Relative efficiency of maize and Imperata cylindrica for haploid induction in Triticum durum following chromosome elimination‐mediated approach of doubled haploid breeding

Intergeneric hybridization in seven diverse durum wheat genotypes was carried out using two composite varieties of Himalayan maize, viz., Bajaura Makka and Early Composite, and a wild grass, Imperata cylindrica, as pollen sources. Observations related to various haploid induction parameters put forth I. cylindrica as significantly better pollen source for haploid induction in durum wheat over maize in terms of pseudoseed formation (46.93%), embryo formation (38.06%), haploid regeneration (40.42%) and haploid formation efficiency (7.44%). The line x tester analysis revealed that both male and female genotypes had significant effects on all haploid induction parameters except haploid formation frequency in later. Among the pollen sources, I. cylindrica emerged as best combiner based on GCA values when compared with the two Himalayan maize composites. Durum wheat genotype, A‐9‐30‐1 was recognized as the best general combiner followed by PDW 314. The present investigation proposed durum wheat × I. cylindrica as a superior technique over maize‐mediated system, and its large‐scale use can open a new horizon in the sphere of durum wheat doubled haploidy breeding programme.     

Relative efficiency of different Gramineae genera for haploid induction in triticale and triticale x wheat hybrids through the chromosome elimination technique

The study was undertaken to evaluate the relative efficiency of different Gramineae genera for haploid induction in triticale (x Triticosecale) and triticale × wheat (Triticum aestivum) hybrids through the chromosome elimination (wheat × maize, Zea mays) system. Eight intergenotypic triticale and 15 triticale x wheat crosses were subjected to hybridization with nine different Gramineae genera viz., Z. mays, Sorghum bicolor, Pennisetum americanum, Setaria italica, Festuca arundinacea, Imperata cylindrica, Cynodon dactylon, Lolium temulentum and Phalaris minor in two separate experiments. This was followed by in vivo auxin treatment of the crossed spikes and subsequent rescue of the haploid embryos to regenerate green haploid plantlets. All the triticale and triticale x wheat crosses resulted in seed set in variable frequencies when hybridized with maize, I. cylindrica, pearl millet and sorghum. Seed set was also obtained with S. italica, F. arundinacea and P. minor in a few crosses in both groups. In general, all the triticale x wheat crosses, except for one in each case, resulted in embryo formation and green haploid plantlet regeneration when hybridizations were carried out with maize and I. cylindrica. However, the latter outperformed the former in embryo formation (25.48% vs. 20.0%) and regeneration (34.17% vs. 15.10%) frequencies, the differences being significant for regeneration frequencies. In the case of triticale hybrids, no significant differences between maize and I. cylindrica were observed for the three parameters of haploid induction. Embryo formation and regeneration were also observed in some of the triticale as well as triticale × wheat F₁ hybrids when hybridized with sorghum and pearl millet.     

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     

Development of novel chromosome doubling strategies for wheat × maize system of wheat haploid production

Two chromosome doubling strategies were evaluated for producing wheat doubled haploids from wheat x maize crosses: (i) in vitro colchicine application to haploid embryos and (ii) colchicine treatment through postpollination tiller injections. In the in vitro approach the haploid embryos were rescued on medium containing colchicine (at concentrations of 0.2, 0.3, 0.4 and 0.5%) and moved to a colchicine‐free regeneration medium 48 h later. Embryos exposed to 0.5% colchicine had 91.67% of their regenerated plants showing chromosome doubling. In the tiller injection approach, different concentrations (0.5, 0.75 and 1.0%) of colchicine solution, which also contained 2,4‐D (100 ppm), were injected into the uppermost inter‐node of crossed tillers 48 and 72 h after pollination. The chromosome doubling efficiency varied from 33 to 100%, with 1% treatment being the most effective. No chimeras of doubled/haploid sectors were observed in the case of the tiller injection treatment and all the florets showed seed set in the doubled plants. Stomatal guard cell length provided rapid, early‐stage and unambiguous analysis of ploidy level on the basis of 10 guard cell observations per plant.     

AFLP analysis indicates no introgression of maize DNA in wheat x maize crosses

Amplified fragment length polymorphisms (AFLPs) were used to follow the possible introgression of maize DNA into haploids of wheat as a side‐effect of exploiting wheat x maize hybridization for haploid production. AFLPs were generated with 64 MseI/ EcoRI and 64 MseI/ PstI primer combinations, and the AFLP profiles of haploids were tested against those of maize and of the regular wheat varieties involved in the crosses. On average, 45.1 and 110.7 fragments were produced per assay with the MseI/EcoRI and MseI/PstI combinations, respectively. Different numbers of fragments were produced for wheat and maize: an average of 81 in the haploid, 80 in the wheat parent, and only 67.1 in maize. No evidence was found for introgression of maize into the wheat genome. Three unique AFLP fragments were detected in haploids, which were not present in the parental wheat genotypes. These ‘novel’ AFLP bands in the haploids could be caused by nucleo‐cytoplasmic interaction in the hybrid zygote. Such instability in the wheat genome is defined as temporal, as it was not detected in further generations when colchicine‐doubled progeny of the haploids was tested for the presence of polymorphic fragments.     

Review of doubled haploid production in durum and common wheat through wheat × maize hybridization

Production of doubled haploids (DHs) is an important methodology to speed the process of breeding and development of mapping populations in crops. The procedure for DH production includes two major steps: haploid induction and chromosome doubling. In recent years, wide hybridization between wheat and maize has become a main approach for haploid production in wheat. In this method, the maize chromosomes are completely eliminated during the early development of the hybrid seeds after wheat spikes were pollinated with maize pollen. Numerous wheat cultivars and mapping populations have been developed using wheat–maize hybridization. In this study, we review the procedures of DH production of durum and common wheat via wide hybridization with maize, the factors which affect the efficiency of DH production, and the mechanism of selective elimination of the maize genome during the early development of the hybrid embryos. We also report a highly efficient protocol for DH production in durum and common wheat, which was established based on the optimal conditions for each of the factors that affect the efficiency of DH production.     

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.     

Molecular and agronomic evaluation of wheat doubled haploid lines obtained through maize pollination and anther culture methods

Although maize pollination (MP) and anther culture (AC) are alternative techniques widely used for wheat doubled haploid (DH) production, there is only limited information on the attributes of the plant materials produced through both methods. This study was conducted to evaluate genetic fidelity, transmission of parental gametes, and to compare field performance of DH populations produced by the MP and AC methods from the F1s of two crosses between spring bread wheat cultivars. The DH populations were compared to single seed descent (SSD) lines created from the same crosses. In total, 76 MP and 122 AC lines of the cross between cultivars of divergent origin were subjected to RAPD and AFLP analysis. Only changes in AFLP banding patterns, at similarly low frequencies, 0.18% (MP) and 0.21% (AC), were detected. The frequency of the DH lines affected by the variation, 14.5% (MP) and 14.8% (AC), was similar in both populations. For most of the DH lines, variation in 1‐2 loci only, out of several hundreds scored, was observed. A total of 14.3% (MP) and 22.2% (AC) marker loci showed the significant segregation distortion from the expected 1 : 1 ratio, but in at least one polymorphic locus the within‐cultivar variation was responsible for the skewed segregation. The field performance of the corresponding MP and AC lines derived from two crosses confirmed the equivalency of both DH populations. In most of the traits analyzed, the MP and AC lines performed the same as the SSD populations created from the same crosses. No, or very small differences in means and ranges, were observed when the best 10% of the lines from all three methods were compared. Moreover, the best 10 % of the lines of the cross between Polish wheat cultivars adapted to the local environment performed significantly better for some traits than different groups of checks used in the study.     

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.     

Recombination of gliadin genes of chromosome 1D in the common wheat hybrid carrying the introgression from Aegilops cylindrica

Recombination of co‐gliadin components of the allele ‘_/’ at the Gli‐D1 locus and the allele at the respective locus from Aegilops cylindrica was revealed in the common wheat hybrid carrying the introgression from Ae. cylindrica. Gliadins of single seeds from F₂ plants of the cross between the winter common wheat variety ‘Albatros Odesskii’ and the common wheat line carrying the introgression from Ae. cylindrica were analyzed by acid polyacrylamide gel electrophor‐esis. The average frequency of recombination detected between the gliadin components was 0.35%.     

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.     

Doubled haploid production in winter wheat and triticale genotypes,using the Hordeum bulbosum system

Summary An attempt was made to produce doubled haploids on 16 winter wheat and six spring and winter triticale genotypes thought to carry genes for interspecific incompatibility. The potential for haploid production was maximized by the use of Hordeum bulbosum genotypes selected for high crossability on crossable wheat genotypes, the use of two post-pollination applications of gibberellic acid and by the pollination of immature florets.A low frequency of seed was set on both the wheat and the triticale genotypes, having mean seed sets of 0.20 per cent and 0.27 per cent respectively. Although the frequency of embryos (seed quality) was high, doubled haploid production was further limited by poor embryo differentiation and regeneration. Haploid plantlets were obtained from the wheat cultivars Moulin and Renard, although successful chromosome doubling and doubled haploid production was achieved in Moulin only.     

Influence of winter and spring wheat genetic backgrounds on haploid induction parameters and trait correlations in the wheat × maize system

The interactive influence of winter and/or spring wheat genetic background on haploid induction parameters and trait correlation was studied by hybridizing five elite and diverse genotypes each of winter and spring wheat and their F₁s (winter × winter, spring × spring, and winter × spring, generated in a diallel design excluding reciprocals) with a single genotype of maize. Data were recorded with respect to per cent seed formation, embryo formation, and regeneration. High genetic variability was present among the wheat genotypes (parents + F₁s) for the three haploid induction parameters. Significant differences were obtained within and between different groups viz., spring wheats, winter wheats, spring × spring wheats, winter × winter wheats, and winter × spring wheats with respect to the three haploid induction parameters based on ANOVA. The winter genotypes (winter parents and winter × winter wheat hybrids) responded better than the spring groups (spring wheat parents, spring × spring and winter × spring wheat hybrids) with respect to embryo formation and winter × spring wheat hybrids yielded significantly the highest numbers of regenerants. Correlation studies amongst the haploid induction parameters indicated that the genes controlling seed formation and haploid plantlet regeneration are negatively correlated when the genetic backgrounds of both ecotypes are combined in winter × spring hybrids. Haploid embryo formation had no association with seed formation and regeneration in all genetic backgrounds, suggesting independent inheritance.     

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 in triticale (×Triticosecale Wittm.) by means of crosses with maize (Zea mays L.) using picloram and dicamba

The aim of the present study of triticale × maize crosses was to find an appropriate growth regulator treatment to improve the yield of triticale haploids and the subsequent production of doubled haploids. The growth effect in unpollinated ovaries of triticale was examined after treatment with 1000 mg/1 indole-3-acetic acid (IAA) or 100 mg/1 solutions of the following auxin analogues: 2,4-dichlorophenoxyacetic acid (2,4-D), 3,6-dichloro-o-anisic acid (dicamba), 4-chloro-o-tolyloxyacetic acid (MCPA), phenylacetic acid (PAA), 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4,5-trichlorophenoxyacetic acid (2,4,5- T), respectively. Dicamba stimulated growth of the ovaries significantly more than picloram and both stimulated more growth than the other growth regulators tested. Neither dicamba nor picloram induced embryo development in unpollinated pistils. Dicamba and picloram solutions, at concentrations of 25, 50, 75 and 100 mg/1, were subsequently applied to pistils of triticale pollinated with maize. On average, between 17.1 and 21.5 embryos/100 fiorets were excised after treatment with 75 or 100 mg/1 solutions of picloram or dicamba but the concentrations of 20 and 50 mg were less effective. The frequencies of excised embryos did not differ between genotypes. Seventy-six green haploids were obtained from 100 embryos rescued in vitro on the 190–2 and modified B5 media, the first medium being superior. The plants were subjected to colchicine treatment at the 3–4 tiller stage. Out of 68 plants brought to maturity, 25 exhibited fertile sectors. In comparison with previous studies, picloram and dicamba significantly improved the efficiency of the triticale × maize crossing. The low dependence on the mother germplasm makes triticale × maize crossing an efficient alternative to the androgenetic methods of doubled haploid production in triticale.     

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.     

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.     

偏凸-柱穗山羊草双二倍体SDAU18 PMC MI染色体数目异常

为探讨偏凸-柱穗山羊草双二倍体SDAU18减数第一分裂中期(PMCMI)染色体丢失特点,利用改良卡宝品红压片法,对SDAU18及其双亲的PMCMI染色体构型进行观察。结果表明:在SDAU18的同一枚花药中,部分PMCsMI出现了不同程度的染色体丢失现象,发生染色体丢失的PMCMI频率平均为4.64%,染色体数目异常的PMCsMI的平均染色体构型为:2n=12.77I+5.26IIR+1.57IIO+0.02III=26.49。其双亲的PMCMI的染色体数目均表现正常,均由14个二价体组成。SDAU18PMCSMI染色体数目变异的特点比较罕见。