Search

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.)

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-I 2 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.

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.)

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 ( R 2 = 0.09 *), ovule fertilization ( R 2 = 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.R 2 = 0.59 *), and seed set ( R 2 = 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.

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.)

The occurring temperature increase in crop production areas worldwide is generating conditions of heat stress that negatively affect crop productivity. Tomato ( Solanum lycopersicum ), a major vegetable crop, is highly susceptible to elevated temperatures. Under such conditions, fruit set is dramatically reduced, leading to significant yield losses. Solanum pimpinellifolium , a wild species closely related to the cultivated tomato, was shown to have beneficial attributes under various abiotic stress growth conditions. We have utilized a new population of backcross inbred lines originated from a cross between S. pimpinellifolium and S. lycopersicum , in order to evaluate its potential as a new genetic resource for improvement of reproductive performance of cultivated tomato under heat stress conditions. This population was screened for various heat stress-related traits, under controlled heat stress and non-stress conditions. Our results show that significant variation exists for all the heat stress related traits that were examined and point at individual lines with better reproductive performance under heat stress conditions that share a common introgression from the wild S. pimpinellifolium parent, suggesting several candidate genes as potential drivers of thermotolerance. Thus, our results place this population as a valuable new resource for the discovery of heat stress related genetic loci for the future development of heat stress tolerant tomato cultivars., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bashary, Miller, Doitsch-Movshovits, Beery, Ouyang and Lieberman-Lazarovich.)

Plant reproduction is highly susceptible to temperature stress. The development of the male gametophyte in particular represents a critical element in the reproductive cycle with high sensitivity to elevated temperatures. Various methods have been used to test the effect of temperature stress on pollen performance or to determine the degree of susceptibility of given species and genotypes. The information gained informs the development of new crop varieties suited to grow under warmer conditions arising through climate change and facilitates predicting the behavior of natural populations under these conditions. The characterization of pollen performance typically employs the terms pollen viability and pollen vigor, which, however, are not necessarily used consistently across studies. Pollen viability is a nominal parameter and is often assayed relying on cellular features as proxy to infer the capability of pollen grains to germinate and complete double fertilization. Alternatively, pollen germination can be determined through in vitro growth assays, or by monitoring the ability of pollen tubes to complete different progamic steps in vivo (ability to reach an ovule, release sperm cells, lead to seed set). Pollen vigor is an ordinal parameter that describes pollen tube growth rate or the efficiency of pollen tube growth as inferred by its morphology or growth pattern. To ensure consistent and relevant terminology, this review defines these terms and summarizes the methodologies used to assess them., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Cryptangieae has recently been revised based on morphology and molecular phylogeny, but cytogenetic data is still scarce. We conducted this study with the aim of investigating the occurrence of holocentric chromosomes and pseudomonads, as well as understanding the mode of chromosomal evolution in the tribe. We performed analyses of meiotic behavior, chromosome counts, and reconstruction of the ancestral state for the haploid number. We present novel cytogenetic data for eight potentially holocentric species: Cryptangium verticillatum, Krenakia junciforme, K. minarum, Lagenocarpus bracteosus, L. griseus, L. inversus, L. rigidus, and L. tenuifolius. Meiotic abnormalities were observed, with parallel spindles being particularly noteworthy. Intra-specific variations in chromosome number were not found, which may indicate an efficient genetic control for the elimination of abnormal nuclei. The inferred ancestral haploid number was n = 16, with dysploidy being the main evolutionary mechanism. At least five chromosomal fissions occurred in Krenakia (n = 21), followed by a further ascending dysploidy event in Lagenocarpus (n = 17). As proposed for Cyperaceae, it is possible that cladogenesis events in Cryptangieae were marked by numerical and structural chromosomal changes., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The present study was carried out to investigate the physio-biochemical and molecular responses of two rice genotypes (Noichi and N22) under drought, heat and combined drought/heat stress conditions. The antagonistic stomatal activity was found under the combined stress conditions; stomata were open under control and heat stress, conversely, stomata remained closed under drought and combined stress levels. Photosynthetic activity and chlorophyll content are decreased by the overproduction of reactive oxygen species and increased lipid peroxidation in both rice genotypes. To prevent oxidative damage, many antioxidant enzymes like catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) are produced in both genotypes under these conditions. Under the single stress conditions, CAT activity were increased in N22, whereas combined stress levels, SOD and APX activity were higher for both genotypes. Proline accumulation was also increased under single as well as combined stress conditions for both genotypes to combat stress injuries. Pollen viability was lost under all stress levels but severe loss was found under combined stress levels, which causes spikelet sterility leading to yield losses for both genotypes. As evident from transcript levels, HSP71.18 and HSP71.10 expressions were higher under single and combined conditions, but HSP72.57 gene expression increased only by individual stress levels. WRKY11, WRKY 55, DREB 2A, LEA3 and DHN1 were positively expressed under all stress levels. Conversely, expression of DREB2B genes was higher only under single stress levels. In summary, these results suggest that the effect of combined stress is different from the single stress and it is more severe than the individual stress., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03980-1., Competing Interests: Conflict of interestThere is no conflict of interest relating to this article., (© King Abdulaziz City for Science and Technology 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

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.)

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 N 2 ), 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 cross-compatibility, 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rane, Thakre, Verma, Kumar, Prakash, Srivastava, P. R., Murukan, Chawla, Mandal, Kumar, Jadhav, Varghese, Patel and Singh.)

Terminal heat during reproductive stages of wheat (Triticum aestivum L.) limits the productivity of the crop. Magnesium (Mg) is an essential macronutrient that is involved in many physiological and biochemical processes to affect photosynthesis and seed weight. The present study comparatively evaluated Mg applied to soil (80 kg MgSO 4 ·7H 2 O ha -1 ) and to plant foliage (4% w/v) in improving wheat performance under terminal heat. Wheat crop was grown in two sets of treatments until the booting stage, and then one set of plants was shifted to a glasshouse (±5 °C) at the booting stage to grow until maturity in comparison to control plants kept under ambient warehouse condition. Heat stress reduced the pollen viability while foliar- and soil-applied Mg improved it by 3% and 6% under heat stress, respectively, compared to the control without Mg treatment. The 100-seed weight, spike length, and biological yield reduced by 39%, 19%, and 50% under heat stress; however, foliar and soil application increased 100-seed weight by 45% and 40%, spike length by 8% and 5%, and biological yield by 35% and 25% under heat stress, respectively. Soil Mg showed maximum SPAD chlorophyll values; however, response was statistically similar to that of foliar Mg as compared to the control without Mg supply. Membrane stability decreased (4%) due to heat stress while foliar and soil treatments improved membrane stability by 8% and 5% compared to that of the control, respectively. Thus, Mg application through soil or plant foliage can be an effective way to reduce negative impacts of terminal heat in wheat by improving pollen viability at anthesis and 100-seed weight that was attributed to increased chlorophyll contents during anthesis., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 R2 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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Female hemp plants are desired in floral hemp operations due to their higher cannabinoid contents. To produce feminized seeds, a critical step of inducing fertile male flowers on female plants is performed. In feminized seed production, freshly mixed STS (silver thiosulfate + sodium thiosulfate) is applied to female plants as an ethylene inhibitor to induce male flowers. However, the short-shelf stability of the STS buffer can cause difficulty in the application and inconsistent results. Alternative methods with improved accessibility and stable buffers will be beneficial for the hemp industry and hemp breeders. A commercially available floriculture product, Chrysal ALESCO ® , contains silver nitrate, the same active ingredient as STS but with increased shelf stability. This study compares Chrysal ALESCO ® to the traditional STS standard methods for male flower induction on female plants and their pollen quality. The two treatments were applied to six female hemp accessions with three replicates investigated, and the male flower counts and pollen quality were compared. No statistically significant difference was discovered in their male flower counts; the STS-treated plant produced an average of 478.18 male flowers, and the Chrysal ALESCO ® -treated plant produced an average of 498.24 male flowers per plant. Fluorescein diacetate (FDA) and acetocarmine stains were used to investigate the pollen quality (non-aborted rate) of two chosen genotypes. FDA-stained pollen of Chrysal ALESCO ® showed a significantly higher non-aborted rate than the pollen of traditional STS-treated plants ( p < 0.001); however, only a marginally higher non-aborted rate was discovered by acetocarmine staining ( p = 0.0892). In summary, Chrysal ALESCO ® performed equally to traditional STS treatment at male flower counts and better or equally in pollen quality. With better shelf stability and easy application, ALESCO ® can be a viable alternative option for stimulating male flowers on female hemp plants.

Pollen grains represent the male gametes of seed plants and their viability is critical for sexual reproduction in the plant life cycle. Palynology and viability studies have traditionally been used to address a range of botanical, ecological and geological questions, but recent work has revealed the importance of pollen viability in invasion biology as well. Here, we report an efficient visual method for assessing the viability of pollen using digital holographic microscopy (DHM). Imaging data reveal that quantitative phase information provided by the technique can be correlated with viability as indicated by the outcome of the colorimetric test. We successfully test this method on pollen grains of Lantana camara , a well-known alien invasive plant in the tropical world. Our results show that pollen viability may be assessed accurately without the usual staining procedure and suggest potential applications of the DHM methodology to a number of emerging areas in plant science., Competing Interests: The authors have no competing interest., (© The Author(s) 2023.)

Paeonia ostii is an important woody oil crop mainly cross-pollinated. However, the low yield has become an important factor restricting the industrial development of P. ostii. Cross-pollination has become one of the important measures to increase the seed yield. Therefore, conservation of pollen with high vitality is crucial to ensure successful pollination of P. ostii . In this study, we found an effective methodological system to assess the viability, ability to germinate, and optimal storage conditions of P. ostii pollen grains. The optimal medium in vitro was 50 g/L sucrose, 100 mg/L boric acid, 50 g/L PEG6000, 100 mg/L potassium nitrate, 300 mg/L calcium nitrate, and 200 mg/L magnesium sulfate at pH 5.4. Optimal germination condition in vitro was achieved at 25 °C for 120 min, allowing easy observation of the germination percentage and length of the pollen tubes. In addition, the viability of pollen grains was assessed by comparing nine staining methods. Among them, MTT, TTC, benzidine-H 2 O 2 , and FDA were effective to distinguish between viable and non-viable pollen, and the results of the FDA staining method were similar to the pollen germination percentage in vitro. After evaluation of pollen storage, thawing and rehydration experiments showed that thawing at 4 °C for 30 min and rehydration at 25 °C for 30 min increased the germination percentage of pollen grains stored at low temperatures. The low-temperature storage experiments showed that 4 °C was suitable for short-term storage of P. ostii pollen grains, while -80 °C was suitable for long-term storage. This is the first report on the in vitro germination, viability tests, and storage of P. ostii pollen grains, which will provide useful information for P. ostii germplasm conservation and artificial pollination.

Commelinaceae is an important component of the Atlantic Forest and its analysis can provide information on the conservation of that species. This study had the objective of analyzing the phenology and floral and reproductive biology of Dichorisandra rhizantha, growing in an Atlantic Forest fragment, to obtain data related to the form of reproduction and pollination mechanism of this species. We monitored the phenophases and reproductive biology of the D. rhizantha in the area studied using different methodologies and microscopy techniques (LM, SEM and FM). The flowering period occurs during the dry season. The species is andromonoecious and has daytime anthesis (4:30 a.m.-1:30 p.m.). It has purple zygomorphic flowers and rimose but functionally poricidal anthers. The pollen viability was 97.6% and the two floral morphs investigated contained a large quantity of pollen grains. The time of greatest stigma receptivity (anthesis) coincided with the pollen viability. Cross-pollination was the only efficient reproductive system of the species, with observation of gametophytic self-incompatibility and pollen tubes growth only as far as the middle or lower third of the pistil. We observed bee species of the genera Xylocopa and Euglossa visiting the flowers and acting as effective pollinators. The immature fruits were formed after about 3.25 ± 1 days, and the presence was noted of ants of the genus Cephalotes acting as dispersers of the seeds, with aril serving as the reward. Although the natural reproductive system is efficient, D. rhizantha is endangered, mainly due to fragmentation of habitat caused by deforestation and urban encroachment., (© 2023 Wiley Periodicals LLC.)

Date palm ( Phœnix dactylifera L.) like other crop species in the arid Mediterranean region is being threatened by genetic erosion and climate change. Therefore, the understanding and assessment of the diversity extent of this species is a primary requisite for preserving these crop resources. This study was designed to quantify the potential of Tunisian male date palms using a set of agro-morphological characteristics i.e. flowering traits, inflorescence morphology and pollen quality. The flowering time traits exhibited a trend of precocious phenotype at emergence spathe trait and the dominance of the full-season phenotype at the opening date. At inflorescence morphology, all observed traits expressed wide ranges which reflected the broad variability of the evaluated male genotypes. Significant difference was recorded for the majority of the examined traits with a high significant variation in the tree quantitative traits: Spathe Total Length, Spathe Maximum Width and Length to the brunched part. Pollen viability ranged from 51.10% to 98.75% while the germination rate was between 0.90% and 70.50%. Our phenotypic investigation has allowed the identification of males with desirable agronomic traits which have been genotyped using 18 nuclear SSR markers and a chloroplast minisatellite for preservation and effective utilization purposes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Major advances have been made in our understanding of anther developmental processes in flowering plants through a combination of genetic studies, cell biological technologies, biochemical analyses, microarray and high-throughput sequencing-based approaches. In this chapter, we summarize widely used protocols for pollen viability staining, investigation of anther morphogenesis by scanning electron microscopy (SEM), light microscopy of semi-thin sections, ultrathin section-based transmission electron microscopy (TEM), TUNEL (terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling) assay for tapetum programmed cell death, and laser microdissection procedures to obtain specific cells or cell layers for transcriptome analysis., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Terminal heat stress during reproductive stage in wheat ( Triticum aestivum L.) causes pollen grain sterility and has a drastic impact on wheat crop production. Finding genotypes with high pollen viability under heat stress is crucial to cope with the impact of climate change through developing heat-tolerant cultivars. To assess the effect of terminal heat stress on pollen viability in a panel of spring wheat genotypes ( N = 200), RCBD (randomized complete block design) field trials were conducted under normal and heat stress conditions for two consecutive years (2020-2021 and 2021-2022). Analysis of variance showed significant variation in genotypes, treatments, and genotype × treatment interaction. Fifty and 46 genotypes were categorized as heat tolerant ( HSI pv < 0.5) over the period of time in many modern wheat varieties. However, some modern wheat varieties released after 2001 such as Janbaz-09 (57%), Ghazi-2019 (57%), and Sindhu-16 (43%) had very low pollen viability under heat stress conditions. The results of phenotypic coefficient of variance (PCV%), genotypic coefficient of variance (GCV%), broad sense heritability (h HSI ), and genetic advance (GA) suggested the major contribution of genetic factors in controlling pollen viability trait. Higher values of h pv and GA under heat stress conditions suggested pollen viability as a heat tolerance trait controlled by additive genetic effects. Taken together, these results suggested pollen viability as a useful trait for selection in early generations under elevated temperatures. The genotypes identified as heat tolerant in both years can be used as genetic resources for breeding cultivars with higher pollen viability under elevated temperature conditions.2 bs ), and genetic advance (GA) suggested the major contribution of genetic factors in controlling pollen viability trait. Higher values of h 2 bs and GA under heat stress conditions suggested pollen viability as a heat tolerance trait controlled by additive genetic effects. Taken together, these results suggested pollen viability as a useful trait for selection in early generations under elevated temperatures. The genotypes identified as heat tolerant in both years can be used as genetic resources for breeding cultivars with higher pollen viability under elevated temperature conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Khan, Wu and Sajjad.)

Pollen grains, the male gametophytes for reproduction in higher plants, are vulnerable to various stresses that lead to loss of viability and eventually crop yield. A conventional method for assessing pollen viability is manual counting after staining, which is laborious and hinders high-throughput screening. We developed an automatic detection tool (PollenDetect) to distinguish viable and nonviable pollen based on the YOLOv5 neural network, which is adjusted to adapt to the small target detection task. Compared with manual work, PollenDetect significantly reduced detection time (from approximately 3 min to 1 s for each image). Meanwhile, PollenDetect can maintain high detection accuracy. When PollenDetect was tested on cotton pollen viability, 99% accuracy was achieved. Furthermore, the results obtained using PollenDetect show that high temperature weakened cotton pollen viability, which is highly similar to the pollen viability results obtained using 2,3,5-triphenyltetrazolium formazan quantification. PollenDetect is an open-source software that can be further trained to count different types of pollen for research purposes. Thus, PollenDetect is a rapid and accurate system for recognizing pollen viability status, and is important for screening stress-resistant crop varieties for the identification of pollen viability and stress resistance genes during genetic breeding research.

Maize pollen is highly sensitive to heat and drought, but few studies have investigated the combined effects of heat and drought on pollen viability. In this study, pollen's structural and physiological characteristics were determined after heat, drought, and combined stressors. Furthermore, integrated metabolomic and transcriptomic analyses of maize pollen were conducted to identify potential mechanisms of stress responses. Tassel growth and spikelet development were considerably suppressed, pollen viability was negatively impacted, and pollen starch granules were depleted during anthesis under stress. The inhibitory effects were more significant due to combined stresses than to heat or drought individually. The metabolic analysis identified 71 important metabolites in the combined stress compared to the other treatments, including sugars and their derivatives related to pollen viability. Transcriptomics also revealed that carbohydrate metabolism was significantly altered under stress. Moreover, a comprehensive metabolome-transcriptome analysis identified a central mechanism in the biosynthesis of UDP-glucose involved in reducing the activity of sucrose synthase SH-1 (shrunken 1) and sus1 (sucrose synthase 1) that suppressed sucrose transfer to UDP-glucose, leading to pollen viability exhaustion under stress. In conclusion, the lower pollen viability after heat and drought stress was associated with poor sucrose synthase activity due to the stress treatments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Both sugar and the hormone gibberellin (GA) are essential for anther-enclosed pollen development and thus for plant productivity in flowering plants. Arabidopsis (Arabidopsis thaliana) AtSWEET13 and AtSWEET14, which are expressed in anthers and associated with seed yield, transport both sucrose and GA. However, it is still unclear which substrate transported by them directly affects anther development and seed yield. Histochemical staining, cross-sectioning and microscopy imaging techniques were used to investigate and interpret the phenotypes of the atsweet13;14 double mutant during anther development. Genetic complementation of atsweet13;14 using AtSWEET9, which transports sucrose but not GA, and the GA transporter AtNPF3.1, respectively, was conducted to test the substrate preference relevant to the biological process. The loss of both AtSWEET13 and AtSWEET14 resulted in reduced pollen viability and therefore decreased pollen germination. AtSWEET9 fully rescued the defects in pollen viability and germination of atsweet13;14, whereas AtNPF3.1 failed to do so, indicating that AtSWEET13/14-mediated sucrose rather than GA is essential for pollen fertility. AtSWEET13 and AtSWEET14 function mainly at the anther wall during late anther development stages, and they probably are responsible for sucrose efflux into locules to support pollen development to maturation, which is vital for subsequent pollen viability and germination., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Premise: Living collections maintained for generations are at risk of diversity loss, inbreeding, and adaptation to cultivation. To address these concerns, the zoo community uses pedigrees to track individuals and implement crosses that maximize founder contributions and minimize inbreeding. Using a pedigree management approach, we demonstrate how conducting strategic crosses can minimize genetic issues that have arisen under current practices., Methods: We performed crosses between collections and compared progeny fitness, including plant performance and reproductive health. We genotyped the progeny and parental accessions to measure changes in diversity and relatedness within and between accessions., Results: The mean relatedness values among individuals within each accession suggest they are full siblings, demonstrating that there was high inbreeding and low diversity within accessions, although less so among accessions. Progeny from the wider crosses had increased genetic diversity and were larger and more fertile, while self-pollinated accessions were smaller and less fertile., Discussion: Institutions that hold exceptional species should consider how diversity is maintained within their collections. Implementing a pedigree-based approach to managing plant reproduction ex situ will slow the inevitable loss of genetic diversity and, in turn, result in healthier collections., (© 2022 The Authors. Applications in Plant Sciences published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

High temperature negatively affects reproductive process significantly, leading to tremendous losses in crop quality and yield. Zhinengcong (ZNC), a crude extract from the endophytic fungus Paecilomyces variotii , has been shown to improve plant growth and resistance to biotic and abiotic stresses. We show here that ZNC can also alleviate heat stress-induced reproductive defects in Solanum lycopersicum , such as short-term heat-induced inhibition on pollen viability, germination and tube growth, and long-term heat stress-induced pollen developmental defects. We further demonstrated that ZNC alleviates heat stress by downregulating the expressions of ROS production-related genes, RBOHs, and upregulating antioxidant related genes and the activities of the corresponding enzymes, thus preventing the over accumulation of heat-induced reactive oxygen species (ROS) in anther, pollen grain and pollen tube. Furthermore, spraying application of ZNC onto tomato plants under long-term heat stress promotes fruit and seed bearing in the field. In summary, plant endophytic fungus extract ZNC promotes the reproductive process and yield of tomato plants under heat stress and presents excellent potential in agricultural applications., Competing Interests: QW and WZ are employed by Shandong Pengbo Biotech Limited Company and Corteva Agriscience, respectively. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cui, Liu, Zhang, Guo, Li, Zhang, Wang, Zeng, Huang, Duan and Cao.)

Alfalfa ( Medicago sativa L.) is an important forage in intercropping or rotation ecosystem, and shading is the principal limiting factor for its growth under the crop or forest. Agronomic studies showed that shading would systematically reduce the biomass of alfalfa. However, little is known about the reproduction of alfalfa under shading conditions. In order to study the effect of shading on the reproductive characteristics of alfalfa, two alfalfa cultivars ("Victoria" and "Eureka") were used to study the effect of shading levels (full light, 56.4% shade, and 78.7% shade) on alfalfa flowering phenology, pollen viability, stigma receptivity, and seed quality. Results showed that shading delayed flowering phenology, shortened the flowering stage, faded the flower colors, and significantly reduced pollen viability, stigma receptivity, the number of flowers, quantity, and quality of seeds. Under shading conditions, seed yield per plant was obviously positively correlated with germination potential, germination rate, pollen viability, and 1,000-seed weight. The number of flower buds, pollen viability, 1,000-seed weight, and germination rate had the greatest positive direct impact on seed yield per plant. Our findings suggested that delayed flowering and reducing reproduction growth were important strategies for alfalfa to cope with shading and pollen viability was the key bottleneck for the success of alfalfa reproduction under shading. However, given that alfalfa is a perennial vegetative-harvest forage, delaying flowering in a weak light environment was beneficial to maintain the high aboveground biomass of alfalfa. Therefore, this should be taken into account when breeding alfalfa cultivars suitable for intercropping. Future research should further reveal the genetic and molecular mechanism of delayed flowering regulating the accumulation and distribution of assimilates between vegetative and reproductive organs of alfalfa under shading, so as to provide a theoretical basis for breeding of shade-tolerant alfalfa cultivars., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Qin, Shen, Li, Qu, Feng, Kong, Teri, Luan and Cao.)

Rising temperatures due to climate change threaten agricultural crop productivity. As a cool-season crop, wheat is heat-sensitive, but often exposed to high temperatures during the cultivation period. In the current study, a bread wheat panel of spring wheat genotypes, including putatively heat-tolerant Australian and CIMMYT genotypes, was exposed to a 5-day mild (34°C/28°C, day/night) or extreme (37°C/27°C) heat stress during the sensitive pollen developmental stage. Worsening effects on anther morphology were observed, as heat stress increased from mild to extreme. Even under mild heat, a significant decrease in pollen viability and number of grains per spike from primary spike was observed compared with the control (21°C/15°C), with Sunstar and two CIMMYT breeding lines performing well. A heat-specific positive correlation between the two traits indicates the important role of pollen fertility for grain setting. Interestingly, both mild and extreme heat induced development of new tillers after the heat stress, providing an alternative sink for accumulated photosynthates and significantly contributing to the final yield. Measurements of flag leaf maximum potential quantum efficiency of photosystem II (Fv/Fm) showed an initial inhibition after the heat treatment, followed by a full recovery within a few days. Despite this, model fitting using chlorophyll soil plant analysis development (SPAD) measurements showed an earlier onset or faster senescence rate under heat stress. The data presented here provide interesting entry points for further research into pollen fertility, tillering dynamics, and leaf senescence under heat. The identified heat-tolerant wheat genotypes can be used to dissect the underlying mechanisms and breed climate-resilient wheat., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Lowe, Hernandez-Leon, Dreisigacker, Reynolds, Valenzuela-Soto, Paul and Heuer.)

The pollen intine layer is necessary for male fertility in flowering plants. However, the mechanisms behind the developmental regulation of intine formation still remain largely unknown. Here, we identified a positive regulator, Arabidopsis novel microgametophyte defective mutant 1 ( AtNMDM1 ), which influences male fertility by regulating intine formation. The AtNMDM1 , encoding a pollen nuclei-localized protein, was highly expressed in the pollens at the late anther stages, 10-12. Both the mutations and the knock-down of AtNMDM1 resulted in pollen defects and significantly lowered the seed-setting rates. Genetic transmission analysis indicated that AtNMDM1 is a microgametophyte lethal gene. Calcofluor white staining revealed that abnormal cellulose distribution was present in the aborted pollen. Ultrastructural analyses showed that the abnormal intine rather than the exine led to pollen abortion. We further found, using transcriptome analysis, that cell wall modification was the most highly enriched gene ontology (GO) term used in the category of biological processes. Notably, two categories of genes, Arabinogalactan proteins ( AGPs ) and pectin methylesterases ( PMEs ) were greatly reduced, which were associated with pollen intine formation. In addition, we also identified another regulator, AtNMDM2, which interacted with AtNMDM1 in the pollen nuclei. Taken together, we identified a novel regulator, AtNMDM1 that affected cellulose distribution in the intine by regulating intine-related gene expression; furthermore, these results provide insights into the molecular mechanisms of pollen intine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mi, Mo, Yang, Liu, Ren, Chen, Long, Jiang, Zhang and Lu.)

Background: In certain unisexual flowers, non-functional sexual organs remain vestigial and unisexuality can be overlooked leading to the ambiguous description of the sexual systems. Therefore, to accurately describe the sexual system, detailed morphological and developmental analyses along with experimental crosses must be performed. Cylindropuntia wolfii is a rare cactus endemic to the Sonoran Desert in southern California and northern Baja California that was described as gynodioecious by morphological analysis. The aims of our project include accurately identifying the sexual system of C. wolfii using histological and functional studies and characterizing the developmental mechanisms that underlie its floral development., Methods: Histological analyses were carried out on different stages of C. wolfii flowers and controlled crosses were performed in the field., Result: Our results identified C. wolfii to be functionally dioecious. The ovule and anther development differed between staminate and pistillate flowers. In vivo pollen germination tests showed that the pollen of staminate and pistillate flowers were viable and the stigma and style of both staminate and pistillate flowers were receptive. This suggests that there are no genetic or developmental barriers in the earlier stages of pollen recognition and pollen germination., Conclusions: Despite being functionally dioecious, we observed that functionally pistillate individuals produced fruits with a large number of aborted seeds. This implies that not only does this species have low reproductive success, but its small population sizes may lead to low genetic diversity., (© 2022. The Author(s).)

Many crop species are cultivated to produce seeds and/or fruits and therefore need reproductive success to occur. Previous studies proved that high temperature on mature pollen at anther dehiscence reduce viability and germinability therefore decreasing crop productivity. We hypothesized that high temperature might affect pollen functionality even if the heat treatment is exerted only during the microsporogenesis. Experimental data on Solanum lycopersicum 'Micro-Tom' confirmed our hypothesis. Microsporogenesis successfully occurred at both high (30 °C) and optimal (22 °C) temperature. After the anthesis, viability and germinability of the pollen developed at optimal temperature gradually decreased and the reduction was slightly higher when pollen was incubated at 30 °C. Conversely, temperature effect was eagerly enhanced in pollen developed at high temperature. In this case, a drastic reduction of viability and a drop-off to zero of germinability occurred not only when pollen was incubated at 30 °C but also at 22 °C. Further ontogenetic analyses disclosed that high temperature significantly speeded-up the microsporogenesis and the early microgametogenesis (from vacuolated stage to bi-cellular pollen); therefore, gametophytes result already senescent at flower anthesis. Our work contributes to unravel the effects of heat stress on pollen revealing that high temperature conditions during microsporogenesis prime a fatal shortening of the male gametophyte lifespan., (© 2021. The Author(s).)

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).)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Generative processes have been evaluated in six European buttercup species in order to verify the hypothesis that the reproduction efficiency of clonal species is lower than that of nonclonal ones. The study covered common species ( Ficaria verna , Ranunculus auricomus , R. bulbosus , R. cassubicus , R. lanuginosus ) and the endangered R. illyricus . The following properties have been assessed: pollen viability (staining method), pollen grain germination and the pollen-tube elongation in pistil tissues (fluorescence microscopy), seed formation efficiency, seed viability (tetrazolium test) and germination ability by introducing factors interrupting dormancy (low temperature and gibberellin application). Additionally, the pistil morphology was documented for R. bulbosus , R. illyricus and R. cassubicus using SEM techniques. It was demonstrated that the reproductive efficiency, expressed as the production of viable seeds able to germinate, was significantly higher in the species reproducing sexually (especially in R. lanuginosus ) compared to the clonal ones. However, the complexity observed leads to separation of an additional group (cluster) of apomictic species: R. auricomus and R. cassubicus , distinguished by the lowest pollen viability and a low ability of the seeds to germinate. In the vegetatively reproducing R. illyricus , the seed formation efficiency was just 13.2% despite the having highest number of pistils in its flowers. The developed seeds of this species observed in our experiment were viable, but in general effective methods to stimulate their germination have not been proposed yet. Here, the first comparative study concerning the biology of sexual reproduction of R. illyricus is presented in the context of its decreasing distribution in natural habitats.

Oryza longistaminata is an African wild rice species that possesses special traits for breeding applications. Self-incompatibility is the main cause of sterility in O. longistaminata , but here we demonstrated that its pollen vitality are normal. Lipid and carbohydrate metabolism were active throughout pollen development. In this study, we used I 2 -KI staining and TTC staining to investigate pollen viability. Aniline-blue-stained semithin sections were used to investigate important stages of pollen development. Tandem mass tags (TMT)-based quantitative analysis was used to investigate the profiles of proteins related to lipid and carbohydrate metabolism in 4-, 6-, and 8.5-mm O. longistaminata spikelets before flowering. Pollen was found to germinate normally in vitro and in vivo . We documented cytological changes throughout important stages of anther development, including changes in reproductive cells as they formed mature pollen grains through meiosis and mitosis. A total of 31,987 RNA transcripts and 8,753 proteins were identified, and 6,842 of the proteins could be quantified. RNA-seq and proteome association analysis indicated that fatty acids were converted to sucrose after the 6-mm spikelet stage, based on the abundance of most key enzymes of the glyoxylate cycle and gluconeogenesis. The abundance of proteins involved in pollen energy metabolism was further confirmed by combining quantitative real-time PCR with parallel reaction monitoring (PRM) analyses. In conclusion, our study provides novel insights into the pollen viability of O. longistaminata at the proteome level, which can be used to improve the efficiency of male parent pollination in hybrid rice breeding applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sun, Wang, Chen, Qin, Li, Ouyang, Peng and He.)

Knowledge of the physiological basis for improved genetic adaptation of common bean ( Phaseolus vulgaris L.) lines to acid soils and high temperature conditions in the Amazon region of Colombia is limited. In this study, we evaluated the differences among 41 common bean lines in energy use, leaf cooling, photosynthate partitioning to pod formation and grain filling, and grain yield over two seasons under acid soil and high temperature stress in the Amazon region of Colombia. Common bean lines evaluated included medium and large seeded interspecific lines of Mesoamerican and Andean gene pools with different levels of adaptation to abiotic stress conditions and some lines are improved for iron and zinc (biofortified) concentration in seeds. We found three bean lines (GGR 147, SMG 21 and SMG 12) that were superior in their photosynthetic response, leaf cooling, photosynthate partitioning ability to pod formation and grain filling, resulting in grain yields exceeding 1900 kg ha -1 under acid soil and high temperature stress conditions. The superior photosynthetic performance was attributed to the efficient use of absorbed energy on the electron level in thylakoids, which is mainly oriented to a higher quantum yield of PSII (ΦII), lower energy dissipation in the form of heat (ΦNPQ), high linear electron flow (LEF) and high fraction of PSI centers in open state (PSIopen). We speculate that these photosynthetic and photosynthate partitioning responses of superior bean lines are part of the genetic adaptation to acidic soils and high temperature stress conditions. Among the evaluated bean lines, three lines (GGR 147, SMG 21 and SMG 12) combined the desirable attributes for genetic improvement of stress tolerance and biofortification. These lines can serve as parents to further improve traits (energy use efficiency and multiple stress resistance) that are important for bean production in the Amazon region.

Background: Decreased spikelet fertility is often responsible for reduction in grain yield in rice (Oryza sativa L.). In this study, two varieties with different levels of heat tolerance, Liangyoupeijiu (LYPJ, heat susceptible) and Shanyou63 (SY63, heat tolerant) were subjected to two temperature treatments for 28 days during the panicle initiation stage in temperature/relative humidity-controlled greenhouses: high temperature (HT; 37/27 °C; day/night) and control temperature (CK; 31/27 °C; day/night) to investigate changes in anther development under HT during panicle initiation and their relationship with spikelet fertility., Results: HT significantly decreased the grain yield of LYPJ by decreasing the number of spikelets per panicle and seed setting percentage. In addition, HT produced minor adverse effects in SY63. The decreased spikelet fertility was primarily attributed to decreased pollen viability and anther dehiscence, as well as poor pollen shedding of the anthers of LYPJ under HT. HT resulted in abnormal anther development (fewer vacuolated microspores, un-degraded tapetum, unevenly distributed Ubisch bodies) and malformation of pollen (obscure outline of the pollen exine with a collapsed bacula, disordered tectum, and no nexine of the pollen walls, uneven sporopollenin deposition on the surface of pollen grains) in LYPJ, which may have lowered pollen viability. Additionally, HT produced a compact knitted anther cuticle structure of the epidermis, an un-degraded septum, a thickened anther wall, unevenly distributed Ubisch bodies, and inhibition of the confluent locule, and these malformed structures may be partially responsible for the decreased anther dehiscence rate and reduced pollen shedding of the anthers in LYPJ. In contrast, the anther wall and pollen development of SY63 were not substantially changed under HT., Conclusions: Our results suggest that disturbed anther walls and pollen development are responsible for the reduced spikelet fertility and grain yield of the tested heat susceptible variety, and noninvasive anthers and pollen formation in response to HT were associated with improved heat tolerance., (© 2021. The Author(s).)

In vitro germination assays are frequently used in screening trials to evaluate the pollen viability of pollinizers. To be effective, screening trials must have defined threshold criteria, from which individuals can then be assessed. However, despite decades of research on pollen viability, no established threshold is available to categorize apple cultivars based on their in vitro pollen tube lengths. This study aimed to identify and characterize the subgroups of cultivars based on their pollen tube growth performance. In vitro pollen tube lengths of 41 individuals were determined by incubating samples on artificial germination media at 15 and 25 °C. A six-number summary statistic was calculated, and hierarchical clustering on principal component (HCPC) analysis was used to determine and characterize subgroups. Furthermore, a decision tree model was used to predict class membership for future datasets. HCPC analysis partitioned the 41 individuals into three subgroups with different performances. The decision tree quickly predicted the cluster membership based on the second quartile at 15 °C and the third quartile at 25 °C. The thresholds from the decision tree can be used to characterize new observations. The use of the methods will be demonstrated using a case study with 29 apple accessions.

Glyphosate is a broad-spectrum herbicide that is widely used in many different commercial formulations. Glyphosate-based herbicides (GBH) are used in forestry operations to reduce populations of plants that compete with merchantable conifers. Past research has found that low-dose GBH applications caused male sterility in agriculturally relevant plants, sparking a need to determine the potential impacts of forestry-related GBH applications on understory plants. We investigated the effects of GBH on the reproductive morphology of Rosa acicularis , a highly prevalent understory shrub within British Columbia, Canada, growing on three operational forestry cutblocks treated with 1.782 kg a.i./ha of glyphosate, in the Omineca Region, and also in a controlled experiment. We analyzed floral and pollen morphology from treated plants and compared these with untreated plants in both scenarios. Pollen viability of treated plants was reduced by an average of 66%, and >30% of anthers were non-dehiscent compared to controls across our three field sites and experimental plants. We also found alterations in pollen and petal morphology in flowers from treated sites and glyphosate residues present in floral tissues 2 years after GBH applications. It is important to fully understand how long GBH-induced change will impact forest vegetation, to preserve natural forest biodiversity and reduce anthropogenic influences on boreal forest ecosystems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Golt and Wood.)

Climate change has created an environment where heat stress conditions are becoming more frequent as temperatures continue to raise in crop production areas around the world. This situation leads to decreased crop production due to plant sensitivity to heat stress. Reproductive success is critically dependent on plants' ability to produce functional pollen grains, which are the most thermo-sensitive tissue. Flavonols are plant secondary metabolites known for their potent antioxidative activity, essential for male fertility in several species including tomato, and implicated in heat stress tolerance. Since flavonols are highly abundant in fruits of the tomato high pigment 2 ( hp2 ) mutant, we tested the level of flavonols in pollen of this mutant, under the hypothesis that increased accumulation of flavonols would render pollen more tolerant to heat stress. Indeed, pollen from two alleles of the hp2 mutant was found to have flavonols levels increased by 18 and 280% compared with wild-type (WT) under moderate chronic heat stress (MCHS) conditions. This mutant produced on average 7.8-fold higher levels of viable pollen and displayed better germination competence under heat stress conditions. The percentage of fully seeded fruits and the number of seeds per fruit were maintained in the mutant under heat stress conditions while decreased in wild-type plants. Our results strongly suggest that increased concentrations of pollen flavonols enhance pollen thermotolerance and reproductive success under heat stress conditions. Thus, the high flavonols trait may help frame the model for improving crop resilience to heat stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rutley, Miller, Wang, Harper, Miller and Lieberman-Lazarovich.)

Abiotic stresses adversely affect crop production causing yield reductions in important crops, including tomato ( Solanum lycopersicum L.). Among the different abiotic stresses, drought is considered to be the most critical one, since limited water availability negatively impacts plant growth and development, especially in arid and semi-arid areas. The aim of this study was to understand how biostimulants may interact with critical physiological response mechanisms in tomato under limited water availability and to define strategies to improve tomato performances under drought stress. We investigated the physiological responses of the tomato genotype 'E42' grown in open fields under optimal conditions (100% irrigation) and limited water availability (50% irrigation) treated or not with a novel protein hydrolysate-based biostimulant (CycoFlow, Agriges, BN, Italy). Plants treated with the protein hydrolysate showed a better water status and pollen viability, which also resulted in higher yield under drought stress compared to untreated plants. The treatment with the biostimulant had also an effect on antioxidant contents and activity in leaves and fruits depending on the level of irrigation provided. Altogether, these results indicate that the application of protein hydrolysates on tomato improved plant performances under limited water availability and in different experimental fields.

In the era of technology, nanotechnology has been introduced as a new window for agriculture. However, no attention has been paid to the effect of cerium dioxide nanoparticles (nCeO 2 ) on the reproductive stage of plant development to evaluate their toxicity and safety. To address this important topic, bean plants ( Phaseolus vulgaris L.) treated aerially with nCeO 2 suspension at 250-2000 mg L -1 were cultivated until flowering and seed production in the greenhouse condition. Microscopy analysis was carried out on sectioned anthers and ovules at different developmental stages. The pollen's mother cell development in nCeO 2 treatments was normal at early stages, the same as control plants. However, the results indicated that pollen grains underwent serious structural damages, including chromosome separation abnormality at anaphase I, pollen wall defect, and pollen grain malformations in nCeO 2 -treated plants at the highest concentration, which resulted in pollen abortion and yield losses. On the ovule side, the progression of development only at the highest concentration was modified in the two-nucleated embryo sac stage, probably due to apoptosis in nuclei. Nevertheless, the findings confirmed the more pronounced vulnerability of male reproductive development under nCeO 2 exposure than female development. The higher concentration decreased seed productivity, including seed set in either pods or whole plant (13% and 18% compared to control, respectively). The data suggested the potential application of nCeO 2 at optimal dosages as a plant productivity ameliorative. However, a higher dosage is considered as an eco-environmental hazard. To our best knowledge, this is the first study analyzing reproductive plant response upon exposure to nCeO 2 .

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.)

Plants adapt to heat via thermotolerance pathways in which the activation of protein folding chaperones is essential. In eukaryotes, protein disulfide isomerases (PDIs) facilitate the folding of nascent and misfolded proteins in the secretory pathway by catalyzing the formation and isomerization of disulfide bonds and serving as molecular chaperones. In Arabidopsis, several members of the PDI family are upregulated in response to chemical inducers of the unfolded protein response (UPR), including both members of the non-classical PDI-M subfamily, PDI9 and PDI10. Unlike classical PDIs, which have two catalytic thioredoxin (TRX) domains separated by two non-catalytic TRX-fold domains, PDI-M isoforms are orthologs of mammalian P5/PDIA6 and possess two tandem catalytic domains. Here, PDI9 accumulation was found to be upregulated in pollen in response to heat stress. Histochemical staining of plants harboring the PDI9 and PDI10 promoters fused to the gusA gene indicated they were actively expressed in the anthers of flowers, specifically in the pollen and tapetum. Immunoelectron microscopy revealed that PDI9 localized to the endoplasmic reticulum in root and pollen cells. transfer DNA (T-DNA) insertional mutations in the PDI9 gene disrupted pollen viability and development in plants exposed to heat stress. In particular, the pollen grains of pdi9 mutants exhibited disruptions in the reticulated pattern of the exine and an increased adhesion of pollen grains. Pollen in the pdi10 single mutant did not display similar heat-associated defects, but pdi9 pdi10 double mutants (DMs) completely lost exine reticulation. Interestingly, overexpression of PDI9 partially led to heat-associated defects in the exine. We conclude that PDI9 plays an important role in pollen thermotolerance and exine biogenesis. Its role fits the mechanistic theory of proteostasis in which an ideal balance of PDI isoforms is required in the endoplasmic reticulum (ER) for normal exine formation in plants subjected to heat stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Feldeverd, Porter, Yuen, Iwai, Carrillo, Smith, Barela, Wong, Wang, Kang, Matsumoto and Christopher.)

Impedance flow cytometry (IFC) is a versatile lab-on-chip technology which enables fast and label-free analysis of pollen grains in various plant species, promising new research possibilities in agriculture and plant breeding. Hazelnut is a monoecious, anemophilous species, exhibiting sporophytic self-incompatibility. Its pollen is dispersed by wind in midwinter when temperatures are still low and relative humidity is usually high. Previous research found that hazelnut can be characterized by high degrees of pollen sterility following a reciprocal chromosome translocation occurring in some cultivated genotypes. In this study, IFC was used for the first time to characterize hazelnut pollen biology. IFC was validated via dye exclusion in microscopy and employed to (i) follow pollen hydration over time to define the best pre-hydration treatment for pollen viability evaluation; (ii) test hazelnut pollen viability and sterility on 33 cultivars grown in a collection field located in central Italy, and two wild hazelnuts. The accessions were also characterized by their amount and distribution of catkins in the tree canopy. Pollen sterility rate greatly varied among hazelnut accessions, with one main group of highly sterile cultivars and a second group, comprising wild genotypes and the remaining cultivars, producing good quality pollen. The results support the hypothesis of recurring reciprocal translocation events in Corylus avellana cultivars, leading to the observed gametic semi-sterility. The measured hazelnut pollen viability was also strongly influenced by pollen hydration ( R adj 2 = 0.83, P ≤ 0.0001) and reached its maximum at around 6 h of pre-hydration in humid chambers. Viable and dead pollen were best discriminated at around the same time of pollen pre-hydration, suggesting that high humidity levels are required for hazelnut pollen to maintain its functionality. Altogether, our results detail the value of impedance flow cytometry for high throughput phenotyping of hazelnut pollen. Further research is required to clarify the causes of pollen sterility in hazelnut, to confirm the role of reciprocal chromosome translocations and to investigate its effects on plant productivity., Competing Interests: TD and EH were employed by the Ferrero Trading Lux (Luxembourg), MD and SK were employed by the Amphasys AG (Switzerland). The Hazelnut Company division of Ferrero Trading Lux (Luxembourg) financed the first author’s Ph.D. grant and coordinated the research. The funder was not involved in data analysis and interpretation, nor in the decision to submit this article for publication. Amphasys AG supplied the cytometer used in the study and provided expert assistance in data interpretation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Ascari, Cristofori, Macrì, Botta, Silvestri, De Gregorio, Huerta, Di Berardino, Kaufmann and Siniscalco.)

Abscisic acid (ABA) is a key signaling molecule that mediates plant response to stress. Increasing evidence indicates that ABA also regulates many aspects of plant development, such as seed germination, leaf development, and ripening. ABA metabolism, including ABA biosynthesis and degradation, is an essential aspect of ABA response in plants. In this study, we identified four cytochrome P450 genes ( CaCYP707A1 , 2 , 3 , and 4 ) that mediate ABA hydroxylation, which is required for ABA degradation in Capsicum annuum . We observed that CaCYP707A-mediated ABA hydroxylation promotes ABA degradation, leading to low levels of ABA and a dehydration phenotype in 35S:CaCYP707A plants. Importantly, seed formation was strongly inhibited in 35S:CaCYP707A plants, and a cross-pollination test suggested that the defect in seed formation is caused by improper pollen development. Phenotypic analysis showed that pollen maturation is suppressed in 35S:CaCYP707A1 plants. Consequently, most 35S:CaCYP707A1 pollen grains degenerated, unlike non-transgenic (NT) pollen, which developed into mature pollen grains. Together our results indicate that CaCYP707A mediates ABA hydroxylation and thereby influences pollen development, helping to elucidate the mechanism underlying ABA-regulated pollen development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Kim, Park, Ma, Park, Yun, Jang and Joung.)

Reproductive stage is very sensitive to various forms of environmental stresses such as drought stress. The proteomic analysis of anther during pollen development in response to drought stress was performed using a label-free quantitative shotgun proteomic technique to define the underlying molecular principles in two contrasting wheat genotypes Shiraz (susceptible) and D-10 (tolerant). Drought stress resulted in around two-fold decline in seed setting capacity and pollen viability in the Shiraz genotype compared to D-10. A Partial Least Square Discriminant Analysis (PLS-DA) of proteomic data revealed the abundance of 131 differentially abundant proteins significantly contributing in separation of drought tolerant and susceptible genotypes under normal and stress conditions. Proteins involved in cellular respiration, carbohydrate metabolism, RNA metabolism, and vesicle trafficking showed completely different responses in two genotypes. These proteins may maintain hexose pool and energy level and control regulation of transcription and transport. Furthermore, different members of functional groups such as protein biosynthesis and degradation, chromatin organization, and cytoskeleton dynamics were differentially abundant in response to stress in both genotypes which suggest their function in both genotypes to maintain minimum pollen viability/ fertility under drought stress. In conclusion, our findings revealed various metabolic and regulatory pathways underlying survival strategies required for pollen fertility and viability. SIGNIFICANCE: Drought caused by global climate change decreases cereal grain productivity worldwide. Yield losses due to water stress have been reported for major small grain cereal including wheat. Our findings highlighted the importance of key proteins in wheat adaptation to drought stress at reproductive stage. The obtained data showed that differentially abundant proteins in drought tolerant wheat genotype was remarkably associated with cellular respiration, carbohydrate metabolism, RNA metabolism, and vesicle trafficking. These results revealed fundamental data to elucidate the complexity of pollen fertility and viability under drought stress condition in wheat., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The macro "PollenCounter" in ImageJ was initially developed to assess pollen viability in grapevine. We set out to see if PollenCounter could be used to assess pollen number and viability in tomatoes.•We tested different optimization scenarios by adjusting the pollen size (100-900, 200-900 pixel 2 ) and circularity of pollen grains (0.4-1, 0.5-1, and 0.6-1) on 31 microscopic images of stained tomato pollen. Both total pollen number and proportion of viable pollen were positively and significantly correlated with the outputs from manual counting. The scenario with 100-900 pixel 2 pollen size and 0.4-1 circularity had the highest association for pollen number ( r  = 0.99) and pollen viability ( r  = 0.86). PollenCounter is 32-fold faster than manual counting.•We added a command to the macro to automatically save the outputs containing the number of total and viable pollen, avoiding transcription errors inherent to manual counting.•We successfully applied the optimized PollenCounter to discriminate tomato genotypes based on pollen number and pollen viability under heat stress. Our results show that PollenCounter, as an open-access macro, can be customized and improved to meet users' needs. The use of PollenCounter can save time and money in pollen quality assessment. We outline the steps to optimize the macro for other samples or crop species. The optimized macro could allow efficient screening of a large germplasm collection for pollen thermo-tolerance and selection of best thermo-tolerant individuals in breeding programs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors. Published by Elsevier B.V.)

Global climate change is increasing the range of temperatures that crop plants must face during their life cycle, giving negative effects to yields. In this changing scenario, understanding the genetic control of plant responses to a range of increasing temperature conditions is a prerequisite to developing cultivars with increased resilience. The current work reports the identification of Quantitative Trait Loci (QTL) involved in reproductive traits affected by temperature, such as the flower number (FLN) and fruit number (FRN) per truss and percentage of fruit set (FRS), stigma exsertion (SE), pollen viability (PV) and the incidence of the physiological disorder tipburn (TB). These traits were investigated in 168 Recombinant Inbred Lines (RIL) and 52 Introgression Lines (IL) derived from the cross between Solanum lycopersicum var. "MoneyMaker" and S. pimpinellifolium accession TO-937. Mapping populations were cultivated under increased temperature regimen conditions: T1 (25°C day/21°C night), T2 (30°C day/25°C night) and T3 (35°C day/30°C night). The increase in temperature drastically affected several reproductive traits, for example, FRS in Moneymaker was reduced between 75 and 87% at T2 and T3 when compared to T1, while several RILs showed a reduction of less than 50%. QTL analysis allowed the identification of genomic regions affecting these traits at different temperatures regimens. A total of 22 QTLs involved in reproductive traits at different temperatures were identified by multi-environmental QTL analysis and eight involved in pollen viability traits. Most QTLs were temperature specific, except QTLs on chromosomes 1, 2, 4, 6, and 12. Moreover, a QTL located in chromosome 7 was identified for low incidence of TP in the RIL population, which was confirmed in ILs with introgressions on chromosome 7. Furthermore, ILs with introgressions in chromosomes 1 and 12 had good FRN and FRS in T3 in replicated trials. These results represent a catalog of QTLs and pre-breeding materials that could be used as the starting point for deciphering the genetic control of the genetic response of reproductive traits at different temperatures and paving the road for developing new cultivars adapted to climate change., (Copyright © 2020 Gonzalo, Li, Chen, Gil, Montoro, Nájera, Baixauli, Granell and Monforte.)