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.     

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.     

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.     

Comparative analysis of in vitro anther- and isolated microspore culture in hexaploid Triticale (X Triticosecale Wittmack) for androgenic parameters

Two haploid induction media (190-0 and W14mi) were tested in isolated microspore culture of two triticale (X Triticosecale Wittmack) genotypes. The W14mi medium proved superior for the production of green plantlets in both genotypes. This basic medium (W14) was used to compare two doubled haploid production methods (isolated microspore culture and anther culture) with the same genotypes. The induction of androgenesis was more effective in isolated microspore culture than in anther culture. The number of embryo-like structures was 9.2 times higher in microspore culture (511.0/100 anthers) compared to anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher (anther culture—20.15/100 anthers; isolated microspore culture—67.6/100 anthers). However, the regenerant plantlets from isolated microspore culture were mainly albinos while predominantly green plantlets were regenerated from anther culture. The production of green plantlets from anther culture (16.8/100 anthers) was 2.9 times higher than from isolated microspore culture (5.8/100 anthers). The efficiency of anther culture was tested with eight winter triticale genotypes. The phenomenon of albinism did not hinder the green plant production in anther culture. Mean green plantlet production was 10.87/100 anthers. This value was two times higher than the number of albinos (5.01/100 anthers) and higher than previously published reports. The anther culture protocol described in this study is an efficient tool for the production of microspore-derived green plantlets in triticale.     

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 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.     

Diploid, tetraploid, and octoploid plants from anther culture of tetraploid orchard grass, Dactylis glomerata L.

This report describes a study of androgenesis in Dactylis glomerata, where the main aim was to find anther culture-responsive clones. Two types of media and two sugars were compared for their effectiveness in anther culture induction and subsequent plantlet production. Embryo formation from the cultured anthers was obtained from 28 of the 108 cloned genotypes using two different substrates, R₂M and FW. Both induction media supported the formation of embryos from the cultured anthers, but around 4.5 times more embryos (0.81 embryos per 100 anthers) were obtained with R₂M compared with FW, and R₂M also gave 5.5 times more green plants (0.054 green plants per 100 anthers) than the FW substrate. In the investigation of a carbohydrate source, responsive clones from the genotype study were tested using maltose as a substitute for sucrose in R₂M. Using maltose instead of sucrose increased embryo formation so that 133 embryos per 100 anthers were obtained compared with 7.1 embryos per 100 anthers obtained with sucrose. The total number of green plants obtained was also improved with maltose compared with sucrose, resulting in 66.3 and 1.9 green plants per 100 cultured anthers, respectively.     

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.     

Anther Culture in Rye: Improved Plant Regeneration Using Modified MS-Media

The use of androgenetic doubled haploids in rye breeding is still limited by low regeneration rates. In this study we tested the influence of two modified MS-media on the number of reacting anthers and on plant regeneration. Picloram and 2,4-D were used as auxin components in the induction media. The highest induction rates (reacting anthers per 100 cultivated anthers) and the highest regeneration rates (number of green plants per 100 cultivated anthers) were reached on a modified MS-medium containing Picloram as auxin source, regardless of the donor plant material. The comparison of the results obtained with the single cross ‘SC 35’ which contains genes of Secale vavilovii and the true Secale cereale donor forms clearly show the genotypic influence of donor plant material on induction and regeneration ability.     

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.     

固体培养基上浸润培养提高花粉植株诱导率的研究

在30×170mm的试管内,倒入25～30ml加琼脂固化的C_(17)培养基,接种冬小麦花药后注入2ml左右不加琼脂而浓度相同的C_(17)培养液浸润培养,冬小麦花药愈伤组织诱导率可从固体培养基的13.94％提高到29.95％,愈伤组织的绿苗分化率还比固体培养基提高10.2％,分化绿苗丛数占接种花药的10.94％,是固体培养的2.37倍。     

Anther-Culture ability in Secale cereale L.

Success in anther culture of rye (Secale cereale L.) has mainly been restricted to genotypes whose genome is partly determined by S. vavilovii Grossh. The aim of this study was to identify true S. cereale genotypes with anther-culture ability. The plant material used included the semi-wild standard genotype SC35, six single crosses of the ‘Petkus’ and 10 of the ‘Carsten’ gene pool, plus a double cross between SC35 and one of the ‘Carsten’ single crosses. Three anther-culture experiments were performed. Several single crosses within the ‘Carsten’ gene pool produced green plants. The ‘Petkus’ single crosses tested did not show anther-culture ability. As expected, the induction and regeneration rates of SC35 were higher than those of all other genotypes tested. The performance of the double cross was intermediate between its parents. The results demonstrate for the first time that anther culture in rye is a valuable tool in plant breeding. The data also show the possibility of transferring anther-culture ability from high- to low-responding genotypes.     

Rye doubled haploids as a research and breeding tool – a practical point of view

Doubled haploid (DH) plants were produced using anther culture from out‐crossing rye, including breeders’ lines, cultivars and F₁ plants with DH parents, to examine the feasibility of using the DH technique for breeding and specifically for developing mapping populations. Only 10–36% of green regenerants produced via anther culture were suitable for research or breeding purposes because of low survival rate or low fertility. Spontaneously arising DH regenerants were more often fertile compared with the colchicine‐treated ones. The fertility of spontaneous DHs varied from sterile to half that found in a normal rye population, which has implications for the design of a crossing scheme and subsequent anther culture. In the reciprocal crosses within one DH population, fertility was the lowest observed, probably because of self‐incompatibility factors, whereas in the DH crosses with normal heterozygous cultivars fertility was the highest. Two mapping populations using DHs were established, the first for out‐crossing rye it would seem. These populations will be used for mapping two important traits, the semi‐dwarf growth habit and preharvest sprouting resistance in rye.     

Anther Culture Derived Homozygous Lines in Hordeum bulbosum

The production of homozygous doubled haploids via anther culture was investigated in the self-incompatible diploid species Hordeum bulbosum. Anthers from three accessions (GBC77, GBC752 and GBC753) were cultured on FHG media using IAA or three levels (1, 50 and 100 mg/l) of the auxin phenylacetic acid (PAA). Four green plants and 63 albino plants were obtained from a total of 1620 plated anthers (540/accession). The best mean anther response, number of embryogenic calli and regenerated plants were obtained with 50 mg/1 PAA. Three of the four green plants survived to maturity and, based on root-tip squashes stained with feulgen, all had 14 chromosomes like the anther donor parent. These anther culture-derived plants contained only some of the parental DNA bands, as observed by PCR analysis, indicating that they are of gametic origin rather than arising from somatic parental tissue. This is the first report of homozygous lines produced from this self-incompatible species.     

Anther culture response of wild Oryza species

Thirteen different wild species of the genus Oryza were investigated for their response to anther culture and plant regeneration. Callus induction from microspores of anthers was found to be strongly dependent on the species. Although large numbers of anthers from wild Oryza species, including O. barthii, O. latifolia, O. australiensis, O. minuta, O. nivara, O. paraguagensis and O. eichingeri, were cultured, no calli could be obtained. However, calli were produced from anthers of O. punctata, O. perennis, O. alta, O. ridleyi and O. rufipogon, although the frequency of callus induction was different. Similar species-dependence was observed in plant regeneration from microspore-derived calli. In total, 62 plants were derived from anther culture, including 47 albino and 15 green plants (of which 26.7% were haploids) from O. perennis; for the first time, six albino plants were obtained from O. ridleyi. Phytohormone combinations in the callus induction medium were found to influence callus induction and different wild Oryza species exhibited their own preferred phytohormone combinations for anther culture. In general, NK medium containing suitable concentrations of auxin and cytokinin may be successfully applied for anther culture of selected wild Oryza species.     

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.     

Anther culture of tropical japonica × indica hybrids of rice (Oryza sativa L.)

Summary Nine japonica × indica F1 hybrids of rice involving 6 indica and 3 japonica tropical varieties, were large scale anther cultured. The frequency of callusing anthers averaged 18.7%. The microspore-derived calli produced green plants with a mean frequency of 8.7%. Albino plants represented 61% of the shoot forming calli. Monitoring of the green and albino plant regenerating capabilities of calli arising between week 4 and week 8 of incubation of the anthers showed no increase of the albino/green ratio and a slow decrease of the shoot forming ability of the transferred calli after the sixth week of culture. Spontaneous doubled haploids (SDH) represented 46% of the regenerated green plants in 4 hybrids. However, a high frequency of partially sterile regenerants was noticed among 132 SDH plants generated from a hybrid.     

Genetic analysis for haploid-regenerations responses of the hexaploid-wheat anther cultures

Genetic variability in response to anther culture was investigated in 49 winter hexaploid wheats, comprising 33 pure lines (F₁₀) derived from a composite cross programme and their 16 parental genotypes. All genotypes were grown in a randomized block design with three replications in a controlled greenhouse. The number of embryoids and total plant regeneration per 100 anthers, as well as the numbers of green and albino plants regenerated per 100 embryoids, were measured. Significant genetic variability was observed among the 49 genotypes for all the traits studied. All traits showed high heritability. Among the genotypes compared, DC²30N and 1BPT-40 gave the best results for the production of embryoids and IBPT-78 had the highest value for the production of green plants. The genotype IBPT-34 developed a large number of albino plants, and it should be useful as a parent in studies to determine the genetic control of albino plants in wheat.     

Evaluation of floral characteristics of barley in the semi-arid climate of north Syria

Previous studies have revealed that, in areas affected by severe drought, yielding ability and stability of barley (Hordeum vulgare L.) could be improved by increasing the level of heterozygosity. One possible approach to increase the heterozygosity level is to improve the outcrossing rate. In view of that, the genetic variability and phenotypic correlation coefficients involving four floral traits related to outcrossing behaviour were investigated in 274 drought adapted barley lines from the International Center for Agricultural Research in the Dry Areas (ICARDA's) current breeding programme. A significant quantitative variation existed in anther length and width, stigma length, and anther extrusion, which indicate that selection for these traits may be effective. Anther extrusion showed weak but highly significant positive correlations with anther and stigma length. Somewhat higher positive correlations were found between anther and stigma length. The results indicate that selection for high anther extrusion and longer anthers and stigmata may increase outcrossing in cultivated barley and consequently improve yielding ability through enhanced heterozygosity.     

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%.