). In places, there was also damage by yellow rust (P. striiformis), septoria (Septoria sp.), and helminthosporium (Helminthosporium sp.), which rarely appear in Hungary.

Breeding.

Five new winter wheat varieties were registered in 1994. The origin of Mv Palma is `F797/Mv 08-82//Mv15.' It is an early variety, with good performance not only in Hungarian trials, but also in Slovenian, Czech, and Romanian trials. It is resistant to powdery mildew and stem rust and moderately resistant to leaf rust. It has category B1 flour quality. Mv Irma (pedigree: T16//Mv15/So6300/3/NSR2//Mv15/So6300) also is early maturing, with shorter stems than Mv Palma and soft grain. It is resistant to stem rust and moderately susceptible to powdery mildew and leaf rust. It was registered because of its great yield potential. Mv Vilma is a sister line of Mv Palma, maturing 2 days later and with better milling quality. Mv Emma is medium early (pedigree: Mv15/Mv 8//MvMA) and has the highest gluten content in the Hungarian variety list. It has a flour quality of A2-B1 and an average yield potential. Mv Szigma is a medium late variety (pedigree: Mv21-85/Mv15) created using anther culture. It has B1 flour quality, good frost resistance, good resistance to stem rust, and moderate resistance to leaf rust and powdery mildew.

Quality.

Wheats from BankFONT SIZE=2 FACE="WP MultinationalA Roman"dt, an East-Hungarian region, played an important role in Hungarian wheat production from the 1930s to the early 1960s. Their good flour quality was characterised mainly by a Brabender farinograph value of A, high gluten content, and generally a 2+12 subunit at the HMW glutenin Glu-D1 locus. The breeding of wheat varieties

with a similar quality serves to broaden the genetic basis for the selection of good flour quality. Our results show that breeding lines with good bread making quality can be selected not only from genotypes with 5+10 Glu-D1 subunits, but also from those with a 2+12 HMW glutenin composition. One example is line MvM 57-91 (registered under name Mv Emma), with similar bread making properties as the old BankFONT SIZE=2 FACE="WP MultinationalA Roman"dt varieties.

Disease.

In greenhouse experiments, 25 powdery mildew races were identified in 1994. The prevalent races and their frequencies were: 72 (30.48 %), 51 (24.28 %), 90 (12.38 %), 46 (7.14 %), and 77 (6.66 %). The number of virulence genes was 5.56. Very few races were virulent to genotypes possessing resistance genes Pm4a and Pm4b. In field experiments with artificial infection, satisfactory protection against leaf rust was provided by resistance genes Lr9, Lr19, Lr23, Lr24, Lr25, Lr27, Lr34, and Lr37 and against stem rust by Sr9b, Sr11, Sr21, Sr24, Sr31, Sr36, and Sr37.

Bunt can be found, to a greater or lesser extent, in all parts of the world where wheat is grown. Among the Tilletia species causing this disease, Tilletia foetida (Wallroth) Liro is the most widespread in Hungary. At present, it does not cause great damage, because seed treatment provides satisfactory protection against the pathogen. If seed treatment was not used, the damage might reach 30-40 %. In artificial infection experiments carried out in the nursery in 1992 and 1993, studies were made on the bunt infection of wheat varieties cultivated in Hungary and containing known resistance genes (Bt1 - Bt10). This was followed in 1993 and 1994 by tests on the winter hardiness and frost resistance, of healthy and bunt-infected wheat seedlings, in the field and in the frost-testing chamber of the phytotron. During the experimental period, the local pathogen population was avirulent to genes Bt8, Bt9, and Bt10 and virulent to Bt7. Varieties containing resistance genes Bt4, Bt5, and Bt6 had good resistance, whereas those varieties with Bt1, Bt2, and Bt3 were only moderately resistant. Among the cultivated varieties, only three proved to be moderately resistant, whereas the remainder were susceptible or very susceptible. The frost resistance of plants infected with bunt was significantly lower than that of healthy plants over the average of the varieties. Different varieties responded to various extents to infection. The degree of bunt infection was closely correlated with a reduction in the frost resistance of infected plants in 75 % of the varieties tested. In 25 % of the varieties, no correlation could be established between the 2-year infection figures and the survival percent of healthy versus diseased plants

Aluminum toxicity.

The effect of aluminum (Al) on the androgenic response of three F₂ population of different `wheat x triticale' crosses were tested in anther culture. Half of each population was used as a control, without in vivo aluminum pretreatment, and selections from the other half were made in a hematoxilin-based seedling test for aluminum tolerance. Only plants showing a satisfactory rate of root regrowth were retained for anther culture. Androgenic response of each population was tested on liquid MN6 media supplemented with 0.26 M maltose at pH 5.8, while for in vitro aluminum treatment, 400 FONT SIZE=2 FACE="WP MathA"FM Al was added to the medium with a pH of 4.2. Based on these results, both in vivo and in vitro treatments significantly influenced the anther culture response of the three F₂ populations. In vivo Al treatment in the seedling stage resulted in significantly higher embryo induction and a slightly higher rate of green plant regeneration, compared to all the control populations studied. On aluminum-containing induction media, embryoid induction dropped significantly, but the embryos exhibited an increase in the frequency to regenerate green plants. The vast majority of embryos obtained from aluminum-containing induction media regenerated green plants, not albinos.

ABA treatment.

ABA treatment increased the frost resistance of the wheat varieties Cheyenne, Martonvasari 14, and Chinese Spring to different extents when they were grown without cold hardening. A survival percent equivalent to that of the cold-hardened control plants was achieved without hardening, if the plants were treated with 20 mg/1 ABA 3 or 6 days

prior to freezing at -6 C. The ABA content in the crown, in terms of fresh weight, was twice that in the leaves even before cold hardening, and this tendency was observed throughout. In both plant organs, a fairly substantial accumulation of ABA occurred during the first 2 days of hardening, after which the concentration dropped and remained relatively constant from the 5th to the 20th day. For both leaves and the crown, the poorly frost-resistant Chinese Spring exhibited the smallest increase in ABA content. Endogenous changes in ABA were correlated only to the hardening level during the initial stages of hardening, and the quantity of endogenous ABA depended on the genetic background of the variety. These facts appear to indicate that ABA plays a role in the initial stages of frost-resistance development, after which its effect weakens.

Climatic change.

Experiments carried out in the phytotron have investigated the effects of an increasing atmospheric concentration of CO₂ and of temperature and rainfall on the hardening processes and overwintering in plants. The experiments were carried out on winter wheat varieties and triticale possessing different degrees of frost resistance and currently cultivated in central Europe. The development of a state of hardening in winter wheat varieties depended, to a great extent, on a gradual decrease in temperature. With one exception, the hardening process in all the varieties tested was favourably influenced by a doubling of atmospheric CO₂ content, so that a significantly larger number of plants survived the frost test (P = 5 %) than when plants of the same varieties were raised under normal conditions. A reduction in the freezing temperature and an increase in soil moisture content caused a slight reduction in the percent survival for varieties with excellent frost resistance and a large reduction in those with medium or poor frost resistance.

Predictions suggest that, in central Europe, as the result of global climatic changes, a reduction in the quantity of winter precipitation, a considerable rise in winter temperatures, and an increase in atmospheric CO₂ concentration will occur. Judging by these experimental results, these changes could improve the overwintering of winter cereals. However, at the same time, a number of factors leading to yield losses must be expected during the vegetative period.

Department of Cell Biology

Agricultural Research Institute of the Hungarian Academy of Sciences,

Martonvasar 2462, Hungary.

B. Barnabas, G. Kovacs, I. Szakacs, I. Takacs, M. Kovacs, and I. Timar.

In vitro fertilization.

During the last year, our research activity was concentrated in two very important fields, namely in vitro fertilization and genetic manipulation of haploid cultures in wheat and some related species. One of the main goals was to extend the earlier established egg cell isolation technique to various wheat genotypes and develop successful fusion and culture systems to obtain fertile in vitro-fertilized plants. According to our present results, the isolation of viable egg cells of several wheat genotypes has been achieved without enzymatic maceration of the ovules. 2,4-D applied to the stigmas at the time of emasculation resulted in soft ovule tissues that enhance disintegration upon mechanical manipulation. The isolated egg cells were viable for more than 2 hours after isolation from the various genotypes. The mean isolation frequency was increased to 40 %. Electrofusion-mediated fertilization of single egg cells of wheat with isolated, individually selected sperm cells was successful. On average, the fusion frequency was 30 %, but under optimal conditions, it could be as high as 55 %. Two days after electric fusion, 60 % of the fusion products started to divide, with 88 % of them forming multicellular structures and, in a few cases, microcalli. The culture of single

unfertilized egg cells, with or without the application of AC field and electric pulses, induced no division. The egg cells and fusion products were cultured in a maize feeder-cell system. Presently, the main question in this research is how we can regenerate fertile plants from the fusion products. For establishing a repetitive protocol, we are working with different feeder-cell systems, optimizing culture conditions, etc. This research is being done in collaboration with the laboratory of Dr. E. Kranz and is supported by a grant from the German-Hungarian Intergovernmental Cooperation for Scientific and Technological Development and by the Hungarian National Scientific Research Fund. We also are trying to extend this technique to barley and durum wheat.

Genetic manipulation of haploid cultures.

Another important question in manipulating reproductive processes is the synchronicity of male and female organ formation and gamete maturation in different wheat varieties and related species by applying ultrastructural techniques. Several species and genotypes were studied in semithin and ultrathin sections originating from different periods of sexual organ development, both in vivo and in vitro, to compare the developmental stages of male and female gametes. In some genotypes, the male gametophyte ripened earlier than the egg cell, whereas in others, the synchronicity was very high. Such knowledge can help in optimizing the efficiency of an in vitro fertilization procedure.

During the last few months, a new technique had been established to regenerate young (5-day-old) zygotic embryos. This work was done to obtain more information on the culture needs of naturally fertilized embryos and to apply this information to the culture conditions needed to obtain optimal IVF fusion products. The young zygotic embryos also serve as targets for gene transfer. Worldwide, young zygotic embryos seem to be the object of choice for particle bombardment. According to the generally applied procedure, scutellar tissue of immature zygotic embryos are the main target of bombardment with DNA-coated particles. After testing (or not) the frequency of transient expression, the embryos are transplanted onto a callus-induction medium. After the induction phase, the growing calli are grown on a selective medium, and only the transgenic calli are regenerated. According to this technique, several transgenic cell lines carrying the GUS and BASTA genes have been established in our laboratory. Some herbicide-resistant plants have been regenerated, but Southern analysis for the presence of foreign genes remains to be done. Using 5-day-old zygotic embryos, another transformation system also was established. The young embryos were bombarded immediately after isolation and germinated directly. This procedure is highly efficient. Without bombardment, 80 % of the isolated embryos developed into fertile plants. After bombardment, the frequency of normally developing plants decreased to 60 %. Some plants showed abnormal morphology, but most of the abnormal plants were fertile. The molecular studies are being done presently. We think that, with the application of direct embryo germination, we will be able to avoid the problems of callus induction and plant regeneration.

Continuing our previous studies on the application of haploid techniques, a highly reproducible cell-plant system has been established for wheat and several wild wheat species. Regenerable, embryogenic, haploid, cell suspensions were initiated from single microspore-originated calli of several wheat genotypes. Repeated callus and cell selections during the culture procedure led to stable haploid suspensions consisting of fine cell clusters each containing 20-50 cells. These suspensions were able to maintain their regenerative capacity during 1-3 years of subculture, and fertile plants have been regenerated frequently with a relatively high frequency. On the basis of this haploid cell suspension system, haploid protoplast culture also has been established. Although the isolation and culture of the haploid protoplast and plant regeneration were carried out successfully, only a few fertile plants were obtained. However, using a new culture protocol, a high frequency of haploid embryos has been induced, but their further development is still problematic.

Established, haploid cell suspensions are the other targets of genetic transformation via particle bombardment. Presently, we have some transgenic cell lines carrying foreign genes, such as GUS, hygromycin, and herbicide resistance. The frequency of stable transgenic cell lines, on the basis of cell clusters bombarded, is more than 30 %. Among them, six subcultured cell lines and 25 plantlets have been regenerated on selective media.

DH production.

For practical purposes, the established anther culture systems also are used for selection to aluminum tolerance. Based on the present results, aluminum toxicity can be induced artificially in anther culture. A 50 or 100 FONT SIZE=2 FACE="WP MathA"FM/l solution of aluminum, applied to the nutrient medium with low pH, considerably reduces callus induction. Higher concentrations entirely destroy the microspore population. On regeneration medium containing the same amount of aluminum, fertile plants can be regenerated. According to seedling test results of grains originating from fertile DH plants, an increase was observed in the variability of aluminum tolerance, even if the aluminum treatment was applied only in the induction phase. The results of the hematoxylin Al-resistance test suggest that resistant DH lines can be obtained by this procedure. The multiplication of progenies and the study of the inheritance of induced aluminum tolerance are now going on.

Utilization of the doubled-haploid method of breeding usually shortens the time to cultivar release, and the method of haploid production needs evaluation in a breeding programme. Several Triticum aestivum and T. durum genotypes were tested for anther culture response and some important lines were found to be recalcitrant. To get haploids from these nonresponsive genotypes, `wheat x maize' crosses were used by applying stored maize pollen. In our laboratory, a successful pollen storage technique has been developed. The combination of this method, with the `wheat x maize' haploid procedure, gives us a very promising technique to introduce recalcitrant genotypes for doubled haploid breeding programmes. When stored maize pollen was used for pollination, the average percentages of haploid embryo formation in `wheat x maize' crosses were 10.7 and 21.6, respectively, whereas, in the case of `durum x maize' crosses, approximately 10 % of the haploid embryos could be regenerated from embryo culture. All plants regenerated from `wheat x maize' crosses were haploids. We did not find any detrimental effect of our pollen storage procedure when compared with the application of fresh maize pollen, and the procedure seems to be more flexible, because we do not have to rely on the synchronization of flowering of the two different species.