Impedance Flow Cytometry for Selection of Pollen Traits Under High Temperature Stress in Pepper

High temperature stress is a major limiting factor for pepper productivity, which will continue to be a problem under climate change scenarios. Developing heat tolerant cultivars is critical for sustained pepper production, especially in tropical and subtropical regions. In fruiting crops, like pepper, reproductive tissues, especially pollen, are the most sensitive to high temperature stress. Typically, pollen viability and germination are assessed through staining and microscopy, which is tedious and potentially inaccurate. To increase efficiency in assessing pollen traits of pepper, the use of impedance flow cytometry (IFC) has been proposed. We conducted three independent experiments to determine the most effective methodology to use IFC for evaluating pollen traits for heat tolerance in pepper. Seven floral developmental stages were evaluated, and stages 3, 4, and 5 were found to best combine high pollen concentration and activity. Flowers in development stages 3, 4, or 5 were then heat treated at 41, 44, 47, 50, and 55 °C or not heat treated (control). The critical temperature to assess heat tolerance using IFC was found to be 50 °C, with a reduction in pollen activity and concentration occurring at temperatures greater than 47 °C. Twenty-one entries of pepper were then accessed for pollen traits using the staining and IFC methods over 2 months, April (cooler) and June (hotter). Growing environment was found to be the greatest contributor to variability for nearly all pollen traits assessed, with performance during June nearly always being lower. PBC 507 and PBC 831 were identified as being new sources of heat tolerance, based on using IFC for assessing pollen. Pollen viability determined by staining and pollen activity determined using IFC were significantly positively correlated, indicating that IFC is an efficient and accurate method to assess pollen traits in pepper. This work provides a basis for further research in this area and supports more efficient breeding of heat-tolerant cultivars.

The Effectiveness of the Sexual Reproduction in Selected Clonal and Nonclonal Species of the Genus Ranunculus

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.

Viability of pollen grains and stigma receptivity in Desert Rose

Adenium obesum (Forssk.) Roem. & Schult., popularly known as desert rose, has become a valuable ornamental plant. In floriculture, the production of hybrids is prioritized. Hence, knowledge on floral biology and sexual reproduction of the target species is fundamental. The objectives of this study were: (1) to test sucrose concentrations and temperatures for in vitro germination of A. obesum pollen grains; (2) to identify the effect of temperature on the viability of A. obesum pollen grains; and (3) to evaluate the viability of pollen grains and stigma receptivity in pre-anthesis, at flower opening, and 72 h post-flower opening in three accessions of A. obesum. A significant relationship between temperatures and sucrose concentrations was observed in the in vitro germination test. The highest percentage of in vitro germination of pollen grains, 39.81%, was observed at an estimated temperature of 26.05 °C. Desert rose accessions maintained in biochemical oxygen demand (BOD) chambers at 30 °C during a 16-h light photoperiod showed faster flowering, and temperatures ≥ 25 °C induced pollen grain viability percentages above 69%. Temperature is one of the most important abiotic factors, influencing mainly in pollen germination, pollen tube growing and in efficiency fertilization. The ICA-wd accession stood out and can be considered a pollen donor in artificial pollination. The stigmas of flowers were receptive from a day before flower opening until three days after. The two parameters presented above, stigma receptivity and pollen viability, allow inferences about the appropriate time for successful pollination and subsequent fertilization in desert roses.

Evaluation of Native and Nonnative Ornamentals as Pollinator Plants in Florida: II. Floral Resource Value

Consumer demand for novel, visually attractive ornamentals has often overshadowed the functional value plants may provide for flower-visiting insects. As native and nonnative species are hybridized for form, color, flowering, and disease resistance, it is important to assess whether some of these alterations influence plant nutrient quality for foraging insect pollinators. A study was conducted to ascertain the resource value of ornamental cultivars compared with their native congeners. The nectar volume and pollen quantity, viability, and protein content of 10 species of popular herbaceous flowering plants, commonly advertised as pollinator-friendly, were evaluated in northcentral Florida. Each genus encompassed a native and nonnative species, apart from pentas. Native species included blanket flower (Gaillardia pulchella), lanceleaf coreopsis (Coreopsis lanceolata), pineland lantana (Lantana depressa), and scarlet sage (Salvia coccinea). Nonnative species included Barbican™ yellow-red ring blanket flower (Gaillardia aristata ‘Gaiz005’), Bloomify™ rose lantana (Lantana camara ‘UF-1011-2’), mysty salvia (Salvia longispicata × farinacea ‘Balsalmysty’), Lucky Star® dark red pentas (Pentas lanceolata ‘PAS1231189’), ruby glow pentas (Pentas lanceolata ‘Ruby glow’) and UpTick™ Gold & Bronze coreopsis (Coreopsis × ‘Baluptgonz’). Floral rewards differed significantly across species. The native scarlet sage exhibited the largest nectar volume per flower in the summer (2.13 ± 0.17 µL), followed by the nonnative mysty salvia (1.26 ± 0.17 µL). In the fall, ruby glow pentas exhibited the largest nectar volume per flower (1.09 ± 0.17 µL) compared with all other ornamentals. The composite flowers of the native and nonnative blanket flower and coreopsis species had the lowest nectar volume per flower regardless of sampling date. Likewise, ruby glow pentas displayed the highest quantity of pollen grains (96.29 ± 0.12) per sample, followed by Lucky star pentas (52.33 ± 0.12), and Barbican blanket flower (50.98 ± 0.12). Pollen viability was similarly high (92% to 98%) among all species, apart from Bloomify rose lantana (20%) and pineland lantana (48%). Pollen protein content was highest in Uptick coreopsis (11.378 ± 1.860 μg/mg dry weight) and Lucky star pentas (10.656 ± 3.726 μg/mg dry weight), followed by lanceleaf coreopsis (7.918 ± 1.793 μg/mg dry weight). These results largely showed that the nonnative ornamentals selected provided resource-rich floral rewards, comparable to native congeners. Still, care should be taken in making similar assessments of other modern floral types.

Effect of Meiotic Polyploidisation on Selected Morphological and Anatomical Traits in Interspecific Hybrids of Brassica oleracea × B. napus

In Brassica, interspecific hybridisation plays an important role in the formation of allopolyploid cultivars. In this study, the ploidy of F1 and F2 generations resulting from interspecific hybridisation between B. oleracea inbred lines of head cabbage (B. oleracea L. var. capitata) (2n = 18) and kale (B. oleracea L. var. acephala) (2n = 18) with inbred lines of rapeseed (B. napus L.) (2n = 38) was examined by flow cytometry analysis and chromosome observation. Furthermore, the effect of meiotic polyploidisation on selected phenotypic and anatomical traits was assessed. The F1 hybrids of head cabbage × rapeseed (S3) and kale × rapeseed crosses (S20) were allotriploids with 2n = 28 chromosomes, and nuclear DNA amounts of 1.97 (S3) and 1.99 pg (S20). These values were intermediate between B. oleracea and B. napus. In interspecific hybrids of the F2 generation, which were derived after self-pollination of F1 hybrids (FS3, FS20) or by open crosses between F1 generation hybrids (FC320, FC230), the chromosome numbers were similar 2n = 56 or 2n = 55, whereas the genome sizes varied between 3.81 (FS20) and 3.95 pg 2C (FC230). Allohexaploid F2 hybrids had many superior agronomic traits compared to parental B. napus and B. oleracea lines and triploid F1 hybrids. In the generative stage, they were characterised by larger flowers and flower elements, such as anthers and lateral nectaries. F2 hybrids were male and female fertile. The pollen viability of F2 hybrids was comparable to parental genotypes and varied from 75.38% (FS3) to 88.24% (FC320), whereas in triploids of F1 hybrids only 6.76% (S3) and 13.46% (S20) of pollen grains were fertile. Interspecific hybrids of the F2 generation derived by open crosses between plants of the F1 generation (FC320, FC230) had a better ability to set seed than F2 hybrids generated from the self-pollination of F1 hybrids. In the vegetative stage, F2 plants had bigger and thicker leaves, larger stomata, and significantly thicker layers of palisade and spongy mesophyll than triploids of the F1 generation and parental lines of B. oleracea and B. napus. The allohexaploid F2 hybrids analysed in this study can be used as innovative germplasm resources for further breeding new vegetable Brassica crops at the hexaploid level.

High Temperature and Humidity Affect Pollen Viability and Longevity in Olea europaea L.

Olea europaea L. is a crop typical of the Mediterranean area that has an important role in economy, society, and culture of this region. Climate change is expected to have significant impact on this crop, which is typically adapted to certain pedo-climatic characteristics of restricted geographic areas. In this scenario, the aim of this study was to evaluate the time-course response of pollen viability to different combinations of temperature and humidity. The study was performed comparing flowering time and pollen functionality of O. europaea from twelve cultivars growing at the same site belonging to the Campania olive collection in Italy. Pollen was incubated at 12 °C, 22 °C, and 36 °C in combination with 50% RH or 100% RH treatments for 5 days. The results highlighted that a drastic loss of pollen viability occurs when pollen is subjected to a combination of high humidity and high temperature, whereas 50% RH had less impact on pollen thermotolerance, because most cultivars preserved a high pollen viability over time. In the ongoing climate change scenario, it is critical to assess the effect of increasing temperatures on sensitive reproductive traits such as pollen viability to predict possible reduction in crop yield. Moreover, the results highlighted that the effect of temperature increase on pollen thermotolerance should be evaluated in combination with other environmental factors such as humidity conditions. The screening of olive cultivars based on pollen thermotolerance is critical in the ongoing climate change scenario, especially considering that the economic value of this species relies on successful fertilization and embryo development, and also that production cycle of Olea europaea can be longer than a hundred years.

Pollen viability and incompatibility in indigenous rice bean (Vigna umbellata (Thunb.) Ohwi & Ohashi)

Pollen viability, germination and compatibility are essential in determining the success of pollination and seed setting of high-valued crops. Rice bean (Vigna umbellata (Thunb.) Ohwi & Ohashi) is an underutilized and unexplored indigenous legume with high potential for commercial production. In this study, pollen quality, viability, germination rate and incompatibility among selected six rice bean (V. umbellata) accessions from Barili, Cebu, Philippines were evaluated to determine the barriers and effective pollination habit for increased productivity while retaining the important traits, including high tolerance in poor soils, superior climatic resilience and resistance to pest and diseases. Results of acetocarmine calorimetric assay showed that rice beans’ (V. umbellata) pollens are highly viable, with accessions VU 004 (56.33 ± 4.91%) and VU 007 (54.34 ± 4.53%) having the optimum viability rate. Brewbaker and Kwack medium treated with 0.2 g.l-1 and 0.3 g.l-1 boric acid (H3BO4) enhanced the germination rate in vitro (11.56 ± 5.53% and 9.47 ± 6.50% respectively). Bud (14.96 ± 1.53%) and post-anthesis pollens (10.28 ± 0.94%) have optimum germination rate in 0.2 g.l-1 boric acid media, while anthesis pollens are suitable in media supplemented with sucrose and boric acid alone (12.20 ± 1.50%) and with 0.1 g.l-1 myo-inositol supplementation (8.49 ± 1.86%). Pollination test revealed that rice bean accessions have high self-compatibility (50.76 + 3.45%) and low cross-compatibility (26.57 + 2.49%). The findings provide an important background in understanding the pollen quality and intraspecific interaction among indigenous rice bean (V. umbellata) accessions in Barili, Cebu to improve production and hybridization.

Integrated Isoform Sequencing and Dynamic Transcriptome Analysis Reveals Diverse Transcripts Responsible for Low Temperature Stress at Anther Meiosis Stage in Rice

Low temperatures stress is one of the important factors limiting rice yield, especially during rice anther development, and can cause pollen sterility and decrease grain yield. In our study, low-temperature stress decreased pollen viability and spikelet fertility by affecting the sugar, nitrogen and amino acid contents of anthers. We performed RNA-seq and ISO-seq experiments to study the genome-wide transcript expression profiles in low-temperature anthers. A total of 4,859 differentially expressed transcripts were detected between the low-temperature and control groups. Gene ontology enrichment analysis revealed significant terms related to cold tolerance. Hexokinase and glutamate decarboxylase participating in starch and sucrose metabolism may play important roles in the response to cold stress. Using weighted gene co-expression network analysis, nine hub transcripts were found that could improve cold tolerance throughout the meiosis period of rice: Os02t0219000-01 (interferon-related developmental regulator protein), Os01t0218350-00 (tetratricopeptide repeat-containing thioredoxin), Os08t0197700-00 (luminal-binding protein 5), Os11t0200000-01 (histone deacetylase 19), Os03t0758700-01 (WD40 repeat domain-containing protein), Os06t0220500-01 (7-deoxyloganetin glucosyltransferase), Pacbio.T01382 (sucrose synthase 1), Os01t0172400-01 (phospholipase D alpha 1), and Os01t0261200-01 (NAC domain-containing protein 74). In the PPI network, the protein minichromosome maintenance 4 (MCM4) may play an important role in DNA replication induced by cold stress.

Ca2+ Participates in Programmed Cell Death by Modulating ROS During Pollen Cryopreservation

Programmed cell death (PCD) is one of the reasons for the decline in pollen viability after cryopreservation. However, the role of calcium ions (Ca2+) in PCD during pollen cryopreservation has not been revealed in the existing studies. In this study, Paeonia lactiflora 'Fen Yu Nu' pollen was used as the research material for investigating the effects of Ca2+ changes on PCD indices and reactive oxygen species (ROS) during pollen cryopreservation. The results showed that after cryopreservation, with the decrease of pollen viability, the Ca2+ content significantly increased. The regulation of Ca2+ content had a significant effect on PCD indices, which showed that the Ca2+ carrier A23187 accelerated the decrease of mitochondrial membrane potential level and increased the activity of caspase-3-like and caspase-9-like proteases and the apoptosis rate. The expression levels of partial pro-PCD genes were upregulated, the anti-PCD gene BI-1 was downregulated, and the addition of Ca2+ chelating agent EGTA had the opposite effect. The addition of the Ca2+ carrier A23187 after cryopreservation significantly increased the ROS content of pollen, the addition of the Ca2+ chelating agent EGTA had the opposite effect, and Ca2+ regulators also had significant effects on the contents of ROS production and clearance-related substances. Ca2+ affected intracellular ROS content by acting on the ROS production and clearance system during the cryopreservation of pollen and is thus involved in the occurrence of PCD.