Differential effects of low and high temperature stress on pollen germination and tube length of mango (Mangifera indica L.) genotypes.

Mango flowering is highly sensitive to temperature changes. In this research, the maximum values of pollen germination rate (PGR), pollen tube length (PTL) and their cardinal temperatures (Tmin, Topt and Tmax) were estimated by using quadratic equation and modified bilinear model under the conditions of 14-36 °C. The pollen germination rate in four mango varieties ranged from 29.1% ('Apple mango') to 35.5% ('Renong No. 1'); the length of pollen tube ranged from 51.2 µm ('Deshehari') to 56.6 µm ('Jinhuang'). The cardinal temperatures ranges (Tmin, Topt and Tmax) of pollen germination were 20.3-22.8 °C, 26.7-30.6 °C and 30.4-34.3 °C, respectively; similarly, cardinal temperatures (Tmin, Topt and Tmax) of pollen tube growth were 20.3-21.2 °C, 27.9-32.1 °C and 30.2-34.4 °C respectively. Of those, 'Renong No. 1' could maintain relatively high pollen germination rate even at 30 °C, however, 'Deshehari' had the narrowest adaptive temperature range. These results were further confirmed by changes of superoxide dismutase, catalase activity and malondialdehyde content. These results showed that mango flowering was highly sensitive to temperature changes and there were significant differences in pollen germination rate and pollen tube length among different varieties. Current research results were of great significance for the introduction of new mango varieties in different ecological regions, the cultivation and management of mango at the flowering stage and the breeding of new mango varieties.

Microfluidics-Based Bioassays and Imaging of Plant Cells.

Many plant processes occur in the context of and in interaction with a surrounding matrix such as soil (e.g. root growth and root-microbe interactions) or surrounding tissues (e.g. pollen tube growth through the pistil), making it difficult to study them with high-resolution optical microscopy. Over the past decade, microfabrication techniques have been developed to produce experimental systems that allow researchers to examine cell behavior in microstructured environments that mimic geometrical, physical and/or chemical aspects of the natural growth matrices and that cannot be generated using traditional agar plate assays. These microfabricated environments offer considerable design flexibility as well as the transparency required for high-resolution, light-based microscopy. In addition, microfluidic platforms have been used for various types of bioassays, including cellular force assays, chemoattraction assays and electrotropism assays. Here, we review the recent use of microfluidic devices to study plant cells and organs, including plant roots, root hairs, moss protonemata and pollen tubes. The increasing adoption of microfabrication techniques by the plant science community may transform our approaches to investigating how individual plant cells sense and respond to changes in the physical and chemical environment.

Membrane receptor-mediated mechano-transduction maintains cell integrity during pollen tube growth within the pistil.

Invasive or penetrative growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading or penetrating cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here, we show that Arabidopsis pollen tubes sense and/or respond to mechanical changes via a cell-surface receptor kinase Buddha's Paper Seal 1 (BUPS1) while emerging from compressing female tissues. BUPS1-defective pollen tubes fail to maintain cell integrity after emergence from these tissues. The mechano-transduction function of BUPS1 is established by using a microfluidic channel device mimicking the mechanical features of the in vivo growth path. BUPS1-based mechano-transduction activates Rho-like GTPase from Plant 1 (ROP1) GTPase to promote exocytosis that facilitates secretion of BUPS1's ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-based mechano-transduction system for cells to cope with the physical challenges during invasive or penetrative growth.

Kinase Partner Protein Plays a Key Role in Controlling the Speed and Shape of Pollen Tube Growth in Tomato.

The rapid and responsive growth of a pollen tube requires delicate coordination of membrane receptor signaling, Rho-of-Plants (ROP) GTPase activity switching, and actin cytoskeleton assembly. The tomato (Solanum lycopersicum) kinase partner protein (KPP), is a ROP guanine nucleotide exchange factor (GEF) that activates ROP GTPases and interacts with the tomato pollen receptor kinases LePRK1 and LePRK2. It remains unclear how KPP relays signals from plasma membrane-localized LePRKs to ROP switches and other cellular machineries to modulate pollen tube growth. Here, we biochemically verified KPP's activity on ROP4 and showed that KPP RNA interference transgenic pollen tubes grew slower while KPP-overexpressing pollen tubes grew faster, suggesting that KPP functions as a rheostat for speed control in LePRK2-mediated pollen tube growth. The N terminus of KPP is required for self-inhibition of its ROPGEF activity, and expression of truncated KPP lacking the N terminus caused pollen tube tip enlargement. The C-terminus of KPP is required for its interaction with LePRK1 and LePRK2, and the expression of a truncated KPP lacking the C-terminus triggered pollen tube bifurcation. Furthermore, coexpression assays showed that self-associated KPP recruited actin-nucleating Actin-Related Protein2/3 (ARP2/3) complexes to the tip membrane. Interfering with ARP2/3 activity reduced the pollen tube abnormalities caused by overexpressing KPP fragments. In conclusion, KPP plays a key role in pollen tube speed and shape control by recruiting the branched actin nucleator ARP2/3 complex and an actin bundler to the membrane-localized receptors LePRK1 and LePRK2.

Intraspecific divergence in floral-tube length promotes asymmetric pollen movement and reproductive isolation.

The causative link between phenotypic divergence and reproductive isolation is an important but poorly understood part of ecological speciation. We studied the effects of floral-tube length variation on pollen placement/receipt positions and reproductive isolation. In a population of Lapeirousia anceps (Iridaceae) with bimodal floral-tube lengths, we labelled pollen of short- and long-tubed flowers with different colour fluorescent nanoparticles (quantum dots). This enabled us to map pollen placement by long- and short-tubed flowers on the only floral visitor, a long-proboscid fly. Furthermore, it allowed us to quantify pollen movement within and between short- and long-tubed flowers. Short- and long-tubed flowers placed pollen on different parts of the pollinator, and long-tubed flowers placed more pollen per visit than short-tubed flowers. This resulted in assortative pollen receipt (most pollen received comes from the same phenotype) and strong but asymmetric reproductive isolation, where short-tubed plants are more reproductively isolated than long-tubed plants. These results suggest that floral-tube length divergence can promote mechanical isolation in plants through divergence in pollen placement sites on pollinators. Consequently, in concert with other reproductive isolation mechanisms, selection for differences in floral-tube length can play an important role in ecological speciation of plants.

Using the sample maximum to estimate the parameters of the underlying distribution.

We propose novel estimators for the parameters of an exponential distribution and a normal distribution when the only known information is a sample of sample maxima; i.e., the known information consists of a sample of m values, each of which is the maximum of a sample of n independent random variables drawn from the underlying exponential or normal distribution. We analyze the accuracy and precision of the estimators using extreme value theory, as well as through simulations of the sampling distributions. For the exponential distribution, the estimator of the mean is unbiased and its variance decreases as either m or n increases. Likewise, for the normal distribution, we show that the estimator of the mean has negligible bias and the estimator of the variance is unbiased. While the variance of the estimators for the normal distribution decreases as m, the number of sample maxima, increases, the variance increases as n, the sample size over which the maximum is computed, increases. We apply our method to estimate the mean length of pollen tubes in the flowering plant Arabidopsis thaliana, where the known biological information fits our context of a sample of sample maxima.

Floral traits influence the opportunity for selection among male gametophytes: independent and combined effects of style length and petal area.

PREMISE: Strong correlations between traits can obscure their independent effects on components of reproduction. Style length (SL) and petal area (PA) vary within species, for example, but their independent effects on the opportunity for selection among pollen genotypes are poorly understood. Previous work in Clarkia detected a positive effect of SL on pollen receipt, potentially intensifying selection. However, this apparent effect of SL may be influenced by a correlated trait, such as PA. Here, we examine the independent effects of these two traits on pollen receipt and performance. METHODS: We collected petals and styles from wild populations of two insect-pollinated Clarkia taxa and estimated the independent and combined effects of SL and PA on pollen receipt and performance. RESULTS: In both taxa, SL and PA are positively correlated. In C. unguiculata, both traits positively and independently affect pollen receipt, but in C. xantiana ssp. xantiana, the two traits act only in combination to affect pollen receipt. In both taxa, pollen receipt positively affects the numbers of pollen tubes entering and penetrating the style, as well as pollen tube attrition. CONCLUSIONS: The effects of SL and PA on pollen receipt and performance are taxon specific. In C. unguiculata, both traits may be independent targets of selection due to their effects on pollen receipt. In C. xantiana ssp. xantiana, by contrast, the combined (but not independent) effects of SL and PA influence pollen receipt. Ecological differences between these taxa require exploration to understand the mechanisms by which these traits affect pollinator behavior.

Effect of flower orientation on the male and female traits of Myrtillocactus geometrizans (Cactaceae).

Intra-individual variation in the production and size of reproductive traits has been documented in columnar cacti, being higher in equator-facing flowers. Such variation is attributed to the high amount of PAR intercepted by stems oriented towards the equator. Most studies focused on this phenomenon have documented the existence of intra-individual variation on traits associated with the female function; however, its impact on traits associated with the male function has been neglected. We tested the hypothesis that equator-facing flowers of Myrtillocactus geometrizans exhibit higher values on traits associated with both male and female functions than flowers facing against it. Number and size of anthers and ovaries, pollen:ovule ratio and number and quality of pollen grains (diameter, germinability, viability and pollen tube length) were estimated from reproductive structures facing north and south, and compared with t-tests between orientations. Number of anthers per flower, number of pollen grains per anther and per floral bud; pollen size, viability and germinability; pollen tube length; ovary length and pollen:ovule ratio were significantly higher in reproductive structures oriented towards the south (i.e. equator). These findings suggest that intra-individual variation in floral traits of M. geometrizans might be associated with different availability of resources in branches with contrasting orientation. Our results provide new evidence of the existence of a response to an orientation-dependent extrinsic gradient. To our knowledge, this is the first study documenting the existence of intra-individual variation on pollen quality and P:O ratio in Cactaceae species.

Structure of the style and pollen tube pathway in the Ziziphoid and Rhamnoid clades of Rhamnaceae.

The ultrastructure of the style and pollen tube pathway before, during and after anthesis were studied in 13 species belonging to the tribes Pomaderreae, Paliureae, Colletieae and Gouanieae (Ziziphoid clade) and Rhamneae (Rhamnoid clade) using light microscopy and transmission electron microscopy. The aim of this study is to provide new morphological characters useful for phylogenetic analysis at suprageneric level in Rhamnaceae. The patterns of pollen tube growth and the ultrastructural changes undergone by cells of the style were also described. Species of Rhamneae (Scutia buxifolia and Condalia buxifolia) have a solid style, with the transmitting tissue forming three independent strands (S. buxifolia) or a central, single horseshoe-shaped strand as seen in transversal section (C. buxifolia) which could derive from the fusion of formerly independent strands. In contrast, Pomaderreae, Gouanieae and Paliureae showed semi-solid styles, while in Colletieae, as previously reported, the style is hollow with two or three stylar canals. The style anatomy and the ultrastructure of the pollen tube pathway show that there is a tendency towards a solid style with a single strand of transmitting tissue within the family. The three-canalled hollow style could be the plesiomorphic state of the character "type of style" in the family, the semi-solid style the synapomorphic state and the solid style with three strands of transmitting tissue the apomorphic state, with the solid style with a single strand of transmitting tissue as the most derived state. Therefore, Colletieae would be the most basal tribe of the Ziziphoid clade.

Effect of mercury on pollen germination and tube growth in Lilium longiflorum.

Pollen development and germination were adversely affected by the presence of mercury, whereas low-concentrations stimulated the whole procedure. Mercury caused morphological anomalies during the tube growth, characterized by irregularly increasing diameters and swelling tips. The main effect was the anomalous cell wall formation at the tip where a substantial number of organelles were found reducing the secretory vesicles. The dense organelle concentration caused a significant reduction of cytoplasmic movement integrity, and the cytosol streaming was gradually reduced or stopped completely. Electron dense, multilamellar myelin-like structures (MMS) of membranous material were frequently present, in close contact with plasmalemma or away from it. A loose network of fibrillar material and spherical aggregates mostly at the tip region were observed which progressively were loosened into the surrounding medium. Elevated mercury concentrations can affect plant reproduction, resulting in anomalies in gamete development and consequently loss of plant biodiversity.

Style morphology and pollen tube pathway.

The style morphology and anatomy vary among different species. Three basic types are: open, closed, and semi-closed. Cells involved in the pollen tube pathway in the different types of styles present abundant endoplasmic reticulum, dictyosomes, mitochondria, and ribosomes. These secretory characteristics are related to the secretion where pollen tube grows. This secretion can be represented by the substances either in the canal or in the intercellular matrix or in the cell wall. Most studies suggest that pollen tubes only grow through the secretion of the canal in open styles. However, some species present pollen tubes that penetrate the epithelial cells of the canal, or grow through the middle lamella between these cells and subepithelial cells. In species with a closed style, a pathway is provided by the presence of an extracellular matrix, or by the thickened cell walls of the stylar transmitting tissue. There are reports in some species where pollen tubes can also penetrate the transmitting tissue cells and continue their growth through the cell lumen. In this review, we define subtypes of styles according to the path of the pollen tube. Style types were mapped on an angiosperm phylogenetic tree following the maximum parsimony principle. In line with this, it could be hypothesized that: the open style appeared in the early divergent angiosperms; the closed type of style originated in Asparagales, Poales, and Eudicots; and the semi-closed style appeared in Rosids, Ericales, and Gentianales. The open style seems to have been lost in core Eudicots, with reversions in some Rosids and Asterids.

Is floral structure a reliable indicator of breeding system in the Brassicaceae?

This study investigated the usefulness of floral characters as a potential indicator of breeding system in the Brassicaceae. Initially, pod set, seed set and pollen tube growth experiments were carried out to confirm the breeding systems of 53 lines representing 25 different cultivated and weedy species from the Brassicaceae. The results of the pod set tests clearly differentiated between self-compatible and self-incompatible species. Floral characters were then evaluated on one or more lines of each of the 25 species. Fourteen floral characters were evaluated including, flower diameter, Cruden's outcrossing index, timing and direction of dehiscence and pollen-ovule ratio. Significant differences between species were evident in all of the floral characteristics evaluated. Flower diameter was generally larger in self-incompatible species than self-compatible species and pollen/ovule ratio was generally higher in self-incompatible species than self-compatible species. However, none of the floral characteristics was able to clearly differentiate the self-compatible and self-incompatible species and allow prediction of the breeding system with absolute confidence. The floral characteristic which was most effective at differentiating the two groups was anther direction at dehiscence.

Cryopreservation of Arecanut (Areca catechu L.) Pollen.

BACKGROUND: Cryopreservation opens new avenues in the field of genetic resource conservation, especially in recalcitrant seeded palms such as arecanut for which field genebanks are exposed to pest and disease attacks and natural calamities. It is only through cryopreservation that the safety of the conserved germplasm can be assured at a relatively low cost for extended periods. OBJECTIVE: The objective of this work was to standardize various aspects of arecanut pollen cryopreservation, viz. collection and desiccation of pollen, in vitro germination, viability and fecundity studies. MATERIALS AND METHODS: Pollens of three arecanut genotypes (Sumangala, Hirehalli Dwarf and Hirehalli Dwarf x Sumangala) were collected in December 2013-February 2014. In vitro viability tests were conducted using fresh and desiccated pollen. Desiccated pollen was cryopreserved by direct immersion in liquid nitrogen and cryostored for different durations (24 hours to 2 years). Viability and fertility studies were conducted using cryopreserved pollen. RESULTS: Pollen extraction was achieved from fully opened male flowers by desiccation at room temperature (33-34 degree C). A medium containing 2.5 g/L sucrose was found to be best for in vitro germination at room temperature. There was no significant difference in germination between desiccated and cryopreserved pollen whereas pollen tube length decreased significantly after cryopreservation. Fertility studies using HD x Sumangala pollen cryostored for various durations (1 month, 1 year and 2 years) showed the setting of 70, 43 and 62%, respectively. Normal nut set was observed using cryopreserved pollen. CONCLUSION: Pollen cryopreservation is a viable option for germplasm conservation and hybridization programmes in arecanut.

Segmentation of Pollen Tube Growth Videos Using Dynamic Bi-Modal Fusion and Seam Carving.

The growth of pollen tubes is of significant interest in plant cell biology, as it provides an understanding of internal cell dynamics that affect observable structural characteristics such as cell diameter, length, and growth rate. However, these parameters can only be measured in experimental videos if the complete shape of the cell is known. The challenge is to accurately obtain the cell boundary in noisy video images. Usually, these measurements are performed by a scientist who manually draws regions-of-interest on the images displayed on a computer screen. In this paper, a new automated technique is presented for boundary detection by fusing fluorescence and brightfield images, and a new efficient method of obtaining the final cell boundary through the process of Seam Carving is proposed. This approach takes advantage of the nature of the fusion process and also the shape of the pollen tube to efficiently search for the optimal cell boundary. In video segmentation, the first two frames are used to initialize the segmentation process by creating a search space based on a parametric model of the cell shape. Updates to the search space are performed based on the location of past segmentations and a prediction of the next segmentation.Experimental results show comparable accuracy to a previous method, but significant decrease in processing time. This has the potential for real time applications in pollen tube microscopy.

Economy, efficiency, and the evolution of pollen tube growth rates.

PREMISE: Pollen tube growth rate (PTGR) is an important aspect of male gametophyte performance because of its central role in the fertilization process. Theory suggests that under intense competition, PTGRs should evolve to be faster, especially if PTGR accurately reflects gametophyte quality. Oddly, we know remarkably little about how effectively the work of tube construction is translated to elongation (growth and growth rate). Here we test the prediction that pollen tubes grow equally efficiently by comparing the scaling of wall production rate (WPR) to PTGR in three water lilies that flower concurrently: Nymphaea odorata, Nuphar advena and Brasenia schreberi. METHODS: Single-donor pollinations on flower or carpel pairs were fixed just after pollen germination (time A) and 45 min later (time B). Mean PTGR was calculated as the average increase in tube length over that growth period. Tube circumferences (C) and wall thicknesses (W) were measured at time B. For each donor, WPR = mean (C × W) × mean PTGR. KEY RESULTS: Within species, pollen tubes maintained a constant WPR to PTGR ratio, but species had significantly different ratios. N. odorata and N. advena had similar PTGRs, but for any given PTGR, they had the lowest and highest WPRs, respectively. CONCLUSIONS: We showed that growth rate efficiencies evolved by changes in the volume of wall material used for growth and in how that material was partitioned between lateral and length dimensions. The economics of pollen tube growth are determined by tube design, which is consequent on trade-offs between efficient growth and other pollen tube functions.

Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

Different heavy metals have various effects on Picea wilsonii pollen germination and tube growth.

Heavy metal pollution has became one of the realistic matters of globality. Previous reports indicated that heavy metals could significantly inhibit pollen germination and tube growth. In the present study, comparative studies on the effects of different heavy metals (As, Hg, Cd, Cr and Cu) on in-vitro picea wilsonii pollen germination and tube growth were carried out. Microscopic evaluation revealed that different heavy metals had various degree of toxicity on P. wilsonii pollen tube development. As showed the most toxic effects on pollen germination, which was followed by Hg and Cd, while Cr and Cu showed relatively lower toxicity. Besides, pollen tubes showed varying shapes in response to different heavy metal stress. Pollen tubes treated with Cd, Hg and As were usually characterized by irregularly increasing diameters and swelling tips with distinct cytoplasimic vacuolation. On the other hand, except for the slightly increased diameters, no obvious abnormal shape were observed in tubes treated with Cr or Cu. Lyso-Tracker Green staining indicated that only Cd-treated pollen tubes showed numerous vacuole-like acidic organelles, though cytoplasmic vacuolization were also observed in pollen tubes treated with Hg and A. In brief, our data indicated that different heavy metals have various effects on Picea wilsonii pollen germination and tube growth, and that in-vitro pollen culture might be used as a competent system for biomonitoring of air pollution.

The pollen tube clear zone: clues to the mechanism of polarized growth.

Pollen tubes usually exhibit a prominent region at their apex called the "clear zone" because it lacks light refracting amyloplasts. A robust, long clear zone often associates with fast growing pollen tubes, and thus serves as an indicator of pollen tube health. Nevertheless we do not understand how it arises or how it is maintained. Here we review the structure of the clear zone, and attempt to explain the factors that contribute to its formation. While amyloplasts and vacuolar elements are excluded from the clear zone, virtually all other organelles are present including secretory vesicles, mitochondria, Golgi dictyosomes, and the endoplasmic reticulum (ER). Secretory vesicles aggregate into an inverted cone appressed against the apical plasma membrane. ER elements move nearly to the extreme apex, whereas mitochondria and Golgi dictyosomes move less far forward. The cortical actin fringe assumes a central position in the control of clear zone formation and maintenance, given its role in generating cytoplasmic streaming. Other likely factors include the tip-focused calcium gradient, the apical pH gradient, the influx of water, and a host of signaling factors (small G-proteins). We think that the clear zone is an emergent property that depends on the interaction of several factors crucial for polarized growth.

Arabinogalactan proteins mark stigmatic receptivity in the protogynous flowers of Magnolia virginiana (Magnoliaceae).

PREMISE OF THE STUDY: Factors affecting floral receptivity in angiosperms remain opaque, but recent studies suggest that the acquisition of stigmatic receptivity associated with cell-wall-related arabinogalactan proteins (AGPs) may be a widespread feature of flowering plants. Here, the time during which a stigma is receptive is evaluated and related to the secretion of AGPs in Magnolia virginiana, a protogynous member of an early-divergent angiosperm clade (magnoliids) with a clearly discernible female receptive phase. METHODS: Magnolia virginiana flower phenology was documented, and histochemical changes in the stigma before and after pollination were examined. Stigmatic receptivity was evaluated in relation to the secretion of AGPs detected in whole mounts and immunolocalized in sectioned stigmas. KEY RESULTS: Protogynous Magnolia flowers had a precise window of stigmatic receptivity, which is concomitant with the secretion of two AGPs labeled for different epitopes. After pollen germination and tube growth, these two AGPs could no longer be detected in the stigmas, suggesting that these AGPs interact with the growing male gametophytes and could be markers of stigmatic receptivity. CONCLUSIONS: These results show that the period of stigmatic receptivity is finely coordinated with the secretion of two arabinogalactan proteins on stigmas of flowers of M. virginiana. This first report of AGP presence in stigmatic tissues in a member of the magnoliids, together with recently described similar patterns in eudicots, monocots, and members of early-divergent lineages of flowering plants, suggests an ancient and widespread role for AGPs on stigmatic receptivity in angiosperms.

Structural and developmental variability in the female gametophyte of Griffithella hookeriana, Polypleurum stylosum, and Zeylanidium lichenoides and its bearing on the occurrence of single fertilization in Podostemaceae.

Angiosperms are characterized by the phenomenon of double fertilization with Podostemaceae as an exception that appears to extend to the entire family. Our earlier work demonstrated the cause of failure of double fertilization and ascertained the occurrence of single fertilization in Dalzellia zeylanica (Tristichoideae, Podostemaceae). In continuation with this work, three more members, i.e., Griffithella hookeriana (Tul.) Warming, Polypleurum stylosum (Wight) Hall, and Zeylanidium lichenoides (Kurz) Engl. (Podostemoideae), have been investigated in the present work. We studied the ontogenetic development of female gametophyte and tracked the path of the two sperm cells from the time of their formation in the pollen tube through their entry into the synergid and gamete fusion. We report the occurrence of a remarkably reduced 3-nucleate, 3-celled mature female gametophyte consisting of an egg cell and two synergids in all the three genera. Interestingly, the central cell is formed during female gametophyte development, but exhibits a species-specific, limited life span, and eventually degenerates prior to the entry of the pollen tube into the synergid, resulting in a failure of double fertilization. Sperm dimorphism on the basis of fluorochrome stainability has been recorded in Z. lichenoides. Further, morphogenetic constraints on the part of male (sperm selection, functional reductionism) and female gametophyte (structural reductionism, inaccessibility of central cell) presumably ensure the failure of double fertilization in these species. Thus, loss of double fertilization in this family is likely a derived condition.