'Florin': A Doubled Haploid Wheat Variety Developed by the Anther Culture Method

The cross from which the doubled haploid wheat variety ‘Florin’ is derived was made in 1978. As early as October 1985, after two years of official tests for registration, ‘Florin’ was licensed. The major advantage of anther culture is the lime saved in comparison with conventional pedigree breeding. Much research remains to be done to understand the anther culture system and to increase the efficiency of producing doubled haploids.     

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.     

Field Performance of Androgenetic Doubled Haploid Spring Wheat Lines in Comparison with Lines Selected by the Pedigree System

A total of 316 doubled haploid lines (DH) of spring wheat were compared with 621 lines selected in a pedigree system (PS) under field conditions in a breeding nursery. The lines originated from 21 crosses and the samples tested represent mean values for variables comprising the main breeding goals such as disease resistance, baking quality, and agronomic traits. In general, the DH lines were later in days to heading and shorter than PS lines of different homozygosity levels. Except for leaf rust (Puccinia recondite), the DHs were more resistant to the artificially infected diseases such as powdery mildew (Erysiphe graminis), stripe rust (Puccinia striiformis), and septoria nodorum blotch (Septoria nodorum). These differences, which were relatively small for practical selection purposes, were probably due to easier and more precise disease assessment of the homozygous DH lines. For quality characters, the DHs had a higher protein level, while the rest of the parameters were similar for both origins. The data analyzed suggest so far that androgentic doubled haploids in spring wheat are very similar to lines selected in a pedigree system in respect to all the agronomic characters tested. However, the DH lines were produced in a much shorter period of time. It is suggested that androgenetic doubled haploids be produced from F₁ hybrids and that the well-established bulk method should continue to allow selection bulk method should continue to allow selection for rare recombinants as soon as homozygosity is reached.     

Production of Doubled Haploids by Anther Culture and Wheat X Maize Method in a Wheat Breeding Programme

Utilization of the doubled haploid method of breeding usually shortens the time to cultivar release, and methods of haploid production need evaluation in a breeding programme. Thirty-eight different three-way crosses were tested for anther culture response. On average 5.8 percent of the anthers cultured produced calli. Three crosses were found recalcitrant for callus induction. Overall, the anther culture method produced 0.6 plantlet per 100 anthers cultured. Five crosses with an average of 5.8 and 2.8 percent of anthers producing calli and plantlets, respectively, were compared using anther culture and wheat × maize crosses. Non-responsive genotypes for callus induction and plantlet formation in the anther culture method proved to be good parental material in wheat × maize crosses. The average percentages of embryo formation and plantlet production in wheat × maize crosses were 10.3 and 4.7, respectively. Anther-derived plants were cytologically unstable, whereas all the plants regenerated from wheat × maize crosses were haploids (n = 21 chromosomes). The chromosome numbers of the polyhaploids were doubled with a colchicine treatment. Improvement of the two haploid production methods to facilitate their efficient use in a breeding programme is discussed.     

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.     

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.     

Variation in Doubled Haploid Plants of Wheat Obtained through Wheat (Triticum aestivum) × Maize (Zea mays) Crosses

Variation was investigated in 110 doubled haploid (DH) lines of wheat derived from wheat × maize crosses. Field observation revealed visible variations in 15 DH2 lines such as extreme dwarfism, low seed fertility, alteration of spike type and stripes. Six agronomic traits, i.e., heading date, spike number/ plant, culm length, spike length, seed fertility and grain weight were statistically analyzed in the DH2 and DH3 generations. Out of the 88 DH2 lines/DH3 groups, 26 %/64 % showed significant differences from the parental variety in the means of one or more traits. Ranges of the DH3 lines were larger than those of the DH2 lines, except for spike number/plant. Furthermore, analyses of variance within and between DH lines showed the presence of heterogeneity/heterozygosity in the DH2 lines/plants. These results indicated the occurrence of gametoclonal variation in the DH lines. It is considered that most of the variations detected were due to the colchicine treatment rather than to the 2,4-D treatment or in vitro culture.     

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.     

The Effect of Sugars and Growth Regulators on Embryoid Formation and Plant Regeneration from Barley Anther Culture

The influence of carbon source and growth regulator composition in induction medium on anther culture response was investigated using spring barley genotypes. Anthers were cultured on BAC3, Ficoll-containing medium, supplemented with one of the following carbohydrates: sucrose, maltose, cellobiosc and melibiose (6 % w/v). The use of either maltose or cellobiose resulted in a significantly higher anther response, calli and/or embryoid production and green plant regeneration compared to the incubation of anthers on a medium containing sucrose. Contrary to these results, the replacement of sucrose by melibiose in BAO medium, drastically reduced the efficiency of anther culture. As an average for the three genotypes tested, the frequency of green plants per 100 anthers plated was 9- to 22-fold higher on medium supplemented with sucrose or cellobiose than on medium containing melibiose as a sole carbohydrate. Among the growth regulators tested, the combination of auxin NAA (2 mg/l) and cytokinin BAP (1 mg/1) performed much better than the employment of auxin 2,4-D combined either with zeatin riboside or BAP as cytokinins. The beneficial effect of medium supplemented with NAA and BAP was associated with better embryoid formation compared to the other growth regulator combinations tested. The hormone-free combination gave a similar anther response and production of calli as any medium supplemented with growth regulators, but the regeneration capacity of calli produced on hormone-free medium was much lower, resulting in the drastic reduction of the number of both total and green regenerants.     

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.     

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.     

Estimation of the Response to Anther Culturing in 60 Genotypes of Different Wheat Species

Sixty genotypes of different wheat species (30 cultivars of common wheat, 12 durum wheat and 18 wild wheat relatives) were estimated for response to anther culturing and capability of plant regeneration. The 4 level of responding anthers out of the total number of anthers cultured was proposed as the criterion for genotype fitness for breeding with the aid of anther culture. Among common wheat cultivars, 13 exceeded this level. Among durum wheat, none of the genotypes investigated exceeded the 4% level. Among other species, 5 cultivars of T. dicoccum as well as T. petropavlovsky and T. dicoccoides spont. exceeded the 4 value.     

The Effect of Gelling Agents on Wheat Anther and Immature Embryo Culture

This study was conducted to determine whether inexpensive starches and various agars could replace Ficoll, which is very expensive, and Bacto-agar, which may be inhibitory, in wheat (Triticum aestivum L.) anther and immature embryo culture. Anthers from Pavon 76’ had significantly greater embryoid-initiation frequencies (number of embryoids per anther) on PI media with corn (0.67) and wheat starch (1.50) than with Ficoll (0.47). Anthers on 85D12 media with Ficoll, corn starch and wheat starch had similar embryoid-initiation frequencies (0.51, 0.50, and 0.52, respectively). The embryoid-regeneration frequencies (number of regenerating embryoids per embryoid plated) on 85D12 media containing corn (0.32) and wheat (0.36) starch were significantly higher than on 85D12 with Bacto-agar (0.14). Embryoids on MS media with Bacto-agar, corn starch, and wheat starch had similar embryoid-regenerating frequencies (0.15, 0.20, 0.22, respectively), which were greater than with some of the other agars tested (0.05 to 0.17). When wheat starch replaced Bacto-agar in immature embryo cultures, the frequency of responding embryos was unchanged, but the frequency of green plants regenerated increased an average of fourfold for two cultivars.     

Influence of Sucrose and Melibiose on Barley Anther Cultures in Starch Media

Anthers from the barley varieties ‘Arra’, ‘Dissa’ and ‘Ingrid’ were cultured in barley starch gelatinized nutrient media. The importance of osmosis in barley starch medium was studied by using sucrose or an inert carbohydrate, melibiose, as an osmoticum. It was found that in the barley starch medium sucrose was not necessary but energy and carbon could be obtained by tissue enzymatically from the starch. Together with the starch medium melibiose had revolutionary effects OB barley anther cultures. This system not only produced extremely high numbers of embryoids and green plantlets but also drastically reduced the number of albinos.     

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.     

Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus

Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process.     

小麦花药培养的基因型效应及优良基因型筛选

为进一步提高小麦花药培养的效率,从而加快花培育种的进程,对140份不同基因型的小麦亲本及其杂种F1和F3代进行花药培养,并对各世代不同基因型小麦的花药培养力进行研究。结果表明：小麦不同基因型及亲本的花药愈伤组织诱导率在2.89%~41.25%之间,平均为3.55%,绿苗分化率在0%~70.00%之间,平均为33.05%;114份不同基因型小麦F1代杂种材料中,有107份材料能够诱导出愈伤组织,愈伤组织诱导率最低为0%,最高为76.25%,平均为11.59%,绿苗分化率在0%~160.00%之间,平均为29.20%;F3代材料均能诱导出愈伤组织,愈伤诱导率在4.58%~80.83%之间,平均为16.39%,绿苗分化率在0%~71.43%之间,平均为34.99%。因此,不同基因型小麦材料的愈伤组织诱导率、绿苗分化率和绿苗产率在各材料间差异很大。依据高培养力组合亲本组配的规律,筛选到可作为桥梁亲本的材料（品种）有：‘宁春4号’、Jinghong No.2、‘鉴63’、Chuanmai 18、Synthderiv.29589、S111、D-27、‘晋匽746-9’和K6-39等。     

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.     

Relative Efficiency of Anther Culture and Chromosome Elimination Techniques for Haploid Induction in Triticale × Wheat and Triticale × Triticale Hybrids

Summary The study was undertaken to evaluate the relative efficiency of anther culture and chromosome elimination (by crosses with maize) techniques of haploid induction in intergenotypic triticale and triticale × wheat hybrids. For this, 15 triticale × wheat and 8 triticale × triticale F₁ hybrids were subjected to anther culture and were also simultaneously crossed with the `Madgran Local' genotype of maize (Zea mays L.) to induce haploids through the chromosome elimination technique. The haploid embryo formation frequency through the chromosome elimination technique was significantly higher in both, triticale × wheat (20.4%) and triticale × triticale (17.0%) F₁ genotypes, as compared to the calli induction frequencies through anther culture (1.6 and 1.4%, respectively). Further, four triticale × wheat and three triticale × triticale F₁ genotypes failed to respond to anther culture, whereas, all the F₁ genotypes formed sufficient number of haploid embryos through the chromosome elimination technique with no recovery of albino plantlets. The haploid plantlet regeneration frequencies were also significantly higher through the latter technique in both triticale × wheat (42.7%) and triticale × triticale (49.4%) F₁s as compared to anther culture (8.2 and 4.0%, respectively), where the efficiency was drastically reduced by several constraints like, high genotypic specificity, low regeneration frequency and albinism. The overall success rates of obtaining doubled haploids per 100 pollinated florets/anthers cultured were also significantly higher through the chromosome elimination technique (1.1% in triticale × wheat and 1.5% in triticale × triticale hybrids), proving it to be a highly efficient and economically more viable technique of haploid induction as compared to anther culture, where the success rates were only 0.2% and 0.1%, respectively.     

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.