Pollination, Fertilization and Seed Development in Inbred Lines and F1 Hybrids of Spring Faba Beans (Vicia faba L.)

When the flowers are not disturbed, the yield of F₁ hybrids of faba beans normally exceeds that in their inbred parents. The basis of this “heterotic autofertility” was investigated by examining flowers for pollination and fertilization throughout the flowering season and assessing the distribution of seeds and pods on mature plants. Six inbred lines and seven of their F₁ hybrids were grown without protection from bee visitation, and their autofertility was estimated’ by comparing their fertilization with that of cytoplasmic male-sterile line. The first direct evidence was obtained that heterotic autofertility acts by increasing; the proportion of flowers which were pollinated and fertilized. The autofertility was not perfect, especially in a closed-flower hybrid, as many ovules remained unfertilized. Several factors contributed to the number of seeds per pod in F₁ hybrids, being superior to parental means: (1) more ovules per flower, (2) greater fertilization of the basal ovules, (3) reduced abortion of the apical ovules and (4) greater overall maturation ovules.     

Cultivar variation for seed development in white clover (Trifolium repens L.)

Summary A controlled environment study was undertaken to clarify the factors responsible for poor seed set and to study seed development, ovule degeneration and seed abortion, both morphologically and cytologically, in three Japanese cultivars of white clover. Although the mean number of ovules per floret was 4.2–5.1, the average number of seeds per floret was found to be only 2.3–2.7. Microscopic examination of carpels from 0 to 28 days following floret maturity and pollination showed that 26–33% and 8–17% of the total seeds lost occurred within the first three days and the third through fifth day following pollination, respectively. Beyond this period occasional seed abortion was observed at all stages of seed development, but this represented a very small proportion (2–7%) of the total seeds lost.A stain clearing technique was used to examine the cytoplasmic state of the embryo sac in intact, unfertilized, mature ovules and embryos of the ovules at 3 and 5-day periods following pollination. It was found that 20–22% of unfertilized and matured ovules were sterile, suggesting that ovule degeneration before fertilization was the major cause for the high percentage of seeds lost within a 0 to 3-day period following pollination. Cytological observations revealed that abortion of developing seed was due to a sudden arrest in embryo growth and that the early development of the embryo of such aborting seed was normal. Either nutrient shortage or meiotic irregularities may be the cause for high ovule sterility or post-fertilization abortion of developing seeds.     

Autofertility and Bee Visitation in Winter and Spring Genotypes of Faba Beans (Vicia faba L.)

Differences in the expression of autofertility and in the evidence of attractiveness to bees were investigated in both autumn-sown and spring-sown faba beans. All six winter stocks and one spring stock expressed very little autofertility, with averages of only 10% of flowers and 7% of ovules fertilized when undisturbed. Three other spring stocks, including a closed-flower population, were moderately autofertile, with 22% of flowers and 17% of ovules fertilized when undisturbed. The remaining two spring stocks, both English cultivars, expressed good autofertility, with 50% of undisturbed flowers and 44% of ovules fertilized. Current levels of autofertility in the winter and closed-flower stocks were inadequate to allow the development of a desirable distribution of pods without substantial bee visitation. If levels of autofertility could be raised simply by selection within populations, then they should have been much higher in these stocks. Therefore, autofertile parents will probably have to be included in programmes for breeding cultivars with improved levels of auto fertility. The closed-flower population received significantly less bee visitation than the other stocks in the spring trial. No variation in attractiveness to bees was found among the open-flowered spring stocks, nor among the winter stocks.     

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.     

Interspecific hybridization between Vicia faba (L.) and Vicia narbonensis (L.): Early pod growth and embryo-sac development

Summary Fertilized embryo-sac development and pod growth was studied in one Vicia faba cultivar, one Vicia narbonensis population and their reciprocal crosses. The initial development of endosperm and embryo was at least four days faster in V. narbonensis than in V. faba. Pods and ovules developed also faster in V. narbonensis than in V. faba. The growth rate of the hybrid pods followed the growth rate of the mother species, but was slower than that of the pods from selfed flowers. In the cross V. narbonensis × V. faba the ovules stopped growing 9 days after pollination, while in the reciprocal cross they stopped growing 15 days after pollination. Hybrid embryo-sacs from V. faba × V. narbonensis were aborted before they reached the stage of 256 endosperm nuclei or 200 embryo cells. Selfed V. faba embryo-sacs reached this stage in less than 9 days after pollination. In the reciprocal cross the embryo-sacs were aborted before they reached the stage of 128 endosperm nuclei or 80 embryo cells. Selfed V. narbonensis embryo-sacs reached this stage at the 4th day after pollination. Given that at these stages the embryo has less than 200 cells it was concluded that an in-ovule embryo culture technique should be developed to obtain hybrid plants.     

Impact of heat stress on pod-based yield components in field pea (Pisum sativum L.)

Elevated temperatures associated with climate change result in crops being exposed to frequent spells of heat stress. Heat stress results in reduced yield in field pea (Pisum sativum L.); it is therefore important to identify cultivars with improved pod and seed retention under heat to mitigate this loss. Objectives were to investigate the effect of heat stress on phenology, yield and pod-based yield components. Sixteen pea cultivars were evaluated at normal and late (hot) seeding dates in the field in Arizona 2012 and in growth chambers with two temperature regimes (24/18°C and 35/18°C day/night temperature for 7 days) during reproductive development. We measured variation in the pattern of pod retention at four-node positions on plants, seed retention by ovule position (stylar, medial and basal) within pods and screened cultivars for pod retention, seed retention and yield. Heat stress reduced seed yield by accelerating the crop lifecycle and reducing pod number and seed size. Heat stress had the most damaging effect on younger reproductive growth (flowers and pods developed later), resulting in ovary abortion from developing flowers. Heat also accelerated seed abortion in all ovule positions within pods. Two high-yielding cultivars under control temperature, “Naparnyk” and “CDC Meadow”, maintained high yield in heat, and “MFR043” had the lowest yield. Cultivars “40-10” and “Naparnyk” retained the most ovules and seeds per pod, and “MFR043” aborted seeds when exposed to heat. In half of the cultivars, ovules at the basal peduncle end of pods were likely to abort while ovules at the medial and stylar end positions developed into seeds. For seven of the field cultivars, ovules at the medial pod position also produced mature seeds. Cultivars “40-10”, “Naparnyk” and “CDC Meadow” had greater pod and ovule retention or maintained high yield under heat stress, and were identified as heat-tolerant cultivars. Our results allow for a better understanding of pod-based yield components in field pea under heat stress and developing heat-tolerant cultivars.     

Genotypic variation and polymorphism of 2S albumins of sunflower

Summary An early postfertilization ovule culture technique in Alstroemeria was described in which young ovules were cultured in vitro two days after pollination. This resulted in the direct production of seedlings in a normally incongruous cross combination. A histological study of the in vitro cultured ovules revealed the development of the embryo inside the ovule in the absence of normal endosperm. The in vivo grown ovules showed a total breakdown of the developing seed within four weeks after pollination due to a postfertilization barrier. This ovule culture technique was applied in a diallel cross of seven Alstroemeria species, five from Chile and two from Brazil. The selfings and the reciprocal crosses between the Brazilian species A. inodora and A. brasiliensis were congruous and gave seed set. The cross A. inodora x A. pelegrina gave viable seeds. In all other 39 combinations no viable seeds were harvested. In those cross combinations both pre-and postfertilization barriers were found. After early ovule culture hybrid plants were obtained in 27 of the 39 incongruous species combinations of the diallel.Abbreviations DAP days after pollination - MS medium medium according to Murashige & Skoog (1962)     

Factors affecting haploid production in wheat using the Hordeum bulbosum system. 3. Post-fertilization effects on embryo survival

Summary An investigation was made of factors affecting the frequency of development of fertilized ovules following wheat x Hordeum bulbosum hybridizations. Seed survival, a term used here to represent the relationship between seed set and fertilization, was shown to vary between the wheat genotypes tested, but was unrelated to the crossability genes present within the wheat genotype. A high seed survival was obtained in crosses involving the variety TH3929 and the single chromosome substitution line, Chinese Spring (Hope 5B). Chinese Spring showed a poor seed survival. Some influence of the H. bulbosum genotype on seed survival was detected. The two environmental factors studied, namely the post-pollination application of gibberellic acid and the ambient temperature, were both shown to affect seed survival. The application of gibberellic acid immediately after pollination or 1 and 2 days after pollination improved seed survival, although the extent of the response was dependent upon the number and timing of the application(s). A genotype-dependent response to the ambient temperature was observed, TH3929 showing a slight but insignificant reduction and Chinese Spring a significant improvement in seed survival with an increase in temperature from 20°C to 26°C. The physiological processes which may be influenced by GA and the ambient temperature and may thereby affect seed survival are discussed.     

Results of multiple pollination in blueberry (Vaccinium elliottii Chapm.)

Blueberry (Vaccinium section Cyanococcus, family Ericaceae) is a shrub that produces multiple-seeded berries in which only a fraction of the ovules develop into viable seeds. A recessive yellowleaf marker gene was used to evaluate the efectiveness of a single pollination versus multiple pollinations in producing seeds in Vaccinium elliottii Chapm. A multiple-pollination experiment was conducted to see if the first pollen applied or the second produced more progeny, and to see if multiple pollinations increased the number of seedlings produced. Flowers of diploid yellowleaf Vaccinium elliottii were pollinated from one to four times at daily intervals. Pollen from redleaf and yellowleaf plants was used to produce two types of seedlings that could be distinguished visually. Generally, percent berry set, seeds per berry, and seed germination were not increased by multiple pollination. After multiple pollination, the pollen applied first always produced the most seedlings. Prior pollination greatly reduced seed set from subsequent pollination, but subsequent pollination caused little or no decrease in the number of seeds set by the first pollination. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pollen tube growth and fertilization after different in vitro pollination techniques of Aechmea fasciata

The effect of cut style and placental pollination on fertilization efficiency was studied for compatible Aechmea fasciata plants. These alternative in vitro pollination techniques resulted in lower penetration rates of the ovules by pollen tubes in comparison to pollination on the stigma. An explanation was found in the intervention of the normal pollination process,through which less pollen-pistil interactions were built up. After cut style pollination the percentage of ovule penetration by pollen tubes increased when a longer style part was left at the ovary. Probably fewer factors that control pollen tube growth are present in the lower style part. Pollen germinated on the ovules after placental pollination but only rarely penetration of the micropyle by a pollen tube occurred. Activation of the ovary, induced by in vivo prepollination for 6 hours, and pollination two days after anthesis did not increase the fertilization percentage. Grafting a style with active growing pollen tubes to ovules on the placenta (placental grafted style pollination) resulted in a higher fertilization percentage. Pollen tube growth through the style was essential for pollen tube guidance to the ovules and penetration of the micropyle. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetics of Six Components of Autofertility in Vicia faba

The inheritance of six components of autofertility was studied in faba beans in two consecutive years (1989 and 1990) by analysing parents, Fl and segregating generations of 14 crosses between preselected pure lines. Genetic analysis was performed by a joint scaling test proposed by Cavalli (1952). Additive gene effects were more important than dominance effects in flowers/node, pods/flower and ovules/ovary. In contrast, characters directly related with the seed such as pods/node, seeds/pod and, to a lesser extent, seeds/ovule showed high positive values of dominance. The three latter traits in general exhibited strong heterosis, probably due to genetic interactions. In order to produce selection gain, any breeding programme has to consider that dominance and heterosis vary according to the character involved. The present study reveals different degrees of dominance expression depending on cross specificity as well as on environmental conditions. The advantages of studying segregating generations instead of diallel crosses and the need for a simultaneous study of many crosses are emphasized in this paper.     

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.     

Water Deficit during the Reproductive Period of Grass Pea (Lathyrus sativus L.) Reduced Grain Yield but Maintained Seed Size

The grain legume grass pea (Lathyrus sativus L.) is adapted to drought‐prone environments, but the extent and mechanisms of its tolerance are not well understood. In a pot experiment, water deficit was imposed on plants by withholding water from first flowering until predawn leaf water potential (LWP_pd) was ?3.12 MPa. Water deficit reduced dry matter, seed yield, harvest index and water use efficiency by 60 %, 87 %, 67 % and 75 %, respectively, when compared with the controls. Flower production stopped when LWP_pd fell to ?1.8 MPa. At LWP_pd?1.5 MPa, only 25 % of flowers resulted in filled pods (compared with 95 % filled pods in the control) with the rest aborted as flowers (48 %) or pods (27 %). Filled pods had more aborted ovules than controls, resulting in 29 % less seeds per pod. Water deficit reduced pollen viability, germination and the number of pollen tubes reaching the ovary by 13 %, 25 % and 31 %, respectively. Emergence from seeds produced from water‐deficient plants was 21 % less than controls, but seedling shoot dry mass was 18 % higher, in accordance with the 19 % higher seed mass. The sensitivity of flowering to drought limited pod numbers but enabled plants to retain existing pods and develop near‐normal seeds with low β‐N‐oxalyl‐l ‐α‐β‐diaminopropionic acid toxin concentrations. This trait is useful for farming systems reliant on harvested seed for the next crop and in cases where seed size influences the value of the product.     

Increased atmospheric humidity post pollination: A possible aid to the production of inbred line seed from mature flowers in the Brussels sprout (Brassica oleracea var. Gemmifera)

Summary In two highly self incompatible inbred lines of Brussels sprouts the effect of increased atmospheric humidity post pollination was examined immediately following 1) hand pollination of green buds and open flowers, and 2) blowfly pollination of open flowers. Data were obtained for mean number of seeds set per pollination, mean number of fruits setting seed, and mean number of seeds produced per fruit which set for both varieties. Measured as number of seeds produced per minute spent pollinating, it was clear that open flower pollination followed by high humidity conditions was a much more efficient method of producing inbred line seed (46 seeds/minute) than green bud pollination (27 seeds/minute).     

The reproductive biology of three perennial Sesbania species (Leguminosae)

Summary The reproductive biology of Sesbania sesban, S. goetzei and S. keniensis (Leguminosae; Papilionoideae) was studied. Fifty percent flowering was observed at 102 to 153 days after germination for S. sesban accessions; 96 to 146 days for S. goetzei and 131 to 176 days for S. keniensis accessions. Flowers opened in the afternoon and remained fresh for 2–3 days. Bee species including Xylocopa sp., Apis mellifera, Megachile bituberculata and Chalicodoma sp. visited the flowers. Hand pollination experiments showed that all three species were self and cross compatible. The percentage of fully developed seeds was greater in pods formed after cross pollination compared to self pollination. No evidence was found for stigmatic or stylar self incompatibility. Outcrossing is probably the common method of reproduction under natural conditions, although in isolated trees substantial seed set by selfing might occur. Pod production under natural conditions was 34% for S. sesban; 49% for S. goetzei and 39% for S. keniensis. Considerable variation was found in pod production under open pollination between accessions of the same species. Selective abortion was observed within pods, with more mature seeds formed at the distal end of the pod.     

Post-pollen-germination barriers to seed set in sweet-potato

Three sweet-potato crossing combinations selected for good pollen germination were characterized by a moderate to high amount of failure of tubes to pass from stigma to style. The number of tubes penetrating the style was about 6 times the number of seeds produced. Ovules of compatibly pollinated flowers were divided into two classes, by size. Larger (probably fertilized) ovules matured into seeds, whereas smaller (probably unfertilized ovules) dried into scales. Besides the incompatibility barrier in sweet potato, which inhibits pollen-germination, a physiological or mechanical barrier between stigma and style is hypothesized. Pollen tube growth is not appreciably inhibited within the style. A third barrier between stylar penetration and mature seed development is postulated but not identified. However, the mechanism does not appear to be due to embryo abortion. Seed abortion leads to small, weak and inviable seeds, and thus represents a further reproductive loss.     

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.     

Optimal pollination conditions for seed set after a self-pollination,an intraspecific cross and an interspecific cross of marrow-stem kale (Brassica oleracea var. acephala)

Summary The effects of different pollination techniques, with and without emasculation and delayed pollination, were investigated to find the conditions for maximum seed set after self-pollination and intraspecific and interspecific crosses of Brassica oleracea var. acephala. The results indicated that the pollination conditions achieving maximum seed set vary with the type of pollination. After controlled self-pollination, the best seed set occurs in bud 3 to bud 10. For the intraspecific cross, the youngest flower and the oldest bud produced the largest number of developed ovules but bud pollination was productive to bud 8. The yields from these two pollination types were best when the female parent was not emasculated. In the interspecific cross with B. campestris cv. Marco the best results came from the youngest flowers and the oldest buds subjected to the standard practice of pollinating directly after emasculation. Possible reasons for these effects are discussed.     

Selection for partial resistance to downy mildew in peas by means of greenhouse tests

Summary A partially downy mildew resistant pea line was back-crossed to a susceptible cultivar with more pods per node and lower seed weight. Breeding lines with different degrees of infection in a greenhouse test were selected. These lines and the two parental lines were investigated in field trials and tested in the greenhouse for four generations. Significant genetic variation among lines was found for infection of seedlings in greenhouse tests, and infection of pods, pod set and seed weight in field-trials. Infection of seedlings in the greenhouse was correlated with infection of pods in the field. In greenhouse tests, the non-genetic variance component was large in comparison with the genetic component and a significant genotype trial interaction was found. Significant repeatability was obtained for downy mildew on seedlings and pods, number of pods per node and seed weight. An unfavourable correlation between susceptibility to downy mildew and number of pods per node was found. No single breeding line showed the ideal combination of good resistance, high number of pods per node and small seeds. However, one line showing better resistance than the susceptible parent, with smaller seeds and more pods per node than the resistant parent was found. The susceptible parent also carries some resistance factor that is not present in the resistant parent.