The assessment of in vitro characters and their influence on the success rates of doubled haploid production in barley

Summary A assessment of in vitro characters: embryo size, differentiation stage and germination time of haploid embryos, was carried out with a view to developing a selection procedure to increase the efficiency of doubled haploid (DH) production using the Hordeum bulbosum system. The differentiation stage of haploid embryos at the time of excision was found to be the most promising character for selection. In the two spring crosses examined 55% of excised embryos were undifferentiated and 45% were differentiated, with overall success rates, in terms of the percentage of DHs produced, of 7.91% and 25.16%, respectively. 17% of excised embryos produced callus. These embryos performed poorly in terms of regeneration and subsequent growth. It is suggested that only differentiated non-callusing embryos are maintained in order to maximize the efficiency of DH production. It is also shown that the overall success rate depends not only on initial stages such as seed setting but also on genotypic differences in response to colchicine treatment at later stages.     

Combining Ability of Doubled Haploids in Coffea canephora P.

Fifty-five doubled haploids (DH) of Coffea canephora were crossed with either heterozygous genotypes or DH in order to study their combining ability. Three agronomic trials were established. Marked hybrid vigour was observed for all characters analyzed including yield. Large differences were evident among top-crosses involving different DH produced from the same parental clone reflecting the high level of heterozygosity of clones. Factorial mating design analysis indicated that all genetic variance was attributable to additive effects in estimates of yield as well as plant height and leaf characteristics. The general combining ability variance component was also predominant for stem girth and susceptibility to leaf rust, although effects due to interaction were detected. Some hybrid combinations had yield comparable to standard clonal varieties. The implications of such results for breeding of Coffea canephora are discussed. Particularly, the development of F₁ hybrid varieties is envisaged.     

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.     

The evaluation of superior Hordeum bulbosum L. genotypes for use in a doubled haploid barley breeding programme

Summary Eight Hordeum bulbosum selections were produced from a cross between Cb 2920/4 and Cb 2929/1, two genotypes widely used in doubled haploid breeding programmes. The selections were hybridized with barley to evaluate their ability to produce high proportions of well-differentiated haploid embryos compared with Cb 2929/1 as control. We report here an initial small-scale investigation followed by a larger-scale test in two different environments to assess seed setting, haploid embryo differentiation rates and VB hybrid formation. These VB embryos contain both parental sets of chromosomes and occur more frequently in the glasshouse during the winter. Two of the eight selections were identified as combining the desirable characteristics of both parents, namely high seed setting on cv. Vada which is partially incompatible with H. bulbosum, large numbers of well-differentiated haploid embryos and a low incidence of VB hybrids. The selections are available for release to interested research workers and plant breedersAbbreviations DH doubled haploid - VB a hybrid from H. vulgare x H. bulbosum which contains both parental sets of chromosomes     

Localisation and combination of resistance genes against soil-borne viruses of barley (BaMMV,BaYMV) using doubled haploids and molecular markers

Barley yellow mosaic virus disease caused by different strains of BaYMV and BaMMV is a major threat to winter barley cultivation in Europe. Different resistance genes against these viruses have been mapped and suitable PCR-based markers have been developed. In this respect doubled haploid (DH) populations proved to be advantageous as they facilitate a repeated test for resistance against all agents of the barley yellow mosaic virus complex and besides this, dominant marker systems are as informative as co-dominant ones in DHs due to the lack of heterozygous genotypes. Using DH populations resistance genes rym4, rym5, rym11, rym13, rym15 and the BaYMV/BaYMV-2 resistance of the barley cultivar ‘Chikurin Ibaraki 1’ have been mapped. DHs are also well suited to pyramiding resistance genes against BaMMV and BaYMV. Since homozygous recessive genotypes are more frequent in DHs than in segregating F₂ populations, DHs can be efficiently used to create broad-spectrum resistance and to extend the usability of partly overcome resistance genes. Results from employing two different strategies for pyramiding, based on one and two DH-steps, respectively, combining three recessive resistance genes, i.e. rym4/rym5, rym9 and rym11, are presented. The faster strategy based on one haploidy step resulted in the identification of all three and two-way combinations of the respective resistance genes.     

The effects of parthenogenesis on wheat embryo formation and haploid production with and without maize pollination

Doubled haploid (DH) lines are important in wheat (Triticum aestivum L.) breeding, and haploids produced via maize pollination precede DH line development. Although maize pollination has proven reliable and broadly applicable to wheat, its success is determined by the wheat and maize genotypes employed. A wheat genotype consisting of nuclear and cytoplasm components predisposing it to parthenogenesis was compared with three other genotypes, each possessing only one or neither component necessary for parthenogenesis. In a glasshouse experiment, each genotype was pollinated with maize and subsequently treated with a2,4-Dichlorophenoxyacetic Acid (2,4-D) solution to determine if parthenogenesis affected embryo formation frequency (EFF)and haploid formation efficiency (HFE). Wheat genotypes were also treated with the2,4-D solution alone to determine if embryos and haploid plants could be produced in vivo without maize pollination. ‘Salmon(K)’, a parthenogenetic genotype consisting of a Salmon 1BL.1RS nucleus in a Ae. kotschyii cytoplasm, had a mean EFF of 32%; whereas, the non-parthenogenetic genotypes had mean EFF calculations ranging from 7 to 21%. Mean HFE for Salmon(K) was not significantly different than the mean HFE for non-parthenogenetic Salmon; however, EFF and HFE calculations for Salmon(K) and Salmon, each with a 1BL.1RS translocation, were generally higher than calculations for genotypes without the translocation. Salmon(K) was the only genotype to produce a 3% or higher EFF and HFE after treatment with 2,4-D alone. Parthenogenesis significantly affected the frequency at which embryos were produced after pollination with maize and the frequency at which embryos and haploid plants were produced after treatment with 2,4-D alone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Discrimination of haploid and diploid maize kernels via multispectral imaging

The use of doubled haploids (DHs) in maize has become ubiquitous in maize breeding programmes as it allows breeders to go from cross to evaluation in as little as 2 years. Two important aspects of the in vivo DH system used in maize are as follows: (i) the identification of haploid progeny and (ii) doubling of the haploid genome to produce fertile inbred lines. This study is focused on the first step. Currently, identification of maize haploid progeny is performed manually using the R1‐nj seed colour marker. This is a labour‐intensive and time‐consuming process; a method for automated sorting of haploids would increase the efficiency of DH line development. In this study, six inbred lines were crossed with the maternal haploid inducer ‘RWS/RWK‐76’ and a sample of seed was sorted manually for each line. Using the VideometerLab 3 system, spectral imaging techniques were applied to discriminate between haploids and hybrids. Using DNA markers to confirm the haploid/diploid state of the tested seed, for the majority of genotypes haploid identification was possible with over 50% accuracy.     

Doubled haploid production in Turkish durum wheats using crosses with maize

Durum wheats cultivated in the Middle Anatolia region of Turkey (Triticum durum cvs ‘Kunduru’, ‘Berkmen’ and ‘Cakmak’) were crossed with maize to evaluate their capacity for haploid embryo and doubled haploid (DH) plant production. A total of 2960 florets were crossed with maize and 13.7% of the florets produced haploid embryos across the three varieties. Haploid embryo and plant regeneration frequencies were highest in ‘Kunduru’, but colchicine doubling was less successful with this genotype. There were statistically significant differences between ‘Cakmak’ and other genotypes for embryo yield (P < 0.01). Overall, 52.3% of all embryos differentiated, but there were no geno-typic differences in differentiation frequencies.     

Histological and inheritance studies of partial resistance in the Brassica napus–Albugo candida host-pathogen interaction

Traditional and doubled haploid (DH) genotypes of oilseed Brassica spp. resistant, partially resistant, moderately susceptible, and susceptible to Albugo candida were compared for phenotypic development of host‐pathogen interaction and histology of host‐pathogen interaction. The partially resistant genotype showed pinhead‐size pustules, mainly on the upper surface of cotyledonary leaves. Relatively less mycelium was observed in the partially resistant genotype compared with the susceptible genotype. In resistant B. napus genotypes, there was neither pustule development nor any mycelial growth. In the moderately susceptible genotype, the pustules were similar to those in the partially resistant genotype in being of pinhead‐size and occasionally coalescing. However, ample mycelial growth in the mesophyll tissue in the moderately susceptible genotype was similar to that in the susceptible control B. rapa cv. ‘Torch’. The susceptible genotype B. rapa cv. ‘Torch’ also showed large coalescing pustules. In the non‐host B. juncea cv. ‘Commercial Brown’, no pustules were formed although some mycelial growth was observed beneath the epidermal cell layer and in the mesophyll cell layer of the cotyledonary leaf tissue. For inheritance studies, two partially resistant B. napus genotypes were crossed with a resistant B. napus genotype. Various generations viz., F1, F1(reciprocal), F₂, and DHs produced from the crosses were inoculated with a zoospore suspension of race 7v of A. candida. The partially resistant phenotype appeared to be controlled by a single recessive gene designated as wpr with variable expression. The simple inheritance of partial resistance has implications for disease resistance breeding against white rust, as this type of resistance can be easily incorporated into elite breeding lines through conventional and DH breeding methods.     

Pleiotropic effects on the ml-o mildew resistance gene in barley in different genetical backgrounds

Summary The effects on quantitative characters of the ml-o5 allele has been investigated by means of doubled haploid lines produced by the Hordeum bulbosum method. In one series of experiments the genetic background in each cross was found significantly to modify the negative pleiotropic effects. Doubled haploids were also produced from a F1 cross and tested in the field during two years, one with strong and one with weak mildew infections. In the first year only an insignificant reduction in grain yield was detected in the mlo lines, in the second it was significantly reduced with 5.9%. Thousand grain weights were, however, higher in the resistant lines. In both series of experiments the possibility to extract very high yielding mlo genotypes was demonstrated. The nature of the genetic background effect is discussed.     

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.     

Variation and heritability of economically important traits in black currant (Ribes nigrum L.) evaluated in a diallel cross design

Summary The inheritance of commercially important characters in black currant (Ribes nigrum L.) was investigated using a complete 5×5 varietal diallel cross. Estimates of the genetic components of variance and broad and narrow-sense heritabilities revealed that additive genetic effects were greater than nonadditive genetic effects for resistance to powdery mildew (Sphaerotheca mors-uvae Berk.), white pine blister rust (Cronartium ribicola Fisher) and leaf spot (Drepanopeziza ribis Kleb.) as well as for bush defoliation and length of strigs. Apart from resistance to leaf spot, narrow-sense heritabilities of these characters exceeded 0.5.Nonadditive genetic effects were greater than additive effects for plant habit, plant vigor, bush size, length of one-year-old shoots, uniformity of berry ripening and fruit yield per bush. Narrow-sense heritabilities of these characters were rather low (0.05–0.42).     

Obtention of haploid embryos and plants through irradiated pollen technique in squash (Cucurbita pepo L.)

The influence of gamma ray doses (25, 50, 75, 100, 200, 300 and 400 Gy) and genotypes (Eskenderany F₁, Acceste F₁, Sakiz, Urfa Yerli) on the induction of haploid embryos obtained by irradiated pollen technique was studied in squash (Cucurbita pepo L.). Different shapes and stages of embryos were derived from seeds extracted from fruits harvested 4–5 weeks after pollination. As a result of the present study, haploid embryos and haploid plants were obtained, with haploid production strongly influenced by gamma ray doses, embryo stages and genotypes. Gamma ray doses of 25 and 50 Gy gave the highest parthenogenetical response. All of the point shape, globular shape, arrow tips and stick-shaped embryos developed into haploid plants. However, only 53.8% of torpedo and 23.1% of heart-shaped embryos gave haploid plants. In contrast, cotyledon-shaped and amorphous-shaped embryos produced only diploid plantlets. The number of embryos per 100 seeds was the highest in ‘Eskenderany F₁’ and ‘Sakiz’ genotypes. After in vitro culture, a total of 93 haploid plantlets were obtained. This revised version was published online in August 2006 with corrections to the Cover Date.     

Improved Techniques for Haploid Production in Wheat using Chromosome Elimination

Wheat × maize and wheat × pearl millet crosses have proved efficient for haploid production using various genotypes of wheat; 22 and 27 % of florets produced embryos. In favourable conditions 6—9 haploid plants per spike were produced. The following simplifications or improvements in technique are recommended: 1. Only a single treatment with an aqueous solution of dicamba or 2,4-D (50–100 ppm) for embryo stimulation in vivo; 2. Application by spraying or dipping the spikes; 3. Application time two to four days after pollination; 4. Embryo rescue 15 to 18 days after pollination; 5. Crosses without emasculation are possible if pollination occurs 1–2 days before anthesis. More than 450 haploids and some doubled haploid (DH) lines (after colchicine treatment in vitro) were produced using these methods. No hybrid plants, chromosome additions or substitutions were found.     

Evaluation of androgenic competence through anther culture in common eggplant and related species

Anther culture is a convenient technique to obtain androgenic haploid and doubled haploid (DH) plants. In common eggplant (Solanum melongena), this technique has been used to develop DH pure lines for producing uniform F1 hybrid seed of some commercial varieties. However, a comprehensive study of the variation of this useful trait among different materials of common eggplant and related species is still lacking. In this work, we studied the androgenic response of 12 accessions of common eggplant and related materials from the primary (eggplant complex) and secondary genepools. We cultured anthers of all the accessions under the same experimental conditions, and studied their competence to produce calli, embryos and plants, as well as the quality and origin of the embryos produced. In our conditions, anthers of 11 out of the 12 accessions produced somatic calli, whereas only 5 also produced microspore-derived embryos, with variable results in terms of embryo quality and of frequency of embryo induction and plant germination. Embryos of responding accessions were initially haploid, and reached the DH status, verified with SSR markers, after a defined period of culture. In addition to other aspects common to many androgenesis-responsive species, our results allowed us to extract conclusions particular to common eggplant and relatives, including the difficulty for finding sources of androgenic competence out of S. melongena, the reduced impact of calli in the production of non-DH individuals, and the need to avoid the occurrence of severe anatomical and functional problems in the apex of most embryos, which seriously reduces their germinative success.     

Harnessing mutant donor plants for microspore culture in Indian mustard [Brassica juncea (L.) Czern and Coss]

Haploid mutagenesis offers several advantages over conventional (seed) approach. However, its potential has not been utilised for Brassica juncea, an important oilseed. In this study, mutant donor plants of three Indian B. juncea genotypes, generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU), were used for microspore culture. The response of mutant donor plants was about 100 times lower than non-mutant controls; a total of 9,411 embryos were produced from the EMS treated donor plants, while microspores isolated from ENU treated donors did not yield any embryos. The lethality of induced mutations demonstrated itself mainly as the induction of abnormal embryos (80%), failure of germination (70%) and failure of plantlet development (70%). Nine doubled haploid (DH) mutant lines and three non-mutant DH lines obtained through this approach were tested for agronomic and biochemical variation over two growing seasons. High variability was observed and stable mutants were recovered for reduced height (125 vs. 168 cm for the control), appressed pod character, altered fatty acid composition higher protein proportion in de-oiled meal (48%) and a lower glucosinolate content in de-oiled meal (59.5 μM/g) relative to controls. The approach demonstrates that despite severe reduction in efficiency of the DH line production, valuable mutants can be recovered from mutated donor plants.     

Detection by simple sequence repeat markers of introgression from Coffea canephora in Coffea arabica cultivars

Microsatellites or simple sequence repeats (SSR) were used to assess polymorphism among 16 Coffea arabica and four Coffea canephora accessions, and to identify DNA introgression fragments from C. canephora in four C. arabica lines. Thirty‐one primer pairs allowed for the identification of 92 polymorphic alleles distributed over 37 loci. The C. arabica accessions derived from the genetic bases ‘Typica’ and ‘Bourbon’ were grouped separately according to their genetic origin. Two genotypes derived from a spontaneous hybrid (C arabica×C. canephora) were classified with the C. canephora accessions from Central Africa. Coffea canephora from West Africa were separated from the other accessions studied. Four alleles related to introgression (i.e. present in C. canephora and introgressed lines, and absent in C. arabica) were identified. The SSR markers were used successfully for characterization of a particular cultivar (‘Veranero’) from Costa Rica, which is known for its late maturity.     

Haploid induction from F1 hybrids between CMS shallot with Allium galanthum cytoplasm and common onion by unpollinated flower culture

The induction of haploid plants from F₁ hybrids between CMS shallot with Allium galanthum cytoplasm and common onion was examined. Starting with 535 unpollinated flowers cultured in B5 medium 25 seedlings from part henogenetic embryos were obtained of which 13 seedlings survived. Eleven seedlings were determined as haploid plants (2n = x = 8) and 2 seedlings were doubled haploid plants (2n = 2x = 16). All haploid and doubled haploid plants preserved chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) from A. galanthum. Segregation in different characters was observed among the haploid plants. The haploid and doubled haploid plants exhibited the different combinations of genes from shallot and common onion. Crossing of the doubled haploid plants with other shallot strains, common onion cultivars or related species may produce excellent F₁ hybrids for bulb production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Segregation of genetic markers among wheat doubled haploid lines derived from wheat x maize crosses

Summary Wheat doubled haploid (DH) lines were produced from the F₁ hybrid, Fukudo-komugi x Oligo Culm, through intergeneric crosses between wheat and maize. F₂ plants and 203 DH lines were analyzed for the segregation of the eight genetic markers, namely, grain proteins, grain esterases, GA-insensitivity and glume traits. The segregation in the F₂ plants fitted to the expected ratios. No deviation was observed among the DH lines, either, except for the glume pubescence. The result indicates the absence of correlation between the markers investigated and the efficiency of embryo formation in the DH lines.     

Double haploids,markers and QTL analysis in vegetable brassicas

Double haploid (DH) plants of Brassica spp. can be produced via anther culture or culture of microspores. This paper reviews the uses of double haploids in crop improvement research in vegetable brassicas (B. oleracea). Applications of DH lines are described for breeding; construction of linkage maps; genetic analysis of quantitative traits and capturing genetic variation. The advantages and disadvantages of DH lines are discussed