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.     

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%.     

Regeneration of fertile doubled haploid plants from colchicine-supplemented media in wheat anther culture

The effect of colchicine added to induction medium for the production of fertile doubled haploid plants after in‐vitro anther culture was studied in wheat, Triticum aestivum L. For this, one winter and two spring wheat varieties were used. Anther cultures of the three genotypes were treated with 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine had no significant effect on anther response and embryoid production of the genotypes examined. However, in the winter wheat genotype ‘Mv Szigma’, colchicine caused a significant reduction in microspore‐derived structures. A significant decrease was also observed in plant regeneration ability of two genotypes (‘Vergina’ and ‘Acheloos’) after colchicine treatment. In addition, a significant reduction of the albinos produced was observed in all genotypes after olchicine treatment. In contrast, the regenerants obtained from the colchicine‐supplemented induction media produced significantly higher percentages of fertile plants in all genotypes. However, the level of fertility, was significantly different among the fertile plants obtained. This, together with the observation that in the case of the winter wheat variety the colchicine treatment resulted in 100% completely fertile plants with a high seed‐setting ability indicate that there is space for further improvement of the method when it is applied to spring cultivars. Finally, the increased number of seeds per 100 plated anthers obtained from all three genotypes after colchicine treatment, clearly demonstrates that the addition of colchicine to induction medium was superior to the conventional anther culture method and it could therefore be introduced into wheat breeding programmes.     

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).     

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 Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

The use of anther culture in breeding winter wheat. II. Production of new doubled haploid lines of winter wheat with 1AL—1RS-translocation A total of 4472 anhers was cultured from 8 F¹ populations of winter wheat with the 1AL—1RS wheat-rye translocation cultivar ‘Amigo’ as one of the crossing arents. When averaged over all populations a frequency of embruoid formation of 10% and of regeneration efficiency of green plants of 1% were observed. In addition to the 45 green regenerated plants, 93 albinos were obtained. 44% of the green plantlets had 21 chromosome in root tips and 29% were spontaneous diploids. Multiple peroxidases were used a biochemical markers in the subsequent characterization of the homogeneous breeding material. The electrophoretic patterns showed that 16 doubled haploids without rye chromosome segments were produced. In addition the features of dh₀- plants showed, that several of the new 1AL—1RS-translocation lines were awnless.     

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.     

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.     

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.     

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.     

Medium-Genotype-Interaction on Androgenetic Haploid Production in Wheat

The present study investigates the effects of media on the rate of green plantlet formation from anther cultures of five different winter and spring wheat cultivars. An increased number of embryos and calli were produced on Ficoll-containing liquid potato-2 medium, whereas the addition of 0.2 M/l maltose increased the rate of regeneration. The genotypes had strong effect on the formation of plantlets, but the developed procedure allows also the regeneration of recalcitrant genotypes. On the best medium sequence, a total of 2430 anthers over all genotypes were plated which developed 1325 macroscopic structures (55 %) of which 159 green plants have developed, i.e. 12 % from the structures and 7 % from the plated anthers, respectively.     

In vitro Selection for Tolerance to Fusarium in F1 Microspore Populations of Wheat

Microspore populations of eight Fhybrids of winter wheat (Triticum aestivum L.) whose parents had different levels of resistance to Fusarium were screened in vitro, using phytotoxins of Fusarium as biochemical probe. Two selection methods were compared for the in vitro selection: either embryoids and calli were first initiated from anthers in toxin-free medium and then grown on medium with 0.3—0.9 %Fusarium toxin; or anthers were immediately cultured in modified liquid potato-2 medium in the presence of 1.5, 2.0 and 2.5ml Fusarium toxin per liter culture medium, a concentration which reduced the number of calli and embryoids to about 10 % compared to the toxin-free controls. Microspores from donor hybrids which were produced from very susceptible cultivars were killed by lower toxin concentrations than micro-spores from hybrids of less susceptible parents. From surviving calli and embryoids, originally initiated from 242,000 anthers in both procedures a total of 375 green lines could be regenerated. The results indicate that it is possible to enrich the fraction of regenerating microspores by those which contain the gene complex responsible for reduced susceptibility to Fusarium by the use of a pathotoxin.     

Potential use of doubled haploid lines for the screening of resistance to yellow rust (Puccinia striiformis) in hexaploid wheat

Doubled haploid lines derived from anther culture of two Iranian spring wheat genotypes‘Ghods’susceptible and‘9106’resistant to yellow rust in Iranian field conditions, and their F₁ hybrids were used in this study. Seedlings of 36 doubled haploid lines, selected out of 96 according to their agronomic traits and the two parental genotypes were inoculated with eight races of yellow rust. The parental genotypes (‘Ghods’and‘9106’) were segregating for some of the races but their doubled haploid lines were either resistant or susceptible to them.‘Ghods’was susceptible to three of the races studied but three doubled haploid lines derived from it were resistant to them. Five selected doubled haploids from the‘9106’genotype and six from F₁ hybrid plants were resistant to all eight races tested. After further investigations in Iranian field conditions it was found that some of these lines can be used as donor genotypes for resistance to yellow rust in wheat breeding programmes. Use of these genotypes should be possible if the French yellow rust races used for selection also represent the dominant races in Iran. It can be concluded that anther culture provides an efficient method for fixing genes of resistance to yellow rust and desirable doubled haploids from F₁ plants can be derived.     

Large-scale production of wheat and triticale double haploids through the use of a single-anther culture method

A total of 257 parental wheat and 38 triticale lines were used for anther culture. On average, 2.1 green wheat haploids were obtained per spike. This response occurred irrespective of the origin of the material (Germany, France, Sweden or UK) and 5 years of testing. Triticale responded with 5.3 green haploids per spike. Using the criterion that one parental line should give at least one green haploid per spike in the screening experiment, green haploids were produced from 88 out of 91 F₁ wheat breeding combinations and from each of 21 F₁ and F₂ triticale breeding combinations. An average of 4.7 green plants were obtained per spike from the wheat production programme, while the triticale programme gave an average of 6.2 green plants per spike. A single medium supplemented with different hormones for anthers and embryos was used for culture of both crops.