Comparison of Anther Culture and the Hordeum bulbosum Method for the Production of Doubled Haploids in Winter Barley

The comparison, between the efficiency of anther culture and the Hordeum bulbosum method in barley was extended to the chromosome number distributions of all planes derived from the two techniques and the proportions of fertile doubled haploid plants which survived until maturity. The frequencies of haploid and spontaneously doubled haploid plants which were useful for practical breeding purposes were found to be around 90 % for both techniques. The remainder consisted of polyploid, mixoploid and aneuploid variants in the case of microspore-derived plants and diploid interspecific hybrids in the progeny of the H. bulbosum method. The ploidy level distributions of the microspore- and H. bulbosum-derived plants appeared to be independent of the genotype of the donor. There were no significant differences between techniques regarding the proportions, of plants which survived a severe winter and the production of fertile doubled haploid plants. Both techniques can therefore complement each other in a breeding programme and their relative merits are discussed. Possible ways of improving doubled haploid production in barley are suggested for increasing its use in breeding schemes.     

Studies on Low Crossabilities Encountered with the Hordeum bulbosum Method for Haploid Production of Barley, Hordeum vulgare L.

Crosses were made between four spring barley (Hordeum vulgare L.) cultivars and five F₁, hybrids with one genotype of Hordeum bulbosum L. in two locations to investigate further previous low crossabilities which had been found in the barley cultivar ‘Apex’ with H. bulbosum. Data at all the main steps of the H. bulbosum technique were recorded and statistically analyzed. Significant differences between barley genotypes were demonstrated for all characters. It was confirmed that ‘Apex’ has poor crossability with H. bulbosum. Out of the three F₁ hybrids having ‘Apex’ as one parent, two exhibited low crossability similarly to ‘Apex’ but one showed significantly higher seed setting than ‘Apex’. The effect of the location was only significant on seed setting, while genotype X location interactions were significant on seed setting, seed quality and rate of haploid plants in relation 10 florets pollinated. Another problem which has influenced the success rate of the H. bulbosum method was found in the cultivar ‘Havilla’. Although seed setting and seed quality were high for this cultivar, embryo differentiation was low. However, this latter problem was found to influence less the overall success rate than poor crossability. Mahalanobis's distances were calculated and the dendrite of the shortest distances between barley genotypes was plotted.     

Components of Response in Barley Anther Culture

Anther culture response with 17 widely-grown varieties and one model variety of barley was studied with one replication from field-grown donor plants and one replication from a growth-chamber. Plants were regenerated from all 18 varieties and green plants were obtained from 16 of them. On average, 1.6 green plants were obtained per 100 cultured anthers from all the material. Estimated variance components for the formation of embryos/callus from the anthers were dominated by the effects of the genotypes and interactions between plant material and environments which together accounted for 60.1 and 17.0 % of the total variation respectively, while environments were nonsignificant for this character. Plant regeneration from embryos/callus were not significantly influenced by either genotype or environments. Components of variance for green plant formation were dominated by the effects of the genotypes, accounting for 73.2 % of the total variation, and a smaller effect from environments accounting for 11.2% of the total variation. Main effects from genotypes on the percentage of green regenerants divided 7 varieties into two distinct groups, indicating that major genetic factors were involved. The genetic basis for green plant regeneration seems different from that governing embryo formation. The results are discussed with respect to the possible prediction of anther culture response for new barley hybrids, as a means for directing the use of barley anther culture towards material that responds well.     

Comparison of doubled haploid lines and F2 bulks for the improvement of barley in the dry areas of North Syria

Drought stress is the main factor limiting barley yields in West Asia and North Africa. This study compares the utility of doubled haploid lines (DHLs) and conventional F₂ plant-derived bulks (F₂Bs) in improving barley in stress environments. Double crosses were made, DHLs were developed by anther culture from double-cross F₁ plants, and F₂Bs were produced by bulking the offspring of F₂ plants. Field tests were conducted in three drought-stressed environments. No major differences were observed in the mean performance of DHLs and F₂Bs. For most traits, both the genotypic and the genotype × location interaction variances were higher in the DHL group, whereas heritabilities were similar. Higher gains from selection were predicted for the DHL group. Regression analysis of yield stability indicated a lower predictability of the DHL performance. The haploid technique can improve breeding populations from which varieties with stable yields can be developed. The costs involved are determined by the DHL production rate, which needs to be improved in many developing countries.     

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.     

An Evaluation of Hordeum vulgare L. ×Psathyrostachys fragilis (Boiss.) Nevski Crosses for Doubled Haploid Barley Production

Investigations were carried out to assess the suitability of the intergeneric cross Hordeum vulgare×Psathyrostachys fragilis for haploid barley production. H. vulgare cvs. ‘Emir’ and ‘Vada’ were each pollinated with P. fragilis P.I. 343192 and plants regenerated from embryos cultured on a modified B5 medium. Seed sets on ‘Vada’ were significantly lower than on ‘Emir’, and all the planes from ‘Vada’×P. fragile remained hybrid. Several of these flowered but there was little pairing between the parental chromosomes. Most of the plants from ‘Emir’×P. fragilis died, as seedlings but 3 plants developed into haploid barley. Because of the practical limitations of pollen availability from P. fragilis and the inconsistencies in haploid plant formation, it is unlikely that the cross will prove as valuable as that between H. vulgare×H. bulbosum for a doubled haploid barley programme.     

Genetic markers for doubled haploid response in barley

In order to analyse the genetic control of anther culture response in barley, a doubled-haploid (DH) population from the cross between a medium responsive cultivar ‘Dobla’ and the model cultivar ‘Igri’ was produced. A linkage map was constructed with 91 markers. A sub-population of 41 lines was characterised for different components of the anther culture response, and was used for quantitative trait loci (QTL) analysis. The vrs1 locus region on chromosome 2H, which determines inflorescence row type, was coincident with the largest putative QTL for number of embryos (nEMB) and albino plants. A region of chromosome 6H was associated with QTLs for nEMB and green plants. QTLs for number and percentage of green plants were located on the long arm of chromosome 5H. Therefore, new QTLs for main components of barley anther culture response were identified on chromosomes 2H, 5H and 6H, indicating that anther culture response in barley could be controlled by relative few genes of large effect. This work is a useful step towards the identification of new regions on the barley genome that could be associated with fundamental biological process implicated in the anther culture response.     

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.     

Somatic in vitro culture response of Lolium perenne L.: genetic effects and correlations with anther culture

Summary Genotypic effects on callus induction and plant regeneration in callus, suspension and protoplast culture, and their correlations with both phenotypic and GCA-values for anther culture response, were studied using 21 genotypes of perennial ryegrass. Differences between genotypes accounted for approximately 40% of the total variation for callus induction and initial callus growth, and 59 and 83% of the variation in callus culture for regeneration percentage and percentage of green plants. Effects of genotypes were less pronounced in suspension culture, where suspensions from the same genotype often behaved differently. Some suspension cultures retained their capacity for green plant regeneration for almost two years, repeatedly producing 80–100% green regenerants during this period. Genotypes with high regeneration percentage and a large proportion of green plants from callus culture were also superior in suspension culture for both regeneration performance and longevity. Regeneration percentage and percentage of green plants were uncorrelated, and probably under different genetic control. While capacity for green plant formation from the different genotypes showed no correlation between anther culture and somatic in vitro culture, a positive correlation was observed between the regeneration percentages in somatic in vitro culture and anther culture (r=0.44^*–0.85^***), suggesting some common genetic control of the two systems.     

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.     

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.     

Comparative Morphological Studies of Microspore Derived and F2 Plants Obtained from an Interspecific Rice Hybrid Oryza sativa Linn. ×O. rufipogon Griff.)*

Anther culture of an interspecific rice hybrid from a cross of Oryza sativa× O. rufipogon was attempted. Of the 117 regenerated pollen clones, 56 could survive to maturity. A majority of these were either haploids or doubled haploids and very few turned out to be chromosomal variants. Comparative study of doubled haploids and the seed derived F₂ plants indicate the distinct advantages of anther culture techniques. (1) Androgenic plants, though few in number, showed greater ariation for all the traits with the exception of ear bearing tillers. (2) Predominance of recombinants with wild traits was observed in F₂ segregation. (3) It was possible to recover indica type recombinants among the anther-derived plants with one or two traits introgressed from O. rufipogon. These results suggest the feasibility and utility of anther culture in distant hybridization for incorporation of alien variation into cultivated rice.     

Genetic Variability for Grain Yield and Protein Content in Barley and Its Modification by Net Blotch

Protein content, yield and 1000-grain weight of healthy and net blotch-diseased plants were studied in a diallel cross programme concerning nine different varieties of barley (Hordeum vulgare L.). F₁ hybrids and their parents were grown in the field in two randomized block design experiments each with three replications (three inoculated and three non-inoculated plots). General and specific combining abilities were determined for yield, protein percentage, 1000 grain weight, protein yield, and non-protein yield. A high genetic variability for the traits studied was observed. Non-protein yield and 1000-grain weight decreased significantly in inoculated plants where protein yield was not affected. Significant general and specific combining abilities were observed for all traits. Values for general and specific combining abilities were, in some cases, significantly modified by net blotch of barley. This indicates that attention must be paid to the danger of drawing conclusions in quantitative genetics studies dealing with both diseased and healthy plants.     

Dihaploid Plant Production from 4 × -Genotypes of Potato by the Use of Efficient Anther Plants Producing Tetraploid Strains (4 × EAPP-Clones) — Proposal of a Breeding Methodology

Tetraploid clones of potato with a superior efficiency in producing androgenetic plants (4 × EAPP-clones) have been obtained by culturing in vitro anthers of 2 × EAPP-clones. The latter were isolated by three cycles of recurrent selection from diploid breeding material (UHRIG 1985 a). In this paper we report on the capacity of 4 × EAPP-clones, when crossed to in vitro unresponsive 4 × genotypes, to transmit to their F₁ their androgenetic potential, Five 4 ×× 4 × F₁ crosses were considered, which produced on average 35 embryoids per flower – a value higher than that of nonresponsive 4 × genotypes (no embryoids obtained), but-also significantly better than the value found for 4× EAPP-clones (9.8 embryoids per flower). The hybrid families behaved differently from each other, with one producing up to 63 embryoids per flower. A range of per plant values was, moreover, found, revealing the existence of a large variability among sister plants belonging to the same F₁ cross. The presented data indicate a rather simple inheritance of dominant genetic factors acting in favor ot androgenesis. They also suggest that the utilized 4× EAPP-clones were possibly heterozygous for such genes. Data are also reported on the ploidy level of anther plants obtained from 4× and 2× EAPP-clones. In this respect 2× EAPP-clones show the interesting capacity of generating, via anther culture, a consistent fraction of tetraploid plants (13,7 %). Based on the findings reported in this paper we propose, for tetraploid S. tuherosum L., a cyclic breeding procedure making use of anther culture and where ploidy level alternates, within a cycle, between 2× and 4×.     

Comparisons between doubled haploid lines produced by anther culture,the Hordeum bulbosum-method and lines produced by single seed descent in barley crosses

Summary Possible negative effects of tissue culture on qualitative and quantitative characters have been investigated in three crosses of spring barley, using doubled haploid lines produced by anther culture (AC, with maltose as a carbon source) or the Hordeum bulbosum-method (HB). In one cross inbred lines produced by single seed descent (SSD) have also been included. Quantitative characters were investigated in a 2-year field experiment at one location. The results show that although the methods in the majority of cases gave similar results, the genotypic arrays produced were not identical. Different markers deviated in the various cross/method combinations. The ranking of methods as well as the frequencies of lines transgressive for grain yield differed between crosses. Thus no consistently negative impact of anther culture in barley has been found, and, although not identical, the various methods may be considered equivalent.Abbreviations AC Anther Culture method - SSD Single Seed Descent method - HB Hordeum bulbosum method     

Anther culture response of F1 durum × bread wheat hybrids after colchicine application

The effect of colchicine on the androgenic response of durum × bread wheat F₁ hybrids was studied. For this, three Greek durum wheat cultivars, exhibiting good yield and pasta quality, were crossed to two bread wheat cultivars with good response to anther culture. Spikes of the resulting hybrids, containing microspores in the mid to late uninucleate stage, were selected and cultured on two different media (W₁₄ and solid potato‐2) with and without colchicine. A negative effect on anther culture response was observed when colchicine was added to the W₁₄ induction medium. A similar effect was observed in two of the hybrids when they were cultured on to potato‐2 induction medium. In both induction media, green plant production was influenced negatively by colchicine treatment. The same was observed in albino plant production. The results of the present study support the view that anther culture response is strongly genotype dependent. Finally, potato‐2 induction medium was the most suitable one for the material studied.     

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.     

Genetic analysis of tissue culture traits in barley cv. 'Lenins'

The barley cultivar ‘Lenins’ was found to be a genotype showing high shoot regeneration ability in cultures derived from immature embryos. Five cultivars different from ‘Lenins’ in shoot regeneration ability were reciprocally crossed with ‘Lenins’ and the inheritance of tissue culture traits was investigated. F₂ plants showed continuous distributions in callus growth and percentage of shoot regeneration, suggesting that these traits were controlled by polygenes. The F₂ population, derived from a cross between ‘Lenins’ and ‘6721′, showed a monogenic segregation for the number of regenerated shoots, and the segregation ratio fitted 1:2:1. Tissue culture traits of ‘Lenins’ were controlled by several genes, whereas the number of regenerated shoots related to the efficiency of shoot regeneration is controlled by one major gene.     

Evaluation of quantitative resistance to yellow rust (Puccinia striiformis f. sp. hordei) in the ICARDA/CIMMYT barley breeding programme

The resistance to yellow rust (Puccinia striiformis f. sp. hordei) of 500 advanced barley lines from the ICARDA/CIMMYT breeding programme in Mexico was evaluated on seedlings in the greenhouse and on adult plants in the field. A high frequency of advanced lines (85.8%) showed a susceptible reaction (infection type ≥ 7) on seedlings after inoculation with isolate Mex-1, representing a Mexican variant of race 24. This indicates the absence of effective hypersensitive resistance. In addition, the same advanced lines showed a large variation in disease severity in the field, ranging from 0 to 95%. More than 76% of the advanced lines with a susceptible reaction in the seedling stage demonstrated low disease severity (10% or less in the adult plant). Consequently, these advanced lines possess high levels of quantitative resistance. Two aspects in the ICARDA/CIMMYT barley breeding programme may explain the large number of advanced lines with high levels of quantitative resistance. First, a recurrent selection approach is applied when advanced (F₅) lines reaching homozygosity are intercrossed. Second, low levels of disease are accepted in the selection process instead of selecting the ultimate green plant. Both aspects combined allow the accumulation of quantitative resistance. Certain cultivars released from South-American national programmes in the late 1970s and early 1980s in Peru (UNA-80), Bolivia (IBTA 80) and Ecuador (Teran) are still resistant, demonstrating the durable nature of quantitative resistance to yellow rust.     

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.