Fertilization Of Egg Cells Research Articles

Behavior of Male Gamete Fusogen GCS1/HAP2 and the Regulation in Arabidopsis Double Fertilization.

In the sexual reproduction of flowering plants, two independent fertilization events occur almost simultaneously: two identical sperm cells fuse with either the egg cell or the central cell, resulting in embryo and endosperm development to produce a seed. GCS1/HAP2 is a sperm cell membrane protein essential for plasma membrane fusion with both female gametes. Other sperm membrane proteins, DMP8 and DMP9, are more important for egg cell fertilization than that of the central cell, suggesting its regulatory mechanism in GCS1/HAP2-driving gamete membrane fusion. To assess the GCS1/HAP2 regulatory cascade in the double fertilization system of flowering plants, we produced Arabidopsis transgenic lines expressing different GCS1/HAP2 variants and evaluated the fertilization in vivo. The fertilization pattern observed in GCS1_RNAi transgenic plants implied that sperm cells over the amount of GCS1/HAP2 required for fusion on their surface could facilitate membrane fusion with both female gametes. The cytological analysis of the dmp8dmp9 sperm cell arrested alone in an embryo sac supported GCS1/HAP2 distribution on the sperm surface. Furthermore, the fertilization failures with both female gametes were caused by GCS1/HAP2 secretion from the egg cell. These results provided a possible scenario of GCS1/HAP2 regulation, showing a potential scheme for capturing additional GCS1/HAP2-interacting proteins.

Egg cell-secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis.

Double fertilization is an innovative phenomenon in angiosperms, in which one sperm cell first fuses with the egg cell to produce the embryo, and then the other sperm fuses with the central cell to produce the endosperm. However, the molecular mechanism of the preferential fertilization of egg cells is poorly understood. In this study, we report that two egg cell-secreted aspartic proteases, ECS1 and ECS2, play an important role in promoting preferential fertilization of egg cells in Arabidopsis. We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20% reduction in fertility, which can be complemented by the full-length ECS1/2 but not by corresponding active site mutants or by secretion-defective versions of ECS1/2. Detailed phenotypic analysis revealed that the egg cell-sperm cell attachment was compromised in ecs1 ecs2 siliques. Limited pollination assays with cyclin-dependent kinase a1 (cdka;1) pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant. Taken together, these results demonstrate that egg cells secret two aspartic proteases, ECS1 and ECS2, to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved. This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana.

Double-seedlings and embryo-free seeds generated by genetic engineering.

Apomixis can fix the heterosis of Hybrid F1, by maintaining its heterozygous genotype, and is an ideal way for the development of hybrid rice. In this paper, we designed an engineering strategy for realizing apomictic reproduction of hybrid rice in the way of induce adventitious embryos. An embryogenesis gene, AtWUS, controlled by the ovule-specific promoter, a ribonuclease gene Barnase driven by the egg cell-specific promoter pDD45, and an inactivation gene ZmAA1 driven by the pollen-specific promoter pG47 were simultaneously integrated into one T-DNA, and co-transformed with the second T-DNA carrying a Barstar gene. Double-seedlings were observed in transgenic line. Whole-genome sequencing and ploidy levels confirmed by flow cytometry showed that one of the double-seedlings was heterozygous diploid and the other seedling was homozygous haploid, which confirmed that embryogenesis in one of the double-seedlings arises from the zygote after fertilization and the other derived from an unfertilized gamete. Meanwhile we obtained embryo-free seeds at frequencies of 2.6% to 3.8% in T1 generation, and 0.75% to 3% in T2 generation. Though we did not obtained adventitious embryos in hybrid rice in this study, the phenomenon of double-seedlings and embryo-free seeds in transgenic line was informative and strongly suggested that endosperm development is an autonomously organized process in rice, independent of egg cell fertilization and embryo-endosperm communication. This provides novel insights into the induction of haploid embryos and lends theoretical support to successful clonal propagation using synthetic apomixis

Phylogenetic and Expression Analysis of CENH3 and APOLLO Genes in Sexual and Apomictic Boechera Species.

Apomictic plants (reproducing via asexual seeds), unlike sexual individuals, avoid meiosis and egg cell fertilization. Consequently, apomixis is very important for fixing maternal genotypes in the next plant generations. Despite the progress in the study of apomixis, molecular and genetic regulation of the latter remains poorly understood. So far APOLLO gene encoding aspartate glutamate aspartate aspartate histidine exonuclease is one of the very few described genes associated with apomixis in Boechera species. The centromere-specific histone H3 variant encoded by CENH3 gene is essential for cell division. Mutations in CENH3 disrupt chromosome segregation during mitosis and meiosis since the attachment of spindle microtubules to a mutated form of the CENH3 histone fails. This paper presents in silico characteristic of APOLLO and CENH3 genes, which may affect apomixis. Furthermore, we characterize the structure of CENH3 by bioinformatic tools, study expression levels of APOLLO and CENH3 transcripts by Real-Time Polymerase Chain Reaction RT-PCR in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of meiosis and before and after fertilization. While CENH3 was a single copy gene in all Boechera species, the APOLLO gene have several polymorphic alleles associated with sexual and apomictic reproduction in the Boechera genera. Expression of the APOLLO apo-allele during meiosis was upregulated in gynoecium of apomict B. divaricarpa downregulating after meiosis until the 4th day after pollination (DAP). On the 5th DAP, expression in apomictic siliques increased again. In sexual B. stricta gynoecium and siliques APOLLO apo-allele did not express. Expression of the APOLLO sex-allele during and after meiosis in gynoecium of sexual plants was several times higher than that in apomictic gynoecium. However, after pollination the sex-allele was downregulated in sexual siliques to the level of apomicts and increased sharply on the 5th DAP, while in apomictic siliques it almost did not express. At the meiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, decreasing in both species after meiosis and keep remaining very low in siliques of both species for several days after artificial pollination until the 4th DAP, when the expression level raised in sexual B. stricta siliques exceeding 5 times the level in apomictic B. divaricarpa siliques. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes. The results obtained may indicate to a role of the CENH3 and APOLLO genes in the development of apomixis in species of the genus Boechera.

Endosperm development is an autonomously programmed process independent of embryogenesis

The seeds of flowering plants contain three genetically distinct structures: the embryo, endosperm, and seed coat. The embryo and endosperm need to interact and exchange signals to ensure coordinated growth. Accumulating evidence has confirmed that embryo growth is supported by the nourishing endosperm and regulated by signals originating from the endosperm. Available data also support that endosperm development requires communication with the embryo. Here, using single-fertilization mutants, Arabidopsis thaliana dmp8 dmp9 and gex2, we demonstrate that in the absence of a zygote and embryo, endosperm initiation, syncytium formation, free nuclear cellularization, and endosperm degeneration occur as in the wild type in terms of the cytological process and time course. Although rapid embryo expansion accelerates endosperm breakdown, our findings strongly suggest that endosperm development is an autonomously organized process, independent of egg cell fertilization and embryo-endosperm communication. This work confirms both the altruistic and self-directed nature of the endosperm during coordinated embryo-endosperm development. Our findings provide insights into the intricate interaction between the two fertilization products and will help to distinguish the physiological roles of the signaling between endosperm and embryo. These findings also open new avenues in agro-biotechnology for crop improvement.

Evaluation of Cytotoxicity and Genotoxicity of Canarium ovatum Engl. (Burseraceae) and Cocos nucifera L. (Arecaceae) Ethanolic Bark Extracts

To further elucidate the potential of the pili and coconut barks in terms of their anticancer activity, their crude extracts were subjected to the sea urchin fertilization assay and Allium cepa test. These tests were conducted in order to determine the cytotoxicity and genotoxicity activities against the test subjects. The generated IC50 values are 0.54 mg/ml and 0.20 mg/ml for Canarium ovatum and Cocos nucifera, respectively. The sea urchin egg cells possess strong sensitivity against toxic agents as well as onion root cells which display the genetic correspondence of the cells affected by noxious substances. Canarium ovatum crude bark extracts exhibited a significant inhibition activity (p < 0.0001) against the sea urchin egg cell fertilization whereas the Cocos nucifera bark extracts did not show any significant inhibition. In terms of its genotoxicity, Canarium ovatum crude bark extracts exhibited a significant inhibition on the mitotic indexes and increased the frequency of aberrations in the onion root cells whereas no significant inhibition on the mitotic indexes of the root cells was observed among those treated with Cocos nucifera. Phytochemical screening results of Canarium ovatum crude bark extracts presented abundant compounds such as flavonoids, phenols, tannins and with traces of saponins. For the Cocos nucifera crude bark extracts, compounds such as saponins were found abundant with traces of flavonoids, phenols, tannins, diterpenes and terpenoids. The results indicated that Canarium ovatum possess anti-proliferative potential as exhibited in the two assays while Cocos nucifera possess toxic potentials at certain concentrations. Further studies are needed to validate the results of the present investigation.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

A Role for Proton Signaling in the Induction of Somatic Cells to Pluripotent Embryonic Stem Cells

The mechanisms of embryonic stem (ES) cell establishment and egg cell fertilization have been studied without examining the interactions of these cell types, perhaps because of the lack of consideration for the similarities between ES cells and fertilized egg cells, especially the lack of calcium signaling. Here we unify the various concepts developed separately so fat in ES cells and fertilized eggs, although both types of cells have the same fate (i.e., to become somatic cells). We discuss the concept of water property changes in the cytoplasm that occur along with the cell cycle, which was initially introduced in the field of biophysics. With this aspect, the similarities and differences between ES cells and fertilized eggs are understandable, and the water property changes help explain why ES cells can have pluripotency, as fertilized eggs do, following a de novo stimulus such as acid treatment. Obokata et al. recently discovered a shortcut for generating pluripotent stem cells which they named “stimulus-triggered acquisition of pluripotency (STAP)” cells from somatic cells at the transient low-pH stimulation [1]. This report has had a significant impact on many developmental biologists and clinicians, because the low-pH treatment seemed an unexpectedly easy way to change somatic cells to pluripotent embryonic stem (ES) cells [2]. However, this method is not so surprising to biophysicists, because several types of cellular stress have been found to induce egg activation associated with a Ca2+ transient increase, such as the change in cytoplasmic pH caused by applying NH4Cl and/or CO2, or even mechanical pricking with a needle [3]. In 2002, Burdon et al. reported that the epiblast cells, which have pluripotency, were obtained from a prolonged culture of ES cells [4]. ES cells have an unusual cell cycle in which the G1 phase that operates in other types of cells is reduced [5]. Such features of ES cells are associated with the deregulated characteristics of the proliferation of tumor cells. Unlike these types of cells, a fertilized egg also starts its own cell cycle with Ca2+ oscillations [6]. This Ca2+ oscillation can be explained by a one- or two-calcium-pool model with the positive feedback of increased Ca2+ and inositol 1,4,5- trisphosphate (IP3) during the rising phase of each Ca2+ increase. It is thus reasonable to speculate that the cell cycle is the time keeper in Ca2+ oscillation in both cultured ES cells in vitro and circadian calcium rhythms in the somatic cells in the body, which were also found to be associated with a slow frequency of Ca2+ oscillation, with 1 cycle/day [7]. The property changes of water state in the cell are another critical issue. Mantre described that the state of water in the somatic cells in the body has ordered-structured bound water (like ice), whereas ES cells as well as cancer cells have free (normal) water in the G1 and G2 phases [8]. Only protons, not other ions, are able to move on the surface of bound water, whereas in free water all types of ions (including protons) can move by diffusion. In this review article, we describe the differences between somatic cells and ES cells regarding proton signaling, which regulates Ca2+ oscillation, and the change in water properties associated with cell cycle. Proton signaling plays a critical role in the nuclear reprogramming to the pluripotent stage.

The cytological changes of tobacco zygote and proembryo cells induced by beta-glucosyl Yariv reagent suggest the involvement of arabinogalactan proteins in cell division and cell plate formation.

BackgroundIn dicotyledonous plant, the first asymmetric zygotic division and subsequent several cell divisions are crucial for proembryo pattern formation and later embryo development. Arabinogalactan proteins (AGPs) are a family of extensively glycosylated cell surface proteins that are thought to have important roles in various aspects of plant growth and development, including embryogenesis. Previous results from our laboratory show that AGPs are concerned with tobacco egg cell fertilization and zygotic division. However, how AGPs interact with other factors involved in zygotic division and proembryo development remains unknown.ResultsIn this study, we used the tobacco in vitro zygote culture system and series of meticulous cell biology techniques to investigate the roles of AGPs in zygote and proembryo cell division. For the first time, we examined tobacco proembryo division patterns detailed to every cell division. The bright-field images and statistical results both revealed that with the addition of an exogenous AGPs inhibitor, beta-glucosyl Yariv (beta-GlcY) reagent, the frequency of aberrant division increased remarkably in cultured tobacco zygotes and proembryos, and the cell plate specific locations of AGPs were greatly reduced after beta-GlcY treatment. In addition, the accumulations of new cell wall materials were also significantly affected by treating with beta-GlcY. Detection of cellulose components by Calcofluor white stain showed that strong fluorescence was located in the newly formed wall of daughter cells after the zygotic division of in vivo samples and the control samples from in vitro culture without beta-GlcY treatment; while there was only weak fluorescence in the newly formed cell walls with beta-GlcY treatment. Immunocytochemistry examination with JIM5 and JIM7 respectively against the low- and high-esterified pectins displayed that these two pectins located in opposite positions of zygotes and proembryos in vivo and the polarity was not affected by beta-GlcY. Furthermore, FM4-64 staining revealed that endosomes were distributed in the cell plates of proembryos, and the localization pattern was also affected by beta-GlcY treatment. These results were further confirmed by subsequent observation with transmission electron microscopy. Moreover, the changes to proembryo cell-organelles induced by beta-GlcY reagent were also observed using fluorescent dye staining technique.ConclusionsThese results imply that AGPs may not only relate to cell plate position decision, but also to the location of new cell wall components. Correlated with other factors, AGPs further influence the zygotic division and proembryo pattern establishment in tobacco.

Expression of lorelei-like genes in aposporous and sexual Paspalum notatum plants

Gametophytic apomictic plants form non-reduced embryo sacs that generate clonal embryos by parthenogenesis, in the absence of both meiosis and egg-cell fertilization. Here we report the sequence and expression analysis of a lorelei-like Paspalum notatum gene, n20gap-1, which encodes a GPI-anchored protein previously associated with apomixis in this species. Phylogeny trees showed that n20gap-1 was evolutionary related to the Arabidopsis thaliana lorelei genes At4g26466 and At5g56170. The lorelei At4g26466 disruption was shown to be detrimental to sperm cell release in arabidopsis. RFLP (Restriction Fragment Length Polymorphism) analysis revealed the occurrence of several homologous sequences in the Paspalum notatum genome, exhibiting polymorphisms genetically linked to apomixis. Real-time PCR showed that lorelei-family genes present a minor activity peak at pre-meiosis and a major one at anthesis. The apomictic genotype analyzed showed a significantly increased activity at pre-meiosis, post-meiosis and anthesis with respect to a sexual genotype. In situ hybridization assays revealed expression in integuments, nucellus and the egg-cell apparatus. Several n20gap-1 alleles differing mainly at the 3' UTR sequence were identified. Allele-specific real-time PCR experiments showed that allele 28 was significantly induced in reproductive tissues of the apomictic genotype with respect to the sexual genotype at anthesis. Our results indicate that P. notatum lorelei-like genes are differentially expressed in representative sexual (Q4188) and apomictic (Q4117) genotypes, and might play a role in the final stages of the apomixis developmental cascade. However, the association of n20gap-1 expression with the trait should be confirmed in significant number of sexual and apomictic genotypes.

Comparative Detection of Calcium Fluctuations in Single Female Sex Cells of Tobacco to Distinguish Calcium Signals Triggered by in vitro Fertilization

Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca(2+)-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca(2+) in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca(2+) signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 microM Fluo-3 for 30 min at 30 degrees C. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca(2+)](cyt) of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.

Identification of a SCAR marker linked to apomixis in buffelgrass ( Cenchrus ciliaris L.)

Apomixis is a mode of asexual reproduction through seeds. The apomictic process bypasses both meiosis and egg cell fertilization, producing offspring that are exact genetic replicas of the mother plant. Cenchrus ciliaris (buffelgrass) is a valuable forage grass, which reproduces through aposporous apomeiosis. We identified one sexual plant in this obligate apomictic population. The objective of this work was to identify molecular markers linked to apospory trait through bulked segregant analysis. Facultative apomictic F1 plants obtained from open pollinated sexual plant were selfed to derive F2 population. Individual F2 plants were classified through embryo sac analysis as sexual, facultative apomictic or obligate apomictic. Two bulks namely, obligate apomictic and sexual were prepared by pooling DNA of individual identified plants and used in PCR analysis. Out of 208 primers tested, 20 were found to be polymorphic. A 600 bp RAPD amplicon specific to apomictic bulk, obtained with OPF-08 primer was cloned and sequenced. Based on the sequence information, a pair of SCAR (sequence characterized amplified region) primers was designed which generated a 600 bp amplicon (named OPF08-600) only in the apomictic plants. Southern hybridization of genomic DNA of apomictic and sexual plants using the OPF08-600 fragment as probe indicated the presence of two copies of the SCAR sequence in only the apomictic plants. Gel elution of restricted DNA fragments and PCR analysis confirmed the presence of duplicated copies of the SCAR sequence in the apomictic plants.

Localization of arabinogalactan proteins in egg cells, zygotes, and two-celled proembryos and effects of -D-glucosyl Yariv reagent on egg cell fertilization and zygote division in Nicotiana tabacum L.

Arabinogalactan proteins (AGPs) have been implicated in a variety of plant development processes including sexual plant reproduction. As a crucial developmental event, plant sexual reproduction generally occurs inside an ovule embedded in an ovary. The inaccessibility of the egg cells, zygotes, and embryos has hindered our understanding of the importance of AGPs in the early events involving fertilization, zygotic division, and early embryogenesis. In this study, the well-established in vitro zygote and ovary culture systems, together with immunofluorescence and immunogold labelling techniques, were employed to investigate the role of AGPs in the early events of sexual reproduction in Nicotiana tabacum. Dramatic changes in AGP content during ovule development were evidenced by western blotting. Subcellular localization revealed that AGPs are localized in the plasma membrane, cell wall, and cytoplasm of pre- and post-fertilized egg cells, and cytoplasm and vacuoles of two-celled proembryos. Abundant AGPs were detected in unfertilized egg cells; however, the level of AGPs substantially decreased in fertilized egg cells. Polar distribution of AGPs in elongated zygotes was observed. The early two-celled proembryos just from zygote division displayed accumulation of AGPs at a low level, while in the elongated two-celled proembryos at the late stage, the AGP content clearly increased. Provision of betaGlcY, a synthetic phenylglycoside that specifically binds AGPs, to the in vitro cultures of isolated zygote and fertilized ovaries increased abnormal symmetrical division of zygotes. In the culture of pollinated but unfertilized ovaries, addition of betaGlcY resulted in arrest of fertilization of the egg cells, but had no effect on fertilization of the central cells. The possible roles of AGPs in fertilization, zygotic division, and proembryo development are discussed.

Heterochronic expression of sexual reproductive programs during apomictic development in Tripsacum.

Some angiosperms reproduce by apomixis, a natural way of cloning through seeds. Apomictic plants bypass both meiosis and egg cell fertilization, producing progeny that are genetic replicas of the mother plant. In this report, we analyze reproductive development in Tripsacum dactyloides, an apomictic relative of maize, and in experimental apomictic hybrids between maize and Tripsacum. We show that apomictic reproduction is characterized by an alteration of developmental timing of both sporogenesis and early embryo development. The absence of female meiosis in apomictic Tripsacum results from an early termination of female meiosis. Similarly, parthenogenetic development of a maternal embryo in apomicts results from precocious induction of early embryogenesis events. We also show that male meiosis in apomicts is characterized by comparable asynchronous expression of developmental stages. Apomixis thus results in an array of possible phenotypes, including wild-type sexual development. Overall, our observations suggest that apomixis in Tripsacum is a heterochronic phenotype; i.e., it relies on a deregulation of the timing of reproductive events, rather than on the alteration of a specific component of the reproductive pathway.

Apomixis for crop improvement

Apomixis is a genetically controlled reproductive process by which embryos and seeds develop in the ovule without female meiosis and egg cell fertilization. Apomixis produces seed progeny that are exact replicas of the mother plant. The major advantage of apomixis over sexual reproduction is the possibility to select individuals with desirable gene combinations and to propagate them as clones. In contrast to clonal propagation through somatic embryogenesis or in vitro shoot multiplication, apomixis avoids the need for costly processes, such as the production of artificial seeds and tissue culture. It simplifies the processes of commercial hybrid and cultivar production and enables a large-scale seed production economically in both seed- and vegetatively propagated crops. In vegetatively reproduced plants (e.g., potato), the main applications of apomixis are the avoidance of phytosanitary threats and the spanning of unfavorable seasons. Because of its potential for crop improvement and global agricultural production, apomixis is now receiving increasing attention from both scientific and industrial sectors. Harnessing apomixis is a major goal in applied plant genetic engineering. In this regard, efforts are focused on genetic and breeding strategies in various plant species, combined with molecular methods to analyze apomictic and sexual modes of reproduction and to identify key regulatory genes and mechanisms underlying these processes. Also, investigations on the components of apomixis, i.e., apomeiosis, parthenogenesis, and endosperm development without fertilization, genetic screens for apomictic mutants and transgenic approaches to modify sexual reproduction by using various regulatory genes are receiving a major effort. These can open new avenues for the transfer of the apomixis trait to important crop species and will have far-reaching potentials in crop improvement regarding agricultural production and the quality of the products.

Non-Mendelian transmission of apomixis in maize-Tripsacum hybrids caused by a transmission ratio distortion.

Apomixis is a mode of asexual reproduction through seeds. The apomictic process bypasses both meiosis and egg cell fertilization, producing offspring that are exact genetic replicas of the mother plant. In the Tripsacum agamic complex, all polyploids reproduce through the diplosporous type of apomixis, and diploids are sexual. In this paper, molecular markers linked with diplospory were used to analyse various generations of maize-Tripsacum hybrids and backcross derivatives and to derive a model for the inheritance of diplosporous reproduction. The results suggest that the gene or genes controlling apomixis in Tripsacum are linked with a segregation distorter-type system promoting the elimination of the apomixis alleles when transmitted through haploid gametes. Hence, this model offers an explanation of the relationship between apomixis and polyploidy. The evolutionary importance of this mechanism, which protects the diploid level from being invaded by apomixis, is discussed.

Non-Mendelian transmission of apomixis in maize–Tripsacum hybrids caused by a transmission ratio distortion

Apomixis is a mode of asexual reproduction through seeds. The apomictic process bypasses both meiosis and egg cell fertilization, producing offspring that are exact genetic replicas of the mother plant. In the Tripsacum agamic complex, all polyploids reproduce through the diplosporous type of apomixis, and diploids are sexual. In this paper, molecular markers linked with diplospory were used to analyse various generations of maize–Tripsacum hybrids and backcross derivatives and to derive a model for the inheritance of diplosporous reproduction. The results suggest that the gene or genes controlling apomixis in Tripsacum are linked with a segregation distorter-type system promoting the elimination of the apomixis alleles when transmitted through haploid gametes. Hence, this model offers an explanation of the relationship between apomixis and polyploidy. The evolutionary importance of this mechanism, which protects the diploid level from being invaded by apomixis, is discussed.

439 PB 218 CHROMOSOMAL VARIATION AMONG PROGENY OF FACULTATIVE APOMICTIC GUAYULE

Progress toward the domestication of guayule (Parrhenium argentatum Gray) as a domestic source of latex, has been slowed because of its complicated reproductive biology. This work was performed to help elucidate the facultative nature of apomictic reproduction in guayule. Ploidy level and six morphological characters were measured in one-hundred and eighty open-pollinated, half-sib progeny derived from twelve different apomictic tetraploid (4n=4x=72) female parents. The number of chromosomes observed among the half-sib progeny varied from 36 to 81, with the majority (77.3%) of the progeny with 72 chromosomes. All of the observed chromosome numbers could be explained either by meiotic reduction in the megaspore mother cells or fertilization of egg cells or both. The frequencies of meiotic reduction and fertilization resulting in these progeny were estimated to be 27.2 and 22.7%, respectively. Among the six measured morphological characters, significant correlations were found only between chromosome numbers and plant height (r=0.37) and leaf area (r=0.31).

Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+

The inwardly rectifying K channel provides the resting K conductance in a variety of cells. This channel acts as a valve or diode, permitting entry of K+ under hyperpolarization, but not its exit under depolarization. This behaviour, termed inward rectification, permits long depolarizing responses which are of physiological significance for the pumping function of the heart and for fertilization of egg cells. Little is known about the outward currents through the inwardly rectifying K channel, despite their great physiological importance, and the mechanism of inward rectification itself is unknown. We have used improved patch clamp techniques to control the intracellular media, and have recorded the outward whole-cell and single-channel currents. We report here that the channel conductance is ohmic and that the well-known inward rectification of the resting K conductance is caused by rapid closure of the channel accompanied by a voltage-dependent block by intracellular Mg2+ ions at physiological concentrations.

MEIOTIC RESTITUTION AND THE ORIGIN OF POLYPLOIDY. I. INFLUENCE OF GENOTYPE ON POLYPLOID SEEDSET IN A TRITICUM CRASSUM × T. TURGIDUM HYBRID

The F1 hybrid (2n = 35) Triticum crassum (2n = 42) × Triticum turgidum cv. Carleton (2n = 28) shows a low but consistent seedset of 28%. Most offspring have twice as many chromosomes (2n = 70) as the F1 hybrid. Cytological studies revealed that a very low number of bivalents (average of 1.7 bivalents) were present at metaphase I; most of the chromosomes were univalents (average of 31.5 univalents). During metaphase I these univalents assembled at the equatorial plate, where the degree of univalent accumulation, determined to be 91.0%, was very high. At anaphase I restitution took place, preventing completion of the first division. In this way many cells obtained one or more large nuclei with or without some micronuclei. Several cells, however, formed one large nucleus containing all 35 chromosomes. These cells were able to proceed through the remaining stages of meiosis without irregularities and produced unreduced, functional gametes with n = 35 chromosomes. It is postulated that autogamous fertilization of egg cells by male gametes, each having n = 35 chromosomes, produced the decaploid offspring with 2n = 70 chromosomes.Since bivalent formation and the high degree of univalent accumulation at the equatorial plate is genetically determined, the occurrence of restitution and the subsequent low set of polyploid seeds are highly influenced by the hybrid's genotype. This was demonstrated in the high reproducibility of the hybrid characteristics under different growing conditions.