Comparative Value of Androgenetic Doubled Haploid and Conventionally Selected Spring Barley Lines

Androgenetic doubled haploid spring barley lines (DH-lines) were compared in field experiments with their pedigree-selected sister lines (PD-lines). The PD-lines resemble a selected proportion of 1 % (10^?2) or less of the original complete F₂-population. By comparison, an average of 10 DH-lines were recovered from 10,000 anthers X > 1,000 microspores, i.e. at least 10⁷ microspores per cross. The average success rate was therefore approximately 10^?6. Both PD- and DH-families showed similar levels of performance with regard to all the characters studied, although no conscious selection had been applied in deriving doubled haploid progeny. Therefore, it is concluded that the regenerated microspore-progenies may be the result of unconscious selection among the large numbers of microspores cultured.     

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.     

Addition of Colchicine to Wheat Anther Culture Media to Increase Doubled Haploid Plant Production

Chromosome doubling is critical for obtaining doubled-haploid plants from wheat (Triticum aestivum L.) anther culture. The most common doubling method applies colchicine to the plant. However, colchicine is phytotoxic and can induce a high frequency of plant death. In this experiment, anthers from two wheat genotypes (“Pavon 76” and ‘Centurk’) were placed on nine embryoid initiation media having three sugar sources (maltose, sucrose, and maltose + glucose) with three colchicine concentrations (0.0, 0.1, and 0.2 g · l^-1). Wheat starch was used as a gelling agent. After three days, the anthers were washed and moved to fresh media without colchicine. Increasing the colchicine concentration decreased the number of embryoids produced from 77.4 embryoids/100 anthers to 29.9 embryoids/100 anthers, but did not significantly affect the frequency of plant regeneration (0.49 green plants/embryoid to 0.40 green plants/embryoid), and increased the frequency of doubled-haploid plants (19.0 doubled-haploid plants/100 green plants to 72.3 doubled-haploid plants/100 green plants). Considering the total number of doubled-haploid plants produced, low levels of colchicine added to the initiation media were very effective.     

'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.     

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.     

Comparative Field Performance of Tissue Culture-Derived Lines and Breeder Lines of HY320 Spring Wheat

This study examines the performance of somaclonal spring wheat (Triticum aestivum L.) lines developed through culture of somatic embryos. Twenty-nine breeder lines of the spring wheat cv. ‘HY320’ were compared to 51 somaclonal lines of the same cultivar. Somaclonal lines were derived from 27 individual embryos (with up to four lines in each “family”). Somaclonal and breeder lines were evaluated in five field experiments in western Canada. Somaclonal lines were more variable than breeder lines for most agronomic, yield component and quality characters, suggesting that variability among somaclonal lines resulted partly from the tissue culture process. Somaclonal lines yielded, on average, 11 % less than breeder lines. Somaclonal lines had 3.8 % fewer spikelets per spike, 6.5 % fewer kernels per spike and kernels which were 2.7 % lighter. Somaclonal lines had greater test weight, protein concentration, and sedimentation values, and harder kernels.     

Distorted Segregation for Mildew Resistance in Doubled Haploid Lines of Spring Barley

The mildew reactions of the second generation of doubled haploid (DH) plants, derived from anther culture of crosses among three spring barley lines carrying different Mla mildew resistance alleles and the cv. ‘Pallas’, were analyzed by using a set of three European and one Israeli mildew isolate. The results indicated, (1) a significant level of distortion segregation in favour of resistant DH genotypes, which was possibly due to linkage of mildew resistance genes on chromosome 5 with genes for plant regeneration and (2) various degrees of dominance for the different resistance genes studied as well as the possible action of modifier genes.     

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.     

Comparison of Spring Barley Lines Produced by Single Seed Descent, Pedigree Inbreeding and Doubled Haploidy

Samples of inbred lines of Hordeum vulgare produced by pedigree inbreeding, doubled haploidy and single seed descent have been compared for a range of agronomic characters. Differences between the means of the in bred s produced by single seed descent and pedigree breeding were due to differing levels of inbreeding. The present study confirmed chat recombinant inbred families which out-perform the higher scoring parent may be produced by either method. Caution should, however, be exercised when simply inherited characters controlled by major genes are fixed in early generations. Differences between the doubled haploid and single seed descent samples for the character thousand grain weight were attributable to coupling linkages involving epistatic genes. The production and evaluation of random inbred lines should be considered as an alternative to pedigree methods of barley breeding.     

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.     

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.     

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.     

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.     

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.     

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).     

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.     

Canopy Temperature Depression Association with Yield of Irrigated Spring Wheat Cultivars in a Hot Climate

This study examines the utility of measurements of canopy temperature depression (TD) below air temperature as an indirect selection criterion for grain yield in breeding of wheat ( Triticum aestivum L.). TD differences can be rapidly assessed using a hand-held infra-red thermometer and may reflect differences in stomatal conductance. Replicated small plot experiments were conducted with 23–24 spring wheat cultivars planted at two dates in the winter cropping cycles of 1991–92 and 1992–93 at a hot low latitude location in Mexico.
Across a two fold range in grain yield at each planting, TD was usually highly significantly correlated with grain yield. Averaging the results for each year, the phenotypic correlation TD vs. yield was 0.84** and 0.89** for 1991–92 and 1992–93, respectively. Correlations were strongest when TD was measured between noon and 4 pm, and were unaffected by crop stage of development (pre-heading, heading, grain fill) or timing relative to irrigation. TD was positively correlated with stomatal conductance. We discuss possible causes of these high correlations, and their implications for wheat breeding.     

Efficient production of doubled haploid wheat plants by in vitro treatment of microspores with trifluralin or APM

Isolated microspore cultures from two doubled haploid (DH) lines of wheat, Triticum aestivum L., were used to develop an in vitro chromosome-doubling protocol. During the initial 24 h or 48 h of culture the microspores were treated with either of the two antimicrotubule herbicides trifluralin or amiprophos-methyl (APM) in concentrations ranging from 0.1 μM to 10μM. Untreated control cultures yielded 209 embryos per 100000 microspores, which is the equivalent of one spike. Among the regenerated plantlets 67% were green, and 15% of the flowering plants were spontaneously chromosome doubled. Treatments with both the herbicides had a significant effect on chromosome doubling, measured as the percentage of fertile regenerants. With the best combination of treatment duration (48 h) and herbicide concentration (10/μM) the percentage of fertile plants among regenerants could be increased up to 74% with APM and up to 65% with trifluralin. The largest numbers of DH plants per spike could be obtained with herbicide concentrations at 1–3 μM. Treatments with either herbicide at these concentrations resulted in an estimated average between the two genotypes of 27 DH plants per 100 000 microspores. These results demonstrate the high potential of APM and trifluralin as chromosome-doubling agents in isolated microspore cultures. The in vitro treatment integrated into tissue culture procedures will constitute an efficient method for chromosome doubling in future wheat breeding     

Inheritance of Resistance to Mycosphaerella graminicola in Hexaploid Wheat

Septoria tritici blotch constitutes a major disease problem of wheat world-wide. To efficiently breed wheat for resistance to this disease, an understanding is required of the inheritance of resistance. Our objective was to study the quantitative inheritance of resistance under field conditions. A nine-parent diallel and a generation mean experiments were conducted at Toluca, México in 1986 and 1987, respectively, to investigate gene effects. General combining ability effects accounted for most of the variation although specific combining ability effects were detected in some crosses. Ias20*5/H567.71, Thornbird, and RPB709.71/Coc contributed the most to reduced disease severity. Reciprocal effects were detected in two of 36 crosses, where RPB709.71/Coc contributed additional reduced disease severity when used as female. The analysis of generation means confirmed results obtained from the diallel. Additive effects were also most important. Dominance effects and epistasis, mostly of the additive × additive type, were found in some crosses. Hence, substantial genetic progress for resistance can be expected among progeny from crosses with resistant parents. However, selection would be most effective if delayed to later generations because of dominance, and choice of the specific female parent may produce a higher level of resistance.