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.     

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。     

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.     

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.     

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     

Efficient haploid induction in wheat by using pollen of Imperata cylindrica

Intergeneric hybridization between wheat ( Triticum aestivum L.) and a wild weedy species, Imperata cylindrica (2n = 20) resulted in the recovery of a high frequency of wheat haploids, which were obtained through the elimination of I. cylindrica chromosomes. Cytological analysis of the root tips revealed the somatic chromosome count of the regenerants equalled 21. Haploid regenerants were also obtained in all the crosses of wheat F1s with I. cylindrica , implying the genotype nonspecific nature of wheat x I. cylindrica hybridization. Variation among wheat F1 hybrids was observed with respect to seed formation (44.9-84.5%), embryo formation (15.1-47.7%) and regeneration (27.0-75.0%) in crosses with I. cylindrica. Comparisons based on the efficiency of I. cylindrica and maize ( Zea mays ) as pollen sources indicated that Imperata-mediated haploid production is equally efficient. There is natural coincidence of flowering period of I. cylindrica with that of wheat under sub-temperate conditions which is advantageous compared with maize, which cannot be grown during the winter season in the sub-temperate regions.     

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.     

Segregation of genetic markers among wheat doubled haploid lines derived from wheat x maize crosses

Summary Wheat doubled haploid (DH) lines were produced from the F₁ hybrid, Fukudo-komugi x Oligo Culm, through intergeneric crosses between wheat and maize. F₂ plants and 203 DH lines were analyzed for the segregation of the eight genetic markers, namely, grain proteins, grain esterases, GA-insensitivity and glume traits. The segregation in the F₂ plants fitted to the expected ratios. No deviation was observed among the DH lines, either, except for the glume pubescence. The result indicates the absence of correlation between the markers investigated and the efficiency of embryo formation in the DH lines.     

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.     

Early diagnosis of ploidy status in doubled haploid production of maize by stomata length and flow cytometry measurements

Reliable discrimination of haploid (H), doubled haploid (DH) and crossing (C) plants in early growth stages could streamline DH production in maize. By detecting in early growth stages undesirable sterile H plants and undesirable heterozygous C plants, a large proportion of resources required for DH production could be saved. The goal of this study was to evaluate the effectiveness of early classification of plants in growth stages V3‐V4 as H, DH or C in the context of DH production using flow cytometry and stomata length. As the reference classification, we used a field score based on plant phenotype commonly applied in DH production and research. Our results show that identification of misclassified C seeds is possible because the overlap in distributions of stomata length between H&DH and C plants is small and the association between flow cytometry and the reference field score is high. In contrast, overlap between H and DH distributions is substantial. Consequently, the main application we see for these classification methods in early growth stages is the identification of C seedlings.     

Genotyping of Anatolian doubled-haploid durum lines with SSR markers

In this study, doubled haploid lines generated from the durum wheat varieties, selections from Middle Anatolian landraces, ‘Çakmak-79’, ‘Berkmen-469’ and ‘Kunduru-1149’ (Savaskan et al., 1997), were analyzed using ten highly polymorphic microsatellite markers for genotyping and evaluation of genetic relationships between and within the doubled-haploid (DH) lines. The average PIC value was found to be 0.531. Populations of doubled-haploid lines of landrace selected cultivars ‘Çakmak-79’ and ‘Kunduru-1149’, were the two most distant populations with (δ μ)² = 1.42. ‘Berkmen-469’ x ‘Çakmak-79’ and ‘Berkmen-469’ x ‘Kunduru-1149’ yielded similar genetic distances, (δμ)² of 0.84 and 0.85, respectively. In addition, the genetic relationship between the progenitors of the DH lines together with other durum wheat varieties was analyzed. A meaningful relationship was obtained based on available pedigree information on the cultivars.     

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.     

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.     

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

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

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.     

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.     

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.     

Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize

Doubled haploid plants are useful in genetic studies and plant breeding, but a consistent and satisfactory frequency of production has been difficult to achieve in durum wheat. Triticum turgidum L., using the maize pollen method. The objective of this study was to develop an objective method of producing doubled haploids in durum wheat. Plant growing and handling conditions, aspects of hormone treatments, wheat genotype and pollen source were considered. The number of caryopses, embryos, haploids, doubled plants and doubled plants that set seed were measured. Although growth conditions, pollen source, method of handling plants and wheat genotype are important considerations, the type of hormone was found to be most significant in the production of doubled haploid plants. When 50mg/l dicamba was substituted for 100 mg/l 2,4‐D the number of doubled haploids per spike increased from 0.2 for the best 2,4‐D treatment to 1.3 for the dicamba treatment. This increased frequency was largely attributed to an increase in the number of caryopses generated for each spike emasculated and from an increased frequency of germination of embryos to haploid plantlets. The best production of caryopses was 0.41 caryopses per florest with 2,4‐D. The best production of haploids per 100 florets was 12 with dicamba and 1.65 with 2,4‐D. The frequency of one doubled haploid per emasculated spike through the use of dicamba is a practical level for generating populations for genetic studies.     

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.     

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。