Seed set,pollen morphology and pollen surface composition response to heat stress in field pea

Pea (Pisum sativum L.) is a major legume crop grown in a semi‐arid climate in Western Canada, where heat stress affects pollination, seed set and yield. Seed set and pod growth characteristics, along with in vitro percentage pollen germination, pollen tube growth and pollen surface composition, were measured in two pea cultivars (CDC Golden and CDC Sage) subjected to five maximum temperature regimes ranging from 24 to 36 °C. Heat stress reduced percentage pollen germination, pollen tube length, pod length, seed number per pod, and the seed–ovule ratio. Percentage pollen germination of CDC Sage was greater than CDC Golden at 36 °C. No visible morphological differences in pollen grains or the pollen surface were observed between the heat and control‐treated pea. However, pollen wall (intine) thickness increased due to heat stress. Mid‐infrared attenuated total reflectance (MIR‐ATR) spectra revealed that the chemical composition (lipid, proteins and carbohydrates) of each cultivar's pollen grains responded differently to heat stress. The lipid region of the pollen coat and exine of CDC Sage was more stable compared with CDC Golden at 36 °C. Secondary derivatives of ATR spectra indicated the presence of two lipid types, with different amounts present in pollen grains from each cultivar.     

Viability of plum ovules at different temperatures

The viability of ovules was studied in five plum cultivars under laboratory conditions at four constant temperatures: 5°C, 10°C, 15°C and 20°C and under field conditions over two years. During 10 days from the onset of full bloom, ovule viability in cvs ‘?a?anska Rana’, ‘?a?anska Najbolja’ and ‘?a?anska Lepotica’ was between 80–100 % at the temperatures of 5°C, 10°C and 15°C, in both years. In the same period, ovule viability in cvs ‘Wangenheims Frühzwetsche’ and ‘Po?ega?a’ was lower, but never below 50%. At the constant temperature of 20°C, all plum cultivars showed a decline in longevity of ovule viability, which was pronounced in cv. ‘?a?anska Rana’. During the 10 days from the onset of full bloom, ovule viability in all five plum cultivars under field conditions showed a high viability, which approximated to the ovule viability of the cultivars at the constant temperatures of 5°C, 10°C and 15°C, in both years. Determination of the longevity of ovule viability in the mentioned plum cultivars is of great importance due to its effect on the effective pollination period and fertilisation success. This paper deals in detail with the interrelations between the temperature effects on ovule viability, pollen tube growth and fertilisation, as well as on fruit setting.     

Freezing injury to ovules, pollen and seeds in winter rape

Winter rapeseed (Brassica napus, cv. Samouraï) flowersearly in spring and, under field conditions, short freezingperiods can occur. Unacclimatized plants were freeze-stressed(–3°C for 4 h) at different developmental stages ofbuds, open flowers and seeds. The dissection of pistils from stressed plants showed that freezingresults in shrivelled ovules. We assessed freezing injury onthe basis of per cent of shrivelled ovules: ovule sensitivitybegins early (8 d before anthesis) but increases up to anthesis.Crosspollination of stressed pistils with non-stressed pollenshowed that recording of freeze-injured ovules is a good methodfor early estimation of the effect of stress on seed yields. On the other hand, stress does not reduce the viability of pollen,except when it was applied at the binucleate pollen stage. Useof frozen pollen x nonstressed pistils has little effect onseed yields. Freeze injury on seeds was assessed by seed filling:seeds are very susceptible just after fertilization until 20d after fertilization (DAF). Freezing seems to inhibit seedfilling. A germination test of stressed seeds during their developmentindicated that embryo viability is not affected if the stressoccurs after 35 DAF. As the embryos develop, resistance to stressincreases. Key words: Brassica napus, rapeseed, freeze injury, pollen and ovule, seed filling     

OVULE PACKAGING IN STOCHASTIC POLLINATION AND FERTILIZATION ENVIRONMENTS

The modular morphology of plants has important consequences for reproductive strategies. Ovules are packaged in discrete structures (flowers) that usually vary stochastically in pollen capture and ovule fertilization, because of the vagaries of pollen transfer by external agents. Different ovule packaging schemes may use limited reproductive resources more or less effectively, so that some number of ovules per flower may be optimal, given the prevailing probabilities of ovule fertilization. I derive a phenotypic model for ovule number per flower that maximizes the expected total ovule fertilizations on a plant when pollination and fertilization vary randomly among individual flowers. This model predicts that, except for small or inexpensive flowers, ovules should be “oversupplied” relative to the mean receipt of pollen tubes, so that pollen limitation of seed set should be common. Published data are congruent with this prediction. Additional hypotheses on the relation of ovule packaging to floral cost, plant size, and variance in pollen receipt are suggested by the model, but few data exist to evaluate these hypotheses.     

Ovule differences between single-kernelled and double-kernelled fruits in almond (Prunus dulcis)

In order to detect differences between almond cultivars that commonly produce one or two seeds, we compared ovule sizes and developmental stages at anthesis and three days after anthesis. The results showed that the presence of only small differences in size between primary and secondary ovules during flowering seemed to be a necessary but not sufficient condition for the production of double‐kernelled fruits. In contrast, differences in the developmental stage of both ovules did not prevent the production of double kernels. Large differences in ovule size at anthesis or in the following days resulted in fruits with single seeds. The behaviour of the secondary ovules in cultivars producing fruit with single seeds was very different, even in closely related cultivars. The degeneration of the secondary ovule seems to be genetically programmed and follows a genotype‐dependent pattern.     

Heat-stress effects on reproduction and seed set in Linum usitatissimum L. (flax)

Heat stress can detrimentally affect the reproductive capacity of many plants. The effect of a 7 or 14 d heat stress on flowering, seed set, pollen viability and germinability of flax (Linum usistatissimum L.) was assessed under growth chamber conditions. An incremental (2 °C/h), cyclical (daytime high 40 °C and night‐time low 18 °C) heat stress was applied 12 d after the initiation of flowering. Although flower formation in flax was not affected by heat stress, boll formation and seed set were reduced with onset of the heat stress. On removal of heat stress the stressed plants showed a compensatory response, flowering and producing bolls at a greater rate than the control plants. Heat stress significantly prolonged flowering by 17 d. Boll weight and seed weight were reduced with heat stress and the number of malformed, sterile seed increased three‐fold after 14 d of heat stress. Pollen viability and appearance were negatively affected after 6 and 10 d of heat stress, respectively. Pollen germinability decreased by the sixth day of heat stress, with no pollen germinating by the tenth day. Effects of heat stress on pollen viability and germinability alone, which did not occur until after the sixth day of the stress, could not account for the decreased boll formation due to heat stress in flax. These observations suggest that a combined effect of heat stress on both pollen and ovules contributes to decreased boll formation and seed set in flax.     

Effect of pollination time,the hour of daytime,pollen storage temperature and duration on pollen viability,germinability, and fruit set of date palm (Phoenix dactylifera L.) cv "Deglet Nour"

Success artificial pollination with viable pollen is crucial process in the production chain of date palms. This study evaluated the impact of pollen storage temperature and duration, pollination time following spathe cracking, and the hour of daytime on pollen viability, germinability, fruit set and yield of 'Deglet Nour' date palm cultivar. In in vitro tests, fresh pollen showed the maximum viability (96.3%) and germination (85%) but it decreased thereafter upon the storage temperature (28, 4 and ?30 °C) and duration (3, 6, 9 and 12 months). In this respect, pollen stored at ?30 °C retained highest viability and germinability followed by those stored at 4 and then at 28 °C. In filed experiments, fruit set was 85, 75, 65, and 45% with pollination using fresh pollen, or pollen stored at ?30, 4 and 28 °C, respectively. Fruit set was 95%, 75%, and less than 50%, for pollination performed on the same day of spathe cracking, 6 and 12 days later, respectively. The highest fruit set percentage and yield/bunch were obtained with pollination performed between 12.0 pm and 15.0 pm in contrast to 8.0–11.0 am or 16.0–17.0.     

Breeding systems in Cyrtanthus (Amaryllidaceae): variation in self‐sterility and potential for ovule discounting

• Breeding systems of plants determine their reliance on pollinators and ability to produce seeds following self‐pollination. Self‐sterility, where ovules that are penetrated by self‐pollen tubes that do not develop into seeds, is usually considered to represent either a system of late‐acting self‐incompatibility or strong early inbreeding depression. Importantly, it can lead to impaired female function through ovule or seed discounting when stigmas receive mixtures of self and cross pollen, unless cross pollen is able to reach the ovary ahead of self pollen (‘prepotency’). Self‐sterility associated with ovule penetration by self‐pollen tubes appears to be widespread among the Amaryllidaceae.
• We tested for self‐sterility in three Cyrtanthus species – C. contractus, C. ventricosus and C. mackenii – by means of controlled hand‐pollination experiments. To determine the growth rates and frequency of ovule penetration by self‐ versus cross‐pollen tubes, we used fluorescence microscopy to examine flowers of C. contractus harvested 24, 48 and 72 h after pollination, in conjunction with a novel method of processing these images digitally. To test the potential for ovule discounting (loss of cross‐fertilisation opportunities when ovules are disabled by self‐pollination), we pollinated flowers of C. contractus and C. mackenii with mixtures of self‐ and cross pollen.
• We recorded full self‐sterility for C. contractus and C. ventricosus, and partial self‐sterility for C. mackenii. In C. contractus, we found no differences in the growth rates of self‐ and cross‐pollen tubes, nor in the proportions of ovules penetrated by self‐ and cross‐pollen tubes. In this species, seed set was depressed (relative to cross‐pollinated controls) when flowers received a mixture of self and cross pollen, but this was not the case for C. mackenii.
• These results reveal variation in breeding systems among Cyrtanthus species and highlight the potential for gender conflict in self‐sterile species in which ovules are penetrated and disabled by pollen tubes from self pollen.

     

Insight into “serpentine syndrome” of Albanian,endemic violets (Viola L., Melanium Ging. section) – Looking for unique,adaptive microstructural floral,and embryological characters

The genus Viola, particularly Melanium section, rich in metallophytes, is an excellent taxon for study of microevolutionary and adaptation processes. Pollen, ovule, and microstructural floral characters were investigated by LM, SEM, and CLSM in seven endemic Albanian violets, five serpentinophytes (Viola albanica, V. dukadjinica, V. albanica × V. dukadjinica, V. raunsiensis, and V. macedonica), two from chalk soil (V. aetolica and V. schariensis), and in their closest relatives (V. lutea ssp. sudetica, V. tricolor ssp. tricolor, and V. arvensis) for their taxonomic usefulness and adaptive value. Three among analyzed characters were common in all Albanian violets however not unique. Serpentinophytes, V. aetolica and V. schariensis possessed hairs deep inside the spur, developed pollen heteromorphism, both increase the chance of pollination in unpredictable conditions and had strongly developed tannin rich layer in the outer integument of the young ovules with a protective role. They also all exhibited high pollen viability (86.9 ± 10.2%), high frequency of normally developed, enlarged (fertilized) ovules in ovary (65.0 ± 24.0%), but also high frequency of degenerations in developing ovules (40.4 ± 9.8%). Several flower characters may be adaptive in the unfavorable, high altitude environment, including serpentine soils. High pollen viability and normally developed fertilized ovules are sufficient for Albanian species maintenance.     

Effect of ovule position within the pod on the probability of seed production in Bauhinia ungulata (Fabaceae)

BACKGROUND AND AIMS: It has been claimed that ovules linearly ordered within a fruit differ in their probabilities of reaching maturity. This was investigated by studying the effect the position of an ovule within the pod has on seed abortion and seed production in Bauhinia ungulata. METHODS: Fruits collected during the dry seasons of 1999, 2000 and 2001 were opened, and the number, position and status of each ovule within the fruit were recorded. A GLM model was used to assess the effects of population, tree identity and ovule position within the pod on ovule fertilization, seed abortion, seed damage and seed maturation in two populations of B. ungulata. KEY RESULTS: Nearly 30% of the ovules were not fertilized in 1999; this percentage dropped to 5% the following two years. Seed abortion (50%) and seed damage (15%) were the same every year during the study period. Only 15% of the initial ovules developed into mature seeds in 1999; this value increased to 35% in 2000 and 2001. However, seed survivorship was dependent on the position of the ovule within the pod; non-fertilized and early aborted ovules were found more often near the basal end of the ovary. The frequency of seed damage was not affected by position. Mature seeds were found mainly in the stylar half of fruits, where ovules are likely to be fertilized by fast pollen tubes. CONCLUSIONS: The pattern of seed production in B. ungulata is non-random but is dependent upon the position of the ovule within the pod. The results suggest that the seeds produced within a fruit might differ in their vigour.     

Pollen-limited seed set in pseudogamous blackberries (Rubus L. subgen. Rubus)

Summary Pseudogamous blackberry species are polyploid and usually exhibit meiotic irregularities causing severe reduction in pollen viability. When species means were compared, relative seed set was strongly correlated with the number of good pollen grains produced per flower divided by the number of ovules (good-pollen/ovule ratio).Partial correlation analysis for percentage viable pollen and relative seed set, controlling for good-pollen/ovule ratio, revealed an almost significant relationship, presumably because meiotic irregularities have a similar effect on both pollen viability and the viability of meiotically derived embryo sacs. Seed germination, on the other hand, showed no relationship with relative seed set or with pollen viability.The automatic selfing capacity exhibited by most of these blackberry species does not appear to be related to either pollen production or relative seed set.     

Flower developmental stage and organ sensitivity of bell pepper (Capsicum annuum L.) to elevated temperature

High temperatures adversely affect crop productivity of several plant species including bell pepper (Capsicum annuum L. var. annuum). The objectives of this study were: (1) to determine whether flower ontogeny is adversely affected by high temperature during different phases of development, including pre‐ and post‐pollination events; (2) to determine the duration of high temperature exposure necessary to cause reduction in fruit set; and (3) to determine whether injury to the pistil or stamen during development is responsible for reduced fruit set during high temperature. We determined that flower buds at <2·5 mm in length, corresponding to microspore mother cell meiosis to tetrad dissolution, and flowers that reached anthesis during the high temperature exposure had reduced fruit set when exposed to 33 °C for 48 or 120 h. Flower buds at <2·5 mm in length also had reduced pollen viability when exposed to 33 °C for 120 h. Morphological examination demonstrated that meiocytes initiated tetrad formation, but after dissolution the microspores remained small and clumped without a thick exine. High temperature exposure at a late‐development, pre‐anthesis stage did not affect pistil or stamen viability, but high post‐pollination temperatures inhibited fruit set, suggesting that fertilization is sensitive to high temperature stress.     

Gibberellic acid impairs fertilization in Clementine mandarin under cross-pollination conditions

We investigated the effect of gibberellic acid (GA₃) in the fertilization of Clementine mandarin cv. ‘Clemenules’ (Citrus clementina Hort. ex Tan.), a parthenocarpic variety that produces seedless fruit due to its self-incompatible nature, but yields seedy fruits when grown under cross-pollination conditions.Experiments were conducted with on-tree ‘Clemenules’ flowers and ‘Fortune’ mandarin pollen (C. clementina Hort. ex Tan. × C. reticulata Blanco), which is sexually compatible with the former. Preanthesis treatment at −2 days after anthesis (−2 DAA) enhanced ovule abortion in both unpollinated and cross-pollinated (at +2 DAA) flowers. In the latter, the number of pollen tubes reaching the ovules was significantly reduced although pollen grains were not treated; thus, fertilization was partially avoided and seed set was reduced. When GA₃ was applied at anthesis (0 DAA) at the time of pollination, ovule abortion was again enhanced, and pollen tube growth was completely arrested; thus, fertilization was prevented and seed set was impeded. When GA₃ was applied 24 h after pollination (+1 DAA in flowers pollinated at anthesis), pollen tube growth was impaired but not arrested and ovule abortion was enhanced; therefore, fertilization was not prevented but impaired.We conclude that, when applied the days around anthesis, GA₃ (10 mg l⁻¹) impairs fertilization by either enhancing ovule abortion or reducing pollen tube growth, in ‘Clemenules’ flowers under cross-pollination conditions. The intensity of the response depends on the physiological flower state at the moment of treatment.     

Production of haploid plants from in vitro culture of unpollinated ovules of Cucurbita pepo

Ovaries from squash plants (cv. Eskandarani) were picked one day before anthesis, and exposed to cold temperature (4 °C) for 0, 2, 4 and 8 days. The ovules were cultured on MS medium with 30 g l⁻¹sucrose, 8 g l⁻¹agar and supplemented with four concentrations of 2,4-D, i.e., 0.1, 1.0, 5 and 10 mg l⁻¹. Then the dishes were incubated at 25 ± 1 °C under 16-h photoperiod for 4 weeks. After that ovules were transferred to growth regulator free MS medium for 4 weeks. Data indicated that the most plantlets per 100 cultured ovules resulted from the ovule of ovaries without cold treatment, when cultured in MS medium supplemented with 1 or 5 mg l⁻¹2,4-D. The cytological study revealed that one third of examined plants were haploid (2n = x = 20) and the others were double haploid (2n = 2x = 40). This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro pollination of isolated ovules of Cichorium intybus L.

 A method for the in vitro pollination of chicory (Cichorium intybus L.) ovules was developed. The purpose was to avoid self-incompatibility after in vitro self-pollination of ovules isolated from flower buds before anthesis and from open flower heads. Seedlings were obtained at a low percentage (0.76%), and the results were explained in terms of pollen viability, pollen germination on the ovule, embryogenesis studies and ploidy analysis. Received: 9 April 1997 / Revision received: 23 July 1997 / Accepted: 6 September 1999     

The relative importance of architecture and resource competition in allocation to pollen and ovule number within inflorescences of Hosta ventricosa varies with the resource pools

Background and Aims

Allocation of resources to floral traits often declines distally within inflorescences in flowering plants. Architecture and resource competition have been proposed as underlying mechanisms. The aim of the present study is to assess the relative importance of resource competition and architectural effects in pollen and ovule production on racemes of Hosta ventricosa, an apomictic perennial herb.

Methods

Combinations of two defoliation treatments (intact and defoliated) and two fruit-set treatments (no-fruit and fruit) were created, and the roles of architecture and resource competition at each resource level were assessed.

Key Results

Pollen and ovule number per flower increased after defoliation, but pollen to ovule ratio per flower did not change. Pollen, ovules and the pollen to ovule ratio per flower declined distally on racemes at each resource level. In the intact treatment, fruit development of early flowers did not affect either pollen or ovule number of late flowers. In the defoliated treatment, fruit development of early flowers reduced both pollen and ovule numbers of late flowers due to over-compensation caused by defoliation. Late flowers on defoliated fruit racemes produced less pollen than intact fruit racemes but the same number of ovules; therefore, the reduction in pollen number was not caused by over-compensation. In addition, the fruit-set rate of early flowers during flowering was higher in intact racemes than in defoliated racemes.

Conclusions

In flowering plants, the relative importance of architecture and resource competition in allocation to pollen and ovules may vary with the resource pools or the overall resource availability of maternal plants.     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.     

Variation of pollen and ovule parameters among different ploidy levels ofCorydalis (Fumariaceae)

The size and number of pollen grains and ovules are compared between 20 populations of different ploidy levels in two self-incompatible species ofCorydalis (Fumariaceae), to test the presence of ploidy-related variation in these reproductive characters. In both species, higher ploidy levels are associated with larger pollen grains, ovules and corolla, but the number of pollen grains and ovules are not different between ploidy levels. The investment per flower is consequently larger at higher ploidy levels, but the flower number per individual is lower, suggesting that the mode of partition of the investment for sexual reproduction varies between different ploidy levels. InC. orthoceras, sex allocation estimated by pollen:ovule ratios in number and volume is more female-biased in polyploids than in diploids. In spite of these variations, the characters studied can not be used as indicators of ploidy level due to the large overlaps between the ploidy levels.     

Differential ovule development following self- and cross-pollination: the basis of self-sterility in Narcissus triandrus (Amaryllidaceae)

Self-pollination results in significantly lower seed set than cross-pollination in tristylous Narcissus triandrus. We investigated structural and functional aspects of pollen–pistil interactions and ovule–seed development following cross- and self-pollination to assess the timing and mechanism of self-sterility. Ovule development within an ovary was asynchronous at anthesis. There were no significant differences in pollen tube behavior following cross- vs. self-pollination during the first 6 d of growth, regardless of style morph type. Double fertilization was significantly higher following cross- vs. self-pollination. Aborted embryo development was not detected following either pollination type up to seed maturity. Prior to pollen tube entry, a significantly greater number of ovules ceased to develop following self- vs. cross-pollination. These results indicate that self-sterility in N. triandrus operates prezygotically but does not involve differential pollen tube growth typical of many self-incompatibility (SI) systems. Instead, low seed set following self-pollination is caused by a reduction in ovule availability resulting from embryo sac degeneration. We hypothesize that this is due to the absence of a required stimulus for normal ovule development. If this is correct, current concepts of SI may need to be broadened to include a wider range of pollen–pistil interactions.     

Breeding systems in Clivia (Amaryllidaceae): late‐acting self‐incompatibility and its functional consequences

Late‐acting (ovarian) self‐incompatibility, characterized by minimal or zero seed production following self‐pollen tube growth to the ovules, is expected to show phylogenetic clustering, but can otherwise be difficult to distinguish from early‐acting inbreeding depression. In Amaryllidaceae, late‐acting self‐incompatibility has been proposed for Narcissus (Narcisseae) and Cyrtanthus (Cyrtantheae). Here, we investigate whether it occurs in the horticulturally important genus Clivia (Haemantheae) and test whether species in this genus experience ovule discounting in wild populations. Seed‐set results following controlled hand pollinations revealed that Clivia miniata and C. gardenii are largely self‐sterile. Self‐ and cross‐pollinated flowers of both species had similar proportions of pollen tubes entering the ovary, and those of C. gardenii also did not differ in the proportions of pollen tubes that penetrated ovules, thus ruling out classical gametophytic self‐incompatibility acting in the style, but not early inbreeding depression. Flowers that received equal mixtures of self‐ and cross‐pollen set fewer seeds than those that received cross‐pollen only, but it was unclear whether this effect was a result of ovule discounting or interactions on the stigma. The prevention of self‐pollination by the emasculation of either single flowers or whole inflorescences in wild populations did not affect seed set, suggesting that ovule discounting is not a major natural limitation on seed production. Flowers typically produce one to three large fleshy seeds from approximately 16 available ovules, even when supplementally hand pollinated, suggesting that fecundity is mostly resource limited. The results of this study suggest that Clivia spp. are largely self‐sterile as a result of either a late‐acting self‐incompatibility system or severe early inbreeding depression, but ovule discounting caused by self‐pollination is not a major constraint on fecundity. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 155–168.