小麦×羊草远缘杂交的受精和胚胎发育

对小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｎＬ）和羊草（ＬｅｙｍｕｓｃｈｉｎｅｎｓｉｓＴ．）杂交的受精和胚胎发育进行了研究。羊草花粉在小麦柱头上萌发较好，花粉管能顺利穿过柱头，进入胚囊。在检查过的３１０个子房中，１７个（５．５％）发生了双受精，产生了胚和胚乳；２个（０．６５％）发生了单卵受精，只产生胚而无胚乳；７个（２．２６％）发生了单极核受精，只产生胚乳而无胚。小麦×羊草虽然受精率为８．４１％，但最后结实率很低，若利用胚培养技术对杂种胚进行早期离体培养，可望提高杂种植株的获得率     

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).     

Controls over pollination and ovule fertilization in Vicia faba

Summary Variation in the frequencies of pollination and ovule fertilization was generated among faba bean (Vicia faba L.) genotypes in experiments which compared self-and open-pollination, natural and extended photoperiods, presence and absence of honey bees and a range of plant populations.There was a near-1:1 relationship between the frequencies of pollination of stigmas and the fertilization of at least one ovule within the pistil. Pollination was the major barrier to fertilization of the pistil, and ovules, since no mechanism was evident in the style to prevent pistil fertilization. The frequency with which single ovules were fertilized in pollinated pistils fell as their distance from the stigma increased, and averaged 83.5% overall.The frequency of pollination was most influenced by the level of autofertility possessed by the genotype, and the activity of pollinators. Provision of honey bees to plants in caged plots showed that high levels of pollination could be achieved under open-pollinated conditions, but this was also influenced by autofertility. Increasing plant population in the field had only a small effect on pollination, but extending photoperiod caused the levels and timing of self-pollination in three genotypes to vary.Large differences were found among genotypes in their early stigma receptivity, that is, the frequency of stigmas found to be pollinated at the erect standard stage, prior to the flower opening and being visited by bees. The implications of early stigma receptivity for producing new cultivars were discussed.     

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.     

Influence of selected wheat and rye genotypes on the direct synthesis of hexaploid triticale

Summary In the synthesis of primary hexaploid triticale, a cross-incompatibility barrier exists when tetraploid wheat (4X) is crossed with diploid (2X) rye. Fertilization may occur, however, abnormal endosperm development usually leads to premature embryo death. Four selected tetraploid wheat lines were crossed as females with seven open-pollinated rye lines and the resulting embryos were rescued in vitro 13–16 days after pollination. The wheat genotypes showed a major influence on crossability (seed set), embryo development and plant recovery. The highest efficiency of amphihaploid plant recovery (18.3 plants per 100 pollinated florets) was obtained from one 4X wheat line originally selected from the cross T. carthlicum × T. dicoccoides. Some of the 3X amphihaploid plants (ABR) derived from two wheat lines showed relatively high level of partial fertility presumably as a result of meiotic restitution. Correlation analysis showed that crossability (seed set), normal hybrid embryo development in vivo and embryo culturability were independent of each other.     

Pollen viability and stigma receptivity in <Emphasis Type="Italic">Lilium</Emphasis> during anthesis

Pollen viability and stigma receptivity are prerequisites for successful pollination and seed set in flowering plants. In this study, the pollen viabilities and stigma receptivities of nine Lilium genotypes (six cultivars and three species native to China) were assayed by in vitro pollen germination and the benzidine-H₂O₂ method, respectively. Embryo sac development during anthesis was observed to further ensure the timing of controlled pollination. In addition, the relationship between stigma secretion and stigma receptivity was studied to estimate the pollination time based on phenotype. Anthers cracked on the day of flowering in all genotypes, but pollen germination during anthesis was not observed in Asiatic hybrids excepted for ‘Tiny pudhye’, which exhibited low pollen viability for a short period of time (from 0 to 1 day after anthesis). In the other genotypes, pollen germination rates were highest on anthesis (five of seven genotypes), 0–1 day after anthesis (L. sulphureum), or 0–2 days after anthesis (one Longiflorum hybrid), and then gradually decreased with days after anthesis. While, stigma receptivity first increased and then decreased during anthesis. For most genotypes, stigmas began to be receptive 1 day after anthesis, and all genotypes exhibited stigma receptivity at 2 days after anthesis. The durations of stigma receptivity and strongest stigma receptivity, were genotype dependent, and were 5–8 days and 1–4 days, respectively. Moreover, on the first flowering day, 6 of 7 genotypes had mature embryo sacs, and at the time at which stigmas began to be receptive, all tested genotypes had mature embryo sacs. Some Lilium genotypes showed stigma secretion, which can be a sign of stigma receptivity. Stigmas became receptive and reached highest receptivity within 1 day of the first appearance of secretion on the surface of the stigma and at peaking, respectively. The results of this study are valuable for the implementation of successful Lilium breeding programs.     

Cytological Evidence for Fertilization in Hexaploid Wheat × Sorghum Crosses

Crosses were made between the hexaploid wheat ‘Chinese Spring’ (2n = 42) and the diploid grain sorghum ‘S9B’ (2n = 20). Sixty-nine out of 100 florets fixed 48 h after pollination contained an embryo, an endosperm, or both, a remarkably high frequency in view of the taxonomic distance spanned by the cross. Percentages of single or double fertilization ranged from 50 % to 91 % for individual spikes. The hybrid origin of the embryos was confirmed by examining zygotes from spikes fixed 25 to 27 h after pollination. Seven of the 8 zygotes in which chromosomes were sufficiently contracted to be counted contained 21 large wheat chromosomes and 10 much smaller sorghum chromosomes. The eighth contained 21 chromosomes from wheat and 20 from sorghum. Sorghum chromosomes did not appear to be attached to the spindle in zygote nietaphases and showed no evidence of movement towards the spindle poles in the single zygote anaphase found. Embryos with two or more cells invariably contained one or more micronuclei and metaphases in embryos with three or more cells contained only 21 wheat chromosomes showing that sorghum chromosomes were rapidly eliminated. Endosperm, when present, was always highly abnormal.     

Factors affecting seed set in the crosses between <Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> (Ramat.) Kitamura and its wild species

Reproductive barriers often exist in the crosses between Dendranthema grandiflorum (Ramat.) Kitamura and its wild species and seriously result in low seed set, consequently reducing breeding efficiency. For the purpose of revealing the factors leading to low seed set, we investigated pollen viability, germination behavior of pollen grains on stigmas and embryo development in the crosses between D. grandiflorum and three wild species, D. nankingense (Nakai) Tzvel., D. indicum (L.) Des Moul. and D. zawadskii (Herb.) Tzvel. using technique of paraffin section, and light, fluorescence and scanning electron microscopy. The results indicated pollen viability of three wild species ranged from 20 to 25%. In the cross between D. grandiflorum and D. nankingense, very few pollen grains germinated on stigmas after pollination and most of them germinated abnormally. In addition, normal embryos were observed in 12% ovaries at 8 days after pollination and thereafter all the embryos aborted. In other two crosses, many pollen grains germinated on stigmas and pollen tubes penetrated stigmas normally after pollination. Moreover, normal embryos were observed in over 50% ovaries from 8 to 15 days after pollination in the cross between D. grandiflorum and D. indicum, and seed set was 59%. In the cross between D. grandiflorum and D. zawadskii, normal embryos were observed in 52% ovaries at 8 days after pollination. After that, however, most embryos degenerated and seed set was only 9%. These data suggest that pollen viability has no significant effects on seed set of the three crosses. Very few germinated pollen grains on stigmas and abnormal growth of most pollen tubes before fertilization, and embryo abortion are the main factors causing failure of the cross between D. grandiflorum and D. nankingense, whereas only embryo abortion is a main factor resulting in low seed set in the cross between D. grandiflorum and D. zawadskii and no barriers occur in the cross between D. grandiflorum and D. indicum.     

Rapid method of determining pollen incompatibility in Chrysanthemum morifolium Ramat

Summary A classification system for predicting high, medium and low seed set from Chrysanthemum morifolium crosses was developed. Stigmas collected and preserved 8 days after pollination were examined with a binocular microscope for adhering pollen grains. Florets from compatible crosses had brown stigmas with pollen grains attached and embryo development while florets from incompatible crosses had white stigmas with no or rarely 1 or 2 pollen grains attached and no embryo development. Using this method the occurrence of seed set was predicted with 78.5% accuracy in one year and 89% in the following. This provides a method of determining the compatibility of a chrysanthemum cross without waiting the 1 to 2 months required for seed ripening.Scientific Journal Series Paper Number 7570 of the Minnesota Agricultural Experiment Station.     

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 Growth in Culture of Detached Ovaries of Wheat (Triticum aestivum L. em. Thell)

Viable embryos have been recovered from fertilized wheat (Triticum aestivum) ovaries detached on the day of, or up to 7 days post-, anthesis and cultured aseptically fur up to 18 days. The most significant factors in determining the yield of embryos was the plating density, age and complexity of the explants. 26% of ovaries excised on the day of anthesis produced viable embryos if cultured as pairs of florets. The potential use of detached ovary cultures in gametophyte microinjection experiments, rescuing embryos from wide crosses and in chemically manipulating the early stages of embryo development are discussed.     

Embryo development and fruit-set in pear induced by untreated and irradiated pollen

Summary Flowers of the pear cultivars Conference, Doyenné du Comice and Gieser Wildeman were pollinated with untreated and 50-krad-irradiated pollen. Fruitlets were sectioned at regular intervals up to 6 weeks after pollination. Tubes of the irradiated pollen grew slower, but within two weeks nearly as many embryos were initiated as in the case of untreated pollen. Thereafter, the proportion of degenerated embryos increased, although the endosperm sometimes persisted. Six weeks after pollination only about 21% of the seeds induced by the irradiated pollen still contained normal embryos. At or before that time in-vitro culture of the immature seeds will be necessary because few embryos survive until harvest. The irradiated pollen stimulated a rather large parthenocarpic fruit-set, presumably associated with endosperm rather than embryo development during the first 4–6 weeks after pollination. The possibilities of haploid production are discussed.     

Cytology of seed development related to floret position in perennial ryegrass (Lolium perenne L.)

In seed crops of perennial ryegrass, Lolium perenne L., yields are low and only 15 to 20% of the florets produce a harvestable seed. This study was conducted to determine if seed abortion was a major cause of low floret site utilization (FSU) and therefore of low seed yield in spaced plants. Spikes of four spaced-planted genotypes of perennial ryegrass were harvested and fixed at one-week intervals one to five weeks after the beginning of anthesis. Of a total of 74 spikes, over 13,000 florets were examined with a binocular microscope and 66 whole spikelets were microtome sectioned. The histology of the pro-embryo is presented in relation to the development of the endosperm. In one low yielding, two intermediate and one high yielding genotype, biological FSU was 8, 72, 73 and 63%, respectively. In all genotypes, 90% of the unproductive florets showed ovule degeneration within a few days after flowering. A few florets were sterile or contained a degenerated embryo sac already before flowering and about 5% of the unproductive florets showed seed abortion later than one week after flowering. Within a spikelet, successful fertilization did not decline from the basal to the distal floret. Although in later stages distal florets had slightly less seeds, unproductive florets were found at all floret positions. Biological FSU decreased mainly by ovule degeneration shortly after flowering. In spaced plants, seed abortion was not important to low seed yield.     

Interspecific hybrids of Zinnia elegans Jacq. and Z. angustifolia HBK: embryology,morphology and powdery mildew resistance

Summary Reciprocal crosses between 13 cultivars of Zinnia elegans Jacq. and one source of Z. angustifolia HBK resulted in interspecific hybrids. Florets examined 14 days after pollination showed that all 13 cross combinations of Z. angustifolia x Z. elegans yielded embryos in 47.2% of the florets pollinated. Six cross combinations of Z. elegans x Z. angustifolia produced embryos in 4.6% of the total Z. elegans florets pollinated. Interspecific crosses resulted in seed-propagated hybrids when Z. angustifolia was the maternal parent. Hybrids were obtained from reciprocal crosses between the two species when embryos were excised and cultured. Morphologically, most hybrids resembled Z. angustifolia but were sterile. Colchicine treatments of selected sterile hybrids resulted in shoots producing fertile inflorescences. Preliminary tests showed that hybrids were resistant to powdery mildew (Erysiphe cichoracearum DC ex Mecat).Scientific Article No. A 3108, Contribution No. 6173 of the Maryland Agricultural Experiment Station, Department of Horticulture.     

The breeding system of rubber (Hevea brasiliensis): an evaluation of controlled hand pollination methods

Summary Controlled hand pollinated pistils of rubber were observed using fluorescence microscopy to assess the efficiency of the universally-employed method for the production of progeny for plant breeding. The controlled hand pollination method conducted in the morning resulted in the deposition of a mean of 15.6 pollen grains on the stigma, with no stray pollination observed. Over 36% of the pistils had the potential to set fruit.Pollinations conducted in the afternoon at the normal time of anthesis, had double the fruit set potential of morning pollinations as measured by penetration of ovules by pollen tubes. Pollinator efficiency also varied, with excessive damage to stigmas resulting in reduced pollen germination and tube growth. There were differences between clones in both female and male fertility, in the proportion of pistils with more than three carpels and in the production of small abnormal stigmas. There was no difference in pollen tube growth following self- or cross-pollination, indicating that the self-sterility mechanism of rubber operates in the ovary. Pollen could be stored for 5 days at 5°C and 75% RH with a 22% loss of fertility.     

Factors affecting oat haploid production following oat × maize hybridization

Doubled haploids (DHs) are becoming increasingly important in crop breeding programmes but methods for producing oat DHs remain inefficient. In this study haploid and DH oat plants were produced using the oat × maize hybridization method. Factors influencing the rate of caryopsis and haploid embryo production including genotype, post‐pollination plant growth regulator application and temperature were investigated. The four growth regulators tested showed significant differences in their capacity to induce caryopsis formation with dicamba producing the highest numbers of caryopses, followed by picloram, 2,4‐dichlorophenoxyacetic acid (2,4‐D) and gibberellic acid (GA₃). No significant differences were observed between these growth regulators for their effect on embryo production. The concentration of dicamba was also important and was found to influence caryopsis but not embryo production, with 50 and 100 mg/l dicamba producing significantly more caryopses than 25 or 5 mg/l. Temperature had a significant impact on both caryopsis and embryo production with the magnitude and direction of response depending on genotype. Rates of haploid embryo production observed were between 0.8% and 6.7% of the pollinated florets. The proportion of haploids, which survived and were successfully doubled with colchicine following transfer to soil was between 72% and 81%.     

An embryological study of a Pisum sativum x Vicia faba cross

Summary Flowers of Pisum sativum plants grown in the field and greenhouse were self-pollinated and pollinated with Vicia faba. Germination of pollen and pollen tube growth were slower in the Pisum x Vicia cross. About 2% fertile ovules in the field and 8% in the greenhouse were obtained from the Pisum x Vicia cross, compared to 26% and 48% for Pisum x Pisum. Development of the Pisum x Vicia embryo was normal in form, though much slower than the Pisum x Pisum embryo, and about the sixth day it began to collapse. The greatest difference between the two types of crosses was observed in the endosperm. While the Pisum x Pisum endosperm nuclei divided normally and endosperm developed throughout the ovule cavity and around the embryo, the Pisum x Vicia endosperm nuclei formed only scattered masses of densely stained nuclei which were not always in close proximity to the hybrid embryo. It was concluded that faillure of some ovules to develop following Pisum sativum x Vicia faba cross pollination was the result of slow germination of pollen and slow growth of the Vicia pollen tubes, with subsequent lack of fertilization. Collapse of fertilized ovules was associated with abnormal and limited development of the hybrid endosperm, possibly leading to lack of nourishment and eventual collapse of the hybrid embryo.     

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.     

Reproductive barriers in the intergeneric hybridization between <Emphasis Type="Italic">Chrysanthemum grandiflorum</Emphasis> (Ramat.) Kitam. and <Emphasis Type="Italic">Ajania przewalskii</Emphasis> Poljak. (Asteraceae)

In order to reveal the causes of low seed set in the intergeneric cross between Chrysanthemum grandiflorum (Ramat.) Kitam. ‘Zhongshanjingui’ (female parent) and Ajania przewalskii Poljak. (male parent), we systematically investigated megasporogenesis and megagametogenesis of female parents, pollen germinability of male parents, pollen–pistil interaction and hybrid embryo development using paraffin section and light, fluorescence and scanning electron microscopy. The results indicate that development of megagametophyte of female parent belongs to Polygonum type and 84.6% ovules developed to normal 8-nucleate embryo sac at the stage of pollination; the pollen germinability in vitro of male parent was 39.3%; a large number of pollen grains germinated normally and no callose deposited on the female stigmas within 24 h after pollination. Furthermore, about 75% ovules were fertilized, whereas only 17.2% ovules developed to normal maturing embryos at 15 days after pollination due to abortion at various developmental stages. Taken together, these results suggest that sterile ovules of female parent, pollen viability and pollen–pistil interaction have no significant influence on seed set in the cross. A high percentage of embryo abortion or degeneration, i.e. post-fertilization barrier, may be the main factor for low seed yield.     

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

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