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Key Message: Upregulation of genes involved in DNA damage repair and sperm cell differentiation leads to restoration of pollen viability in synthetic allotetraploid B. carinata after chromosome doubling. Apart from the well-known contribution of polyploidy to crop improvement, polyploids can also be induced for other purposes, such as to restore the viability of sterile hybrids. The mechanism related to viability transition between the sterile allodiploid and the fertile allotetraploid after chromosome doubling are not well understood. Here, we synthesised allodiploid B. carinata (2n = 2x = 17) and allotetraploid B. carinata (2n = 4x = 34) as models to investigate the cytological and transcriptomic differences during pollen development. The results showed that after chromosome doubling, the recovery of pollen viability in allotetraploid was mainly reflected in the stabilisation of microtubule spindle morphology, normal meiotic chromosome behaviour, and normal microspore development. Interestingly, the deposition and degradation of synthetic anther tapetum were not affected by polyploidy. Transcription analysis showed that the expression of genes related to DNA repair (DMC1, RAD51, RAD17, SPO11-2), cell cycle differentiation (CYCA1;2, CYCA2;3) and ubiquitination proteasome pathway (UBC4, PIRH2, CDC53) were positively up-regulated during pollen development of synthetic allotetraploid B. carinata. In summary, these results provide some refreshing updates about the ploidy-related restoration of pollen viability in newly synthesised allotetraploid B. carinata., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Main Conclusion: Four cultivars of Paeonia lactiflora pollen have a different viability after cryopreservation, and that the difference of pollen viability is related to calcium ions and cell wall deposition. Cryopreservation is a vital technique for preserving germplasm resources, offering extensive application prospects. Understanding the factors influencing pollen viability after cryopreservation is crucial for the permanent preservation and exchange of pollen resources. This study investigated pollen from four Paeonia lactiflora cultivars with varying viability after cryopreservation, aiming to determine whether calcium ions (Ca 2+ ) and cell wall deposition affect these viability changes. The results showed that Ca 2+ -ATPase activity and cytoplasmic Ca 2+ of all four cultivars exhibited an increasing trend after cryopreservation; the calmodulin (CaM) content varied with cultivars. Correlation analysis showed that fresh pollen viability was significantly negatively correlated with cytoplasmic Ca 2+ content and positively correlated with Ca 2+ -ATPase activity, while pollen viability after cryopreservation exhibited a significantly negative correlation with cytoplasmic Ca 2+ content and a positive correlation with CaM content. The pollen cell wall of the cultivar 'Zi Feng Chao Yang' (ZFCY), which showed increased viability after cryopreservation, contained significantly higher levels of low-temperature tolerance-related phospholipids and proteins compared to other cultivars. Additionally, all cultivars maintained a clear Ca 2+ gradient at the tips of pollen tubes after cryopreservation, without significant callose accumulation. These findings suggest that differences in Ca 2+ signaling and cell wall components deposition influence changes in pollen viability after cryopreservation, and the Ca 2+ gradient and callose at the tip of pollen tubes are not responsible for preventing pollen tube growth., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

To gain a deeper understanding of the flowering pattern and reproductive characteristics of Epimedium sagittatum, to enrich the research on the flower development of E. sagittatum and its reproductive regulation, and to screen the methods suitable for the rapid detection of pollen viability of E. sagittatum and to promote its cross-breeding. The characteristics of its flower parts were observed, recorded and measured, and the pollen viability of E. sagittatumwas determined by five methods, including TTC staining, I 2 -KI staining, red ink staining, peroxidase method and in vitro germination method. The flowering process of E. sagittatum can be divided into five stages: calyx dehiscence, bract spathe, petal outgrowth, pollen dispersal, and pollination and withering. The results of I 2 -KI staining and peroxidase method were significantly higher than those of other methods; the in vitro germination method was intuitive and accurate, but the operation was complicated and time-consuming; the red ink staining method was easy to operate and had obvious staining effect, and the results were the closest to those of the in vitro germination method; and it was found that the pollen of E. sagittatum was not as effective as the in vitro germination method at the bud stamen stage, the flower stigma and the flower bud. It was also found that the pollen viability and germination rate of E. sagittatum pollen were higher in the three periods of bud spitting, petal adductor and pollen dispersal. Comparing the five methods, the red ink staining method was found to be a better method for the rapid detection of pollen viability; the best pollination periods of E. sagittatum were the bud stamen stage, petal adductor stage, and pollen dispersal stage of flowers at the peak of bloom. This study on the flowering and fruiting pattern of E. sagittatum, and the related mechanism of sexual reproduction, can be used as a reference for the next step of research on the breeding of E. sagittatum., (© 2024. The Author(s).)

Programmed cell death (PCD) is an important factor to reduces the viability of plant germplasm after cryopreservation. However, the pathways by which PCD occurs is not fully understood. To investigate whether there is a mitochondrial pathway for pollen PCD after cryopreservation, the pollen of Paeonia lactiflora two cultivars with different PCD levels after cryopreservation was used as test material and the changes of mitochondrial calcium ions (Ca 2+ ), structure, function and their relationship with PCD were compared. The results showed that compared with fresh pollen, the PCD of 'Feng Huang Nie Pan' was significantly reduced after cryopreservation. Their mitochondrial Ca 2+ content decreased by 74.27%, mitochondrial permeability transition pore (MPTP) opening reduced by 25.41%, mitochondrial membrane potential slightly decreased by 5.02%, cardiolipin oxidation decreased by 65.31%, and oxygen consumption remained stable, with a slightly ATP production increase. On the contrary, compared with fresh pollen, 'Zi Feng Chao Yang' showed severe PCD after cryopreservation. The decline in mitochondrial Ca 2+ -ATPase activity led to an accumulation of excessive Ca 2+ within mitochondria, triggering widespread opening of MPTP, significantly affecting mitochondrial respiration and energy synthesis. These results suggest the mitochondrial pathway of PCD exists in pollen cryopreservation., Competing Interests: Declaration of Competing Interest This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. We have read and understood your journal’s policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The pollen viability directly affects the pollination process and the ultimate grain yield of rice. Here, we identified that the MORN motif-containing proteins, OsMORN1 and OsMORN2, had a crucial role in maintaining pollen fertility. Compared with the wild type (WT), the pollen viability of the osmorn1 and osmorn2 mutants was reduced, and pollen germination was abnormal, resulting in significantly lower spikelet fertility, seed-setting rate, and grain yield per plant. Further investigation revealed that OsMORN1 was localized to the Golgi apparatus and lipid droplets. Lipids associated with pollen viability underwent alterations in osmorn mutants, such as the diacylglyceride (18:3_18:3) was 5.1-fold higher and digalactosyldiacylglycerol (18:2_18:2) was 5.2-fold lower in osmorn1, while the triacylglycerol (TG) (16:0_18:2_18:3) was 8.3-fold higher and TG (16:0_18:1_18:3) was 8.5-fold lower in osmorn2 than those in WT. Furthermore, the OsMORN1/2 was found to be associated with rice cold tolerance, as osmorn1 and osmorn2 mutants were more sensitive to chilling stress than WT. The mutants displayed increased hydrogen peroxide accumulation, reduced antioxidant enzyme activities, elevated malondialdehyde content, and a significantly decreased seedling survival rate. Lipidomics analysis revealed distinct alterations in lipids under low temperature, highlighting significant changes in TG (18:2_18:3_18:3) and TG (18:4_18:2_18:2) in osmorn1, TG (16:0_18:2_18:2) and PI (17:2_18:3) in osmorn2 compared to the WT. Therefore, it suggested that OsMORN1 and OsMORN2 regulate both pollen viability and cold tolerance through maintaining lipid homeostasis., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

One of the most vulnerable phases in the plant life cycle is sexual reproduction, which depends on effective pollen transfer, but also on the thermotolerance of pollen grains. Pollen thermotolerance is temperature-dependent and may be reduced by increasing temperature associated with global warming. A growing body of research has focused on the effect of increased temperature on pollen thermotolerance in crops to understand the possible impact of temperature extremes on yield. Yet, little is known about the effects of temperature on pollen thermotolerance of wild plant species. To fill this gap, we selected Lotus corniculatus s.l. (Fabaceae), a species common to many European habitats and conducted laboratory experiments to test its pollen thermotolerance in response to artificial increase in temperature. To test for possible local adaptation of pollen thermal tolerance, we compared data from six lowland (389-451 m a.s.l.) and six highland (841-1,030 m a.s.l.) populations. We observed pollen germination in vitro at 15 °C, 25 °C, 30 °C, and 40 °C. While lowland plants maintained a stable germination percentage across a broad temperature range (15-30 °C) and exhibited reduced germination only at extremely high temperatures (40 °C), highland plants experienced reduced germination even at 30 °C-temperatures commonly exceeded in lowlands during warm summers. This suggests that lowland populations of L. corniculatus may be locally adapted to higher temperature for pollen germination. On the other hand, pollen tube length decreased with increasing temperature in a similar way in lowland and highland plants. The overall average pollen germination percentage significantly differed between lowland and highland populations, with highland populations displaying higher germination percentage. On the other hand, the average pollen tube length was slightly smaller in highland populations. In conclusion, we found that pollen thermotolerance of L. corniculatus is reduced at high temperature and that the germination of pollen from plant populations growing at higher elevations is more sensitive to increased temperature, which suggests possible local adaptation of pollen thermotolerance., Competing Interests: The authors declare that they have no competing interests., (© 2024 Jackwerth et al.)

Key Message: Fast-drying and cooling induce fast intracellular water loss and reduced ice-crystal formation, which may promote the formation of intracellular glasses that might improve the likelihood of wheat pollen survival. Long-term storage of pollen is important for the fertilization of spatially or temporally isolated female parents, especially in hybrid breeding. Wheat pollen is dehydration-sensitive and rapidly loses viability after shedding. To preserve wheat pollen, we hypothesized that fast-drying and cooling rates would increase the rate of intracellular water content (WC) removal, decrease intracellular ice-crystal formation, and increase viability after exposure to ultra-low temperatures. Therefore, we compared slow air-drying with fast-drying (dry air flow) and found significant correlations between pollen WC and viability (r = 0.92, P < 0.001); significant differences in WCs after specific drying times; and comparable viabilities after drying to specific WCs. Fast-drying to WCs at which ice melting events were not detected (ΔH = 0 J mg -1 DW, < 0.28 mg H 2 O mg -1 DW) reduced pollen viability to 1.2 ± 1.0%, but when drying to 0.39 mg H 2 O mg -1 DW, some viable pollen was detected (39.4 ± 17.9%). Fast cooling (150 °C min -1 ) of fast-dried pollen to 0.91 ± 0.11 mg H 2 O mg -1 DW induced less and a delay of ice-crystal formation during cryomicroscopic-video-recordings compared to slow cooling (1 °C min -1 ), but viability was low (4.5-6.1%) and comparable between cooling rates. Our data support that the combination of fast-drying and cooling rates may enable the survival of wheat pollen likely due to (1) a reduction of the time pollen would be exposed to drying-related deleterious biochemical changes and (2) an inhibition of intracellular ice-crystal formation, but additional research is needed to obtain higher pollen survival after cooling., (© 2022. The Author(s).)

The future impact of climate change and a warmer world is a matter of great concern. We therefore aimed to evaluate the effects of temperature on pollen viability and fruit set of Mediterranean orchids. The in vitro and controlled pollination experiments were performed to evaluate the ability of pollinia stored at lower and higher temperatures to germinate and produce fruits and seeds containing viable embryos. In all of the examined orchids, pollen stored at -20 °C remained fully viable for up to 3 years, reducing its percentage germination from year 4 onwards. Pollinia stored at higher temperatures had a drastic reduction in vitality after 2 days at 41-44 °C, while pollinia stored at 47-50 °C did not show any pollen tube growth. The different levels of pollen viability duration among the examined orchids can be related to their peculiar reproductive biology and pollination ecology. The germinability of pollinia stored at lower temperatures for long periods suggests that orchid pollinia can be conserved ex situ. In contrast, higher temperatures can have harmful effects on the vitality of pollen and consequently on reproductive success of the plants. To our knowledge, this is the first report demonstrating the effects of global change on orchid pollen, and on pollen ability to tolerate, or not, higher air temperatures. Although vegetative reproduction allows orchids to survive a few consecutive warm years, higher temperatures for several consecutive years can have dramatic effects on reproductive success of orchids., (© 2020 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

Colour phenotypes are often involved in communication and are thus under selection by species interactions. However, selection may also act on colour through correlated traits or alternative functions of biochemical pigments. Such forms of selection are instrumental in maintaining petal colour diversity in plants. Pollen colour also varies markedly, but the maintenance of this variation is little understood. In Campanula americana, pollen ranges from white to dark purple, with darker morphs garnering more pollinator visits and exhibiting elevated pollen performance under heat stress. Here, we generate an F2 population segregating for pollen colour and measure correlations with floral traits, pollen attributes and plant-level traits related to fitness. We determine the pigment biochemistry of colour variants and evaluate maternal and paternal fitness of light and dark morphs by crossing within and between morphs. Pollen colour was largely uncorrelated with floral traits (petal colour, size, nectar traits) suggesting it can evolve independently. Darker pollen grains were larger and had higher anthocyanin content (cyanidin and peonidin) which may explain why they outperform light pollen under heat stress. Overall, pollen-related fitness metrics were greater for dark pollen, and dark pollen sires generated seeds with higher germination potential. Conversely, light pollen plants produce 61% more flowers than dark, and 18% more seeds per fruit, suggesting a seed production advantage. Results indicate that light and dark morphs may achieve fitness through different means-dark morphs appear to have a pollen advantage whereas light morphs have an ovule advantage-helping to explain the maintenance of pollen colour variation., (© 2020 European Society For Evolutionary Biology.)

The number of pollen grains is a critical part of the reproductive strategies in plants and varies greatly between and within species. In agriculture, pollen viability is important for crop breeding. It is a laborious work to count pollen tubes using a counting chamber under a microscope. Here, we present a method of counting the number of pollen grains using a cell counter. In this method, the counting step is shortened to 3 min per flower, which, in our setting, is more than five times faster than the counting chamber method. This technique is applicable to species with a lower and higher number of pollen grains, as it can count particles in a wide range, from 0 to 20,000 particles, in one measurement. The cell counter also estimates the size of the particles together with the number. Because aborted pollen shows abnormal membrane characteristics and/or a distorted or smaller shape, a cell counter can quantify the number of normal and aborted pollen separately. We explain how to count the number of pollen grains and measure pollen size in Arabidopsis thaliana, Arabidopsis kamchatica, and wheat (Triticum aestivum).

Pollen viability affects the probability that a pollen grain deposited on a plant's stigma will produce a viable seed. Because a mature seed is needed before a gene flow event can occur, pollen viability will influence the risk of escape for genetically engineered (GE) crops. Pollen viability was measured at intervals for up to 2 h following removal of the pollen from the anthers. It was quantified at three temperatures and for different alfalfa varieties, including both conventional and Roundup Ready (RR) varieties. Pollen viability was assessed using in vitro germination. Time since removal from the anthers was the most prevalent factor affecting pollen viability in alfalfa. Pollen viability declined with increasing time at all three temperatures and for all varieties tested. Pollen viability was not affected by temperatures ranging between 25 and 37 °C and did not vary among plant varieties, including conventional and RR varieties. Bee foraging behaviour suggested pollen viability within the first 10 min following pollen removal from a flower to most affect seed production. Pollen longevity was predicted to have little impact on seed set and gene flow. Linking pollinator behaviour to pollen viability improved our understanding of its impact on gene flow risk., (© 2019 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

Field pea (Pisum sativum), a major grain legume crop, is autogamous and adapted to temperate climates. The objectives of this study were to investigate effects of high temperature stress on stamen chemical composition, anther dehiscence, pollen viability, pollen interactions with pistil and ovules, and ovule growth and viability. Two cultivars ("CDC Golden" and "CDC Sage") were exposed to 24/18°C (day/night) continually or to 35/18°C for 4 or 7 days. Heat stress altered stamen chemical composition, with lipid composition of "CDC Sage" being more stable compared with "CDC Golden." Heat stress reduced pollen viability and the proportion of ovules that received a pollen tube. After 4 days at 35°C, pollen viability in flower buds decreased in "CDC Golden," but not in "CDC Sage." After 7 days, partial to full failure of anthers to dehisce resulted in subnormal pollen loads on stigmas. Although growth (ovule size) of fertilized ovules was stimulated by 35°C, heat stress tended to decrease ovule viability. Pollen appears susceptible to stress, but not many grains are needed for successful fertilization. Ovule fertilization and embryos are less susceptible to heat, but further research is warranted to link the exact degree of resilience to stress intensity., (© 2018 John Wiley & Sons Ltd.)

High temperature exposure is widely used as a physical mutagenic agent to induce 2n gametes in Populus. However, whether high temperature exposure affects induced 2n pollen viability remains unknown. To clarify whether high temperature exposure affected the induced 2n pollen viability, 2n pollen induced by 38 and 41 °C temperatures, pollen morphology, 2n pollen germination in vitro, and crossing induced 2n pollen with normal gametes to produce a triploid was, based on observations of meiosis, conducted in Populus canescens. We found that the dominant meiotic stages (F = 56.6, p < .001) and the treatment duration (F = 21.4, p < .001) significantly affected the occurrence rate of induced 2n pollen. A significant decrease in pollen production and an increase in aborted pollen were observed (p < .001). High temperature sometimes affected in ectexine deposition and some narrow furrows were also analysed via details of ectexine structure. However, no significant difference in 2n pollen germination rate was observed between natural 2n pollen (26.7%) and high-temperature-induced 2n pollen (26.2%), and 42 triploids were created by crossing high-temperature-induced 2n pollen, suggesting that 38 and 41 °C temperatures exposure will not result in dysfunctional induced 2n pollen., (© 2018 John Wiley & Sons Ltd.)

The development of different pollen morphs by one specimen - pollen heteromorphism - occurs in ca. one-third species of the genus Viola. Melanium section species (pansies) stand out in producing the widest range of apertures among Viola species. Aperture number decreases with elevation increase, and faster germination of five-aperturate pollen as compared with three-aperturate has previously been postulated. We re-examined pollen heteromorphism in the context of its viability, and made correlations with elevation (>1500 m a.s.l. versus <1500 m a.s.l.), soil type (metalliferous versus non-metalliferous; MET versus NMET) and chromosome number based on selected study criteria of ca. 20% karyologically and morphologically strongly differentiated but genetically closely related pansies. A total of 79% of analysed species were heteromorphic, forming three- to six-colp or ate pollen per individual flower. Mean aperture number and pollen viability were not affected by soil type (MET versus NMET). Mean aperture number was also not influenced by elevation or species chromosome number. Positive correlations were established between aperture number and pollen viability, negative between pollen viability and elevation (increasing altitude of 100 m decreased pollen viability by 0.4%) and lack of correlation between chromosome number and pollen viability. The varied frequencies of different pollen morphs among species are not under the general pressure of ecological conditions, as previously postulated for the species of Melanium section. Rather, this trait in pansies, similar to other floral characters (e.g. long, curved nectar spur, 'landing platform', posterior petals with nectar guides), is adaptive but dependent on the breeding system (inbreeding versus outbreeding) of the individual species., (© 2018 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

The evolution of flower color, especially petal pigmentation, has received substantial attention. Less understood is the evolutionary ecology of pollen pigmentation, though it varies among and within species and its biochemical properties affect pollen viability. We characterize the distribution of pollen color across 24 populations of the North American herb Campanula americana, and assess the degree to which this variation is genetically based. We identify abiotic factors that covary with pollen color and test whether germination of light and dark pollen is differentially affected by variable temperature and UV. Pollen color varies from white to deep purple in C. americana and is genetically determined. There was a longitudinal cline whereby pollen was darkest in western populations. Accounting for latitudinal variation, western populations experience elevated temperature and UV irradiance. Germination of light-colored pollen was reduced by 60% under high temperature, but dark pollen was unaffected. Exposure to UV reduced germination of light and dark pollen similarly. The cline in pollen color across the range may reflect adaptation to heat stress. This study supports thermal tolerance as a novel function of pollen pigmentation and contributes to growing evidence that abiotic factors can drive floral diversity., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Pollen storage and viability are very important for pollination, breeding, biodiversity, biotechnology, conservation, and other biological and non-biological studies of the date palm. Optimizing procedures and duration of storage are important for effective and long-term date palm pollen storage and viability. Here we describe pollen storage methods, such as room temperature (25-30 °C), refrigeration (4 °C), storage at 4 °C in desiccators, deep freezer (-20 °C), and cryopreservation (-196 °C). Based on pollen viability by staining and in vitro germination methods, cryopreservation is the best method for long-term storage without any significant effect on pollen viability (75-84%); however, the percentage of pollen viability depends on the storage period.

Analyzing pollen quality in an efficient and reliable manner is of great importance to the industries involved in seed and fruit production, plant breeding, and plant research. Pollen quality parameters, viability and germination capacity, are analyzed by various staining methods or by in vitro germination assays, respectively. These methods are time-consuming, species-dependent, and require a lab environment. Furthermore, the obtained viability data are often poorly related to in vivo pollen germination and seed set. Here, we describe a quick, label-free method to analyze pollen using microfluidic chips inserted into an impedance flow cytometer (IFC). Using this approach, pollen quality parameters are determined by a single measurement in a species-independent manner. The advantage of this protocol is that pollen viability and germination can be analyzed quickly by a reliable and standardized method.

Dichogamy is generally thought to be a mechanism that prevents self-fertilization in flowering plants. This study aims to investigate the relationships between floral age and stigma receptivity, style length and pollen viability, and define how floral characters avoid self-pollination in a gynodioecious Chinese plant, Elsholtzia rugulosa. We assessed the relationships between flower age and style length, stigma receptivity, and pollen viability in E. rugulosa. This species produces 2 forms with plants bearing either hermaphrodite flowers (H) or female flowers (F). Corolla length in F flowers was shorter than the corolla length of H flowers and produced no pollen. H flowers were protandrous, pollen release of H flowers occurred before stigma receptivity. Stigma receptivity was significantly positively correlated with style length in both F flowers and H flowers. Pollen viability in H flowers declined significantly with floral age. Our results suggest that self-pollination in H flowers is likely reduced by dichogamy because stigma receptivity and pollen viability were effectively separated in time. However, because H inflorescences typically have multiple flowers open at the same time means that geitonogamous selfing is not avoided.

In flowering plants, pollen development is a highly programmed process, in which a lot of genes are involved. In this study, a gene, designated as GhMYB24, encoding R2R3-MYB-like protein was isolated from cotton. GhMYB24 protein is localized in the cell nucleus and acts as a transcriptional activator. Northern blot analysis revealed that GhMYB24 transcripts were predominantly detected in anthers. It was further found that strong expression of GhMYB24 was mainly detected in pollen and was regulated during anther development by in situ hybridization. Overexpression of GhMYB24 in Arabidopsis caused flower malformation, shorter filaments, non-dehiscent anthers and fewer viable pollen grains. Further analysis revealed that the septum and stomium cells of anthers were not broken, and fewer fibrous bands were found in the endothecium cells in transgenic plants. A complementation test demonstrated that GhMYB24 was able to recover partially the male fertility of the myb21 myb24 double mutant. Expression levels of the genes involved in the phenylpropanoid biosynthetic pathway and reactive oxygen species homeostasis were altered in GhMYB24-overexpressing transgenic plants. Furthermore, the genes involved in jasmonate biosynthesis and its signaling pathway were up-regulated in the transgenic plants. Yeast two-hybrid assay indicated that GhMYB24 interacted with GhJAZ1/2 in cells. Taking the data together, our results suggest that GhMYB24 may play an important role in normal anther/pollen development.

Pollen grains are central to sexual plant reproduction and their viability and longevity/storage are critical for plant physiology, ecology, plant breeding, and many plant product industries. Our goal is to present progress in assessing pollen viability/longevity along with recent advances in our understanding of the intrinsic and environmental factors that determine pollen performance: the capacity of the pollen grain to be stored, germinate, produce a pollen tube, and fertilize the ovule. We review current methods to measure pollen viability, with an eye toward advancing basic research and biotechnological applications. Importantly, we review recent advances in our understanding of how basic aspects of pollen/stigma development, pollen molecular composition, and intra- and intercellular signaling systems interact with the environment to determine pollen performance. Our goal is to point to key questions for future research, especially given that climate change will directly impact pollen viability/longevity. We find that the viability and longevity of pollen are highly sensitive to environmental conditions that affect complex interactions between maternal and paternal tissues and internal pollen physiological events. As pollen viability and longevity are critical factors for food security and adaptation to climate change, we highlight the need to develop further basic research for better understanding the complex molecular mechanisms that modulate pollen viability and applied research on developing new methods to maintain or improve pollen viability and longevity. [ABSTRACT FROM AUTHOR]

Flower bud differentiation is the commencement of plant reproductive development. Flower organ specialization follows flower bud differentiation. This biological mechanism controls whether plants reproduce successfully via seed. Caucasian clover (Trifolium ambiguum) is used in mixed temperate pastures. However, its low natural seed setting rate contributes to a low seed yield. This study aimed to elucidate how the reproductive biology of Caucasian clover could affect seed production. Caucasian clover flower bud differentiation and flower organ development were systematically examined. Flower bud differentiation has six consecutive stages: pre‐differentiation, differentiation of the inflorescence, the floret, the perianth, the stamen, and finally the pistil primordium. The anther has two chambers. Pollen grains have six development phases: the tetraspore, early‐uninucleate, late‐uninucleate, vegetative cell and germ‐cell form, binucleate, and maturation phase. The microspore tetrad is tetrahedral, and the mature pollen grains are bicellular. Pollen grain viability was found 35%. The Caucasian clover monocarpelate ovary contains 1–2 ovules that are covered with a 2‐layer integument. The ovule is anatropous. The embryo sac development type is a polygonum. The floral formula is "⚥↑K(5) C1+1+2 A(9)+1G(1:1:1−2)". These genetic traits of two pollen sacs and low viability pollen grains may be responsible for the low natural seed setting. Poor seed setting may also be due to embryo sac dysplasia that leads to ovule sterility. Therefore, the breeding aim should be to improve pollen viability, to decrease embryo sac dysplasia and to reduce embryo abortion that could be targeted for genetic improvement to enhance seed yields. [ABSTRACT FROM AUTHOR]

Cannabis (Cannabis sativa) is a versatile crop belonging to the Cannabaceae family, and is dioecious, typically with separate male and female plants. The flowers of female plants, especially the trichomes, accumulate relatively higher contents of cannabinoids compared with those of male plants. For this reason, to obtain seeds that are genetically female, it is desirable to induce the development of male flowers on a female plant that produces genetically female haploid gametes. Silver thiosulfate (STS) is a highly effective chemical for male flower induction. We investigated male flower induction in three commercial cultivars of female cannabis (Spectrum303, SuperwomanS1, and CBGambit) regarding the treatment frequency, stage of application, and concentration of STS applied as a foliar spray. All three cultivars showed adequate induction of male flowers in response to 1.5 mM STS applied at the early reproductive stage. In particular, SuperwomanS1 was most highly responsive to induction of male flowers, even when treated with 0.3 mM STS at the early reproductive stage. Treatment with three applications of STS was more effective compared with a single application, but a single application of 1.5 mM STS at the early reproductive stage was sufficient for male flower induction. A single STS application during the middle stage of reproductive growth was inadequate for induction of male flowers. However, 6 weeks after three applications of STS, CBGambit exhibited approximately 54% male flower induction at 0.3 mM STS, Spectrum303 showed approximately 56% induction at 3 mM STS, and SuperwomanS1 yielded approximately 26% induction at 1.5 mM (expressed as percentage of total number of individuals with the induced male flowers). Pollen stainability tests using KI-I2 solution and Alexander's staining showed high pollen viability with over 65% at different single STS concentrations, indicating that pollen grains induced by STS have sufficient viability for the self-pollination. This study demonstrated that different cultivars of cannabis respond diversely to different STS concentrations and highlighted the potential benefits of three STS applications during the middle reproductive stage for cannabis breeding. [ABSTRACT FROM AUTHOR]

Supra-optimal temperature adversely affects plant growth and development. Among different growth phases, the reproductive stage of plants is highly sensitive to high temperature stress. The heat tolerance of plants is reliant on a complex interplay of various factors, including the activation of heat stress responses, downstream signaling pathways, and biosynthesis of plant hormones. This intricate network of mechanisms results in the production of several secondary metabolites, which play a crucial role in enhancing the plant's ability to cope with high temperature stress. Many of these secondary metabolites are crucial for the reproductive performance of plants, as they are involved in the attraction of pollinators, pollen germination, pollen tube growth, pollen viability, pollen fertility, and fertilization. Reproductive tissues of plants are highly vulnerable to heat stress, but increased biosynthesis of secondary metabolites (flavonoids, polyamines, glutathione, etc.) induces reproductive thermotolerance through reactive oxygen species homeostasis, protecting the cellular membranes, improving pollen fertility, reducing anther abortion, regulating cellular signaling and activation of genes/transcription factors associated with metabolic pathway network. The identification and engineering of some genes controlling the secondary metabolic pathways network have proved effective in inducing terminal heat stress tolerance in transgenic plants. This review highlights the effect of heat stress on plant secondary metabolism and the role of different plant secondary metabolites in contributing to heat stress tolerance in reproductive tissues. The engineering of genes involved in plant secondary metabolic pathway and their effect on heat stress tolerance has also been discussed. [ABSTRACT FROM AUTHOR]

In flowering plants, sexual reproductive success depends on the production of viable pollen grains. However, the mechanisms by which QUA QUINE STARCH (QQS) regulates pollen development and how transcriptional activators facilitate the transcription of QQS in this process remain poorly understood. Here, we demonstrate that INDUCER OF CBF EXPRESSION 1 (ICE1), a basic helix–loop–helix (bHLH) transcription factor, acts as a key transcriptional activator and positively regulates QQS expression to increase pollen germination and viability in Arabidopsis thaliana by interacting with INDETERMINATE DOMAIN14 (IDD14). In our genetic and biochemical experiments, overexpression of ICE1 greatly promoted both the activation of QQS and high pollen viability mediated by QQS. IDD14 additively enhanced ICE1 function by promoting the binding of ICE1 to the QQS promoter. In addition, mutation of ICE1 significantly repressed QQS expression; the impaired function of QQS and the abnormal anther dehiscence jointly affected pollen development of the ice1‐2 mutant. Our results also showed that the enhancement of pollen activity by ICE1 depends on QQS. Furthermore, QQS interacted with CUT1, the key enzyme for long‐chain lipid biosynthesis. This interaction both promoted CUT1 activity and regulated pollen lipid metabolism, ultimately determining pollen hydration and fertility. Our results not only provide new insights into the key function of QQS in promoting pollen development by regulating pollen lipid metabolism, but also elucidate the mechanism that facilitates the transcription of QQS in this vital developmental process. [ABSTRACT FROM AUTHOR]

Fritillaria spp., comprising perennial bulbous plants of significant medicinal and ornamental value, face high endangerment in their natural habitats. Despite their importance, the reproductive characteristics and adaptive evolution mechanisms of these species remain incompletely understood. This study focused on the pollination strategies and reproductive biology of Fritillaria imperialis L. The research was conducted at the Erzincan Horticultural Research Institute in Türkiye. Our investigation categorized the flowering process of F. imperialis into nine distinct phases. Through comprehensive assessments of the pollen/ovule ratio, self-incompatibility index (SII), and ex situ pollination experiments, we observed high levels of self-incompatibility and allogamy in F. imperialis. Our findings revealed that pollination of F. imperialis primarily relied on pollen vectors, with Apis mellifera and Bombus terrestris identified as the most effective pollinators. Furthermore, average seed set rate, seed production, and seed viability were quantified at 80.5%, 228 seeds, and 86.3%, respectively. The average numbers of pollen viability and pollen grains were measured at 93% and 702,000, respectively. This comprehensive analysis of the reproductive biology of F. imperialis provides crucial insights for the conservation and genetic management of this highly valuable species. The findings contribute to a deeper understanding of the reproductive strategies employed by Fritillaria spp., which may inform future conservation efforts and breeding programs for these endangered plants. [ABSTRACT FROM AUTHOR]

Lavender (Lavandula spp.) is one of the most widely grown essential oil crops in the world. This study aimed to determine the nuclear DNA contents using flow cytometry, pollen viability using TTC (2,3,5-Triphenyl Tetrazolium Chloride) and IKI (Iodine Potassium Iodide) tests, pollen production quantities using hemacytometric method, and seed retention rates per spike and flower of lavender (L. angustifolia var. Raya) and lavandin (L. intermedia var. Super) grown under ecological conditions in Isparta province of Turkey. The nuclear DNA contents were 2.11 and 2.54 pg 2C-1, respectively in the lavender and lavandin cultivar. The flowers of the lavender cultivar produced abundant pollen grains (average 5800 pollen per flower and 1450 pollen per anther) with high viability (60.65-65.05%) and seed retention rate per spike (91.57% on average). The lavandin cultivar, which had very low pollen viability (1.08-3.32%) and pollen grains (average of 2350 pollen per flower and 587.5 pollen per anther) gave very low seed retention rates per spike (0.60% on average). While each flower had four ovaries with the potential to produce four nutlets, lavandin flowers produced only trace numbers (0.15% on average) of seeds. As a result, the lavandin cultivar had more nuclear DNA content, longer stem and spike, smaller size but more numerous flowers, less and lighter anthers, lower pollen grains and viability, and vey few inviabile seeds compared to the lavender cultivar. It has been observed by eye that honey bees do not visit lavender and lavandin flowers for collecting pollen, but solely for collecting nectar. [ABSTRACT FROM AUTHOR]

Pollen fertility is a primary regulator of grain yield and is highly susceptible to cold and other environmental stress. We revealed the roles of rice cell wall invertase gene PWIN1 in pollen development and chilling tolerance. We uncovered its preferential expression in microspores and bicellular pollen and identified its knock‐down and knock‐out mutants. pwin1 mutants produced a higher proportion of abnormal pollen than wild‐type plants. The contents of sucrose, glucose, and fructose were increased, while ATP content and primary metabolism activity were reduced in the mutant pollen. Furthermore, the loss of function of PWIN1 coincided with an increase in SnRK1 activity and a decrease in TOR activity. Under chilling conditions, pwin1 mutants displayed significantly reduced pollen viability and seed‐setting rate, while overexpressing PWIN1 notably increased pollen viability and seed‐setting rate as compared with the wild‐type, indicating that PWIN1 is essential for rice pollen development and grain yield under cold stress. This study provides insights into the molecular mechanisms underlying rice pollen fertility during chilling stress, and a new module to improve chilling tolerance of rice at the booting stage by molecular design. [ABSTRACT FROM AUTHOR]

Understanding pollen and ovule fertility as factors influencing fruit and seed set is important in cassava breeding. Extended daylength with red light (RL) and plant growth regulators (PGRs) have been used to induce flowering and fruit set in cassava without any reference to effects on pollen viability or ovule fertilizability. This study investigated the effects of field-applied RL and PGR on pollen viability and ovule fertilizability. Panels of cassava genotypes with early or moderate flowering responses were used. RL was administered from dusk to dawn. Two PGRs, 6-benzyl adenine (BA), a cytokinin and silver thiosulphate (STS), an anti-ethylene, were applied. Pollen viability was assessed based on pollen grain diameter, in vitro stainability, in vivo germinability, ovule fertilizability, and ploidy level. Treating flowers with RL increased the pollen diameter from 145.6 in control to 148.5 µm in RL, 78.5 to 93.0% in stainability, and 52.0 to 56.9% in ovule fertilizability in treated female flowers. The fruit set also increased from 51.5 in control to 71.8% in RL-treated female flowers. The seed set followed a similar trend. The ploidy level of pollen from RL-treated flowers increased slightly and was positively correlated with pollen diameter (R2 = 0.09 *), ovule fertilization (R2 = 0.20 *), fruit set (R2 = 0.59 *), and seed set (R2 = 0.60 *). Treating flowers with PGR did not affect pollen diameter but increased stainability from 78.5% in control to 82.1%, ovule fertilizability from 42.9 to 64.9%, and fruit set from 23.2 to 51.9% in PGR-treated female flowers. Combined BA + STS application caused the highest ovule fertilizability, fruit, and seed set efficiency. These results show that RL and PGR treatments increase pollen viability and ovule fertilizability. This is important for planning pollination strategies in cassava breeding programmes. [ABSTRACT FROM AUTHOR]

• The pompom weed is likely pollinated by Apis mellifera and Hypolimnas misippus. • Gametophytic apomixis with autonomous endosperm development in triploids. • The co-occurrence of apomixis and polyploidy may be facilitating its invasiveness. • There is high pollen grain viability despite apomictic reproduction. • Low genetic diversity within and among the sampled triploid populations. Polyploidy and apomixis enable successful plant invasions into new regions. Campuloclinium macrocephalum (pompom weed) is a successful alien invasive species in South Africa that has been shown to comprise multiple ploidy levels. Despite the identification of triploid and tetraploid cytotypes in South African populations, the primary reproductive strategy was unknown. We investigated the reproductive strategy used by triploid populations of the pompom weed. We assessed whether insects contributed to pollen transfer using pollinator exclusion experiments, estimated genetic diversity, evaluated pollen tube growth in the styles, and estimated male fertility using pollen grain size and viability. We found evidence that strongly suggests that the pompom weed can reproduce via gametophytic apomixis with autonomous endosperm development. Pollinator exclusion experiments showed that while insects may positively influence reproductive success, the pompom weed can set lower quantities of seed in the absence of pollinators and that overall seed viability did not differ between open and excluded pollinator treatments. Further, while individuals are capable of uniparental reproduction, seed set appears to be independent of fertilization because we did not observe pollen tubes growing down the styles and/or reaching the ovules, despite high pollen viability. The sampled triploid populations also showed low genetic diversity, which may be a result low recombination frequencies. The low genetic variation should result in all of the sampled populations being equally susceptible to the biocontrol agents; however, this is not being observed in the sampled populations. Only one out of the four sampled populations showed a significant reduction in seed set and germination percentages due to the damage from Puccinia eupatorii (rust fungus) and Liothrips tractabilis (thrips). Nevertheless, the combination of an apomictic reproductive strategy and polyploidy is likely contributing to making pompom weed a formidable invader in South Africa. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the fertility of "Huajin 6" and the effect of exogenous methyl jasmonate on its fertility. In this study, "Huajin 6", "Huajin 6" treated with methyl jasmonate and "Damaohua" were used as the research objects, the stamen phenotypes and the shape of pollen grains were observed, pollen viability and stigma receptivity were measured. The results showed that the pistil structure and function were normal, and although the stamen anthers did not dehisce, they were still capable of producing pollen with a certain amount of vigor. Methyl jasmonate could promote the opening of the flowers of "Huajin 6" and improve the development of pollen grains to a certain extent, but it could not promote anthers dehiscence of "Huajin 6". This study can provide theoretical guidance for the cultivation of new honeysuckle varieties using "Huajin 6". [ABSTRACT FROM AUTHOR]

This study aims to explore the pollen morphology of 19 plant species from the Khewra Salt Range by using light microscopy (LM) and scanning electron microscopy (SEM). Plant specimens were collected from various sites in the Khewra Salt Range, identified, and deposited in the Herbarium of Pakistan. Pollen morphology was studied using LM and SEM. Quantitative characteristics, including polar and equatorial diameter, exine thickness, colpus and spines length and width were studied using LM. The studied pollen taxa exhibited extensive variation in size and sculpture, with potential taxonomic implications. Pollen shape of most of the studied species was sub-spheroidal but pollen sizes ranged from 0.98 µm to 14.3µm. Symmetry and polarity of pollen grains were observed, contributing to plant species identification. The P/E ratio varied from Dichanthium annulatum (0.56) to Medicago polymorpha (1.15). Scanning electron micrographs revealed diverse exine sculpturing patterns, such as scabrate and echinate. At the same time, Nerium oleander (0.7-2.1µm) showed high exine thickness and large size colpus with a length of 7.5µm and width of 8.26µm. The largest spines were observed in Vicia sativa (3.7µm long and 1.38 µm wide). The taxonomic key based on palyno-morphological characters offers a tool for quick and precise identification. Pollen viability values were determined. Highest pollen viability was noted for Capparis decidua (97%) that show the species adaptation in Salt range. Palynological features can aid in resolving taxonomic problems and contribute to further karyological and taxonomic evaluations. This study contributes valuable insights into the palynological diversity of plant species of salt range, emphasizing the importance of pollen morphology in understanding and conserving salt-affected ecosystems. [ABSTRACT FROM AUTHOR]

Understanding the regulation of autotetraploid sterility is essential for harnessing the strong advantages in genomic buffer capacity, biodiversity, and heterosis of autotetraploid rice. miRNAs play crucial roles in fertility regulation, yet information about their reproductive roles and target genes in tetraploid rice remains limited. Here, we used three tetraploid lines, H1 (fertile), HF (fertile), and LF (sterile), to investigate cytological features and identify factors associated with autotetraploid sterility. LF showed abnormal meiosis, resulting in low pollen fertility and viability, ultimately leading to scarce fertilization and a low-seed setting compared to H1 and HF. RNA-seq revealed 30 miRNA-candidate target pairs related to autotetraploid pollen sterility. These pairs showed opposite expression patterns, with differential expression between fertile lines (H1 and HF) and the sterile line (LF). qRT-PCR confirmed that miR9564, miR528, and miR27874 were highly expressed in the anthers of H1 and HF but not in LF, while opposite results were obtained in their targets (ARPS, M2T, and OsRPC53). Haplotype and expression pattern analyses revealed that ARPS was specifically expressed in lines with the same haplotype of MIR9564 (the precursor of miR9564) as LF. Furthermore, the Dual-GFP assay verified that miR9564 inhibited the fluorescence signal of ARPS-GFP. The over-expression of ARPS significantly decreased the seed setting rate (59.10%) and pollen fertility (50.44%) of neo-tetraploid rice, suggesting that ARPS plays important roles in autotetraploid pollen sterility. This study provides insights into the cytological characteristic and miRNA expression profiles of tetraploid lines with different fertility, shedding light on the role of miRNAs in polyploid rice. [ABSTRACT FROM AUTHOR]

An in vitro pollen storage study was conducted using pollen from three seeded citrus plant genotypes, viz., ‘Mexican lime’, ‘W’. Murcott’ and ‘Mosambi’ and five seedlesscitrus genotypes, viz., ‘Lisbon lemon’, ‘Jaffa’, ‘Clementine’, ‘Hamlin’ and ‘Mukaku Kishu’. Pollen viability and germination percentage were evaluated at different storage temperature treatments, i.e., at room temperature (in anhydrous calcium chloride) (control), in a refrigerator at 4°C, in a freezer (-20°C), and in a freeze drier (-80°C). The viability of the pollen plants was tested with an acetocarmine stain (2%). Among all the tested sucrose concentrations (0, 5, 10, 15, 20, and 25%) for in vitro pollen germination, the 15% sucrose concentration had the highest effect on pollen germination. The results showed significant differences in pollen viability and germination under different storage temperature conditions. The pooled data revealed that, among the seeded genotypes, W. Murcotts showed the maximum mean viability and germination percentage (67.86% and 60.88%, respectively) after 48 weeks of storage at -80°C, and the minimum values were observed for Mexican lime (46.57% and 33.71%, respectively). However, in the low-seeded genotype, Mukaku Kishu had the maximum mean pollen viability and germination (71.52% and 64.07%, respectively) after 48 weeks of storage at -80°C, and the lowest values were observed in Jaffa (39.36% and 28.08%, respectively). The results indicate that the freeze drier storage temperature (-80°C) had the greatest effect on retaining pollen viability and germination in both the seeded and low-seeded genotypes. However, a progressive decrease in pollen viability and germination rate was observed with increasing duration at all storage temperatures, reaching a minimum at 48 weeks after storage. However, the reduction in pollen storage ability was greatest at room temperature and 40°C. Pollen grains stored at low temperatures (-80°C and -20°C) showed good viability and germination percentage compared with those stored at room temperature and 4°C. [ABSTRACT FROM AUTHOR]

Pollen, the male gametophyte of seed plants, is extremely sensitive to UV light, which may prevent fertilization. As a result, strategies to improve plant resistance to solar ultraviolet (UV) radiation are required. The tardigrade damage suppressor protein (Dsup) is a putative DNA-binding protein that enables tardigrades to tolerate harsh environmental conditions, including UV radiation, and was therefore considered as a candidate for reducing the effects of UV exposure on pollen. Tobacco pollen was genetically engineered to express Dsup and then exposed to UV-B radiation to determine the effectiveness of the protein in increasing pollen resistance. To establish the preventive role of Dsup against UV-B stress, we carried out extensive investigations into pollen viability, germination rate, pollen tube length, male germ unit position, callose plug development, marker protein content, and antioxidant capacity. The results indicated that UV-B stress has a significant negative impact on both pollen grain and pollen tube growth. However, Dsup expression increased the antioxidant levels and reversed some of the UV-B-induced changes to pollen, restoring the proper distance between the tip and the last callose plug formed, as well as pollen tube length, tubulin, and HSP70 levels. Therefore, the expression of heterologous Dsup in pollen may provide the plant male gametophyte with enhanced responses to UV-B stress and protection against harmful environmental radiation. [ABSTRACT FROM AUTHOR]

East African diploid cooking bananas, commonly called Mchare, are a staple crop for millions of subsistence farmers in Tanzania, particularly in the Pangani region in northern Tanzania. Several pathogens constrain Mchare production significantly and threaten food security. Sources of resistance to these pathogens have been identified; however, partial male and female sterility impedes successful resistance introgression, complicating the breeding process. Mchare cultivars are also the only known surviving representatives of a diploid banana subgroup that contributed unreduced gametes to many of the most widely grown and successful triploid dessert bananas (‘Cavendish’, ‘Gros-Michel’, ‘Silk’, and ‘Prata’). As such, they represent an essential intermediate step in the conventional improvement of bananas worldwide. We assess the amount and viability of pollen among Mchare and wild genotypes to identify the most fertile Mchare cultivars that can be used in conventional banana improvement. Pollen was collected from 14 banana genotypes for quantification and viability testing over 7 months, and the optimal time for pollen collection was determined to be 0800 HR. Significant variation among banana genotypes in terms of both overall pollen production and percentage of pollen viability was observed. The wild-type bananas ‘Calcutta 4’ (International Musa Germplasm Transit Center (ITC) 0249] and ‘Borneo’ (ITC0253) had the greatest overall pollen production (> 31,000 pollen grains/anther) and viability (~74%), whereas ‘Ijihu Inkundu’ (ITC1460; Mchare genotype) was the least productive (almost completely sterile), with an average pollen production of a few hundred grains per anther and a viability of 7%. There were significant differences among months in terms of pollen viability, with the greatest average viability observed in May, April, and February (> 51%), and the lowest average pollen viability in July (41%). Significant differences were observed among the Mchare genotypes, with ‘Huti-White’, ‘Huti green bell’ (ITC1559), and ‘Mchare Laini’ consistently producing more substantial amounts of total pollen and an overall more significant proportion of viable pollen. This information is vital to improve Mchare bananas and the global breeding of dessert bananas. The choice of Mchare banana used in improvement programs could affect fertility and the likelihood of breeding success. [ABSTRACT FROM AUTHOR]

The genus Dyckia (Bromeliaceae) is endemic to South America, and its species show important ecological roles in xeric environments. The flowering phenology as well as floral and reproductive biology were monitored monthly in Dyckia dissitiflora at two sites (Bela Vista: 11°42′03.4″ S 39°05′21.7″ W and Barra do Vento: 11°37′58.1″ S 39°03′33.7″ W) in the municipality of Serrinha, Bahia, Brazil. Flowering occurs in the early austral summer with a peculiar time of beginning of anthesis (10:00–11:00), offering resources 18 h after the first petal movements. Pollen viability was 98%, with a high production of pollen grains. Stigma receptivity is continuous from flower opening to senescence, and nectar production initiates at 04:00 and extends until 18:00. The species is self‐compatible and autogamous, but not agamospermic. Pollen tubes developed and reached the micropyle within 24 h in all of the reproductive tests. The hummingbird Chlorostilbon lucidus was the only floral visitor and pollinator (frequency = 0.82 ± 0.02). Fruiting occurs 3 days after anthesis, and the seeds are wind‐dispersed. Self‐compatibility and autogamy are essential strategies for the reproductive success of D. dissitiflora in light of the low number and visit frequency of its pollinators, and it is favored by slow floral movements and the position of the stigmas in relation to stamens. [ABSTRACT FROM AUTHOR]

Crossbreeding is a multi-stage process with inherent challenges and risks in developing new varieties. Success hinges on selecting highly fertile parents. In species like lisianthus, uncertainty persists regarding the optimal methods for assessing pollen quality, which is crucial for evaluating pollen parent fertility. This study seeks to identify the most reliable techniques for this purpose. Fresh and dead pollen from four lisianthus (Eustoma grandiflorum) varieties was used. The dead pollen was obtained by thermal inactivation. Five chemical staining methods (iodine-potassium iodide, 2,3,5-triphenyltetrazolium chloride - TTC, lactophenol cotton blue, safranin, acetocarmine) were employed to assess pollen viability, and two biological methods (Petri dishes, hanging drops) were used to determine the germination rate. Four solid medium cultures were employed in Petri dishes, while the hanging drop utilised four liquid medium cultures. Thirteen tests were conducted for each variety, evaluating fresh and dead pollen. The study found significant variations in pollen quality among lisianthus varieties and methods. Fresh pollen showed viability rates ranging from 56.87% to 99.41% and germination rates from 0.20% to 45.11%. TTC exhibited the lowest viability rate across all varieties, while the highest germination rate was observed in the liquid culture medium with only boric acid and PEG1500. Notably, TTC was the sole viability method that did not stain dead pollen, and no germination occurred in any method for dead pollen. TTC is the most reliable staining method, and a liquid culture medium with boric acid and PEG1500 effectively determines lisianthus pollen quality. Varying boric acid and PEG1500 concentrations are advisable. [ABSTRACT FROM AUTHOR]

The knowledge of pollen morphology, suitable storage condition, and species compatibility is vital for a successful grapevine improvement programme. Ten grape genotypes from three different species, viz., Vitis vinifera L., Vitis parviflora Roxb., and Vitis champini Planc., were studied for their pollen structure and pollen storage with the objective of determining their utilization in grape rootstock improvement programs. Pollen morphology was examined through the use of a scanning electron microscope (SEM). The viability of the pollen was assessed using 2,3,5-triphenyltetrazolium chloride (TTC). In vitro pollen germination was investigated using the semi-solid medium with 10 % sucrose, 100 mg/L boric acid, and 300 mg/L calcium nitrate. The results revealed variations in pollen micro-morphology in 10 genotypes, with distinct pollen dimensions, shapes, and exine ornamentation. However, species-wise, no clear difference was found for these parameters. Pollen of V. parviflora Roxb. and Dogridge was acolporated and did not germinate. The remaining eight genotypes exhibited tricolporated pollen and showed satisfactory in vitro pollen germination. Storage temperature and duration interactions showed that, at room temperature, pollen of most of the grape genotypes can be stored for up to 1 day only with an acceptable pollen germination rate (>30 %). However, storage for up to 7 days was successfully achieved at 4 °C, except for 'Pearl of Csaba'. The most effective storage conditions were found to be at -20 °C and -196 °C (in liquid N2), enabling pollen storage for a period of up to 30 days, and can be used for pollination to overcome the challenge of asynchronous flowering. Four interspecific combinations were studied for their compatibility, among which V. parviflora Roxb. × V. vinifera L. (Pusa Navrang) and V. parviflora Roxb. × V. champini Planc. (Salt Creek) showed high crosscompatibility, offering their potential use for grape rootstock breeding. However, V. parviflora Roxb. × V. vinifera L. (Male Hybrid) recorded the lowest compatibility index among studied crosses. In the case of self-pollinated flowers from V. parviflora Roxb. and V. parviflora Roxb. × V. champini Planc. (Dogridge), pollen failed to germinate on the stigma due to male sterility caused by acolporated pollen. As a result, the flowers of these genotypes functioned as females, which means they are ideal female parents for grape breeding without the need for the tedious process of emasculation. [ABSTRACT FROM AUTHOR]

The grain yield is closely associated with spikelet fertility in rice (Oryza sativa L.) under high temperatures, and nitrogen (N) plays a crucial role in yield formation. To investigate the effect of panicle N application on yield formation under high temperatures at the panicle initiation stage, two rice varieties [Liangyoupeijiu (LYPJ, heat susceptible) and Shanyou63 (SY63, heat tolerant)] were grown and exposed to high daytime temperature (HT) and control temperature (Control) during the panicle initiation stage. Low (LPN) and high (HPN) panicle N applications were conducted. HT markedly decreased the yields by 87% at LPN and 48% at HPN in LYPJ and 31% at LPN and 36% at HPN in SY63. The decrease in grain yield under HT was primarily attributed to the decline in spikelet fertility, HPN increased spikelet fertility. HT resulted in the abnormal development of anthers, which included disordered, enlarged, and broken anther wall layers, degraded and irregularly shaped microspores, delayed tapetum degradation, less vacuolated microspores per locule, abnormal and aborted pollen grains; however, HPN improved the development of anthers under HT, particularly in LYPJ. A high rate of evapotranspiration resulted in an approximately 1°C decrease in panicle temperatures at HPN compared with that at LPN in both varieties under HT. Overall, these results demonstrate that the increased panicle N application favors normal anther development in LYPJ by decreasing the panicle temperature, which results in high pollen viability and spikelet fertility, and consequently less yield loss under HT. [ABSTRACT FROM AUTHOR]

Introgression allows polyploid species to acquire new genomic content from diploid progenitors or from other unrelated diploid or polyploid lineages, contributing to genetic diversity and facilitating adaptive allele discovery. In some cases, high levels of introgression elicit the replacement of large numbers of alleles inherited from the polyploid's ancestral species, profoundly reshaping the polyploid's genomic composition. In such complex polyploids, it is often difficult to determine which taxa were the progenitor species and which taxa provided additional introgressive blocks through subsequent hybridization. Here, we use population-level genomic data to reconstruct the phylogenetic history of Betula pubescens (downy birch), a tetraploid species often assumed to be of allopolyploid origin and which is known to hybridize with at least four other birch species. This was achieved by modeling polyploidization and introgression events under the multispecies coalescent and then using an approximate Bayesian computation rejection algorithm to evaluate and compare competing polyploidization models. We provide evidence that B. pubescens is the outcome of an autoploid genome doubling event in the common ancestor of B. pendula and its extant sister species, B. platyphylla , that took place approximately 178,000–188,000 generations ago. Extensive hybridization with B. pendula , B. nana , and B. humilis followed in the aftermath of autopolyploidization, with the relative contribution of each of these species to the B. pubescens genome varying markedly across the species' range. Functional analysis of B. pubescens loci containing alleles introgressed from B. nana identified multiple genes involved in climate adaptation, while loci containing alleles derived from B. humilis revealed several genes involved in the regulation of meiotic stability and pollen viability in plant species. [ABSTRACT FROM AUTHOR]

Background: Most Rubiaceae species are hermaphroditic with outcrossing often promoted by distyly and self-incompatibility, as well as by andromonoecy and dioecy in some species. However, cleistogamy has been reported in three genera, including Richardia. Aims: To characterise the reproductive biology of Richardia brasiliensis (Rubiaceae), a ruderal herb native to southern USA, Central America and large parts of South America, and determine if it exhibits preanthesis cleistogamy. Methods: In three natural populations of R. brasiliensis in Argentina, floral processes were monitored in the flower-bud stage and during anthesis until senescence. In addition, stigma receptivity and pollen viability were determined at these different floral stages in two populations, and experimental controlled pollinations were performed to check for spontaneous self-pollination, natural pollination and apomixis. Floral visiting insects were observed and collected for identification. Results: Autonomous self-pollination occurred when flowers were in bud and stigmas were receptive, with ovules fertilised prior to anthesis. These flowers were protogynous and functionally cleistogamous, though later passed through a chasmogamous phase when flowers opened and were visited by diverse insects. Conclusions: Our study demonstrates that preanthesis cleistogamy occurs in flower-buds of R. brasiliensis, indicating that this mode of self-pollination and self-fertilisation may be more common in Rubiaceae than previously realised. KEY HIGHLIGHTS: Flowers of Richardia brasiliensis exhibit two striking features in the flower-bud stage: stigmatic receptivity and self-pollination causing preanthesis cleistogamy to occur. Pollen grain germination on distinctly enlarged papillae cells and pollen tube growth in the style to the ovary were observed in 85% of fixed flower-buds. During anthesis, flowers are visited by a diversity of insects collecting pollen and nectar (Orders Hymenoptera (Apidae, Formicidae and Trigonalidae) and Diptera (Syrphidae)), but due to preanthesis cleistogamy most reproduction occurs via self-fertilisation. [ABSTRACT FROM AUTHOR]

Pollen cells require large amounts of sugars from the anther to support their development, which is critical for plant sexual reproduction and crop yield. Sugars Will Eventually be Exported Transporters (SWEETs) have been shown to play an important role in the apoplasmic unloading of sugars from anther tissues into symplasmically isolated developing pollen cells and thereby affect the sugar supply for pollen development. However, among the 17 CsSWEET genes identified in the cucumber (Cucumis sativus L.) genome, the CsSWEET gene involved in this process has not been identified. Here, a member of the SWEET gene family, CsSWEET5a, was identified and characterized. The quantitative real-time PCR and β-glucuronidase expression analysis revealed that CsSWEET5a is highly expressed in the anthers and pollen cells of male cucumber flowers from the microsporocyte stage (stage 9) to the mature pollen stage (stage 12). Its subcellular localization indicated that the CsSWEET5a protein is localized to the plasma membrane. The heterologous expression assays in yeast demonstrated that CsSWEET5a encodes a hexose transporter that can complement both glucose and fructose transport deficiencies. CsSWEET5a can significantly rescue the pollen viability and fertility of atsweet8 mutant Arabidopsis plants. The possible role of CsSWEET5a in supplying hexose to developing pollen cells via the apoplast is also discussed. [ABSTRACT FROM AUTHOR]

Wheat pollen, which is characterized by its short lifespan, exhibits rapid germination after anthesis. The preservation of wheat pollen is contingent upon environmental factors including temperature, relative humidity, light, and wind. The aim is to explicate the process for efficiently storing wheat pollen, particularly with regard to breeding. The short longevity of wheat pollen grains renders it impractical to conduct tests for pollen viability and in vitro germination on a large scale. Herein, the impact of storage temperatures and duration was assessed on pollen viability and in vitro germination in order to optimize storage conditions for preserving pollen viability. Pollen grains from 50 diverse spring wheat genotypes, each with three replicates, were harvested and stored at temperatures of 22 °C, −20 °C, and 4 °C. Subsequently, pollen viability and in vitro germination rates were determined after storage for 1, 3, and 6 days. The results revealed that storage temperatures, durations, genotypes, and their interactions had a statistically significant impact on both pollen viability and in vitro germination. Notably, when pollen was kept at 22 °C, almost all genotypes exhibited a loss of pollen viability and in vitro germination after 1, 3, and 6 days of storage. Likewise, storage at −20 °C failed to extend pollen germination. However, at a storage temperature of 4 °C, the pollen of 36 wheat genotypes exhibited a range of 6–14% for in vitro pollen germination and even remained viable for 6 days. The ANOVA revealed a substantial variation in grain number per spike between wheat genotypes, thereby highlighting the significant influence of genetic variations on grain yield. Moreover, a slight positive association between the viability of wheat pollen and the number of grains was found in the current study, suggesting that a variety of factors affect the number of grains produced. Simple linear regression analysis further revealed a significant negative correlation between pollen viability, in vitro germination, and storage time and temperature. In conclusion, our findings underscore that 4 °C is the optimal temperature for preserving pollen viability and in vitro pollen germination in spring wheat for up to 6 days. The results of the present study suggests that the pollen viability of wheat is dependent on genotype, storage temperature, and storage duration. Thus, the 36 wheat genotypes identified during the present work could be efficiently maintained at 4 °C for short-term storage (6 days) and could be further used for genetic and breeding purposes. [ABSTRACT FROM AUTHOR]

Characterizing the shape of pollen is crucial for plant breeding, taxonomy, and conservation, and studies on pollen viability can help breeding initiatives. This study examined the male fertility and pollen morphology of six Cucurbitaceae species with potential for both economic and nutritional benefit. Pollen grains were acetolyzed for morphological study, and a light compound microscope and an LEICA digital microscope were used to determine the exine stratification as well as other characteristics of pollen grains. The Cucurbitaceae family is eurypalynous, as evidenced by the noticeable differences in pollen physical characteristics between species. The shapes of pollen might be oblate, sub-oblate, or oblate-spheroidal. Pollen viability percentage was determined by using different staining methods- Acetocarmine test, Lugol’s test and Aniline blue test respectively. M. dioica, or spiny gourd, had the highest pollen viability percentage (94.4%), while L. acutangula, or Chinese okra, had the lowest percentage (80.83%) when it was fresh. Using Lugol's test, six Cucurbitaceae species' pollen grains were found to be starchy by nature. In Lugol's test, M. dioica showed the highest percentage of pollen viability, whereas M. charantia showed the lowest percentage of pollen viability following the anthesis of the flowers (at fresh). Species identification relies heavily on pollen morphology, particularly in this diverse family where many species exist. High viability rates encourage seed production and fertilization, which are necessary for effective hybrid development and conservation. [ABSTRACT FROM AUTHOR]

Aquilaria sinensis (Lour.) Spreng is a known medicinal plant producing agarwood. To date, studies on the floral biology of A. sinensis have been limited. In this study, the floral micro- and ultra-structures, pollen viability, stigma receptivity, and artificial pollination of A. sinensis were investigated. The results show that the flower's development can be divided into five stages, including the flower bud differentiation stage (2–7 d), the flower bud stage (7–13 d), the flowering stage (14 d), the pollination stage (14–15 d), and the fruiting stage (15–25 d). The floral organs mainly include: 4–6 split calyces, 10 petals, 10 stamens, and 1 pistil. The anther is oblong, with four pollen sacs the pollen is round, with maximum viability 6 h after flowering; and stigma receptivity is at its best 6 h before flowering. Artificial pollination is successful in the field. These findings will provide useful information for producing and breeding A. sinensis. [ABSTRACT FROM AUTHOR]

One of the most vulnerable phases in the plant life cycle is sexual reproduction, which depends on effective pollen transfer, but also on the thermotolerance of pollen grains. Pollen thermotolerance is temperature-dependent and may be reduced by increasing temperature associated with global warming. A growing body of research has focused on the effect of increased temperature on pollen thermotolerance in crops to understand the possible impact of temperature extremes on yield. Yet, little is known about the effects of temperature on pollen thermotolerance of wild plant species. To fill this gap, we selected Lotus corniculatus s.l. (Fabaceae), a species common to many European habitats and conducted laboratory experiments to test its pollen thermotolerance in response to artificial increase in temperature. To test for possible local adaptation of pollen thermal tolerance, we compared data from six lowland (389–451 m a.s.l.) and six highland (841–1,030 m a.s.l.) populations. We observed pollen germination in vitro at 15 °C, 25 °C, 30 °C, and 40 °C. While lowland plants maintained a stable germination percentage across a broad temperature range (15–30 °C) and exhibited reduced germination only at extremely high temperatures (40 °C), highland plants experienced reduced germination even at 30 °C–temperatures commonly exceeded in lowlands during warm summers. This suggests that lowland populations of L. corniculatus may be locally adapted to higher temperature for pollen germination. On the other hand, pollen tube length decreased with increasing temperature in a similar way in lowland and highland plants. The overall average pollen germination percentage significantly differed between lowland and highland populations, with highland populations displaying higher germination percentage. On the other hand, the average pollen tube length was slightly smaller in highland populations. In conclusion, we found that pollen thermotolerance of L. corniculatus is reduced at high temperature and that the germination of pollen from plant populations growing at higher elevations is more sensitive to increased temperature, which suggests possible local adaptation of pollen thermotolerance.

Interspecific hybridization has long been used to produce Phalaenopsis hybrids with superior traits such as flower size, color, lip morphology, heavy pigmentation spots, spike number, plant height, floret number and size, inflorescence angle, and branching. For a successful breeding program, superior parents with desirable horticultural traits must be selected for hybridization based on market preferences. However, many existing hybrids produce triploids or aneuploids and are almost completely sterile. A database of the cytogenetic backgrou nds of species and cultivars is essential for formulating systematic breeding programs. However, this information is limited to the Phalaenopsis cultivars. Therefore, chromosome composition, pollen viability, and meiotic abnormality of two Phalaenopsis yellow cultivars, namely Phalaenopsis ‘Fuller’s Sunset’ and Phalaenopsis ‘Geumgongju,’ were analyzed in this study. Cytogenetic analyses revealed that both cultivars had hypotetraploid and asymmetrical chromosomes. Assessment of pollen viability using the 2,3,5-triphenyl tetrazolium chloride (TTC) staining test and in vitro pollen germination in five flower developmental stages demonstrated that P. ‘Fuller’s Sunset’ pollinia were viable because it were stained in all stages with pollen tube growth. In contrast, unstained pollinia and the absence of pollen tubes were observed in P. ‘Geumgongju.’ Sporad stage analysis showed that P. ‘Geumgongju’ contained predominantly abnormal sporads with micronuclei compared to those of P. ‘Fuller’s Sunset.’ The results of this study provide vital information for breeding programs for Phalaenopsis cultivars and emphasize the importance of selecting appropriate parents for hybridization. [ABSTRACT FROM AUTHOR]

Exacum bicolor (Roxb.) is an herbaceous perennial endemic to peninsular India and enlisted as an endangered plant species. Since a thorough insight into the reproductive biology of a species is necessary to aid conservation and crop improvement, the present study was formulated to assess the floral biology and reproductive behaviour of the plant. It was found that the bisexual flowers of this plant possess pollen grains that remain viable for nine days after anthesis exhibiting maximum viability on the first day of anthesis (84.61%) and 84.18 per cent viability on the previous day of anthesis. Stigma was found to be receptive upto four days after anthesis. Maximum seed weight per capsule (17.21 mg) was realised from flowers pollinated on the third day of flower opening. There was 100 per cent fruit set in flowers that were open pollinated as well as in the flowers which were either controlled out-crossed or self-pollinated under control. Fruit set was not observed on bagging the flowers to avoid natural out-crossing, and on open pollination. Hence, it became evident that Exacum bicolor (Roxb.) is primarily a cross-pollinated species, although no self-incompatibility mechanism operates in the species. Also, natural self-pollination is not favoured for procreation; reproduction via apomixis is absent in the species. [ABSTRACT FROM AUTHOR]

In vitro pollen germination is considered the most efficient method to assess pollen viability. The pollen germination frequency and pollen tube length, which are key indicators of pollen viability, should be accurately measured during in vitro culture. In this study, a Mask R-CNN model trained using microscopic images of tree peony (Paeonia suffruticosa) pollen has been proposed to rapidly detect the pollen germination rate and pollen tube length. To reduce the workload during image acquisition, images of synthesized crossed pollen tubes were added to the training dataset, significantly improving the model accuracy in recognizing crossed pollen tubes. At an Intersection over Union threshold of 50%, a mean average precision of 0.949 was achieved. The performance of the model was verified using 120 testing images. The R 2 value of the linear regression model using detected pollen germination frequency against the ground truth was 0.909 and that using average pollen tube length was 0.958. Further, the model was successfully applied to two other plant species, indicating a good generalizability and potential to be applied widely. [ABSTRACT FROM AUTHOR]