Some aspects of cross-pollination in wheat (Triticum aestivum L.) 2. Anther extrusion and ear and plant flowering pattern and duration

Summary In the glasshouse the five spring wheat varieties Gaby, Jufy I, Opal, Orca and Peko were investigated for differences in number of anthers extruding during florescence. A maximum of 4 ears per plant was maintained. The mean of the absolute number of extruding anthers per ear varied from 105 (Peko) to 170 (Gaby). Also, significant differences were established in the absolute number of extruding anthers divided by the number of seeds set in each ear; this value ranged on an average of 2.27 (Opal) to 3.58 (Gaby). It is remarkable that a variety-ear number interaction manifests itself for both mentioned data: as far as Jufy is concerned the first developing ear produces least, whereas in Orca and Peko it produces most. No significant differences in this respect occur between the first 4 ears of a plant of the varieties Gaby and Opal. A simple relationship between number of extruding anthers and pollen donor capacity on the field could not be demonstrated.The flowering pattern and duration of ears and plants of these five varieties varies considerably. The mean flowering duration of each individual ear runs from 4.92 (Opal) to 6.35 days (Jufy). In addition to this a difference in flowering pattern is found, i.e. a certain number of days after the beginning of flowering Jufy appears to have turned out the smallest percentage of its total production of extruding anthers, while Opal had produced the greatest (Fig. 2). As regards this phenomenon, the first ear of a plant generally appears to lag significantly behind the second and third. Trying to explain these phenomena, on the one hand it was taken into account that in the case of varieties which extrude many anthers, florets of the third rank and higher will be more involved; these are not scored until later in the flowering period of the ear, which implies a proportionally smaller share for the first flowering days. On the other hand a slower or faster physiological flowering process is supposed.There appears to be a much bigger difference in outset of flowering between the first and the second ear of a plant than between the second and the third. For Jufy the first mentioned difference is bigger than for the other varieties. The mean flowering duration per plant of these five varieties varies (for 3 ears per plant) from 8.6 (Opal) to 10.7 days (Jufy).     

Electron-microscopy on anther tissue and pollen of male sterile and fertile wheat (Triticum aestivum L.)

Summary 1. Anther tissue and pollen grains of cytoplasmic male sterile and fertile wheat were studied with the aid of an electron-microscope for differences in cytoplasmic structures.2. Clear and consistently occurring cytoplasmic differences were not observed, although unidentifiable particles were discovered in both the sterile and the fertile material.3. Male sterility finds expression in the small number of organelles in the sterile pollen grains which consequently are little or hardly physiologically active and thus subject to a rather rapid degeneration. On the contrary the fertile pollen grains are full of cell organelles and therefore well equipped for a complete development.4. The fertile pollen grain contains a clear intine which is thicker than the exine. Only a very thin intine was observed in the sterile material.5. Male sterility has no visible influence on exine formation. Ubisch bodies occur in the anthers of both male sterile and fertile plants. There is no sign of any contribution to exine formation on the part of the Ubisch bodies. There was more evidence of exine formation from within than from without. The normality of exine formation of sterile pollen grains need not indicate exine formation from without; it suggests rather that exine is largely formed before degeneration.     

Some aspects of cross-pollination in wheat (Triticum aestivum L.). 4. Seed set on male sterile plants as influenced by distance from the pollen source,pollinator: Male sterile ratio and width of the male sterile strip

Summary The literature dealing with seed set on male sterile wheat plants by open pollination is critically reviewed with special attention to the influence of the distance from the pollen source and the pollinator: male sterile ratio. It is suggested to indicate seed set only as a percentage of the fertile analogue.Own investigations in Wageningen, the Netherlands, indicated that seed set on male sterile wheat plants on various distances from a pollen source of 6×6 m of a mixture of three spring wheat varieties amounted to maximally about 36%, but decreased quickly at increasing distances. Apart from an evident effect of the wind direction, in 1968 average seed set became less than 10 % after 1 m distance from the pollen source; in 1969 and under more favourable experimental conditions for the first meter from the pollen source a decrease to 25% took place, becoming 10% only after 3 m. It is recognized that for practical conclusions the pollen field size applied is too small.Both in 1971 and 1972 the seed set increased with increasing pollinator: male sterile ratio. This increase always was relatively small and, consequently, the kg yield hybrid seed per ha to be expected always decreased when this ratio increased. Applying a female strip width of 2 m and pollinator (mixture of five spring wheat varieties): male sterile ratios of 1:2, 1:1 and 2:1, in 1971 the latter ratio yielded only 60% of the former. In 1972 the same ratios were tested, but besides three male sterile strip widths were applied: 2, 3 and 4 m. In this year the calculated percentage seed set was much higher than that of the previous years (maximum almost 80%), probably as a consequence of more and larger pollinator areas and a more adequate way of establishing the 100% value of seed set. Again the kg yield hybrid seed per ha strongly decreased with an increase of the pollinator: male sterile ratio. The efficiency of the different ratios depends on the width of the female strip. Unfortunately, but according to the expectation, the kg yield hybrid seed per ha decreases, apart from the male to female ratio, fixing the 2 m value at 100%, to about 88% at 3 m and 72% at 4 m female strip width.     

Relationship between anther culture response and aluminium tolerance in wheat (Triticum aestivum L.)

A combination of in vivo and in vitro selection methods were used to increase aluminium tolerance in wheat using wheat x triticale crosses. Both in vivo and in vitro aluminium treatments significantly influenced the anther culture response. In vivo selection at the seedling stage resulted in significantly higher embryo induction. On induction media containing aluminium, the embryoid induction frequency dropped significantly, but there was an increase in the green plant regeneration frequency. In spite of this effect, all doubled haploid (DH) lines were more tolerant to aluminium in seedling tests than the winter wheat parent. The application of in vivo aluminium selection, before the start of anther culture, increased the probability of obtaining DH lines with significantly higher tolerance, compared to the original population. After three selection cycles of the original populations, there was a significant difference in the root regrowth rate of tolerant and sensitive plants. Both sensitive and tolerant plants showed a decrease due to the presence of aluminium in the induction media, with a greater decrease occurring in sensitive plants. Correlation between the rate of root regrowth in the seedling test and the change in embryo induction was positive, but moderate, emphasising the fact that plants with higher root regrowth tended to be more tolerant of the presence of aluminium in the induction medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

The crossing of common wheat (Triticum aestivum L.) with cultivated rye (Secale cereale L.). I. crossability,pollen grain germination and pollen tube growth

Summary The crossing of common wheat (Triticum aestivum L.) with rye (Secale cereale L.), and especially the action of the crossability genes of wheat, was studied using the readily crossable wheat cv Chinese Spring (genotype kr ₁ kr ₁ kr ₂ kr ₂ >), the poorly crossable wheat cv Hope (genotype Kr ₁ Kr ₁ Kr ₂ Kr ₂ ), as well as the disomic substitution line of chromosome 5B of Hope into Chinese Spring (CS/Hope 5B, genotype Kr ₁ Kr ₁ kr ₂ kr ₂ ). By comparing crossability and actual fertilization, the poor crossability with rye of both cv Hope and the CS/Hope 5B substitution line was shown to result from absence of fertilization. Studies of pollen grain germination and pollen tube growth showed that the dominant alleles of the crossability genes manifested themselves through retardation and eventually inhibition of pollen tube growth at the style base and in the ovary wall. In Hope the growth of all pollen tubes was inhibited, whereas in CS/Hope 5B rarely fertilization was achieved. The recessive alleles of the crossability genes do not seem to have an influence on the growth of rye pollen tubes in wheat pistils.     

Chromosomal regions associated with green plant regeneration in wheat (Triticum aestivum L.) anther culture

Genetic capacity for green plant regeneration in anther culture were mapped in a population comprising 50 doubled haploid lines from a cross between two wheat varieties ‘Ciano’ and ‘Walter’ with widely different capacity for green plant regeneration. Bulked segregant analysis with AFLP markers and composite interval mapping detected four QTLs for green plant percentage on chromosomes 2AL (QGpp.kvl-2A), 2BL (QGpp.kvl-2B.1 and QGpp.kvl-2B.2) and 5BL (QGpp.kvl-5B).The three QTLs detected on chromosome 2AL and 2BL all derived their alleles favouring green plant formation from the responsive parent ‘Ciano’.The remaining QTL on chromosome 5BL had the allele favouring green plants from the low responding parent ‘Walter’. In a multiple regression analysis the four QTLs could explain a total of 80% of the genotypic variation for green plant percentage. None of the chromosomal regions with QTLs for green plant percentage showed significant influence on either embryo formation or regeneration frequencies from the anther culture. The three major QTLs located on group two chromosomes were fixed in a second DH population derived from two parents ‘Ciano’ and ‘Benoist’,both with high capacity to produce green plants. A QTL explaining31.5% of the genetic variation for green plant formation were detected on chromosome 5BL in this cross as well. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of anther culture-derived cell suspensions exclusively regenerating green plantlets in wheat (Triticum aestivum L.)

A reproducible procedure for deriving highly regenerable cell suspensions that can readily and consistently regenerate green plantlets in wheat is described. Initiation and selection of the right type of callus from anther cultures, which consisted of friable early embryogenic portions that can easily disperse in liquid medium was important for the establishment of rapidly growing embryogenic suspensions. Using this type of inoculum no significant variation between three different independent replications was noted when cell suspensions from eleven specially recombined doubled haploid lines were maintained on General medium supplemented with dicamba and a predominance of amino acid nitrogen. This approach also enhanced a long-term embryogenic competence of the cell cultures, with some of the suspensions retaining their morphogenic capacity over a period of more than 15 months. Depending on the medium composition high frequencies of embryogenesis (over 70%) and green plantlet regeneration (repeatedly producing 90–100% of green regenerants) were obtained from the cell aggregates for most of the embryogenic cell lines. Potential advantages of anther culture-derived embryogenic cell suspensions for transformation purposes are the high number of cell lines which can be established routinely and the apparent maintenance of a stable haploid genome by the regenerants in culture. It is anticipated that an increased use of anther or microspore derived doubled haploid techniques in future wheat breeding programmes may favour selection in the breeding material of plant types generally responsive to such protocols. This revised version was published online in July 2006 with corrections to the Cover Date.     

Some aspects of cross-pollination in wheat (Triticum aestivum L.) 1. Pollen concentration in the field as influenced by variety,diurnal pattern,weather conditions and level as compared to the height of the pollen donor

Summary For the large-scale production of hybrid seed the cross-fertilizing capacity of wheat must be increased. Breeding good pollinators is, therefore, a necessity. Five spring wheat varieties were examined for differences in pollen shedding capacity under normal field conditions. The examination was carried out by collecting pollen on object slides every hour from 06.00 till 20.00 h during flowering. In general, the variety Peko proved to be the best shedder of pollen, while Opal was the worst. Gaby, Jufy I and Orca were intermediate. Consistent results were hard to obtain mainly due to varying weather conditions.It was established that pollen is released throughout the whole period of daylight. The maximum release of pollen, however, occurred from 08.00 till 11.00 h. A less pronounced and much smaller peak was noticed from about 15.00 till 18.00 h. Most pollen appeared to be released at a temperature of 16–20°C and a relative humidity of 70–75%. Although there seems to be some relationship between the diurnal pattern of the pollen release and the indicated values of these meteorological factors, one cannot say that the former is entirely determined by the latter.It has been suggested in the literature that a male-sterile parent that is shorter than its pollinator would be most likely to produce good seed set in the hybrid wheat seed production field. Investigations showed that a greater concentration of pollen was found below the ear level of pollinator plants than at or above ear level.     

Identification of powdery-mildew-resistance genes in common wheat (Triticum aestivum L.). VI. Wheat cultivars grown in China

A total of 26 common wheat cultivars and advanced breeding lines grown in China were tested with a set of 11 differential powdery-mildew isolates. Seven cultivars were susceptible. Another seven cultivars showed the response pattern of resistance gene Pm2, either individually or in combination with genes Pm3d or Pm4a. Five cultivars expressed the resistance of gene Pm4b singly or in combination with Pm6. Another four cultivars exhibited the response patterns of genes Pm5, Pm6 and Pm8, respectively. Three cultivars, which included one breeding line with a pair of substituted chromosomes from Haynaldia villosa, presumably carrying the resistance gene Pm21, showed resistance-response patterns to all the isolates tested.     

Spike pretreatments, anther culture conditions, and anther culture response of 17 German varieties of spring wheat (Triticum aestivum L.)

The aim of this work was to establish an in vitro regeneration system from anther cultures of different German varieties of spring wheat (Triticum aestivum L.). Using ‘Nandu’ the most widely grown spring wheat cultivar in Germany, different culture conditions were investigated with regard to their influence on anther culture response. The best results were obtained when applying a cold pretreatment to the donor spikes and using the synthetic L3 induction medium, liquid or solidified with gelrite. The highest rates obtained in these experiments with ‘Nandu’ were 8.6% responding anthers, 22.3% embryoid induction, 15.3% albino regeneration and 5.5% green plant regeneration (all rates related to the number of cultured anthers). Of the ‘Nandu’ plants analysed, 51.1% were haploid and 44.3% were diploid, probably as a consequence of spontaneous chromosome doubling. When screening a further 16 commercial German varieties of spring wheat, 10 exhibited good anther culture response and four of these (‘Eta’‘Jondolar’, ‘Mieka’, and ‘Star’) proved to be highly responsive, reaching embryoid induction rates between 4.3 and 10.3% and rates of green plant regeneration between 5.4 and 10.7%.     

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.     

The effect of colchicine treatment on microspore division and microspore-derived embryo differentiation in wheat (Triticum aestivum L.) anther culture

Summary The relationship between division symmetry andin vitro microspore embryo genesis was studied using two winter wheat varieties of high embryogenic capacity. Anther cultures were treated with colchicine added to the induction media at concentrations of 0.01%, 0.02% and 0.04%. As a result of the colchicine treatment, the rate of symmetrical divisions increased significantly which was followed by a significant increase in the microspore-derived embryo frequency. The effect of colchicine was not dependent on the concentrations used. On the basis of this it can be supposed that there is a clear relationship between symmetric divisions and microspore-derived embryo differentiation.     

The crossing of common wheat (Triticum aestivum L.) with cultivated rye (Secale cereale L.). II. Fertilization and early post-fertilization developments

Summary Fertilization and early seed development was studied in the variety Chinese Spring of common wheat (Triticum aestivum L.) after pollination with rye (Secale cereale L.) and selfing, and in the common wheat variety Hope after selfing. In all three combinations the first pollen tube reached the micropyle in about 40 min after pollination. When pollinated with rye the migration of the sperm nuclei to the egg cell and the polar nuclei was delayed by about an hour. In the subsequent development until 72 hours after pollination the average cellular and nuclear doubling times of embryo and endosperm were about 16 and 8 hours for the interspecific combination. 18 and 9 hours for Chinese Spring selfed and 20 and 12 hours for Hope selfed.     

Growth habit in common wheat (Triticum aestivum L. em. Thell.)

Summary A study of the Vrn genotypes of 642 spring wheats supports the theory that only Vrn1, Vrn2 and Vrn3 exist in Tricticum aestivum. In none of the varieties investigated Vrn4 was present. Seven varieties, which according to literature carry Vrn4, showed to carry Vrn1, Vrn2 and/or Vrn3. Some varieties were mixtures of Vrn-genotypes, which could mislead geneticists in pooled data analysis. Other causes for misinterpretation of the data could be hybrid necrosis, hybrid dwarfness or a wrong determination of plants with a winter habitus. Only Hope was dominant on another Vrn locus. Its haploid Vrn-genotype is Vrn1 vrn2vrn3 Vrn5.     

Effects of desiccation and a change in temperature on germination of immature grains of wheat (Triticum aestivum L.)

A study was made of the effects of desiccation and a change in temperature on the germination of wheat grains harvested 20 days after anthesis. When the germination test was performed immediately after harvesting, germination percentages ranged from 5.2% to 10.7%. Germination percentages increased to 48.2% to 90.3% after grain had been desiccated at 20 °C and then hydrated at 10 °C. This increase occurred even if grains had been desiccated in an atmosphere of high relative humidity. The germination percentage of non-desiccated grains depended on the germination temperature. When the pericarp and testa were removed from embryos, the germination percentage of grains incubated at 20 °C and then at 10 °C was higher than that of grains incubated at 10 °C. In general, a low germination temperature is believed to be effective in breaking dormancy of wheat grains. However, a change in temperature stimulated germination to a greater extent than a constant low temperature. The germination percentage of 5-day cycle alternating temperature was greater than that with 1-day, 2-day and 10-day cycles. Although the germination of immature wheat grains was stimulated by both high and low germination temperatures, it is likely that cycles shorter and, also, longer than a critical period induce limited germination. Loss of dormancy commonly occurs when development of wheat grains proceeds at a high temperature, with imbibition at a low temperature. However, germination ability of non-desiccated immature wheat grain was enhanced by a change in temperature during germination. This revised version was published online in August 2006 with corrections to the Cover Date.     

Preliminary Diallel Analysis of Anther Culture Response in Wheat (Triticum aestivum L.)

A four-parent complete dialleld mating-set of three Triticum aestivum L. cultivars ‘Edwall’, ‘Chris’ and ‘Spillman’, and an alloplasmic line of ‘Selkirk’ in Aegilops variabilis Eig. cytoplasm was used. Genetic components among total variation for callus induction, green plant percentage and green plant yield werr 92%, 80% and 77%, respectively, but lower (36%) for plant regeneration. General combining ability (GCA) variance components and narrow-sense heritabilities estimated for callus induction, green plant pereentnge and green plant yield were 74%, 66%, and 53%, and 0.68, 0.54 and 0.43. respectively. Specific combining ability (SCA) variancw was calculated as 85%) of total genctie variation for plant regeneration. Significant reciprocal effects for callus induction. green plant percentage and green plant yield were observed.     

Germination of pollen of weed rye (Secale segetale L.) on wheat (Triticum aestivum L.) stigmas and the growth of the pollen tubes

Summary Two gene pairs have been found regulating the crossability of wheat and rye. It is concluded from our work that the same genes regulate crossability between wheat and weed rye. The crossing barrier was not observed in the stigma, style or ovary wall because pollen tubes of weed rye were seen in these tissues irrespective of the wheat variety used as the female parent.Pollen grains germinated within 6 min after pollination. The tubes reached the region of the micropyle after about 40 min.     

Studies on multilines in wheat (Triticum aestivum L.).12. Breeding of a multiline variety by convergence of breeding lines

Summary The variation among the component lines of the KSML 3 (a multiline based on cultivar Kalyansona, spring bread wheat) was studied for agronomic characteristics. For days to earing and plant height the variation was small. This helped in imparting uniformity to the multiline. The lines had an improved tillering ability and had larger seeds. This partially explained the increased yield potential of the multiline as compared with Kalyansona.All the lines were susceptible only to one or two races of the yellow and brown rust. In no case any race was virulent against all the lines. In the field all the lines were resistant to both rusts.     

Substitution analysis of drought tolerance in wheat (Triticum aestivum L.)

Chromosome substitution lines of the wheat variety ‘Cappelle Desprez’ into ‘Chinese Spring’ were tested for drought tolerance in growth chambers in the Martonvásár phytotron. Three different moisture regimes were created: E1, fully irrigated control; E2, mid-season water stress (preanthesis); and E3, terminal water-stress during grain filling. Data were analysed to estimate the chromosomal location of the genes controlling relative water-content (RWC), relative water-loss (RWL), drought-susceptibility index (DSI) and phenotypic stability in each substitution line. Simultaneous consideration indicated that most of the genes controlling these characters are located on chromosomes 1A, 5A, 7A,4B, 5B, 1D, 3D and 5D.     

Evidence for genetic control and media effect on haploid regeneration in the anther culture of hexaploid wheat (Triticum aestivum L.)

Crosses were made between seven hexaploid wheat genotypes. Twenty-one F₁ hybrids and their parents were grown in a greenhouse with 16 h day/8 h night at 25°C and 15°C, respectively. The experiment was a complete randomized block design with three replications. Each replication consisted of one pot with three plants. Anther culture was performed in two different induction media (CHB and W14) and androgenetic traits were studied. Statistical analysis was carried out separately for each induction medium. Genetic variation was highly significant for androgenetic traits and the best parent (IBPT 19) produced 68 embryos and 9.3 green plants per 100 anthers in CHB medium. Genetic components were affected by induction media and some components were significant in one medium and non significant in the other. General combining ability (GCA) was significant for all androgenetic traits, except for albino plant regeneration in both media and total plant regeneration in CHB medium, whereas specific combining ability (SCA) was not significant for the traits studied. Narrow sense heritability was high for embryo induction frequency and green and total plant regeneration. All our results indicate that androgenetic parameters can be improved in hexaploid wheat by genetic means.