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.     

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.     

Variation of the crossability of durum wheat with maize

With the aim of examining crossability of durum wheat with maize, two sets of durum wheat genotypes and a set of D-genome chromosome substitution lines of the durum wheat variety ‘Langdon’ were crossed with maize, and followed by 2,4-dichlorophenoxyacetic acid (2,4-D) treatment in detached-tiller culture. In crosses of 25 durum wheat genotypes (breeding lines) with maize, percent frequencies of embryo formation increased from 1.4% to 2.8% by adding silver nitrate to the detached-tiller culture solution. In crosses of 32 durum wheat genotypes (advanced lines and varieties) with maize using the silver nitrate addition, frequencies of embryo formation ranged from 0.0% to 15.8%; seven genotypes showing more than 6.0% embryo formation frequency were related in their pedigrees. In crosses of a set of chromosome substitution lines with maize, higher frequencies of embryo formation were obtained in substitution lines with chromosomes 1D, 3D, 4D and 7D. These results suggest that 1) adding silver nitrate to the 2,4-D treatment increases overall frequency of embryo formation but is not effective enough to induce the development of seeds and embryos from all durum wheat genotypes, and 2) some D-genome chromosomes substituted in a durum wheat genetic background may enhance crossability with maize in combination with homoeologous chromosomes of durum wheat. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Genetic Variability for Haploid Production in Crosses Between Tetraploid and Hexaploid Wheats with Maize

Crosses were made between 14 wheat genotypes (11 tetraploid, 3 hexaploid) and a single Fl hybrid of maize that was used as the male parent. The experimental design consisted of randomized blocks with three replications. Plants were grown under controlled greenhouse conditions (day length 16 h and temperature 25 °C/15 °C, day/night). To enhance embryo survival, 2, 4-D treatment (10 mg/1) was applied to spikes 24 h after pollination with maize. Embryos were recovered from all tetraploid and hexaploid wheats at a rate of 2.09 to 26.76 per 100 pollinated florets. Haploid and doubled haploid plants were obtained from all hexaploid genotypes (T. aestivum) and from 5 of 11 tetraploid genotypes (T. turgidum var.). The most important point of these experiments was the ability to produce haploid plants from tetraploid wheat for two reasons: firstly, anther culture cannot be applied in tetraploid wheat (T. turgidum var.) due to the inefficiency of embryo formation and the high proportion of albino plants. Secondly, to date, crosses between tetraploid wheat and maize have resulted in embryo formation, but not in haploid plants.     

A simple technique for the identification of embryo-carrying seeds from wheat × maize crosses prior to dissection

Embryo rescue is an important step in the wheat x maize system of induced haploidy in wheat. On average, only one-third of the seeds carry embryos, but they all have to be dissected because no morphological distinction is available to distinguish between seeds with and without embryos. We here report a simple technique in which immature seeds from wheat × maize crosses are placed on a transparent surface and illuminated from above. The free-floating embryo, settled at the bottom of the seed, can be seen from below or in an image reflected on a plane mirror. Using this technique 97.8% of the embryo-containing seeds could be detected prior to dissection. The technique will be useful in reducing the time and labour involved in embryo culture, thereby scaling up haploid production.     

Effect of parental genotypes on haploid embryo and plantlet formation in wheat × maize crosses

Genotypic influence of both male and female parents on haploid production through interspecific crosses was studied using eight wheat and four maize genotypes. The average numbers of embryos and green haploid plantlets obtained per pollinated floret were 17.6% and 10.1%, respectively. Clear genotypic influence of the wheat genotype was detected, but heterozygosity of the wheat did not affect haploid production. Analogous response to anther culture and interspecific crossing was observed, still a wheat variety which did not respond to anther culture, produced 1.1 plantlets per pollinated spike upon maize pollination. This appears to be a major advantage of interspecific crossing compared to anther culture technique in wheat. Circumstantial evidence is presented for specific wheat × maize interaction on haploid plantlet formation. Rye chromatin enhanced haploid production but only in a complete 1B/1R substitution line. Ovaries with an embryo were found to be dispersed evenly all over the wheat spike, suggesting that within certain limits the developmental stage of ovaries and thus time of pollination within a spike are not as important as it was previously assumed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of spikelet position on the success frequencies of wheat haploid production using the maize cross system

Barbela is an old Portuguese landrace of wheat that is highly genetically heterogeneous. Different Barbela populations when subjected to aluminium stress show variable levels of tolerance. In order to study the inheritance of this character, doubled haploid (DH) lines were developed. These DH were obtained by intergeneric crosses of 14 different lines of Barbela with maize. During this process the efficiency of the technique was evaluated and suggestions for its improvement were obtained. Several parameters were studied in the crosses: % of crossability, % of embryos per florets pollinated and % of embryos per seed set. The different genotypes of Barbela showed significant variation for the parameters analysed. When the reciprocal crosses were analysed, no differences were found, indicating that cytoplasm differences do not influence the parameters of DH production. However, different spikelet positions (lower, middle and upper) gave highly significant differences in all parameters analysed. Highest success frequencies were obtained for pollinated spikelets in the middle of the spike. This can indicate that concentrating on the middle part of the spike can increase the frequency of DHs obtained using inter generic crosses of wheat with maize. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Production of haploids in bread wheat, durum wheat and hexaploid triticale crossed with pearl millet

Pearl millet is an efficient alternative to maize as a pollen source for haploid production in bread wheat. To compare haploid production frequencies in other Triticeae species, the crossabilities of two genotypes each of bread wheat, durum wheat and hexaploid triticale with four pearl millet genotypes and a maize control were examined. Embryos were obtained from crosses of all three species with both pearl millet and maize. However, significant differences in crossability were found among the three species (10.5–79.8% seed development and 1.4–15.8% embryo formation), as well as among genotypes of durum wheat (7.2–23.7% and 2.1–6.4%) and hexaploid triticale (0.3–20.6% and 0.1–2.7%). Crossability of bread wheat with pearl millet was relatively high. Haploid plants were regenerated from crosses of all three species with pearl millet. As in the case of maize crosses, low crossabilities of durum wheat and hexaploid triticale with pearl millet can be attributed to the absence of D-genome chromosomes.     

Identification of the spontaneous 7BS/7RL intergenomic translocation in one F1 multigeneric hybrid from the Triticeae tribe

The F1 AABBRH^ch hybrids studied here were produced by crosses between the Portuguese triticale cultivar 'Douro' (AABBRR) and the tritordeum line HT9 (AABBH^chH^ch). Fluorescent in situ hybridization performed with genomic DNA probes genomic in situ hybridization (GISH) from rye and Hordeum chilense allowed the unequivocal parental genomes discrimination in all hybrids. Among 55 plants, one presented a spontaneous wheat–rye translocation which was successfully detected after GISH. Recombinant chromosomes identification was made after reprobe with pTa71 and pSc119.2. Nine rDNA loci were detected by pTa71 and pSc119.2 identified the chromosome arms involved in the translocation, after comparing the observed hybridization patterns with those described by several authors. We identified the spontaneous wheat–rye translocation as being the 7BS/7RL. Many wheat–rye translocations have been found (e.g. 1BL.1RS and 1AL.1RS), but as far as we know, this is the first time that this translocation is reported. We considered it helpful for wheat breeding programmes as it could provide the transference of interesting agronomic characteristics from rye (e.g. leaf rust resistance) to wheat.     

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.     

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.     

利用小麦ⅹ玉米诱导小麦单倍体形成的研究

摘 要：利用小麦(Triticum aestivum L.)与玉米(Zea mays L.)进行远缘杂交,是获得小麦单倍体的一条重要途径,也是获得双单倍体(Double haploid ,DH)植株、构建小麦遗传群体的重要途径之一。本研究为创建小麦抗吸浆虫DH群体,在温室条件下对5个小麦抗、感吸浆虫杂交组合进行了单倍体诱导试验。结果表明：小麦不同基因型对诱导单倍体胚形成有较大影响;小麦授粉后2,4-D适宜的点药时间为6－36小时;适宜的2.4-D药液为 10－30mg/L;授粉后适宜的剥胚培养时间为14－16天,授粉后茎秆离体培养有较好的诱导效果,培养温度以20-250C为宜。本文提出了在温室条件下高效诱导小麦单倍体植株的方法及条件。     

小麦-黑麦二体代换系间杂交诱导染色体易位的研究

摘 要：小麦-黑麦二体代换系间杂交,是诱导小麦-黑麦染色体易位的重要途径,有理论与应用价值。本研究利用DS1R/1D与DS5R/5A、DS6R/6A杂交,结果表明,F1减数分裂有19个二价体、4个单价体,有部分同源染色体配对和染色体易位现象;F2 对带有黑麦性状的24株普通小麦类型植株进行原位杂交检测,共检测出10株有染色体易位,易位频率为41.6%。易位株系为：06-16-7、06-16-3、06-16-5、06-16-7、06-16-8、06-17-7、0617-11、06-17-15、06-17-16、06-18-5。由于亲本选配、检测植株有一定的目的性,易位植株均表现有一定的应用价值。     

利用普通小麦、硬粒小麦种间杂种优势选育高千粒重新种质研究

为了提高千粒重,连续8年对普通小麦(T.aestivum)和硬粒小麦(T.durum)的正反交后代进行种质创新研究。结果表明:利用普通小麦、硬粒小麦种间杂种优势,是实现种质创新,培育高千粒重新种质的一条有效途径,在入选的319个子粒饱满度一级的株系中,260个株系千粒重超全国小麦黄淮区域试验对照品种石4185(亲本),占入选株系的81.5%,其中,最高千粒重为61.22 g,超亲优势为46.28%;正交、反交后代在该性状上无差异,表明子粒千粒重与细胞质无关;目前已选育出抗旱、抗病、农艺性状优良,千粒重40.1~61.22 g的株系263个,可直接用于小麦遗传育种研究及新品种选育。     

寒地多年生小麦的选育与细胞遗传学分析

通过杂交方法获得八倍体小偃麦与中间偃麦草杂种后代，对该杂交后代进行了形态学观察和细胞遗传学分析。杂交当代结实率为10%~39%；F₁表现为两亲中间型，多年生，抗小麦多种病害，生长的第2和第3年结少量种子，结实率为2%~3%；F₂分离复杂，出现八倍体小偃麦类型和中间偃麦草类型的多年生材料；F₃和F₄代出现一些普通小麦类型的多年生小麦,表现多分蘖、多小穗、抗病、抗寒。F₁根尖减数分裂中发现49条染色体，在减数分裂中期I形成14~17个二价体和4~21个单价体；而F₂和F₃代减数分裂时形成14~21个二价体和9~17个单价体。杂种后代结实率逐代恢复。F₁植株已在田间自然条件下生长5年。从F₄代中获得了4个植株高大(140 cm)、分蘖丰富(60个以上)、小穗多(25~30个)的饲草型多年生小麦株系，它们不仅具有良好的刈割再生能力，而且兼抗多种病害，抗寒性好，草质与中间偃麦草相似。还获得了一些普通小麦类型的多年生株系，有待进一步改良。这些结果为多年生小麦的遗传研究和利用提供了信息和材料基础。     

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.     

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.     

Relative Efficiency of Anther Culture and Chromosome Elimination Techniques for Haploid Induction in Triticale × Wheat and Triticale × Triticale Hybrids

Summary The study was undertaken to evaluate the relative efficiency of anther culture and chromosome elimination (by crosses with maize) techniques of haploid induction in intergenotypic triticale and triticale × wheat hybrids. For this, 15 triticale × wheat and 8 triticale × triticale F₁ hybrids were subjected to anther culture and were also simultaneously crossed with the `Madgran Local' genotype of maize (Zea mays L.) to induce haploids through the chromosome elimination technique. The haploid embryo formation frequency through the chromosome elimination technique was significantly higher in both, triticale × wheat (20.4%) and triticale × triticale (17.0%) F₁ genotypes, as compared to the calli induction frequencies through anther culture (1.6 and 1.4%, respectively). Further, four triticale × wheat and three triticale × triticale F₁ genotypes failed to respond to anther culture, whereas, all the F₁ genotypes formed sufficient number of haploid embryos through the chromosome elimination technique with no recovery of albino plantlets. The haploid plantlet regeneration frequencies were also significantly higher through the latter technique in both triticale × wheat (42.7%) and triticale × triticale (49.4%) F₁s as compared to anther culture (8.2 and 4.0%, respectively), where the efficiency was drastically reduced by several constraints like, high genotypic specificity, low regeneration frequency and albinism. The overall success rates of obtaining doubled haploids per 100 pollinated florets/anthers cultured were also significantly higher through the chromosome elimination technique (1.1% in triticale × wheat and 1.5% in triticale × triticale hybrids), proving it to be a highly efficient and economically more viable technique of haploid induction as compared to anther culture, where the success rates were only 0.2% and 0.1%, respectively.