Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

Anther culture in the breeding process of winter wheat. I. Ability of 1B—-1R wheat-rye translocation forms for androgenesis 45 winter wheat varieties or F₁ hybrids, F₂ populations and lines with 1B—1R wheat-rye translocation were tested for their anther culture ability. A total of 48058 anthers was cultured on Potato-2 medium. When averaged over all genotypes and the two experimental years frequencies of embryoid formation of 5.4—6.8 per 100 anthers were observed. Plant regeneration efficiency from embryoids ranged from 5.3—9.1 % or a mean of 4—5 green plants per 1000 anthers plated. The results confirmed the preferential regeneration frequency of gametes with the 1BL—1RS chromosome compared to the gametes with the 1BL—IBS chromosome. Multiple peroxidase were used as marker. The effect of cold pretreatment or of media on the androgenetic response and productivity was not important. On the contrary the variability between the anther response from single ears of the same genotype was noticeable. Examples are presented for the transferability of the androgenetic ability to breeding material. Most green plants obtained were haploid or spontaneous doubled haploid. By cloning it was guaranteed, that progenies were obtained from most of the haploids after colchicine treatment.     

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

Anther culture in the breeding process of winter wheat. III. Ability of winter wheat F₁ populations with the two heterozygous 1AL–IAS/1AL–IRS and 1BL–1BS/1BL–IRS chromosome pairs Application of anther culture to four F₁ hybrids between the IBL–IRS (‘Amigo’) and several 1BL–IRS wheat-rye translocation forms yielded 129 green pollen plants in an average embryo induction frequency of 17.6 %. A total of 2632 anthers was inoculated. 25 % and 42 % of the regenerated plants were haploid and spontaneously doubled haploid, and 33 % had abnormal chromosomal structure. After chromosome doubling treatment 87% of all pollen plants set seeds. By means of multiple peroxidases and Giemsa C-banding patterns, the anther culture progeny could be further classified into 16 plants without the short arm of IR-chromosome of rye, 21 IAL–IRS and 50 1BL–IRS translocation lines and into 16 IAL–IRS, IBL–IRS double translocation lines according to the four possible characteristic types of F₂ gametes of the tested F₁ hybrids. Advantages of the haploid technique for the selection of desirable traits and the meaning of the IRS genes in wheat are discussed.     

Multiple Endopeptidasen als biochemische Marker für die Resistenz von Winterweizen gegenüber Pseudocercosporella herpotrichoides (Fron) Deighton

Multiple endopeptidases as biochemical marker for resistance of winter wheat to Pseudocercosporella herpotnchoides (Fron) Deighton. Electrophoretic patterns of primary leaf endopeptidases in breeding material derived from crosses between different winter wheat genotypes and amphidiploids (Triticum turgidum×Aegilops ventricosa) were compared with those of cultivars susceptible to Pseudocercosporella herpotrichoides. The results indicate that the multiple endopeptidase EP-1 coded by Wheat chromosome 7 D is absent in the international known lines ‘VPM 1’ and ‘Roazon’ and in all 24 winter wheat selections with increased resistance to P. herpotrichoides. A close relationship between the absence of EP-1 and the introduction of Aegilops ventricosa resistance is assumed. The use of this biochemical marker in wheat breeding is proposed.     

Characterization of wheat-triticale doubled haploid lines by cytological and biochemical markers

Five wheat-triticale doubled haploid (DH) lines— M08, V209, DH220-14-2, DH696-3-4 and M16 —derived from anther culture of F₁s resulting from crosses involving hexaploid or octoploid triticale × hexaploid wheat, were characterized by cytological and biochemical markers. Cytological evidence from genomic in situ hybridization and C-banding indicated that DH lines M08 and V209 (2n= 42) each contained a pair of 1BL/1RS translocation chromosomes. DH220-14-2 (2n= 42) was also a translocated line with two pairs of chromosomes containing small fragments of rye. One of the translocation fragments carried the Sec-1R gene originating from the satellite region of 1RS; the origin of the other one remains unknown. DH696-3-4 (2n= 42) contained a 3D(3R) substitution. In M16 (2n= 44), three pairs of rye chromosomes, 3R, 4R and 6R, were present, 4R as an addition and 3D(3R) and 6D(6R) as substitutions. Biochemical, isozyme and storage protein markers confirmed the cytological conclusions. The advantages of transferring alien chromosomes or chromosome fragments into wheat and creating alien aneuploid lines by anther culture of hybrid F¹s are discussed.     

Investigations on the Anther Culturability of Four German Spring Wheat Cultivars and the Influence of Light on Regeneration of Green vs. Albino Plants

Homozygous doubled-haploid plantlets derived from anther culture of wheat (Triticum aestivum L.) and triticale (×Triticosecale Wittmack) are useful breeding materials. However, efficiency of an-drogenesis needs improvement. We used media (basic components, are the same as 85DI2) each containing one of the seven auxins [2,4,5-trichlorophenoxyacetic acid (2,4,5–T), P-chloraphenoxyacetic acid (pCPA), 3,6-dichloro-o-anisic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-butrytic acid (IBA), indole-3-acctic acid (IAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) as a control] in combination with 6-furturyl-aminopurine (kinetin). In addition, each of the four cytokinins [6-benzylaminopurine (6-HA), 2-isopenlylnyl adenine (2-ip), 6-(4-hydroxy-3-meihylbut-2-enylamino) purinc (zeatin), and kinetin as a control] was tested in combination with 1-naphthalene acetic acid (NAA). Anthers containing microsporcs at miduninucleatc stage from live wheat cultivars (Angus, Centurk, Chris, K.itt, and Pavon 76) and two octoploid trilicale lines (T81, T82) were tested mainly for callus induction and polyhaploid production on each of the 11 media. The cultivar × medium interaction was not significant, When averaged over all growth regulators, Pavon was (he best cultivar which produced 14.4 % calli and 23 % polyhaploid plantlets. Averaged over all cultivars, the medium containing 2, 4-U produced the highest calli (13.9 %). Undifferentiated calli were regenerated on 87T1 medium, which contained IAA (1 mg/1) and kinetin (2 mg/1).     

Ertragsstruktur alter und neuer Winterweizensorten unter dem Aspekt der Einlagerung und Remobilisation von Fruktanen im Weizenhalm

Yield components in old and new german winter wheat varieties with respect to the storage and remobilization of fructan in the wheat stem
Yield components and changes in carbohydrate content of four old german winter wheat varieties, bred before 1950, were compared with six new varieties over two years in field trials. In both years the old varieties built up more biomass than the new varieties. In the latter a better harvest-index (0,56) as compared to the old varieties (0,43) was obtained, resulting in higher grain yields for the new varieties. Changes in water-soluble carbohydrate (WSC) content were mainly due to variations in fructan content. Maximum of fructan content was mostly reached at the stage of early milk ripeness. In the new varieties a faster and more complete remobilization of fructan occurred; especially in the year with the short grainfilling period.     

Genetic Variation for Anther Callus Inducibility in Crosses of Highly Culturable Winter Wheats

Heritability studies suggest that variation among genotypes for callus inducibility should respond to selection. The objective of this study was to determine if crosses among parents with high callus initiation frequencies would produce segregation from which genotypes with improved callus inducibility could be selected. The winter wheat (Triticum aestivum L.) cultivars‘Centurk 78’, ‘Norstar’ and ‘Norwin’ were chosen as parents, based on their high callus initiation frequencies and diverse parentage. F₂ populations from crosses between each of these parents were evaluated for callus initiation frequencies and callus vigor as determined by growth rate and healthy appearance. Heterosis was detected for callus initiation in the‘Norwin’בNorstar’ cross and for callus vigor in all crosses. Broad sense heritability estimates of 0.25 and 0.37 for callus initiation in the ‘Centurk 78’בNorstar’ and ‘Norwin’בNorstar’ crosses, respectively, suggest this trait can be improved by selection. Heritability estimates for callus vigor were less promising, ranging from 0.11 to 0.24.     

Die Ertragsbildung von Dinkel (Triticum spelta L.) und Weizen (Triticum aestivum L.) unter verschiedenen Umweltbedingungen im Freiland

Dry Matter Production of Spelt ( Triticum spelta L.) and Wheat ( Triticum aestivum L.) under Different Environmental Conditions in the Field
The primary aim of our work was to obtain information regarding the germination characteristics of spelt under different environmental conditions in field experiments, and to trace possible differences between spelt and wheat, with regard to crop production. For the present study two spelt varieties ( Ostro/Rouquin) and two wheat varieties ( Arina/Probus) were used. In addition the spelt cultivars Altgold, Oberkulmer and Hercule were included in some of the experiments. To obtain information on the influence of the glumes, spelt was planted in the hulled (SP +) and dehulled (SP-) form.
Field emergence of spelt SP- was up to 40 % lower than that of SP+, Under water logged soil, the percentage of germination was higher for spelt SP+ than for wheat.
Spelt produced more tillers per plant than wheat. However, tiller reduction was also higher for spelt so finally both species had similar numbers of ears per plant. Plants with a high tiller production might be more flexible in response to unfavourable growing conditions.
Grain yield of spelt and wheat was developed in different ways. Spelt produced fewer but larger kernels per ear, whereas wheat produced more but smaller kernels. In 1986 grain yield was up to 20 % higher in wheat than in spelt. In 1987, an unfavourable year for cereal crop production in marginal areas, grain yield was higher for spelt than for wheat. For the farmer, the "husked" form of spelt is economically important. This was equal or higher than the grain yield of wheat in both years.
The high germination rate under unfavourable conditions, in combination with a high tillering capacity and the production of large kernels appear to be the major reason for the stable yielding ability of spelt.     

Gliadins as biochemical markers for Aegilops turcomanica genes in wheat lines

An Aegilops turcomanica-typical gliadin was discovered in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of ethanol-soluble seed proteins from wheat lines Tr. 476, 482 and 492, which had been derived from a direct cross of Ae. turcomanica and Triticum aestivum and which revealed powdery mildew resistance due to a putative Ae. turcomanica-introgression. This Ae. turcomanica-derived gliadin was tested for its suitability as biochemical marker. For this purpose, doubled-haploid lines were produced via anther culture from crosses of Tr. 482 and Tr. 492 with actual winter wheat cultivars and breeding lines. Until now, 173 lines with Ae. turcomanica-gene(s) have been selected from 297 doubled haploid wheat lines.     

Effect of parental genotypes and colchicine treatment on the androgenic response of wheat F1 hybrids

The effect of the parental genotypes and colchicine treatment on the androgenic response of wheat (Triticum aestivum L.) F1 hybrids was studied. For this, anthers from three F1 hybrids and their parents were cultured on W14 initiation medium and W14 supplemented with 0.03% colchicine. The number of responding anthers, microspore‐derived structures/100 anthers, green plants/embryos cultured, green plants/100 anthers and albino plants/100 anthers were recorded. It was observed that embryo formation and plant regeneration ability were genetically controlled and genotype dependent. In both treatments the variety Kavkaz had a significantly higher percentage of responding anthers, microspore‐derived structures and green plants/100 anthers than the other genotypes. On the other hand, the variety Myconos also demonstrated high microspore‐derived structure production and green plant regeneration when treated with colchicine. The good response observed in these two varieties indicates the importance of colchicine treatment only for certain genotypes. Green plant production capacity of the hybrids was intermediate to that of the parental varieties. As one parent with a high or even an intermediate response to anther culture could lead to the production of sufficient (for breeding purposes) green plants from the F₁ hybrids, it was concluded that screening the inbred lines for the response to anther culture with and without colchicine treatment could contribute to utilization of breeding material with a low response to anther culture via the proper hybrid combinations.     

Androgenesis in Hexaploid Spring Wheat F2 Populations and Their Parents Using a Multiple-Step Regeneration System

The main goals of the wheat androgenesis experiment were to study the main phenomena of in vitro androgenesis in anther culture of F₂ populations (10) and their parents (6), to compare the usage of P-4 and C-17 media for the formation of embryo/callus and to demonstrate a new plant regeneration system. The P-4 induction medium was found to be significantly better than the C-17 in the number of responsive anthers (RA) and calli induced (CI) at the 1 % and 0.1 % level, respectively. Genotypic effect was evident in both RA and CI. The yields of F₂ populations in RA and CI were significantly higher than those of their parents regarding both media. The data confirmed the existence of heterosis for RA and CI in F₂ populations. The ratio of green/albino plant regeneration was more favourable in the C-17 derived embryo/calli than in the P-4 derived ones. The frequency of plantlet regeneration was enhanced in the group of unresponsive calli by the application of the multiple-step regeneration system. In this system the calli lacking well developed morphogenic structure were transferred to a new regeneration medium, containing a higher concentration of the same cytokinin, other cytokinin or basic medium, before the occurrence of irreversible changes in their physiology.     

The Response of Anther Culture in a Genetically Wide Material of Winter Wheat (Triticum aestivum L.)

In a screening experiment with 215 different winter cultivars of Triticum aestivum L. for response in anther culture, pollen embryos or callus were obtained from 200 different cultivars and green plants from 93 different cultivars. On average, from the whole material, 1.3 green plants were obtained per 100 anthers cultured. Variance components estimated from replicates with selected parts of the plant material indicated that for the formation of pollen embryos from anthers, interactions between genotypes and replications were dominating, accounting for 45 to 50 per cent of the variation. Main effects from genotypes were less prominent, accounting for 25 to 30 per cent of the variation, and the replications showed relatively little effect, accounting, for about 4 to 12 per cent of total variation. Regeneration of plants from pollen embryos or callus was not significantly influenced by either genotype or replicates. The frequencies of plants regenerated being green, however, were influenced from both genotype and environments contributing 42.5 and 34.9 per cent of the total variation respectively, while only minor interactions between genotype and environments were indicated for this character.     

Aneuploid Analysis of Anther Culture Response in Wheat

Genetic stocks of Triticum aestivum including the disomic, 8 ditelosomic and 3 nullisomic-tetrasomic ‘Chinese Spring’ wheat lines were employed to ascertain the chromosomal arm locations of the genes acting on the production of embryos from micro-spores and on the regeneration capacity (green and albina) of the microspore-derived embryos. All these aneuploid lines differed significantly from the parental line ‘Chinese Spring’ for embryo production. Our results confirmed or in most cases established that genes affecting embryo production are located on several chromosomal arms: IBS, 1BL, 3AS, 3AL, 5AS, SAL, 5BS, 5BL, 7DS, 7DL. Whereas most of the chromosomal arms stimulate the production of embryos from the microspores, IBS and 1BL reduce it. The results of plant production from microspore-derived embryos suggest that the genes increasing regeneration ability are located on CS5A chromosome and are likely associated to a gene increasing green plant frequency. On the contrary, the 1BL arm increases the albina frequency.     

Studies of the Genetic Relationship between Anther Culture and Somatic Tissue Culture Abilities in Wheat

A population of thirty-eight doubled haploid lines, developed from the F₁ between two wheat parents differing in anther culture and somatic tissue culture responses, ‘was used to examine the genetical control of responses to these in vitro systems. During anther culture genetic variation between lines was exhibited for frequencies of callus induction., embryo production and embryo regeneration rates. In addition the relative frequencies of green and albino plants was shown to be genotype dependent. However, there was no correlation, between the frequencies of embryo production and the regeneration rate of those embryos suggesting an independent genetic control of these two components. Transgressive segregation for performance was observed for all components indicating that at least two genes are involved in the response of each, and lines for improved performance, combining high ernoryo production rates and good regeneration capacity were identified. No genetic variation for frequencies of callus induction from immature embryos was observed in this cross. However, genetic variation for the regeneration frequencies of plants was observed. Lines with an improved tissue culture response over the two parents were identified. There was no correlation between the performance of lines in anther culture and somatic tissue culture, indicating separate genetical control, and lines with alternative levels of response to the two systems were identified.     

Efficient application of in vitro anther culture for different European winter wheat (Triticum aestivum L.) breeding programmes

The efficiency of our anther culture protocol was tested with high‐ and low‐responding genotypes, ‘Svilena’ and ‘Berengar’, and 93 F₁ winter wheat crosses in 2010 and 2011. Based on data for these genotypes, the effect of genotype influenced the number of embryo‐like structures, regenerated plantlets and green plantlets, while the number of albino plantlets was affected by genotype, year and environmental factors. Although genotype also influenced the production of green plantlets from breeding crosses, with green plantlets per 100 anthers ranging from 0.04 to 28.67, the average regeneration rate over all crosses was 5.3 green plantlets/100 anthers, which resulted in a total of 11 416 well‐rooted green plantlets. The survival rate of green plantlets following acclimatization was 97.21% in 2010 and 96.34% in 2011. In this study, the phenomenon of albinism and genotype dependency did not hinder the production of more than five thousand green plantlets each year. In our experiments, anther culture proved to be an efficient method in winter wheat breeding programmes with lower costs than alternative technologies.     

Spike pretreatments, anther culture conditions, and anther culture response of 17 German varieties of spring wheat (Triticum aestivum L.)

The aim of this work was to establish an in vitro regeneration system from anther cultures of different German varieties of spring wheat (Triticum aestivum L.). Using ‘Nandu’ the most widely grown spring wheat cultivar in Germany, different culture conditions were investigated with regard to their influence on anther culture response. The best results were obtained when applying a cold pretreatment to the donor spikes and using the synthetic L3 induction medium, liquid or solidified with gelrite. The highest rates obtained in these experiments with ‘Nandu’ were 8.6% responding anthers, 22.3% embryoid induction, 15.3% albino regeneration and 5.5% green plant regeneration (all rates related to the number of cultured anthers). Of the ‘Nandu’ plants analysed, 51.1% were haploid and 44.3% were diploid, probably as a consequence of spontaneous chromosome doubling. When screening a further 16 commercial German varieties of spring wheat, 10 exhibited good anther culture response and four of these (‘Eta’‘Jondolar’, ‘Mieka’, and ‘Star’) proved to be highly responsive, reaching embryoid induction rates between 4.3 and 10.3% and rates of green plant regeneration between 5.4 and 10.7%.     

Anther and isolated microspore culture response of wheat lines from northwestern and eastern Europe

Hexaploid wheat genotypes from north-western Europe show low responses to current anther culture techniques. This phenomenon was investigated on 145 north-western European wheat lines. Twenty-seven lines from eastern Europe were included to observe the response pattern of wheat from an area, where the technique has been used successfully. On average, eastern European wheat lines produced 3.6 green plants per 111 anthers, while only 1.4 green plants per 111 anthers were obtained in north-western European lines. This difference was due to the high capacity for embryo formation among the eastern European lines, while the ability to regenerate green plants was widespread in both germplasm groups. Isolated wheat microspore culture performed on 85 of these wheat lines gave an average 3.7-fold increase in green plants per anther compared with the anther culture response. The increased recovery of green plants was due to improved plant regeneration and increased green plant percentage from embryos derived from isolated microspore culture.     

Single and combined effects of colchicine, L-proline and post-inoculation low temperature on anther culture of wheat, Triticum aestivum L.

This investigation was undertaken to study the effects of colchicine, L-proline and a post-inoculation cold temperature of 14°C for 7 days, either as a single or as combined treatments, on the parameters of anther culture of the DH83Z118.32 wheat genotype. Results indicate that the addition of 100 mg/1 colchicine to the induction medium for a period of 3 days and at an incubation temperature of 28°C yielded the highest success index. This treatment, although it significantly reduced embryogenesis, improved embryoid quality so that not only the total regeneration, but also the percentage of green plantlets increased significantly, In addition, it raised the percentage of doubled haploids and, consequently, led to a significantly better final success index. Combining the colchicine treatment with a reduced post-inoculation temperature did not intensify its beneficial effect, although a treatment of reduced temperature alone was superior to normal temperature (28°C) for most parameters studied. The beneficial effects of adding L-proline (200 mg/1) to the induction medium, combined with a low temperature (10°C for 4 days) treatment, were diminished when this treatment was combined with a colchicine treatment.     

Agronomic Performance of Wheat Doubled-Haploid Lines Derived from Cultivars by Anther Culture

To determine the level of gametoclonal variation among doubled-haploid lines (DHLs) of Triticum aestivum L. developed using anther culture techniques and its effect on agronomic performance, 70 anther culture-derived DHLs of ‘Kitt’ were compared for 7 agronomic traits to 50 single-seed descent-derived lines (SSDLs) of ‘Kitt’ and to the cultivar ‘Kitt’. In a second experiment, 26 DHLs of ‘Chris’ were compared for 7 agronomic traits to 29 SSDLs of ‘Chris’ and to the cultivar ‘Chris’. Each experiment was grown as a randomized complete block design with three replications in three environments. For ‘Kitt’, the DHLs averaged significantly lower grain yields than the comparable SSDLs. For ‘Chris’, the DHLs averaged lower, but not significantly lower, grain yield than the SSDLs. In both ‘Kitt’ and ‘Chris’, the genetic component of variance for yield of the DHLs was significantly larger than that of the SSDLs indicating the presence of gametoclonal variation. The lower average grain yield of the DHLs was explained by a larger group of low-yielding DHLs than was present in the SSDLs. Six ‘Kitt’ DHLs and 3 ‘Chris’ DHLs were lower yielding than the lowest yielding SSDL, respectively. Elite DHLs and SSDLs were similar for mean grain yield performance. Though the DHLs and SSDLs were significantly different for some yield components, the affected yield component changed with the cultivar and there was no consistent effect. Significant genotype × environment interactions were detected for some traits which were caused by changes in the magnitude of differences, rather than reversals in ranking, indicating that low yielding DHLs could be culled on the basis of visual selection or single-environment testing. Hence, gametoclonal variation was induced by the anther culture techniques used in this study, tended to be deleterious for grain yield, and was sensitive to the growing environment. However, as the DHLs and SSDLs had similar expected population means based upon expected gains from selection, this gametoclonal variation should not be a major hindrance to wheat breeding.