Development of photoperiod-sensitive cytoplasmic male sterile (PCMS) wheat lines showing high male sterility under long-day conditions and high seed fertility under short-day conditions

A “two-line system” using photoperiod-sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm under long-day photoperiods (≧15 h) has been proposed as a means of producing hybrid varieties in common wheat (Triticum aestivum). The PCMS line is maintained by self-pollination under short-day conditions, and hybrid seeds can be produced through outcrossing of the PCMS line with a pollinator line under long-day conditions. Our previous studies revealed that PCMS lines showing complete male sterility under long-day conditions are necessary for practical hybrid wheat breeding, especially to obtain high hybrid purity in F₁ seeds. Furthermore, practical PCMS lines should have high seed fertility under short-day conditions, which is associated with female fertility. Wheat cv. Norin 26 with Ae. crassa cytoplasm exhibits high seed fertility under short-day conditions, and cv. Fujimikomugi with Ae. crassa cytoplasm shows high male sterility under long-day conditions. Here we developed practical PCMS lines derived from the F₁ generation of Norin 26 and Fujimikomugi (with Ae. crassa cytoplasm) that were then backcrossed to elite wheat lines.     

Photoperiod-sensitive cytoplasmic male sterility in wheat with Aegilops crassa cytoplasm

Summary Triticum aestivum cv. Norin 26 with Aegilops crassa, Ae. juvenalis or Ae. vavilovii cytoplasm (all D² type) has been studied relative to its photoperiodic response of male sterility and fertility restoration patterns. Alloplasmic lines of Norin 26 with a D² type cytoplasm showed almost complete male sterility under long-day conditions (15 h), but high male fertility under short-day conditions (14.5 h). No significant influence of temperature on reduction in male fertility was observed. Thus, this type of male sterility is called photoperiod-sensitive cytoplasmic male sterility (PCMS). The PCMS is expressed in the form of pistillody of stamens. Histological studies revealed that there were incomplete ovule-like structures instead of tapetal cells and pollen grains in the pistillate stamens. The floret differentiation stage of the plant is the stage that is sensitive to photoperiod. The PCMS can be used as a new means for hybrid wheat production, named two-line system. The PCMS line is maintained and multiplied by self-fertilization under short-day conditions, and hybrid seed can be produced by crossing the PCMS line with a pollinator line under long-day conditions. In contrast to the system of hybrid wheat production using the T. timopheevi cytoplasm, the present system requires only PCMS and pollinator lines.Abbreviation CS Chinese Spring - N26 Norin 26 - PCMS photoperiod-sensitive cytoplasmic male sterility     

Comparison of two fertility restoration systems against photoperiod-sensitive cytoplasmic male sterility in wheat

A ‘two‐line system’ using photoperiod‐sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm under a long‐day photoperiod ( 15 h) has been proposed as a new means of producing hybrid varieties in common wheat. The PCMS line is maintained by self‐pollination under short‐day conditions, and hybrid seeds can be produced through outcrossing of the PCMS line with a pollinator under long‐day conditions. Two kinds of fertility restoration systems against the PCMS are known. One is involved with a set of multiple fertility‐restoring (Rf) genes in the wheat cultivar ‘Norin 61’ located on (at least) chromosomes 4A, 1D, 3D and 5D. The other is controlled by a single dominant major Rf gene, Rfd1, located on the long arm of chromosome 7B in the wheat cultivar ‘Chinese Spring’. To examine the degree of fertility restoration by these two systems, nine PCMS lines were crossed with ‘Norin 61’ and ‘Chinese Spring’ as the restorer lines, and the F₁ hybrids were investigated. The degree of fertility restoration was estimated by comparing the seed set rates in the F₁ hybrids having the Ae. crassa cytoplasm and those with normal cytoplasm. The results revealed that the fertility restoration ability of a set of multiple Rf genes in ‘Norin 61’ was higher than that of the Rfd1 gene in ‘Chinese Spring’.     

Genetic analysis of fertility restoration against photoperiod-sensitive cytoplasmic male sterility in Triticum aestivum cv. Norin 61

Triticum aestivum cv. Norin 26 with the Aegilops crassa cytoplasm becomes almost completely male sterile when grown under a long-day condition (15 h of light or longer), but is highly male fertile under a short-day condition (14.5h or less). This type of male sterility is called photoperiod-sensitive cytoplasmic male sterility (PCMS). Genetic analyses were made of the fertility-restoring (Rf) genes effective against PCMS that are present in T. aestivum cv. Norin 61. Conventional and monosomic studies indicated that restoration of fertility is controlled by multiple Rf genes located on at least four chromosomes: 4 A, 1D, 3D and 5D. The genetic mechanism of fertility restoration by the genes of‘Norin 61’differs from the mechanisms reported for‘Chinese Spring’and a‘Norin 26’mutant line.     

An EMS-induced wheat mutant restoring fertility against photoperiod-sensitive cytoplasmic male sterility

Triticum aestivum cv. 'Norin 26' with Aegilops crassa cytoplasm shows photoperiod-sensitive cytoplasmic male sterility (PCMS). This alloplasmic line is almost completely male-sterile under long-day conditions (≥ 15h), but highly male-fertile under short-day conditions (≤ 14.5h). To obtain male—fertile mutants against PCMS, seeds of the alloplasmic line were treated with ethyl methane sulfonate (EMS). The M₃ generation was evaluated for PCMS expression, and one fertility-restoring (FR-mutant) line showing high male fertility under the long-day conditions was selected. Reciprocal F, hybrids between the FR-mutant and the alloplasmic 'Norin 26' showed male sterility under the long-day conditions, and continuous segregation with respect to the degree of fertility restoration occurred in their F₂ generations. These results indicate that multiple recessive mutations with minor effects, induced in the nuclear genome, are involved in the fertility restoration. In fact, no restriction-fragment-length polymorphisms of mitochondrial DNA between the FR-mutant and the alloplasmic 'Norin 26' are found.     

A comparison of cytoplasmic and chemically-induced male sterility systems for hybrid seed production in wheat (Triticum aestivum L.)

A comparison of two male sterility systems was carried out in wheat for hybrid seed production and hybrid performance. Seventeen hybrid combinations based on Triticum timopheevi cytoplasm were compared with their genetically equivalent CHA-facilitated combinations. The same set of restorer lines was used as parents in CMS- and CHA-based hybrids to maintain genetic equivalence. In the first experiment aimed at study of female line behavior and crossed seed production, the CHA treated lines showed significantly shorter heights whereas CMS lines were similar to the control. The two systems were equally effective in sterilizing rate. The outcrossing percentage of the CMS lines was almost twice that of the CHA treated lines. Thousand-grain weight of the crossed seeds on CMS lines was greater than on the CHA treated lines and control. On average, the germination percentage of seeds on CMS lines was double that of the CHA treated lines and the percentage of effective outcrossed seeds in CMS lines was 3 times more than that from CHA treated lines. The second experiment was conducted to examine the yield performance of the hybrids derived from the two systems of male sterility. A total of 40 entries including 20 hybrids and 20 parents were evaluated in the experiment. The mean grain yield of the CMS-based hybrids was greater than that of the CHA-based hybrids,the B and R lines. All the CMS-based hybrids showed significantly higher grain yields than their better parents whereas all the CHA-based hybrids, except two,showed no significant yield increases over their better parents. Possible reasons for differences in CMS- and CHA-based hybrid performance are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic and cytogenetic studies with a male sterile mutant of common wheat (Triticum aestivum)

Summary Genetic and cytogenetic studies were done on a male sterile mutant of the wheat variety Probus. Association of the 4A chromosome carrying the ms gene was studied in the F₁ of the male sterile Probus with Chinese Spring ditelo 4AS, with Transec and with line T4AS-DRS respectively. The presumption that the genetic male sterility of the mutant was due to a terminal deletion of the short arm of chromosome 4A could be confirmed.Linkage studies showed that the ms gene was at 17 map units from the dwarfing gene (Rht3) of Minister dwarf. This allows selection of short male sterile plants at the seedling stage.     

Physiological studies on grass-dwarf selection lines in wheat (Triticum aestivum L.)

Summary Low power heating wires insulated in a flexible plastic strip were used to heat the shoot meristematic region of grass-dwarf genotypes to a precise temperature. The results indicate that the shoot apical meristem is the region requiring 26°C for the initiation of reproductive development in these genotypes. A secondary effect of the reproductive growth induced by high temperature treatments, was a reduction in the high levels of peroxidase enzymes found in vegetative grass-dwarf plants. The heating wire provides a precise method to identify Type I, II and III grass-dwarf genotypes according to their temperature requirements, of 26°C, 21°C and 16°C respectively, for reproductive growth under the same temperature (16 C) and photoperiod (12 hours).     

Effects of nucleus,cytoplasm and male sterile nucleus-cytoplasm combinations on callus initiation in anther culture of wheat

Summary Nuclear and cytoplasmic factors affect tissue culture response in wheat (Triticum aestivum), and cytoplasmic male sterility may enhance callus initiation in anther culture. Three wheat nuclear genotypes, each in normal and two alien cytoplasms conferring cytoplasmic male sterility, were evaluated for callus initiation frequency in anther culture. Nuclear genotype had the greatest effect on callus initiation, but cytoplasm and nucleus X cytoplasm interaction also produced significant effects. The nuclear genotype of Chris outperformed Butte and Coteau in all cytoplasms. Ordinary wheat and Triticum timopheevi cytoplasms outperformed Aegilops speltoides cytoplasm. Cytoplasmic male sterility did not increase callus initiation. This suggests manipulating the nuclear genotype is the best strategy for improving the capacity of wheat to initiate callus in anther culture.     

Chemically induced sterility in wheat for hybrid seed production

Summary The gametocidal effects of RH-531, RH-532, and RH-2956 were studied with three rates and times of foliar application on two spring wheat (Triticum aestivum L.) varieties, Anza and Yecora 70. RH-531 and RH-532 applied at or before booting stage caused rather large reduction in plant height while RH-2956 had a small effect on height. Application of any of the three chemicals on Anza at meiotic stages reduced self-pollination (SP) fertility by 97–99%. If 90% SP fertility reduction is acceptable, the application time could vary from premeiosis to postmeiosis for Anza. Yecora 70 was less sensitive than Anza to gametocide treatments. The lowest SP fertility obtained in Yecora 70 with RH-531, RH-532, and RH-2956 was by treatment at booting (4 kg/ha), at premeiosis (2 kg/ha), and heading (8 kg/ha), and the corresponding SP fertility reductions were 74, 89, and 71%. Pollen stainability-determined by aceto-carmine, IKI, and Alexander's stains-was not affected by gametocide treatment and was not useful in evaluating sterility induced by these chemicals. For the RH-531-treated Anza and Yecora 70 and the RH-532-treated Anza, more than 94% of the seeds produced by open-pollination on treated plants were from self-or sib-pollination as determined by progeny testing. However, RH-532-treated Yecora 70 and RH-2956-treated Anza and Yecora 70 did result in more crossed seeds, with a maximum of 20% outcrossing. It was believed that spike compactness induced by the chemicals and the related poor flower opening were the major limitations for outcrossing. Artificial pollinations made to the test varieties showed that RH-531 induced both male and female sterility and was undesirable for practical use. RH-532, however, could be applied at the booting stage to avoid severe female sterility, and RH-2956 clearly was the best of the three chemicals, with the female fertility very close to those of the untreated checks of both Anza and Yecora 70. Of the three chemicals, RH-2956 gave the highest percentage of hybrid plants in the progeny of hand-or open-pollinated spikes. Its practical use for hybrid seed production, however, will depend largely on improvement of methods to increase cross pollination in wheat.     

Development of new cytoplasmic source of male sterile lines in rice for shallow lowland

Two shallow lowland male sterile lines, MotiA and PadminiAwith Miz.4 cytoplasmic source were developed throughintervarietal hybridization and recurrent backcrossing withtheir respective parents. In all probability, MotiA and PadminiA of Miz.4 source may be genetically different from the Wild abortive CMS source (V.20A). These two CMS lines are semitall in stature, photosensitive, of late duration in flowering with easy fertility restoration ability, and thus could be useful for developing hybrid rice suitable for shallow lowland areas.     

Induction of a mutation in the male fertility gene of the preferentially transmitted Aegilops sharonensis chromosome 4S1 and its application for hybrid wheat production

Summary Wheat plants nullisomic for chromosome 4B are male sterile due to the absence of the male fertility gene Ms1. However, plants in which chromosome 4B has been substituted by the preferentially transmitted chromosome 4S¹ of Ae. sharonensis are male fertile due to the compensating effect of Ms4 on the alien chromosome. This substitution line has been mutated and three recessive mutation of Ms4 have been selected. Plants homozygous for these mutations are male sterile. The implication of these mutations for hybrid wheat production is discussed.     

Pollen sterility induced by ethrel and its utilization in hybridization of wheat

Summary In 1974 and 1975 gametocidal effects of Ethrel were investigated under field conditions in the spring wheat cv. Zlatka. In experimental plants grain setting per spikelet and ear as well as ability to be cross-pollinated were ascertained.As compared with the control, treated variants achieved more than 90% sterility. Female fertility was not affected and cross-pollination was possible. Genetical check showed that the best variants, when cross-pollinated, produced 50–55% hybrids; grain setting, however, was brought down by more than 37%. As a result of Ethrel treatment haulm length was reduced and the vegetation period was somewhat prolonged.     

Mutations blocking sensitivity to gibberellic acid promote ethylene-induced male sterility in wheat

Summary Ethylene is known to perturb normal reproductive development in wheat, particularly the development of functional pollen. Two experiments were carried out to test the hypothesis that increasing insensitivity to gibberellic acid (GA), conferred by the Rht genes, would be associated with increased male sterility in ethrel or Cerone^®-treated wheat. Wild type (WT=rht1/rht1, rht2/rht2), single dwarf (SD=Rht1/Rht1, rht2/rht2 or SD=rht1/rht1, Rht2/Rht2), and double dwarf (DD=Rht1/Rht1, Rht2/Rht2) near-isogenic lines in six genetic backgrounds were treated with ethrel or Cerone^® at the late tetrad to early uninucleate stage of pollen grain development. Ethrel induced pollen abortion in all genotypes but was highest for DD (41% above background) followed by SD (20%), and then WT genotypes (10%). Spikelet fertility decreased as the number of Rht alleles increased in response to ethrel or Cerone^® treatments. Expressed as a percent of controls, spikelet fertility was 56% for WT, 42% for SD, and 29% for DD. The consistent linear relationship between the number of Rht alleles and sensitivity of ethylene-induced male sterility suggests that GA and its recognition may exert a stabilizing effect in pollen development in the presence of stress or an ethylene shock.Paper No. 764 of the Department of Plant Breeding and Biometry, Cornell Agricultural Experiment Station.     

Induction of male sterility in wheat using organic ligands with high specificity for binding copper

Summary Because copper is extremely important to the development of normal polllen, an attempt was made to induce male sterility in wheat by applying specific copper-binding ligands to wheat plants. Four different chelates were used at two rates in three methods of application. All four chelates, cupferron, neocuproine, benzotriazole and cuprizone, reduced grain yield at high concentration applied to the soil at sowing but benzotriazole was most effective, even when applied at late tillering to either soil or foliage, and it also reduced yield to a lesser extent when applied at low concentration. At high concentration of benzotriazole (50 mg kg^-1 of dry soil) the percentage of pollen staining with I₂/KI was very low (0–7%) depending on method of chelate application), and this soil treatment resulted in complete male sterility. The appearance of the pollen, anthers, grain, ears and leaves in many cases mimicked that of normal copper deficiency, and also that caused by other recognised gametocides. These results raise the question of whether binding of copper or some other disturbance of copper metabolism may be the mechanism by which andro-gametocidal chemicals work and if so, dictate a theoretical basis for selecting such chemicals for testing.     

Evaluation of three sources of pollen fertility restoration genes and natural outcrossing of cytoplasmic male-sterile wheat

Summary Fertility restoration genes in Triticum aestivum L. in Texas Restorer Composite (TRC), D6301, and four CIMMYT restorer lines were studied, and selection was made for higher fertility in TRC. Mean-while, outcrossing percentages of seed set for 27 spring habit cytoplasmic male sterile (cms) varieties were evaluated for 3 to 5 years at Davis. The winter-habit TRC material did not restore reasonably good fertility, and the response to selection for higher fertility seemed to be slow. This poor fertility could be partly due to its late winter growth habit causing flowering at a period of high temperature and low humidity at Davis. The highest F₁ fertility was 46.6% in the cross cms Ramona x TRC-6, and its F₂ segregated into the ratio of 15 fertile to 1 sterile, with fertility ranging from 3.2 to 100%. Suggested for its improvement was intensive selection in the original TRC material and in the segre-gating F₂ population, followed by intercrossing. D6301 has 2 fertility restoration genes with different strengths which restore fertility up to 45.2% when both genes are heterozygous. D6301 is quite likely heterogeneous for these genes. Four CIMMYT restorer lines, D7464, D7465, D7466, and D7467, had satisfactory F₁ fertility restoration after crossing with cms Ramona 50. In 1975, the fertilities of the F₁'s ranged from 71 to 85% and were over 90% in 1976. The F₂ population of the cross cms Ramona 50 × D7464 segregated into a ratio of 3 fertile to 1 sterile, indicating that D7464 has a single dominant gene for fertility restoration. The F₂'s of crosses cms Ramona 50 × D7465, cms Ramona 50 × D7466, and cms Ramona 50 × D7467 gave a ratio of 15 fertile to 1 sterile, indicating that two gene pairs in these three lines were responsible for the fertility restoration. The best of this group was D7467 which restored fertility fully after being crossed with cms Ramona 50 (T. timopheevi cytoplasm).The early-flowering cms male-sterile varieties had higher outcrossing rates (16 to 38%) than late varieties (6 to 30%) over a 5-year period. This was due to hot and dry weather during the late growing season as well as to the rarity of windborne pollen. In 1970, 1971, 1972, and 1976, the variation among varieties was rather great. Some of them such as Roque 66 and Bajio 67, had consistently high outcrossing rates. This outcrossing ability seemed to be inherited and probably associated with the open-flowering characteristics of each variety.     

Chromosomes responsible for sensitivity of embryo to abscisic acid and dormancy in wheat

The sensitivity of the embryo to abscisic acid (ABA) has been reported toplay an important role in seed dormancy. Using ditelocentric lines of wheatcv. Chinese Spring (CS, nondormant and ABA insensitive), F₂ seedsbetween monosomic lines of CS and a wheat line Kitakei-1354 (dormant,ABA sensitive) and deletion lines of CS chromosome 4A, germinability ofseeds and embryo-half seeds incubated in water and ABA were examined. The results indicated that the long arm of chromosome 4A carried majorgene(s) for the embryo sensitivity to ABA and dormancy. Chromosome2D might be also involved in the sensitivity to ABA.     

Genetic variation for nitrogen use efficiency in winter wheat (Triticum aestivum L.)

Summary The new European Common Agricultural Policy and environmental considerations are certainly to change agricultural practices toward low input cultivation systems. Nitrogen is one of the main inputs of winter wheat in northern France and it contributes highly to phreatic water pollution. A research programme has then been set up in order to study whether it is possible to breed for winter wheat cultivars using more efficiently N fertilisers. Less nitrogen would be applied, decreasing pollution risks and operational costs. It has been shown that a large variation exists for N related traits and for the resistance against N deficiency. On the one hand the cv Arche is very resistant to N deficiency, its yield on low N conditions (with no N fertiliser) is on average 89% of its yield on high N conditions (with a high N application). On the other hand, cv Récital is very susceptible to N deficiency as this same percentage is only 61%. A study on 10 hybrids showed that heterosis for grain yield was higher at low N level than at high N level. This was due to a higher number of grains per m².     

Diversity among sources of cytoplasmic male sterility in sunflower (Helianthus annuus L.)

Summary Ten cytoplasmic male sterile (CMS) sunflower (Helianthus annuus L.) lines were crossed with nine maintainer or male fertility restorer lines in a diallel crossing scheme. Based on fertility restoration of the F₁ generation, CMS lines were divided into four groups. At least two new sources of CMS, CMS PET2 and CMS GIG1, were found to be potentially useful for commercial production of hybrids. Environment had an influence on fertility restoration of one CMS line, CMS MAX1. Effective restoration of male fertility for CMS RIG1, CMS ANN2, and CMS ANN3 was not found.     

The effect of gibberellic acid on male fertility in bread wheat

Summary The effect of gibberellic acid (GA₃) on male fertility was investigated in wheat. Greenhouse and field experiments were carried out. GA₃ induced high levels of male sterility both in gibberellin (GA)-sensitive and GA-insensitive genotypes. The optimum concentration was 2000 ppm of GA₃ when applied at successive sprays. The critical period for GA₃ treatment, in the general sense, extended from glume differentiation to premeiotic interphase in the oldest florets of the spike, though differences were found between GA-sensitive and GA-insensitive genotypes in the extension of this period. The effect of GA₃ on plant height was also studied. The potential use of GA₃ as a chemical hybridizing agent in wheat breeding is discussed.Abbreviations GA gibberellin - GA₃ gibberellic acid - ms male sterility Scientific Paper GEN-810 of the Instituto de Gen]'etica, CICA, INTA, Castelar, Argentina