The significance of the methods of stigmatal and placental pollinationin vitro inAntirrhinum majus L.; seed and callus formation on placentae

A study was made on certain problems connected with the application of methods of stigmatal and placental pollinationin vitro in the snapdragon. Germinable pollen without microbial contamination could be obtained only from the flowers which were left, after surface sterilization, on the stem till the dehiscence of anthers. The germination of pollen, and especially the growth of pollen tubes was better in a 0.3 M lactose solution with 10^?3 per cent H₃BO₃ than in usual sucrose media. The seedsin vitro could be obtained only after normal pollination and the successive artificial cultivation of the entire pistil (the method of stigmatal pollinationin vitro). The seeds did not differ either in their morphology or in their size from those developed under natural conditions, they were viable and without dormancy. Though the pollinated pistils were cultivated in the medium without growth substances, callus formation from uncovered ovules and placenta was observed in some cases. A dependence was revealed of the proliferation on pollination and on the cultivar employed. When using the method of placental pollinationin vitro we failed to obtain seeds. The main difficulty in the use of this method seems to consist in the fact that pollen tubes are not capable of growing from the surface of ovules and placenta into the micropyle.     

Perpetuation of the self-incompatible rare species Swainsona laxa R. BR by pollination in vitro and in situ

Seed set in the rare plant Swainsona laxa grown in the glasshouse was prevented by self-incompatibility and the lack of cross pollination due to loss of pollen viability before the flowers opened. Fertilization of decapitated, excised pistils in vitro overcame the incompatibility and resulted in viable seed production. Presence of the calyx was not necessary. Manual cross pollination in situ using pollen collected from flower buds before opening also produced seed.     

Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae) and its consequences on fruit and seed set

Turnera hermannioides is a ruderal distylic subshrub, native to NE-Brazil. In the Catimbau National Park, situated within the semi-arid Caatinga region, we studied the pollination ecology of this species, emphasizing (1) effective pollinators; (2) characteristics of short- and long-styled flowers; (3) intra- and intermorph pollen flow; and (4) fruit and seed set. Short and long-styled morphs differ in pollen size and ornamentation, stigmatic surface, style and stamen length and nectar production. The flowers are obligate intermorphic outcrossers and depend on animals for pollination. The flowers of T. hermannioides attracted insect visitors of 25 species, among them butterflies, beetles, but mainly bees. Polylectic bees, such as Apis mellifera, stingless bees, and solitary Callonychium brasiliense were the most frequent visitors and the principal pollinators. The frequency of visits, however, was very low (on average 1.9 visits/flower/day), resulting in a low and unbalanced legitimate pollen flow, i.e. a much lower number of pollen grains from short-styled flowers reaching stigmas of long-styles than vice versa, which in turn compromised fruit and seed set. Surprisingly, Protomeliturga catimbaui and P. turnerae, both oligolectic on flowers of the Turneraceae-clade of the Passifloraceae s.l., were very rare flower visitors and did not contribute significantly to the pollination of Turnera hermannioides. We discuss how the low visitation rate and the inefficiency of pollinators (i.e. inefficiency in promoting legitimate pollination) compromise pollen flow and, consequently, fruit and seed set in this species.     

Pollen and resource limitations to lifetime seed production in a wild population of the endangered plant Disanthus cercidifolius var. longipes H. T. Chang (Hamamelidaceae)

Disanthus cercidifolius Maxim. var. longipes H. T. Chang, a plant species that only occurs in a few counties in Hunan, Jiangxi and Zhejiang Provinces and with a relatively small number of individuals, is ranked as a second Class endangered species for conservation in China. We have studied the effect of pollen and resources available to female reproduction, and the reproductive mechanism of “excess flowers with low fruit set” in Disanthus cercidifolius Maxim. var. longipes H. T. Chang was discussed. Results are as follows Pollen from different sources has significant effects on fruit set and seed set of Disanthus cercidifolius Maxim. var. longipes H. T. Chang. The pollen source rather than pollen numbers significantly affected reproduction of this species. In wild populations, producing one fruit needs about 54.8 flowers, and one satiation seed needs about 6.60 flowers or 83.19 ovules. After fertilizing, which was propitious to flower development, the abortion rate of flower buds was decreasing, but the flowering rate was increasing. The fruit set and seed set was also significantly increasing, while abortion rate of fruit was significantly decreasing. With the increasing percentages of cutting leaves, the fruit set decreased, but the abortion rate of fruit shows no significant differentiation among treatments. After cutting branches that were puny, broken and insectin-fested branches, the flower number seemed to be decreasing, but the fruit set and seed set all increased significantly. After removing some flowers, the fruit set was calculated with respect to the number of flowers remaining after the treatment increased with increasing of percentages of flower removal, whereas fruit set calculated with respect to the initial number of flowers remained constant, and the mean weights of per fruit and per seed all decreased significantly. Sufficient spatial or temporal heterogeneities in nutrient levels might allow limitation of seed set by resources and pollen in a natural population, while supplying resources may indirectly affect pollination by increasing attraction of the flowers to pollinators. There were very low fruit and seed sets in natural populations of Disanthus cercidifolius Maxim. var. longipes H. T. Chang. Different factors may have interacted to effect a low fruit set. A joint adoption of the “selection abortion hypothesis”, “ovary reserve hypothesis” and “male function hypothesis” seems to be the most likely explanation for the reproductive strategy of “excess flowers with few fruit sets” in Disanthus cercidifolius Maxim. var. longipes H. T. Chang.     

Seed formation in Narcissus pseudonarcissus L. After placental pollination in vitro

Viable seeds of Narcissus were obtained after the pollination of excised placentas in vitro. They manifested normal dormancy as the seeds formed in nature. The amount of seed set was lower when sucrose in the medium was replaced by invert sugar. On the other hand, it was greatly improved by changing the incubation temperature from 25°C to 15°C. Both the process of fertilization and the development of fertilized ovules seem to be favourably affected by the low temperature. A significantly higher number of seeds per ovary was obtained on placentae excised from flowers collected 2 days after anthesis as compared with those taken on the day of anthesis.     

Seed set after pollination with in-vitro-matured,isolated pollen of Triticum aestivum

Summary Immature pollen of two varieties of Triticum aestivum, at the stage right after the first pollen mitosis, was isolated from individual anthers and cultured in microcultures of microliter droplets. In a specifically designed medium, some of the pollen grains developed to maturity. These were applied to excised stigmas on agar, where they produced pollen tubes. Application to flowers in vivo led to seed set. Pollen was matured in vitro from a variety that produced a different protein banding pattern on SDS-PAGE as compared to the variety that was pollinated. The protein banding in the produced seeds showed the hybrid pattern, demonstrating that the seeds were not produced by self-pollination in this in-breeding species but by pollination with the in-vitro-matured pollen.     

Flower formation in excised tobacco stem segments; I. Methodology and effects of plant hormones

The formation of flowers has been studied in stem tissue excised from flowering plants of Nicotiana tabacum variety Wisconsin No. 38, and cultured in vitro on Murashige and Skoog nutrient medium. A procedure for quantitative evaluation of factors influencing floral expression has been developed and effects of the growth substances, indole-3-acetic acid (IAA), kinetin and gibberellic acid (GA₃), on the process are reported.     

华北驼绒藜自然种群结实的花粉和资源限制

运用人工授粉、补充无机营养、去叶处理以及疏花处理研究了华北驼绒藜自然种群花粉和资源有效性对结实的影响,并进一步探讨了该植物的选择性败育现象和繁殖对策。结果表明:花粉来源而不是花粉数量对华北驼绒藜结实存在显著影响;同枝授粉结籽率和结实率均低于自然授粉,异株授粉则显著高于同枝授粉和自然授粉;异株授粉种子千粒重显著高于自然授粉和同株异枝授粉,极显著高于同枝授粉。华北驼绒藜结实存在资源限制,补充施肥提高了单枝开花数和结籽率。随摘除叶片数量的增加,结籽率明显降低,摘除叶片处理与对照之间均形成显著差异,摘除1/2叶、3/4叶以及去全叶处理使种子千粒重显著低于对照。人工疏花实验表明华北驼绒藜存在选择性败育现象。华北驼绒藜可根据花粉和资源的可利用性来调整性分配、授粉方式和结实以获得最大适合度,具有复杂的繁殖对策,表现出较好的环境适应性。     

A comparison of in vitro with in vivo flowering in Gentian

Young nodal explants of Gentiana triflora Pall. var. axillariflora were cultured in a woody plant medium (WPM) supplemented with B5 vitamins, sucrose (3%) and kinetin (2.0 μM). A novel observation was made in that in vitro flowering occurred following development of the axillary bud of the cultures. A comparison was made between in vitro and in vivo flowers. Although smaller in size, the in vitro flowers were morphologically comparable to the in vivo ones. Flowers from both sources were semi-opened or not opened. The colour of the in vitro flowers was paler than those in vivo. Stigma development was generally poor in both in vitro and in vivo flowers. Pollen viability was over 90% in both types of flowers. About 11% and 34% of pollen from in vitro and in vivo flowers, respectively, germinated on WPM containing 100 g l⁻¹ sucrose solidified with 10 g l⁻¹ agar. Hand pollination of stigma could raise viable seed production in in vivo-flowering plants from 0.3 o/o (i.e. without aided pollination) to 3% but none in in vitro-flowering plants where only seed-like structures, probably unfertilised ovules, were found. This revised version was published online in August 2006 with corrections to the Cover Date.     

Pollination intensity and potential seed set in Passiflora vitifolia

Summary Initial seed set and fruit set were pollen-limited in a Costa Rican population of Passiflora vitifolia, a self-incompatible species with 200–350 ovules per flower. Pollination intensity was measured by counting the number of allogamous pollen grains on stigmas of the large one-day flowers. Hand-pollinations demonstrated that 25–50 pollen grains are required for fruit set, and >450 are needed for maximum seed set, with a pollen:seed ratio of about 1.6:1.0. Hummingbirds (Phaethornis superciliosus) delivered sufficient allogamous pollen for maximum seed set to only 28% of the flowers examined. Naturally pollinated flowers yielded fewer fruits and fewer seeds per fruit than those pollinated by hand. Most pollen transferred by humming-birds was self-incompatible; emasculated flowers yielded higher seed set than flowers with intact anthers. Visitation rates did not provide a good index of effective pollination.There were significant differences in ovule number, maximum seed set, and maximum per cent seed set among individual vines. More than half of an individual's flowers failed to set fruit, whether pollinated by birds or by hand. In this population, maximum reproductive potential may be limited by maternal resources for fruit development, but seed set varies with pollination intensity. Pollen-limited seed set may be a disadvantage of self-incompatibility, especially in species with many-seeded fruits.     

Why is there variation in mean seed size among plants within single populations? test of the fertilization efficiency hypothesis

We tested the fertilization efficiency hypothesis, which attempts to explain mean seed size variation among plants within single populations, by comparing the patterns of seed size variation between chasmogamous (CH) flowers and cleistogamous (CL) flowers in Impatiens noli-tangere and Viola grypoceras, respectively. The fertilization efficiency hypothesis predicts that larger plants produce larger seeds if the number of pollen grains captured by a plant increases with increased allocation of resources to its attractive structures (e.g., corolla and nectar), but with diminishing gains. Thus, seed size should depend on plant size in seeds from CH flowers because of the diminishing gains of capturing pollen in these flowers, whereas seed size should not depend on plant size in seeds from CL flowers because CL flowers need not capture outcross pollen. We found significant positive correlations between mean seed size per plant and plant size for seeds from CH flowers in both species. However, there was no significant positive correlation between these two factors for seeds from CL flowers of both species. The results of the present investigations were thus consistent with the fertilization efficiency hypothesis.     

Effects of boron on pollen viability in wheat

Grain set failure in wheat, caused by boron (B) deficiency, is associated with poorly developed pollen and anthers. This paper presents results of a study of the effect of B on pollen viability when it was supplied "internally" through the roots and externally in an agar medium for in vitro germination.There was no major effect of B supply to wheat plants on the number of pollen anther^-1 or the percentage of pollen with positive reaction to iodine. Pollen germination in the medium was, however, responsive to both internal and external B supply. When B was not added to the medium, germination was poor, regardless of the level of B supplied to the plant, in both a B deficiency sensitive (SW41) and a B deficiency tolerant (Sonora 64) genotypes. The percentage of germinated pollen and length of the pollen tube increased with increasing medium B. With 20–100 mg H₃BO₃ L^-1 in the medium, the percentage of germinated pollen and length of the pollen tube responded positively to increasing B supply to the plant.No difference was found between sensitive and tolerant genotypes in the effect of B on their pollen viability. On the other hand, without added B in the nutrient solution applied to the plant, grain set was depressed in the B deficiency sensitive SW41 and not in the B deficiency tolerant Sonora 64. A difference in B supply to the germinating pollen in the stigma and style is one possible explanation for this variation in the response to B among wheat genotypes.     

Within-population spatial variation in pollinator visitation rates,pollen limitation on seed set,and flower longevity in an alpine species

Pollen limitation through insufficient pollen deposition on stigmas caused by too infrequent pollinator visitation may influence the reproductive outcome of plants. In this study we investigated how pollinator visitation rate, the degree of pollen limitation, and flower longevity varied spatially among three sites at different altitudes within a population of the dwarf shrub Dryas octopetala L. in alpine southern Norway. Significant pollen limitation on seed set only occurred at the mid-elevation site, while seed set at the other sites appeared to be mainly resource limited, thus indicating a spatial variation in pollen limitation. There was no association between the spatial variation in the extent of pollen limitation and pollinator visitation rate to flowers. However, pollinator visitation rates were related to flower longevity of Dryas; sites with low visitation rates had long-lived flowers and vice versa. Thus, our results suggest within-population spatial co-variation between pollinator visitation rates, pollen limitation, and a developmental response to these factors, flower longevity.     

Asymmetrical conspecific seed-siring advantage between Silene latifolia and S. dioica

Background and Aims

Silene dioica and S. latifolia experience only limited introgression despite overlapping flowering phenologies, geographical distributions, and some pollinator sharing. Conspecific pollen precedence and other reproductive barriers operating between pollination and seed germination may limit hybridization. This study investigates whether barriers at this stage contribute to reproductive isolation between these species and, if so, which mechanisms are responsible.

Methods

Pollen-tube lengths for pollen of both species in styles of both species were compared. Additionally, both species were pollinated with majority S. latifolia and majority S. dioica pollen mixes; then seed set, seed germination rates and hybridity of the resulting seedlings were determined using species-specific molecular markers.

Key Results

The longest pollen tubes were significantly longer for conspecific than heterospecific pollen in both species, indicating conspecific pollen precedence. Seed set but not seed germination was lower for flowers pollinated with pure heterospecific versus pure conspecific pollen. Mixed-species pollinations resulted in disproportionately high representation of nonhybrid offspring for pollinations of S. latifolia but not S. dioica flowers.

Conclusions

The finding of conspecific pollen precedence for pollen-tube growth but not seed siring in S. dioica flowers may be explained by variation in pollen-tube growth rates, either at different locations in the style or between leading and trailing pollen tubes. Additionally, this study finds a barrier to hybridization operating between pollination and seed germination against S. dioica but not S. latifolia pollen. The results are consistent with the underlying cause of this barrier being attrition of S. dioica pollen tubes or reduced success of heterospecifically fertilized ovules, rather than time-variant mechanisms. Post-pollination, pre-germination barriers to hybridization thus play a partial role in limiting introgression between these species.     

Nectar dynamics and reproductive success in Saponaria officinalis (Caryophyllaceae) in southern Germany

Nectar production and flower visitors of the night-flowering Saponaria officinalis L. (Caryophyllaceae) were studied in relation to the reproductive success. Nectar production was worthwhile for nocturnal flower visitors. Nectar standing crop was about 267 μg sugar per flower, and comparison of nectar offering of covered and freely exposed flowers revealed that main nectar secretion time is mainly during the night up to the morning hours. In both covered and freely exposed flowers nectar volumes decreased over the day. In covered flowers, nectar volume, sugar concentration, and sugar amount per flower increased up to the third day; in older flowers sugar secretion ceased. In 1996 Autographa gamma (Noctuidae) was the exclusive nocturnal flower visitor, but pollen transfer experiments proved that A. gamma (Noctuidae) is a very ineffective pollinator of S. officinalis. In 1999 up to 50% of the observed visitors were Sphingidae, which resulted in a significantly higher seed set. Fruit set was constantly high independent of pollinator availability. In the nectar manipulation experiments seed set was highest in non-emasculated flowers filled with unnaturally high concentrated sucrose solutions. Differences to seed set on stalks treated with a sucrose solution mimicking naturally concentrated nectar were significant. Lowest fruit and seed set were found on inflorescences with emasculated flowers filled with a sucrose solution mimicking naturally concentrated nectar.     

Enhanced seed development in the interspecific cross Allium cepa x A. sphaerocephalon through ovary culture

Allium sphaerocephalon pollen tubes grew into styles and penetrated micropyles of Allium cepa, but ovules started to degenerate about 16 days after pollination and no seeds developed. Seeds developed in vitro in ovaries excised from flowers 4 and 7 days after pollination. Seven weeks after culture initiation, seeds had grown in 4 of 96 excised ovaries, cultured on BDS medium supplemented with GA₃. Although the culture medium supported seed maturation within excised ovaries of self-pollinated A. cepa flowers, no viable hybrid seeds were recovered from crosses with A. sphaerocephalon. Extended post-fertilization barriers may have restrained development of hybrid embryos in vitro. Ovary culture followed by in ovulo embryo rescue may be feasible for distant-species hybridization in Allium.     

Pollination and late-acting self-incompatibility in Cyrtanthus breviflorus (Amaryllidaceae): implications for seed production

Background and Aims

Animal pollination is typically an uncertain process that interacts with self-incompatibility status to determine reproductive success. Seed set is often pollen-limited, but species with late-acting self-incompatibility (SI) may be particularly vulnerable, if self-pollen deposition results in ovule discounting. Pollination is examined and the occurrence of late-acting SI and ovule discounting assessed in Cyrtanthus breviflorus.

Methods

The pollination system was characterized by observing floral visitors and assessing nectar production and spectral reflectance of flowers. To assess late-acting SI and ovule discounting, growth of self- and cross-pollen tubes, and seed set following open pollination or hand pollination with varying proportions of self- and cross-pollen, were examined.

Key Results

Native honeybees Apis mellifera scutellata pollinated flowers as they actively collected pollen. Most flowers (≥70 %) did not contain nectar, while the rest produced minute volumes of dilute nectar. The flowers which are yellow to humans are visually conspicuous to bees with a strong contrast between UV-reflecting tepals and UV-absorbing anthers and pollen. Plants were self-incompatible, but self-rejection was late-acting and both self- and cross-pollen tubes penetrated ovules. Seed set of open-pollinated flowers was pollen-limited, despite pollen deposition exceeding ovule number by 6-fold. Open-pollinated seed set was similar to that of the cross + self-pollen treatment, but was less than that of the cross-pollen-only treatment.

Conclusions

Flowers of C. breviflorus are pollinated primarily by pollen-collecting bees and possess a late-acting SI system, previously unknown in this clade of the Amaryllidaceae. Pollinators of C. breviflorus deposit mixtures of cross- and self-pollen and, because SI is late-acting, self-pollen disables ovules, reducing female fertility. This study thus contributes to growing evidence that seed production in plants with late-acting SI systems is frequently limited by pollen quality, even when pollinators are abundant.     

Flowering and pollination patterns of Magnolia denudata with emphasis on anatomical changes in ovule and seed development

Magnolia denudata is an excellent ornamental and ecologically important tree that exhibits low fecundity because of seed abortion. To better understand the cause of this low sexual reproduction, pistil receptivity, and pollen quality were investigated using culture medium and histochemical staining, with emphasis on pollen viability at different flowering stages. Cross-pollination, geitonogamy, and self-pollination were undertaken on flowers of sample trees. We found that flowers produce a large number of pollen grains available for effective pollination. Pollen viability varied at different stages, but was highest during stage 3, in which the stamens detach from the axis, the anthers dehisce, and the gynoecium stigma starts to fade. The duration of pistil receptivity was approximately 5 h. These results indicate that poor fertilisation may occur if the optimum period for pollination is missed or the stigma receives poor-quality pollen grains from stages other than stage 3. Although dichogamy avoids selfing, it has led to considerable geitonogamy. Our anatomical analysis of ovule and seed development further revealed that fertilisation occurred in samples with geitonogamy, but that the embryo degenerated in the torpedo stage, demonstrating that aborted seeds were produced by fertilisation, rather than by pseudogamy. Our results provide new insights into the mechanism of reduced seed set under natural conditions.     

Reproductive biology of two species of Canavalia DC. (Fabaceae)—A non-conventional wild legume

The reproductive biology of two important species of Canavalia, i.e. Canavalia gladiata and Canavalia virosa, was investigated in detail by studying floral phenology, floral biology including fruit and seed set, breeding system and pollinator's activity. Both the species flower and set their seed primarily from August to December. The study of pollen–-pistil interaction indicated the existence of morphological protandry in both species, and pollen germination occurred only after rupture of the stigmatic surface. This suggests that some form of self-incompatibility operates in these species. Ants were the common vectors tripping the stigma and transporting some foreign pollen. Campylomma verbasci (large black ants) were only seen on the flowers of C. virosa, while Monomorium minimum (small black ants) were restricted to the flowers of C. gladiata. Inadequacy of reliable pollinators and high rate of bud/flower drop may be the main factors for low fruit and seed set in both the species.     

The Growth Activity and Fertilizing Capacity of Submersely Cultivated Pollen Tubes of Nicotiana tabacum L.

Tobacco pollen was cultivated in shaken suspension culturesand the growth estimated by weighing the mass of germinatedpollen separated from the nutrient solution. A formula for calculatingmean pollen tube length from the weight of the culture has beenderived for this pollen species. The growth of pollen tubes in vitro is shown to have a rhythmiccharacter. The rapid growth comparable to the mean growth ratein styles is limited to short time intervals alternating withprogressively extending periods of very depressed growth, whichceased entirely after 10–12 h of cultivation. When tested by placental pollination in vitro, the fertilizingcapacity of the pollen culture was found to increase duringthe first hour of cultivation but to decrease steadily thereafter.On the other hand, with the application of pollen tubes fromculture on stigma, a short precultivation period and even themere wetting of pollen had a negative effect on the seed set.With pollen cultivated longer than 4 h, no seed formation wasobserved.