Effect of pollinator on haploid production in durum wheat crossed with maize and pearl millet

The aim of this work was to analyse the influence of the male parent on the production of embryos and haploid plants in durum wheat crossed with maize and pearl millet, to find a proper trait to identify the most efficient pollinators and to evaluate the mixtures of pollen. Two genotypes of durum wheat, low and high responding, were crossed with eight pollen samples: (i) three maize hybrids, (ii) three pearl millet inbred lines, (iii) a mixture of maize pollen and (iv) another mixture of pearl millet pollen. No significant differences on embryos and haploid plant production were observed among the four samples of maize pollen, but there were clear genotypic differences for the production of haploids between genotypes of pearl millet. The best pearl millet genotype produced significantly more haploid plants than the other two and the mixture of pollen. There was no correlation between the production of embryos and haploid plants. Therefore, the production of haploid plants must be the criterion to identify superior pollinators. In addition, a mixture of pollen is inappropriate except when using genotypes previously identified as good pollinators.     

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 polyhaploids of hexaploid wheat using stored pearl millet pollen

The effects of drying and freezing on viability of pearl millet pollen were examined with the aim of using stored pollen in polyhaploid production of hexaploid wheat. Freshly collected pollen of pearl millet line NEC 7006 with 55% water content, germinated at a frequency of 80%. Pollen that was dried for two hours to 6% water content showed 50% germination frequency and maintained similar frequencies after the freezing process. In crosses of hexaploid wheat variety Norin 61 with fresh pearl millet pollen, embryos were obtained at a frequency of 27.6%. In crosses with pollen stored at -196 °C, -80 °C and -20 °C for one month, embryo formation frequencies ranged from 27.5 to 17.4%. After five and twelve months of storage, the frequencies ranged from 29.7 to 14.6% at storage temperatures of -196 °C and -80 °C, and from 8.0 to 3.2% at -20 °C, indicating significant differences among storage temperatures. However, no significant frequency difference was found among pollen water contents at the time of collection. All plants regenerated from crosses with pearl millet pollen stored for five months were wheat polyhaploids. These results suggest that stored pearl millet pollen is an efficient medium for producing polyhaploids in hexaploid wheat. This revised version was published online in August 2006 with corrections to the Cover Date.     

Morphological and physiological differences in the response of cereals to zinc deficiency

Greenhouse and growth chamber experiments were carried out using seven bread wheat (Triticum aestivum), three durum wheat (T. durum), two rye (Secale cereale), three barley (Hordeum vulgare), two triticale (x Triticosecale Wittmack) and one oat (Avena sativa) cultivars to study response to zinc (Zn) deficiency and Zn fertilisation in nutrient solution and in a severely Zn deficient calcareous soil. Visual Zn deficiency symptoms, such as whitish-brown necrotic patches on leaf blades, developed rapidly and severely in the durum wheat and oat cultivars. Bread wheat showed great genotypic differences in sensitivity to Zn deficiency. In triticale and rye, visual deficiency symptoms were either absent or appeared only slightly, while barley showed a moderate sensitivity. When grown in soil, average decreases in shoot dry matter production due to Zn deficiency were 15% for rye, 25% for triticale, 34% for barley, 42% for bread wheat, 63% for oat and 65% for durum wheat. Differential Zn efficiency among and within cereal species was better related to the total amount of Zn per shoot, but not to the Zn concentration in the shoot dry matter. However, in leaves of Zn efficient rye and bread wheat cultivars, the activity of Zn-containing superoxide dismutase was greater than in Zn inefficient bread and durum wheat cultivars, suggesting higher amounts of physiologically active Zn in leaf tissue of efficient genotypes. When grown in nutrient solution, there was a poor relationship between Zn efficiency and release rate of Zn-chelating phytosiderophores from roots, but uptake of labelled Zn (⁶⁵Zn) and its translocation to the shoot was higher in the Zn efficient rye and bread wheat cultivars than in inefficient bread and durum wheat cultivars. The results demonstrate that susceptibility of cereals to Zn deficiency decline in the order durum wheat > oat > bread wheat > barley > triticale > rye. The results also show that expression of high Zn efficiency in cereals was causally related to enhanced capability of genotypes to take up Zn from soils and use it efficiently in tissues. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

Comparison of rye,triticale, durum wheat and bread wheat genotypes for Fusarium head blight resistance and deoxynivalenol contamination

Small-grain winter cereal crops can be infected with Fusarium head blight (FHB) leading to mycotoxin contamination and reduction in grain weight and quality. Although a number of studies have investigated the genetic variation of genotypes within each small-grain cereal, a systematic comparison of the winter crops rye, triticale, durum and bread wheat for their FHB resistance, Fusarium-damaged kernels (FDK) and deoxynivalenol (DON) contamination across species is still missing. We have therefore evaluated twelve genotypes each of four crops widely varying in their FHB resistance under artificial infection with one DON-producing F. culmorum isolate at constant spore concentrations and additionally at crop-specific concentrations in two environments. Rye and triticale were the most resistant crops to FHB followed by bread and durum wheat at constant and crop-specific spore concentrations. On average, rye accumulated the lowest amount of DON (10.08 mg/kg) in the grains, followed by triticale (15.18 mg/kg) and bread wheat (16.59 mg/kg), while durum wheat had the highest amount (30.68 mg/kg). Genotypic variances within crops were significant (p ≤ .001) in most instances. These results underline the differing importance of breeding for FHB resistance in the different crops.     

Effects of D-genome chromosomes on crossability of hexaploid triticale (X Triticosecale Wittmack) with maize

With the aim of producing polyhaploids of hexaploid triticale, 20 genotypes from a CIMMYT breeding programme and eight D-genome chromosome substitution lines of ‘Rhino’ were crossed with maize. In crosses between 20 triticale genotypes and maize, 15 lines produced embryos. Frequencies of embryo formation ranged from 0.0 to 5.4%, with an average of 1.1%. From a total of 200 pollinated spikes, 62 plants were regenerated. Most regenerated plants were polyhaploids with 21 chromosomes, and few aneuhaploids with 22 chromosomes were found. In crosses of triticale substitution lines with maize, all the lines produced embryos, while ‘Rhino’ produced no embryos at all. Higher frequencies of embryo formation were obtained in substitution lines with chromosomes 2D and 4D. These results suggest that D-genome chromosomes in a triticale genetic background have the effect of increasing the frequency of polyhaploid production in triticale x maize crosses.     

Low relative humidity increases haploid production in durum wheat X maize crosses

Haploidization is a useful tool for genetic analysis and plant breeding, but a consistent and satisfactory protocol for haploid production has been difficult to achieve in durum wheat. The objective of this study was to analyze the influence of the relative humidity of the environment, when culturing detached tillers during the production of haploids plants in durum wheat by the maize method. Thirty‐eight F3 lines from eight crosses of durum wheat were pollinated with bulked pollen from three commercial maize hybrids. A mixture of 2‐4D and dicamba was used as a hormone treatment. The numbers of caryopses, embryos and haploids plants were scored. When 65‐85% (light‐dark) humidity was substituted for 55‐65% the number of haploids per spike increased notably. This increased frequency was largely attributed to a rise in the production of generated caryopses. On average, 15.2 vs. 9.3 caryopses, 5.0 vs. 2.8 embryos, and 3.1 vs. 0.6 haploid plants, per spike, were produced under low and high humidity regimes, respectively.     

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.     

Effects of preharvest sprouting on the genetic structure of durum wheat 'landraces'

Preharvest sprouting that occurs in wheat might affect seed viability and cause genetic erosion during periodical rejuvenation of durum wheat accessions in a gene bank. Two durum wheat landraces (MG 7713 and MG 7805) that had been rejuvenated for several years and did show a high percentage of presprouted seeds in the lot from the fourth rejuvenation cycle were identified. The frequency of durum and bread wheat genotypes and the distribution of the two species in three seed classes (ungerminated seeds, seeds with swollen embryo and germinated seeds) were studied. The modified phenol test was used to identify durum and bread wheat seeds and the genotypic frequencies within each species were assessed on the basis of acid polyacrylamide gel electrophoretic patterns of gliadin storage proteins. In these two landraces, durum wheat was more susceptible to preharvest sprouting than bread wheat and the frequency of bread wheat seeds significantly increased over the three rejuvenation cycles examined. Despite this, preharvest sprouting did not cause significant changes in the genotypic frequencies observed within species or loss of some genotypes that could not be attributed to susceptibility to sprouting.     

Wheat EST-SSRs for tracing chromosome segments from a wide range of grass species

Transferability of 116 common wheat expressed sequence tag–simple sequence repeats (EST-SSR) markers was investigated on 168 accessions representing 18 grass species to identify new alleles useful for wheat improvement. Transferability among the Triticeae ranged from 73.7% for Aegilops longissima to 100% for wheat subspecies ( Triticum compactum ) but was also good for less related species such as rye (72.8%) or maize (40.4%). On average, the number of alleles/locus detected by EST-SSR markers was 3.1 for hexaploid wheat. The polymorphism information content (PIC) values simultaneously estimated for Triticum aestivum and Triticum durum were similar for the two species (0.40 and 0.39, respectively). The allelic diversity within allogamous species was higher (0.352–0.423) compared with that of T. aestivum and T. durum (0.108 and 0.093, respectively). T. aestivum and T. durum shared the largest number of alleles (74.6%) while among the three ancestral diploid species of bread wheat, Aegilops tauschii had the highest percentage of alleles with T. aestivum (57.4%). These results indicate that grass orphan species can be studied using wheat EST-SSRs and can serve as a source of new alleles for wheat genetic improvement.     

Genetic characterization of spontaneous diploid androgenetic wheat and triticale plants

The homozygosity of spontaneous hexaploid plants derived from anther culture was evaluated in wheat and triticale by means of 18 and 22 microsatellite markers, respectively. Most of the spontaneous hexaploid plants were homozygous for all the loci tested and had chromosomes recombining for parental alleles. Only 12% of the spontaneous hexaploid wheat plants, 11% of the artificially doubled wheat plants and 4.4% of the spontaneous hexaploid triticale plants were heterozygous at one to three of the loci studied. This showed that spontaneous hexaploid plants mostly came from normal haploid cells, obtained after meiosis, that underwent spontaneous chromosome doubling. However, first or second division restitution, producing unreduced gametes, cannot be completely excluded to explain the origin of the spontaneous hexaploid plants. Cytomixis, involving only one chromosome, or development of aneuploid trisomic gametes, followed by chromosome doubling for the spontaneous hexaploid plants, or transposition events could also explain some of the results obtained.     

Durum wheat × maize crosses for haploid wheat production: Influence of parental genotypes and various experimental factors

To improve haploid plant production in durum wheat, the haplomethod involving intergeneric crossing with maize followed by embryo rescue was used. The influence of parental genotypes and various experimental factors were studied. Ten cultivars of Triticum turgidum ssp. durum (female parent) were crossed with eight genotypes of Zea mays. After pollination, plant stems were either maintained in situ or cut near the base and kept in a 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐sucrose solution. Ten to 18 days after pollination, embryos were excised from developed ovaries and cultured on one of MS, MS/2, or B5 media. Haploid embryos and plants were obtained (78 green haploid plants regenerated in 0 year). The wheat genotype was significant for ovary development, embryo and plant formation, whereas the maize genotype was significant only for embryo formation. Detailed results of all crosses showed the best crossing partner for each wheat genotype. Cutting the plant stems after pollination gave better results than maintaining them in situ. The optimal stage for embryo rescue was 14 days and B5 and MS/2 media were more efficient than MS for embryo culture.     

Enhancing Fusarium crown rot resistance of durum wheat by introgressing chromosome segments from hexaploid wheat

Compared with hexaploid wheat, tetraploid durum is more susceptible to Fusarium crown rot (FCR) infection. The feasibility of enhancing FCR resistance in durum wheat by introgressing chromosome segments of hexaploid wheat was investigated by generating and analysing a backcross population derived from a susceptible durum wheat variety ??Bellaroi?? (recurrent parent) and a resistant hexaploid genotype ??CSCR6?? (donor parent). Together with a few scattered segments on various chromosomes, segments of a large section of the donor chromosome 6B showed a significant effect in enhancing FCR resistance in the durum background. However, a known major locus on the donor 3BL conferring high level of resistance to FCR in hexaploid wheat failed to provide any improvement in resistance than that of the genome average once it was introduced into the durum wheat. A small proportion of the backcross population gave similar resistance to the bread wheat variety ??Kennedy??, a level of FCR resistance acceptable to durum growers. These lines share a 4B segment from the hexaploid donor, although the segment was not among those with the largest individual effect across the whole population. These results show that it is feasible to improve FCR resistance of durum wheat by exploiting hexaploid chromosome segments, although resistance loci of the hexaploid wheat may not function properly in durum backgrounds.     

Analysis of adaptation of barley,triticale, durum and bread wheat under Mediterranean conditions

Summary Yield data obtained from a comparative small grain cereals trial, grown for five consecutive growing seasons at a total of 23 environments in Cyprus, were subjected to regression analysis. Within each environment, yield trials consisted of a standard set of three cultivars or elite lines of barley, triticale, durum and bread wheat. The regression coefficient (b) of crop mean on the environmental index (I) and the mean square deviation from regression (sd²) were calculated for each crop. Each crop tended to have its own characteristic value of sd² and its magnitude was an excellent indicator of specific crop-environment interaction. The causes of large sd², for two of the four crops, were the susceptibilith of barley to lodging, when favourable conditions were encountered at high yielding environments, and triticale dependence on late season precipitation. Durum wheat and triticale had an average response to different yielding environments (b>1.19) and both were significantly different from those of bread wheat (1.08) and barley (0.54). Hence, barley, bread and durum wheat are specifically adapted to low, average and high yielding Mediterranean environments, respectively. The cultivation of triticale at the expence of durum wheat is not feasible. Furthermore, interactions between crops and environments demonstrated by the regression parameters, should constitute the basis for decision making, regarding crop adaptation in a region. The average yield in all environments should not be considered as a proper criterion for adaptation. In this study, triticale had a similar mean grain yield (3,842 kg/ha) to that of bread wheat, but was significantly higher yielding than barley or durum wheat (5 and 7%, respectively).     

Wheat x pearl millet hybridization: consequence and potential

Summary Eight grain pearl millet (2n=14) accessions were crossed as male to hexaploid spring wheat cv. Fukuho (2n=6x=42). An average of 80% wheat pistils showed pearl millet pollen tube entry in the ovules, compared to 56% in wheat x maize cv. Seneca 60 cross. Of the 15 embryos, obtained through in vitro immature seed culture from wheat x pearl millet crosses, 3 plantlets were produced and grown to maturity. These three were of the somatic chromosome constitution 2n=42, 21 and 22, respectively. Haploid wheat plant (2n=21) apparently originated from pearl millet chromosome elimination during embryogenesis. The 22 chromosome plant had retained a single pearl millet chromosome at tillering stage, but this chromosome was eliminated from pollen mother cells prior to and also during gamete formation. The significance and potential uses of this wide cross is discussed.     

The presence of three groups of Scalavatis and other hexaploid bread wheat plants contaminating durum wheat fields in Cyprus

Summary The mutual resemblance of 81 hexaploid wheat plants contaminating durum fields on Cyprus was investigated by determining their phenotypes of gliadins, glutenins and peroxidase. Three groups were generated by clustering programs. Group 1 consists of plants with ears resembling those of durum plants, plants of groups 2 and 3 have a bread wheat appearance. Suggestions on the origin of these three groups are made. Group 1 may derive from (back)crosses between hexaploid and durum plants since time immemorial. If so, the hexaploid plants would have served as donor of the D genomes, and durum plants as the recurrent parent. Groups 2 and 3 may derive from two introductions, either from two countries, or from the same country, but then in different periods.     

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.