Microspore Pollen Research Articles

Decision Support Tool to Predict Panicle Initiation in Aerobic Rice

Aerobic rice cultivation offers the potential to reduce irrigated water use. A multitude of challenges, such as cold sterility, drought stress, and labor shortages, limit its adoption in temperate rice-growing regions. Increasing the duration and extent of soil moisture tension between irrigation events has been demonstrated to slow crop development. Delaying panicle initiation (PI) beyond the optimal window can expose rice to cold nighttime temperatures during the cold sensitive early pollen microspore, severely reducing yield. Tools to assist Australian temperate farmers and researchers in the irrigation management of aerobic rice to ensure PI occurs during the optimal window do not yet exist. Using data collected from an aerobic rice experiment conducted in temperate Australia in 2020–2021 and 2021–2022, a predictive model was built to assist in forecasting PI based on the timing of irrigation. Estimation of the area on an hourly basis of the cumulative evapotranspiration with rainfall subtracted from pre-emergent irrigation to PI, defined as the irrigation deficit integral, was used to account for the frequency, duration, and extent of soil moisture deficit between irrigation events. The relationship between the irrigation deficit integral and the number of days from pre-emergent irrigation to PI (R2 = 0.91) was used to build a model to predict PI with a root mean square error of 1.8 days for the validating data set. Furthermore, an example is provided of how the model can be used as a decision support tool to assist researchers and growers to schedule irrigation of aerobic rice to ensure PI occurs in a timely manner. This will increase the likelihood of high-yielding aerobic rice and may enhance the adoption of water-saving rice cultivation.

Effect of Soil Moisture Deficit on Aerobic Rice in Temperate Australia

Declining water availability is pressing rice growers to adopt water-saving irrigation practices such as aerobic rice to maintain profitability per megalitre (ML) of water input. Irrigators require well-defined irrigation thresholds to initiate irrigation to maximise water productivity. Such thresholds do not exist for temperate rice regions. Adopting a strategy that has been reported to succeed in non-temperate environments may fail in temperate climates, and therefore, needs investigation. This study aimed to investigate, in a temperate Australian environment, the effect of increasing soil moisture deficit during the rice vegetative period on crop physiological development, grain yield and water productivity. The study was conducted in a commercial farm using a randomised complete block design in the 2020/21 and 2021/22 growing seasons. Automated gravity surface irrigation technologies were adopted to enable high-frequency irrigation. Extending soil moisture deficit beyond 15 kPa was found to significantly delay panicle initiation by at least 13–14 days, exposing rice to cold temperatures in Year 1 during the cold-sensitive early pollen microspore period. This reduced yield by up to 55% (4.5 t/ha) compared to the 15 kPa treatment that was not impacted by cold sterility. In the absence of cold sterility, irrigated water productivity and total water productivity ranged between 1.02 and 1.61 t/ML, and 0.84 and 0.93 t/ML, respectively. The highest yields (8.1 and 7.5 t/ha) were achieved irrigating at a soil tension of 15 kPa in growing seasons 2020/21 and 2021/22. This research demonstrates that sound water productivity can be achieved with aerobic rice cultivation in temperate climates, providing cold temperatures during early pollen microspore are avoided. The quantification of the delay in crop development caused by increasing soil moisture deficit provides rice farmers greater confidence in determining the irrigation strategy and timing of pre-emergent irrigation in regions at risk of cold sterility. However, due to the high labour demand associated with aerobic rice, the adoption of aerobic rice at a commercial scale in this Australian environment is unlikely without adopting automated irrigation technology.

Assessment of the mutagenic potential of the water of an urban river by means of two Tradescantia-based test systems

River pollution can be caused by anthropogenic or natural factors. When testing water quality for the presence of toxic substances, higher plants as bioindicators for the genotoxic effects of complex mixtures are effective and appropriate. Hence, in this work the Tradescantia (clone 02) stamen hair mutations (Trad-SHM) and Tradescantia micronuclei (Trad-MCN) were used to determine mutagenic and clastogenic potential of an urban river. A significant increase in the level of all studied endpoints as well as morphological changes, including pink cells (PC) and colorless cells (CC) in stamen hairs, stunted hairs (SH), tetrads with micronuclei (MN) and MN in tetrads of pollen microspores in the Tradescantia was observed compared to the negative control (tap water). As an example riverine system, part of the River Hrazdan (Armenia) flowing through a highly urbanized and industrial area was studied. The positive control (10 mM CrO3) showed the highest genotoxicity for the SHM assay (PC: 5.1 / 1000, CC: 17.9 / 1000) and for the MCN assay (12 MN / 100 tetrads and 9.4 ± 0.53 tetrads with MN). Genetic responses were analyzed in conjunction with the concentrations of select elements in the riverine water. Reasons for observing such a high level of genetic markers in the riverine water and applicability of the Tradescantia (clone 02) test-systems in environmental risk assessment and biomonitoring are discussed.

Clastogenecity evaluation of water of Lake Sevan (Armenia) using Tradescantia micronucleus assay

The clastogenic effects of water samples in seven locations of Lake Sevan (Armenia) with the application of Trad-MCN (micronuclei) bioassay using Tradescantia (clone 02) were investigated. A significant increase in the frequency of micronuclei in tetrads of pollen microspores and tetrads with micronuclei exposed to the test samples compared to the control has been revealed. A multivariate analysis indicated linkage between the frequencies of occurrence of micronuclei in the cells and Ni and Co ions. The results were compared with the endpoints of another Tradescantia-based test system (stamen hair mutation test) performed on the same water samples and generation of the plant: occurrences of micronuclei in sporogenic cells coincided with that of non-surviving stamen hair.

Sex-biased gene expression in dioecious garden asparagus (Asparagus officinalis).

Sex chromosomes have evolved independently in phylogenetically diverse flowering plant lineages. The genes governing sex determination in dioecious species remain unknown, but theory predicts that the linkage of genes influencing male and female function will spur the origin and early evolution of sex chromosomes. For example, in an XY system, the origin of an active Y may be spurred by the linkage of female suppressing and male promoting genes. Garden asparagus (Asparagus officinalis) serves as a model for plant sex chromosome evolution, given that it has recently evolved an XX/XY sex chromosome system. In order to elucidate the molecular basis of gender differences and sex determination, we used RNA-sequencing (RNA-Seq) to identify differentially expressed genes between female (XX), male (XY) and supermale (YY) individuals. We identified 570 differentially expressed genes, and showed that significantly more genes exhibited male-biased than female-biased expression in garden asparagus. In the context of anther development, we identified genes involved in pollen microspore and tapetum development that were specifically expressed in males and supermales. Comparative analysis of genes in the Arabidopsis thaliana, Zea mays and Oryza sativa anther development pathways shows that anther sterility in females probably occurs through interruption of tapetum development before microspore meiosis.

Compositional Analysis of Grain and Forage from MON 87427, an Inducible Male Sterile and Tissue Selective Glyphosate-Tolerant Maize Product for Hybrid Seed Production

Conventional maize hybrid seed production has historically relied upon detasseling using either manual methods or semiautomated processes to ensure the purity of the hybrid cross. Monsanto Co. has developed biotechnology-derived MON 87427 maize with tissue-selective glyphosate tolerance to facilitate the production of hybrid maize seed. MON 87427 utilizes a specific promoter and intron combination to drive expression of CP4 EPSPS protein in vegetative and female reproductive tissues, conferring tolerance to glyphosate. This specific combination of regulatory elements also results in limited or no production of CP4 EPSPS protein in two key male reproductive tissues: pollen microspores, which develop into pollen grains, and tapetum cells that supply nutrients to the pollen. Thus, MON 87427 induces a male sterile phenotype after appropriately timed glyphosate applications. To confer additional benefits of herbicide tolerance and/or insect resistance, MON 87427 was combined with MON 89034 and NK603 by conventional breeding to develop MON 87427 × MON 89034 × NK603. The work described here is an assessment of the nutrient, antinutrient, and secondary metabolite levels in grain and forage tissues of MON 87427 and MON 87427 × MON 89034 × NK603. Results demonstrated that MON 87427 is compositionally equivalent to a near-isogenic conventional comparator. Results from this analysis established that the compositional equivalence observed for the single-event product MON 87427 is extendable to the combined-trait product, MON 87427 × MON 89034 × NK603. With increasing global demand for food production, the development of more efficient seed production strategies is important to sustainable agriculture. The study reported here demonstrated that biotechnology can be applied to simplify hybrid maize seed production without affecting crop composition.

An Unusual Aquaporin-Like Metalloid Boric Acid Channel in Arabidopsis

The Major Instrinsic Protein/aquaporin superfamily is an ancient family with a conserved protein fold that forms a channel that mediates the bidirectional transport of water and uncharged solutes. The evolution of land plants was accompanied by a major diversification of the MIP gene family that resulted in the acquisition of new transport functions. Among these divergent plant specific MIP subfamilies are the “nodulin 26 intrinsic proteins” (NIPs), based on structural and functional homology to soybean nodulin 26. NIPs are divided into three subfamilies (NIP I, II and III) which show low to absent water permeability but have acquired the ability to transport a variety of uncharged solutes ranging from glycerol to boric acid to protonated lactic acid. In the present work we characterize the transport and gating properties of an unusual NIP II protein from Arabidopsis pollen microspores, AtNIP7;1 which is related to the boric acid channels AtNIP6;1 and 5;1, necessary for the uptake of this critical micronutrient. Functional characterization of NIP7;1 shows that unlike NIP5;1 and NIP6;1 which form constitutive boric acid channels, the intrinsic boric acid transport activity of NIP7;1 is extremely low. Molecular modeling suggests that a conserved tyrosine residue (Tyr 81) located in the transport pore stabilizes a closed conformation of the pore. Molecular dynamics simulation suggests that the closed conformation is stabilized by hydrogen bonding between the Tyr81 hydroxyl group and Arg 220 of the canonical “aromatic-arginine” selectivity filter. Since boric acid is both essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr 81 modulates transport and provides an additional level of regulation of uptake of boric acid in male gametophyte development.

Rice growth, yield and water productivity responses to irrigation scheduling prior to the delayed application of continuous flooding in south-east Australia

Abstract The majority of rice grown in south-east Australia is continuously flooded for much of its growing season, but reduced irrigation water availability brought about by a combination of drought and environmental flow legislation has presented a need to maintain (or even increase) rice production with less irrigation water. Delaying the application of continuous flooding until prior to panicle initiation can increase input water productivity by reducing non-beneficial evaporation losses from free water and the soil. A field experiment was conducted over two growing seasons, 2008/9 and 2009/10, comparing a conventional dry seeded treatment (the control – continuous flooding from the 3 leaf stage) with delayed continuous flooding (10–20 days prior to panicle initiation) with several irrigation scheduling treatments prior to flooding commencement. In the first year, the delayed water treatments were irrigated at intervals of 40, 80 and 160 mm of cumulative reference evapotranspiration (ETo) prior to delayed continuous flooding, thereby imposing differing degrees of crop water stress. In year 2, the 80 and 160 mm treatments were modified by use of a crop factor (Kc) when the plants were small and the 40 mm treatment was replaced with a continuously flooded treatment throughout the crop duration. Decreases in net water input (irrigation + rain − surface drainage) and increases in input water productivity were achieved by reducing the flush irrigation frequency during the pre-flood period. Savings of 150 and 230 mm (10 and 15%) were achieved in Year 1 from the 80 and 160 mm cumulative ETo irrigation frequency treatments, respectively, in comparison to the control. In the second year, net water input savings of 230 and 330 mm (15 and 22%) were achieved with the 80/Kc and 160/Kc mm treatments, respectively. Input water productivity of the 160 mm treatment was 0.06 kg/m 3 (8%) higher than the control in Year 1, while in Year 2 a 0.15 kg/m 3 (17%) increase in input water productivity above the control was achieved by the 160/Kc mm treatment. Delaying the application of continuous flooding in the second year greatly extended the period of crop growth suggesting the need for earlier sowing (by 7–10 days) to ensure pollen microspore still occurs at the best time to minimise yield loss due to cold damage. Nitrogen fertiliser management is an important issue when delaying continuous flooding, and nitrogen losses appeared to increase with the frequency of irrigation prior to continuous flooding. This was likely due to increased denitrification from alternate wetting and drying of the soil. Further research is required to determine the most appropriate nitrogen management strategies, and to also better define the optimal pre-flood irrigation frequency.

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.

Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen and tapetum of rice.

To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of IAA, gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the mature anther were analyzed. We also analyzed the global expression profiles of genes related to seven phytohormones, namely auxin, GAs, CKs, brassinosteroids, ethylene, ABA and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser microdissection technique (LM-array analysis). IAA and GA4 accumulated in a much higher amount in the mature anther compared with the other tissues, while CKs and ABA did not. LM-array analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of IAA and GA4 in the mature anther. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was down-regulated at the later stages of MS/POL development. In the case of auxin signaling genes, such mirror-imaged expression observed in GA synthesis and signaling genes was not observed. IAA receptor genes were mostly expressed during the late stages of MS/POL development, and various sets of AUX/IAA and ARF genes were expressed during the different stages of MS/POL or TAP development. Such cell type-specific expression profiles of phytohormone biosynthesis and signaling genes demonstrate the validity and importance of analyzing the expression of phytohormone-related genes in individual cell types independently of other cells/tissues.

Effect of pollen load size and source (self, outcross) on seed and fruit production in highbush blueberry cv. ‘Bluecrop’ (VACCINIUM CORYMBOSUM; Ericaceae)

Reproductive fitness of a plant is ultimately determined by both number and quality of seed offspring. This is determined by sexual selection of pollen microspores and ovules during pollination and fertilization. These processes may include pollen competition and seed abortion, which reduce the number of microspores and ovules available for final seed production. Thus, even an excess of pollen microspores to ovules does not result in fertile seeds equal to ovule number. We investigated pollen requirements of highbush blueberry (Vaccinium corymbosum cultivar 'Bluecrop') for maximal seed production and how fertile seed number translates into fruit quality, since fruit quality would ultimately determine the dispersal of its offspring. We demonstrate that individual blueberry flowers with a mean of 106 ovules reach their maximum fruit set and mass and minimum time to ripen when 125 outcross pollen tetrads pollinate a flower, compared to 10 or 25. Three hundred tetrads resulted in the increase of fertile seeds, but did not result in a further increase of fruit mass or fruit set, or decrease in time to ripen. We also examined the effect of pure and mixed loads of self and outcross pollen (25 and 125 tetrads), and found no differences in fertile seed number, fruit mass, or percentage fruit set when pollen loads were either 25 self or outcross pollen tetrads, although number of days to ripen was significantly shorter by 8 d with 25 outcross tetrads. When the pollen load of 125 tetrads consisted of self or a 50:50 mixture of self and outcross pollen, fruit mass, days to ripen, and percentage fruit set were not different from loads of 125 outcross pollen. In addition, a pollen load of 25 outcross tetrads resulted in fertile seed number and fruit quality in between that of 25 self, and 125 self, 125 mixed, or 125 outcross tetrads. Large, small, and flat seed types were identified, and only large seeds (length = 1.7 mm) were fertile. These results improve our understanding of pollen load size and source requirements of a crop plant and the limits to pollen transfer when translated to fruit growth.

Interspecific genomic rearrangements in androgenic plants derived from a Lolium multiflorum×Festuca arundinacea (2n=5x = 35) hybrid

Pollen microspores from a hybrid between Lolium multiflorum (2n=4x=28) and Festuca arundinacea (2n=6x=42) were cultured and over 200 androgenic green plants established. In the initial hybrid one chromosome from each of the five homoeologous groups was labelled by a distinct PGI/2 homoeoallele. Segregation of PGI/2 alleles in the male gametes and in the androgenic plants indicated chromosome pairing preferences in the hybrid at meiosis. The pentaploid Festulolium hybrid genome comprised two homologous sets of Lolium (Lm) chromosomes and the polyhaploid set of F. arundinacea, namely one genome of F. pratensis (Fp) and two of F. glaucescens(Fg and Fg1). There was a high degree of preferential chromosome pairing between the two homologous sets of L. multiflorum chromosomes, and each androgenic plant contained a Lolium PGI/2 allele. Results supported earlier work indicating that gamete viability depended on the presence of a complete Lolium genome. Genomic in situ hybridization (GISH) was carried out on mitotic chromosome preparations of two androgenic plants using the total genomic DNA of L. multiflorum as a probe. Recombinants were observed between chromosomes of all three genomes in the pentaploid hybrid, demonstrating the efficacy of the hybrid as a starting point in introgression breeding programmes.

Deep floodwater protects high-nitrogen rice crops from low-temperature damage

A field experiment was conducted to investigate the interaction of nitrogen (N) status, sowing date, and water depth on rice yield in the Murray Valley, New South Wales, where low minimum temperatures often lead to pollen sterility and low yield in high N status crops. The experiment combined 4 N rates (0, 75, 125, 250 kg N/ha) applied as urea just before permanent flood; 3 cultivars (Jarrah, early maturing; Amaroo and Doongara, midseason); 2 sowing dates (26 September, 31 October 1991); and 2 water depths (5, 20 cm) at the microspore stage. The early-sown crops and the later sown Jarrah crop encountered minimum air temperatures of 19�C at the cold-sensitive microspore stage, compared with 15�C for the other 2 later sown crops. Total dry matter production was little affected by either water depth or sowing date, but increased from 16 to 22 t/ha with N application. Shallow water depth and delayed sowing date affected yield through reduced harvest index. Grain yield response to N fertiliser was dependent on sowing date, water depth, and variety. For the early-sown crops grown in deep water, yields of Amaroo and Jarrah-increased from 7 to 13 t/ha with increasing N supply, while the yield response of Doongara plateaued at 9 t/ha. With shallow water, the yields of all varieties decreased from 7 to 3 t/ha with increasing N. For the later sowing date, Jarrah growing in deep water yielded up to 13 t/ha at high N, but yields of Amaroo and Doongara decreased from 7 to 2 t/ha with increasing N supply. For the later sowing date, yields of all varieties growing in shallow water decreased to <2 t/ha with applied N. In the deep water crops, developing microspores were submerged or partially submerged and so avoided low air minimum temperatures. In the early-sown crops, the microspore stage occurred during a period of relatively warm nights. Harvest index was successfully modelled using panicle temperature and N content at early pollen microspore. The experiment shows that high N applications can lead to high rice yields provided the microspores are protected from low temperatures by the use of deep water at the stage of microspore development. As a result, recommendations for N fertiliser application need to be adjusted for sowing date and expected water depth at early pollen microspore

Fine-Structural Study on the Formation of the Generative Cell Wall and Intine-3 Layer in a Growing Pollen Grain of Lilium longiflorum

At the end of mitosis in the lily pollen microspore, the fan-shaped cell plate gives rise to a cell wall delineating a hemispherical cell. At first, the cell wall of the newly formed generative cell and the intine-3 layer of the pollen grain wall are inseparable. Gradually, the wall of the generative cell near the pollen grain wall becomes thicker and wall segments are formed between the thickened zones, and these make a network system by which the generative cell becomes suspended and separated from the pollen grain wall. After the separation, the intine-3 layer is formed inside the intine 2. The generative cell wall and the intine-3 layer are formed by coated vesicles, polysaccharide particles and rough ER.

FINE‐STRUCTURAL STUDY ON THE FORMATION OF THE GENERATIVE CELL WALL AND INTINE‐3 LAYER IN A GROWING POLLEN GRAIN OF LILIUM LONGIFLORUM

At the end of mitosis in the lily pollen microspore, the fan‐shaped cell plate gives rise to a cell wall delineating a hemispherical cell. At first, the cell wall of the newly formed generative cell and the intine‐3 layer of the pollen grain wall are inseparable. Gradually, the wall of the generative cell near the pollen grain wall becomes thicker and wall segments are formed between the thickened zones, and these make a network system by which the generative cell becomes suspended and separated from the pollen grain wall. After the separation, the intine‐3 layer is formed inside the intine 2. The generative cell wall and the intine‐3 layer are formed by coated vesicles, polysaccharide particles and rough ER.

Effect of Minimization of Culture Medium on the Process of Androgenesis in vitro (Nicotiana tabacum L. var. White Burley, Datura innoxia Mill.)

Summary The medium consisting of agar and sucrose is sufficient for the development of ernbryoids into globular stage, whereas for the development of complete plants it is necessary to add chelated iron. The highest androgenic embryogenesis occurred on AS medium (0.8% sugar, 2% sucrose). The specifirity of culture medium is not decisive for the ini tiation of androgenesis. The initiation of androgenic embryogenesis is assumed to be an autonomous function of extirpated anthers. A continual androgenic development of pollen microspores into globular embryoids takes place on both minimal medium without FeEDTA and complete medium without FeEDTA. Isolated embryoids transferred to the medium consisting of agar, sucrose and FeEDTA markedly change their morphology but do not develop into complete plants, whereas in a complete medium, the complete plants are formed. Sucrose concentration of 0.5- -l% in complete medium does not influence the transformation of isolated embryoicls into green plantlets. In minimal medium, the wall of cultured anther can replace nutritionally the complete medium, but cannot replace the morphoregulatory effect of FeEDTA. The nutritional capacity of the another wall is limited.