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.     

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.     

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.     

High Frequencies of Fertilization and Haploid Seedling Production in Crosses Between Commercial Hexaploid Wheat Varieties and Maize

Nineteen commercial hexaploid wheat varieties were crossed with the maize F₁ hybrid ‘Seneca 60’. Fertilization frequencies ranged from 32.1 % to 47.5 % of pollinated florets (mean 39.5 %) in the 14 winter wheat varieties and from 40.7 % to 51.4 % (mean 47.8 %) in the five spring wheat varieties. In some cases only an endosperm was formed and the frequencies of embryo formation were therefore slightly lower, being 28.2 % to 45.9 % (mean 36.4 %) for winter wheats and 39.8 % to 48.6 % (mean 45.1 %) for spring wheats. Mean values were significantly higher in the spring wheats but no significant variation was found between varieties within the spring or winter categories. In the five spring wheats the mean yield of embryos, and hence the potential yield of haploid plants, was 3.4-fold higher than with the tetraploid Hordeum bulbosum clone PB179. For the 14 winter wheats the figure was 10.9-fold higher. These differences were highly significant (p < 0.001) in all varieties. A single 2,4-D treatment given to spikes one day after pollination with maize enabled embryos to be recovered from all 19 varieties. A total of 311 embryos were recovered from 950 florets (an average of 7.3 embryos per spike) of which 191 germinated, giving an average yield of one haploid plant for every 5.0 florets pollinated (4.4 haploid plants per spike).     

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.     

In vitro chromosome doubling with colchicine during microspore culture in wheat (Triticum aestivum L.)

Isolated microspores of two DH lines of wheat were treated with 8 different colchicine concentrations up to 3 mM for either 24 h or 48 h during microspore culture. Untreated control cultures produced on average 220 embryos per spike (100,000 microspores), 68% of the regenerated plantlets were green, and 15% of the flowering plants were fertile. The colchicine treatments had a significant effect on chromosome doubling as measured by the percentage of fertile regenerants. Using colchicine concentrations around 1 mM the percentage of fertile plants among the regenerants was increased up to 53%. The highest number of embryos and regeneration rates were observed after 24 h colchicine treatment, while the highest frequencies of green plants and fertile plants were obtained with 48 h colchicine treatments. The highest number of DH plants per spike was found after treatment with colchicine concentrations of 300 to 1000 μM. Such treatments resulted in an estimated average between the two genotypes of 23 doubled haploid plants per spike. This revised version was published online in July 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.     

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.     

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.     

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 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.     

利用单倍体育种技术快速培育糯性小麦新品系

该研究利用关东 10 7和白火麦为亲本做常规杂交 ,用玉米花粉对其杂种F1进行单倍体诱导 ,单倍体加倍后得到 50个DH系 ,对这些DH系进行SDS—PAGE电泳分析 ,有 6个株系是糯性小麦 ,其基因型为wx -aabbdd ;同时进行了这些DH株系种子的I2 -KI染色 ,上述 6株系种子胚乳染色均为棕红色。这 2种方法同时证明该 6个DH系为纯合糯性株系 ,说明SDS -PAGE电泳和I2 -KI染色方法鉴定糯性小麦是可靠的和准确的。该文还分析了 6个小麦株系在株高、单株穗数、千粒重及粒质地等性状上的异同。糯性小麦由于具有支链淀粉含量高、高峰粘度高和膨胀势高等特点 ,成为面条专用小麦 ;支链淀粉在轻工、医药等行业应用广泛。因此 ,糯性小麦的培育成功为小麦品质育种研究和支链淀粉的开发利用开辟了一条新途径     

Large-scale production of wheat and triticale double haploids through the use of a single-anther culture method

A total of 257 parental wheat and 38 triticale lines were used for anther culture. On average, 2.1 green wheat haploids were obtained per spike. This response occurred irrespective of the origin of the material (Germany, France, Sweden or UK) and 5 years of testing. Triticale responded with 5.3 green haploids per spike. Using the criterion that one parental line should give at least one green haploid per spike in the screening experiment, green haploids were produced from 88 out of 91 F₁ wheat breeding combinations and from each of 21 F₁ and F₂ triticale breeding combinations. An average of 4.7 green plants were obtained per spike from the wheat production programme, while the triticale programme gave an average of 6.2 green plants per spike. A single medium supplemented with different hormones for anthers and embryos was used for culture of both crops.     

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     

小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性

小麦与玉米杂交是诱导小麦单倍体最有效的途径之一, 但单倍体和双单倍体产生频率不稳定影响了该技术的应用。选用13个小麦杂种F₁代单交组合与玉米杂交, 研究了不同小麦生长环境、生长素处理、培养基和壮苗处理对单倍体及双单倍体产生频率的影响。小麦生长在大田, 去雄后割穗培养与玉米杂交平均得胚率为23.9%, 每个杂交穗平均得胚数6.8个, 均是返青后从大田移回冷温室盆栽的3倍以上;不同小麦杂交组合间胚产生频率存在明显差异。生长素Dicamba蘸穗处理平均得胚率是21.5%, 与2,4-D处理得胚率(21.1%)无显著差异, 但不同杂交组合间差异显著。B5培养基幼胚萌发率为70.9%~88.3%, 平均82.0%;1/2 MS培养基胚萌发率为70.0%~86.0%, 平均76.6%;两种培养基平均胚萌发率无显著差异。试管苗经壮苗培养基壮苗处理与试管苗经移栽壮苗处理后加倍效率分别是67.6%和8.6%。移栽壮苗处理的苗分蘖少, 生长较弱, 加倍处理后存活率低和加倍率低是其单倍体加倍效率低的原因。     

2,4-D浓度对玉米花粉诱导小麦产生单倍体幼胚得胚率的影响

在玉米花粉诱导普通小麦产生单倍体技术中,2,4-D浓度是影响幼胚得胚率的重要因素之一.为了确定2,4-D的适宜浓度,利用9个玉米材料和16个小麦材料进行了研究.在小麦散粉前和散粉后,授玉米花粉后12h和36 h分别用150、200、250、300、350 mg/L的2,4-D蘸穗2次,调查数据显示,散粉前处理得胚率平均值分别为6.74%、7.19%、8.44%、7.53%和6.61%;散粉后分别为2.53%、3.11%、4.70%、2.81%和2.67%.结果表明,小麦散粉前授玉米花粉后用250 mg/L的2,4-D处理得胚率最高.     

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.     

Harvest Index and Spike Index in Crosses between Wheat Genotypes with Different Spike Architecture

Crosses between vulgare wheat genotypes with different spike architecture were examined for total biomass per plant, grain mass per plant, and harvest index. The genotypes with branched spikes (turgidum type) and tetrastichon spikes were equal or superior to those with normal spikes in total biomass per plant but inferior in grain mass per plant. Consequently, they had significantly lower harvest indices. In the F₁ and F₂ generation, high heterosis for total biomass per plant occurred in all crosses between the genotypes with branched and normal spikes. There was heterosis for grain mass per spike in most crosses, but it was not as high as for total biomass. This investigation confirmed earlier findings that genetic changes towards the branched or the tetrastichon spike do not increase the potential for grain yield. Since, however, the crosses between the genotypes with branched and normal spikes showed high heterosis for grain mass per plant, lines with highly fertile but normal spikes may be expected in the segregating generations as a result of a genetic change in sink capacity. The genotypes with branched and tetrastichon spikes produced higher biomass per spike but lower spike index than the genotypes with normal spike. This may be an indication that the increase in vegetative area of the spike does not necessarily have a positive effect on grain mass per spike.     

Comparative Field Performance of Tissue Culture-Derived Lines and Breeder Lines of HY320 Spring Wheat

This study examines the performance of somaclonal spring wheat (Triticum aestivum L.) lines developed through culture of somatic embryos. Twenty-nine breeder lines of the spring wheat cv. ‘HY320’ were compared to 51 somaclonal lines of the same cultivar. Somaclonal lines were derived from 27 individual embryos (with up to four lines in each “family”). Somaclonal and breeder lines were evaluated in five field experiments in western Canada. Somaclonal lines were more variable than breeder lines for most agronomic, yield component and quality characters, suggesting that variability among somaclonal lines resulted partly from the tissue culture process. Somaclonal lines yielded, on average, 11 % less than breeder lines. Somaclonal lines had 3.8 % fewer spikelets per spike, 6.5 % fewer kernels per spike and kernels which were 2.7 % lighter. Somaclonal lines had greater test weight, protein concentration, and sedimentation values, and harder kernels.     

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.