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.     

Wheat x pearl millet hybridization: consequence and potential

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

不同贮藏温度条件下3个烟草品种花粉活力、形态及生理指标变化

以烟草品种N. rustica、Maryland 609和云烟85花粉为试验材料,研究不同贮藏温度条件下参试烟草品种花粉的活力、形态及相关生理指标的变化。以烟草品种N. rustica冻存花粉活力检测结果为例,在-196℃和-80℃保存条件下,花粉贮藏16个月后活力分别为0.64±0.06和0.65±0.09,与贮藏前0.66±0.04相比,未出现显著下降,而4℃贮藏的花粉在保存5个月后即丧失活力。花粉电镜图片显示,在-196℃和-80℃贮藏条件下,花粉形态仍然规则饱满,而4℃条件下贮藏16个月的花粉则发生皱缩、畸形,萌发沟扭曲。-196℃贮藏条件下N. rustica的花粉可溶性糖含量（309.276mg/g）、可溶性蛋白含量（55.216mg/g）、游离氨基酸总量（15.817μmol/mg）、脯氨酸含量（1.596mg/g）、超氧化物歧化酶（SOD）活性（4.284U/g）均显著高于4℃贮藏条件下的花粉;-80℃贮藏条件下花粉脯氨酸含量（1.626mg/g）和SOD活性（5.546U/g）高于-196℃（液氮）条件下贮藏的花粉,可能与不同温度下花粉冻结速度有关。结果表明,-196℃和-80℃条件下保存对烟草花粉的活力保持更有利。     

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.     

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.     

Effects of short-term storage on germinability of pistachio pollen

The effects of short‐term storage under room conditions (constant 25°C and 35% relative humidity), refrigeration (4°C) and freezing (‐20°C) on the germinability of pollen of pistachio, Pistacia vera L., were studied and the effects of desiccation and pollen age on its germinability were reassessed. It was found that: (1) weight loss as a result of desiccation was positively correlated with germinability; (2) pollen grains stored in room conditions only germinated after prehydration, which restored germinability even after 7 days of exposure; (3) refrigerated (4°C) pollen grains retained their germinability for at least a week; (4) frozen pollen grains irreversibly lost most of their germinability; (5) 2‐day‐old pollen grains, within the anthers, retained their germinability under room conditions. These findings will contribute to the improvement of pistachio pollen preservation and help to find a simple and inexpensive method for its short‐term storage, while retaining its germinability.     

Storage of avocado pollen

Summary Avocado pollen was stored at a range of temperatures and relative humidities (RH) for up to one year and the pollen was tested for viability in vivo.Pollen stored for one month was capable of germination on the stigma and penetrating the ovule when stored at 4°C with <1,23,55 and 75% r.h. and at -196°C with 0% r.h. Most pollen samples stored at 25 and -15°C at a range of r.h. would germinate on the stigma but none would penetrate the ovule.After one year of storage, pollen at 4°C and <1 and 23% r.h. would germinate on the stigma but would not penetrate the ovule. There was no germination of pollen stored at 4°C and 55 and 75% r.h. Only pollen stored at -196°C and 0% r.h. would penetrate the ovule, but thawing and refreezing once during the year destroyed the viability.     

Low temperature storage of pistachio pollen

Summary Pollen from four male pistachio (Pistacia vera L.) clones was stored at –196°C and –20°C for up to 12 months and tested for ability to germinate in vitro following a period of hydration at high humidity. Germination of fresh pollen was high (>80%) for each clone. At –196°C, pollen of cv. Peters survived freezing, storage and thawing with no loss of germinability; pollen of the other three clones had sharp declines in germination possibly attributable to cellular lesions incurred during freezing or thawing. When the relative humidity of the –20°C storage environment was maintained at or near 33%, Peters pollen had high rate of germination through 12 months storage. Without control of relative humidity, Peters pollen germination was high at 4 months, but declined at 12 months. Germination requirements became more exacting for pollen stored at –20°C for 12 months at suboptimal humidity conditions. Pollen of the other three clones did not tolerate storage at –20°C as well as Peters pollen regardless of the storage humidity environment.     

Cryopreservation of Delphinium pollen at –30 °C

Pollen of garden varieties and species of Delphinium that was cryopreserved at –30 ^°C after 3 hours of air drying and storage on silica gel, had high germination and pollen tube growth in vitroeven after 180-day storage. On the other hand, pollen stored at 25 ^°C showed a marked decrease in the germination rate within 10 days. The best in vitro germination of Delphinium pollen was on a 1% water agar containing 15% sucrose and 0.005 to 0.01% boric acid and at 15 to 20 ^°C. Field pollination with the cryopreserved pollen showed higher fruit and seed set than pollination with pollen stored at 25 ^°C, and was not significantly different from fresh pollen. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Establishment of a long-term storage method for soft X-ray irradiated pollen in watermelon

A new method for preserving viable soft X-ray irradiated pollen for the production of seedless watermelons (Citrullus lanatus L.) from diploid plants was established by storing the pollen under N₂ at −25°C for 1 year. Pollen stored at 4°C had the ability to germinate until 28 days under all atmospheres tested except O₂. However, after storage at this temperature for 3 months it did not germinate in any treatment conditions. Pollen stored at −25°C for 1 year under N₂, CO₂ or under a vacuum had a germination rate of about 50%. Pollen stored in O₂ or in air had a much lower germination rate, less than 20% and 10%, respectively. Thus, it is important to store watermelon pollen at a low temperature when it is being stored for a long time. Nitrogen and CO₂ were effective in extending the life of watermelon pollen. On the other hand, it has been demonstrated that the viability of pollen was reduced by storage under O₂. The N₂ and CO₂ are thought to be effective because they are essentially inert to the living pollen. Also, a lot of pollen tubes were observed in a middle portion of an ovary pollinated with pollen stored at −25°C under N₂; however, pollen tubes were not observed in ovaries pollinated with pollen stored under a vacuum. It was revealed in 1 year storage experiments that viability of pollen stored under N₂ was higher than that stored under a vacuum. Seedless fruits produced with pollen stored at −25°C under N₂ had the same characteristics as the control fruit. Authors did this research in ‘Research and Development Program for New Bio-Industry Initiatives’.     

Storage of maize (Zea mays L.) pollen at −196°C in liquid nitrogen

Summary The water content of pollen has a decisive influence on its storability in liquid nitrogen. Pollen with an initial high water content cannot be stored successfully at extremely low temperatures, so a certain degree of drying must be carried out before storage. Provided the viability of the pollen is not significantly reduced during drying, the pollen remains viable and fertile when kept at –196°C.     

人工合成小麦与普通小麦杂交后代衍生群体的Rht8基因分析

【目的】四倍体小麦与节节麦杂交培育的人工合成小麦已广泛应用于国内外小麦品种改良。通过研究人工合成小麦与普通小麦杂交后代的Rht8基因型,有助于提高分子标记育种效率,也有助于Rht8 基因型的多态性研究,并为人工合成小麦在中国小麦品种改良和分子标记育种中的应用提供依据和方法;【方法】以引自CIMMYT的人工合成小麦分别与中国四川成都平原主栽普通小麦品种杂交、回交的BC2F2:6后代群体中选育的113份优良高代系和川麦38、川麦42、川麦43和川麦47育成品种为材料,采用特异引物的PCR 扩增和改进的聚丙烯酰胺凝胶电泳对其Rht8基因型进行了研究;【结果】在以syn768、Syn769、Syn780和Syn786人工合成小麦为亲本的117份后代衍生群体检测材料中,Rht8基因型频率为77.78%。从每一个人工合成小麦形成的小的后代衍生群体看,Rht8基因型频率各不相同。以syn768为亲本的后代衍生群体,Rht8基因型频率最高,为96.70%;在以syn769为亲本而育成的优良高代系和川麦38、川麦42与川麦43育成品种中,Rht8基因型频率最低,为71.64%;以Syn780为亲本的后代衍生群体中,Rht8基因型频率为73.68%,分离比率约为3:1;以Syn786为亲本育成的材料只有川麦47,该品种不含有Rht8该基因;【结论】不论父本或母本的Rht8的基因型状况如何,它们所产生的杂交后代材料Rht8基因的遗传是随机的。     

Effect of Cultivar, Incubation Temperature, and Stage of Microspore Development on Anther Culture in Wheat and Triticale

Effects of incubation temperature and developmental stage of microspores on polyhaploid production in three wheat cultivars‘Pavon 76′, ‘Kitt’, and ‘Chris’ and one triticale cultivar, ‘T81′, were studied using a one-step medium. Calli failing to differentiate on the one-step medium were placed on a medium containing 1 mg/l indole-3-acctic acid (IAA) and 2 mg/1 6-furfurylaminopunne (KIN). Anthers containing either early- or late-uninucleate microspores were incubated in dark at 26, 28 or 32°C lor 3 days prior to transfer to 26°C. Averaged over temperatures and microspore stages, frequency of calli and green plantlets were 8.9 % and 3.4 %, respectively, for wheat cultivar‘Pavon 76′, 8.4 % and 1.6 % for cultivar ‘Kitt’, 4.5 % and 0.25 % for cultivar ‘Chris’, and 2.9 % and 0.12 % for the triticale cultivar‘T81′. However, cultivar-by-developmental-stage interaction was significant for frequency of callus induction. Temperature had no significant effects on callus induction and plantlet regeneration. Anthers containing early-unmucleate microspores produced no polyhaploids.     

Cryopreservation of pollen from two rose cultivars

Summary An investigation was undertaken on the storage characteristics of pollen collected from two English rose cultivars. A rapid decline in viability was observed in pollen stored at +4° C and –20° C, whereas the viability of pollen, stored at ultra-low temperature (–196° C), remained constant. Cryopreserved pollen was shown to retain its ability for fertilisation. The effects of the stage of flower development and anther dehiscence were assessed on both pre-and post-cryopreservation viabilities. Successful long-term storage of pollen will facilitate hybridisation of rose species and cultivars that do not flower synchronously.     

Genetic Variability for Haploid Production in Crosses Between Tetraploid and Hexaploid Wheats with Maize

Crosses were made between 14 wheat genotypes (11 tetraploid, 3 hexaploid) and a single Fl hybrid of maize that was used as the male parent. The experimental design consisted of randomized blocks with three replications. Plants were grown under controlled greenhouse conditions (day length 16 h and temperature 25 °C/15 °C, day/night). To enhance embryo survival, 2, 4-D treatment (10 mg/1) was applied to spikes 24 h after pollination with maize. Embryos were recovered from all tetraploid and hexaploid wheats at a rate of 2.09 to 26.76 per 100 pollinated florets. Haploid and doubled haploid plants were obtained from all hexaploid genotypes (T. aestivum) and from 5 of 11 tetraploid genotypes (T. turgidum var.). The most important point of these experiments was the ability to produce haploid plants from tetraploid wheat for two reasons: firstly, anther culture cannot be applied in tetraploid wheat (T. turgidum var.) due to the inefficiency of embryo formation and the high proportion of albino plants. Secondly, to date, crosses between tetraploid wheat and maize have resulted in embryo formation, but not in haploid plants.     

The viability and storage of strawberry pollen

Investigations were carried out in 1990 and 1991 to estimate the pollen viability of five strawberry genotypes and their suitability for storage. Pollen viability was assessed by acetocarmine staining, in vitro germination, and in vivo assays. Pollen was stored in darkness at ?18°C for 1 year, and pollen viability was estimated every 4 months during storage. The highest percentage of stained and in vitro germinated pollen grains, respectively, was shown by fresh pollen of cv. ‘Dukat’ (91 and 45%). The lowest values of these characteristics were observed in pollen of cv. ‘Paula’ (48% and 20%, respectively). The best response to storage at ?18°C occurred in pollen of the breeding clone ‘B-302’ and the cv. ‘Redgauntlet’. ‘Paula’ pollen responded least favourably to this. Pollen of the cvs. ‘Dukaf’. ‘Senga Sengana’, and clone ‘B-302’ after storage for 4 months, still had sufficient capacity for fruit set after cross-pollinations.     

Effects of Tillage and Cropping Systems on Soil Moisture Balance and Pearl Millet Yield

A 2-year study was conducted to determine the effects of tillage and cropping systems on soil moisture balance, growth and yield of pearl millet (Pennisetum glaucum (L.) R.Br.). Three tillage treatments, viz. minimum tillage (one harrowing), conventional tillage (two harrowing, cross) and deep tillage (ploughing followed by two har-rowings), and four cropping systems, viz. monoculture of pearl millet, pearl miliet-clusterbean (Cyamopsis tetra-gonoloba (L.) Taub.) rotation, monoculture of pearl millet with 5 t ha⁻¹ farm yard manure (FYM), and intercropping of pearl millet and clusterbean, were compared. Deep tillage improved the soil moisture storage, water use efficiency and grain yield of pearl millet while consumptive use of water was higher with minimum tillage. Total dry matter yield with deep tillage and conventional tillage was 23.2 and 10.2% higher than minimum tillage in the season 1, and the corresponding values for season 2 were 30.7 and 13.3%. The Pearl millct-clusterbean rotation and monoculture of pearl millet with the application of 5 t ha⁻¹ FYM gave 17.2 and 6.1% higher yield than monoculture of pearl millet, respectively. Maximum water use efficiency was observed in rotation followed by FYM application.     

Analysis of reproductive isolation between pearl millet (Pennisetum glaucum (L.) R.Br.) and P. ramosum, P. schweinfurthii, P. squamulatum, Cenchrus ciliaris

Crosses between pearl millet lines and Pennisetum ramosum, P. schweinfurthii, P. squamulatum or Cenchrus ciliaris were observed for the frequency and development of zygotes, the possibility of embryo rescue, and the fertility of F1 hybrids obtained. Eight per cent of the ovules from diploid millet × P. ramosum crosses showed small embryos which could not be rescued. However, 59% of the ovules from tetraploid millet × P. ramosum crosses showed well-developed embryos that were easy to rescue 14 days after pollination. F1 hybrids were male sterile but female fertile when pollinated by diploid millet. Both diploid and tetraploid millet ovules showed the presence of hybrid zygotes after pollination with P. schweinfurthii at rates ranging from 25% to 45%. The diploid millet× P. schweinfurthii hybrid zygotes often developed almost normal seeds giving, without embryo rescue, totally sterile plants. The tetraploid millet × P. schweinfurthii hybrid embryos were normal but the endosperm was severely defective. A hybrid obtained by embryo rescue was totally sterile. A diploid millet-P. schweinfurthii amphidiploid was obtained by somatic embryogenesis associated with colchicine treatment during callogenesis. This amphiploid plant was male sterile, but gave many seeds when pollinated by a tetraploid millet and few seeds when pollinated by a diploid millet. P. squamulatum pollinating diploid millets produced proembryos with large undifferentiated endosperms in 73% of the ovules. A normal seed set was observed on tetraploid millets pollinated by P. squamulatum and the resulting F1 hybrids were partially male and female fertile. Backcrosses of these hybrids were much more fertile when pollination was from a tetraploid millet rather than from a diploid millet. C. ciliaris pollinating a diploid millet showed, in 60% of the ovules, proembryos and endosperms similar to those observed with P. squamulatum and no hybrid could be rescued. Crosses with a tetraploid millet could not be attempted due to the pistil-pollen incompatibility of tetraploid millets available with C. ciliaris. Ploidy levels of mating partners do not seem to influence pistil-pollen compatibility, but play a major role in post-zygotic abortion. With adequate ploidy levels of parents, and embryo rescue, it seems that the pearl millet gene pool can be considerably enlarged by germplasm from many other species.     

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.