Early post-pollination events in hexaploid wheat × maize crosses

Summary Cytological events in the first 12 h after pollination were studied in crosses between the hexaploid wheat genotype Chinese Spring and the maize genotype Seneca 60. A pollen tube was first observed in the embryo sac 4 h after pollination, and maize sperm nuclei were first observed in the embryo sac after 5 h. On 29 occasions two, and on 1 occasion three, pollen tubes penetrated the embryo sac. Four categories of aberration limiting the frequency of fertilization were identified: (1) in 20% of florets no pollen tube reached the embryo sac; (2) in at least 1.9% the pollen tube severely damaged the wheat egg cell and polar nuclei; (3) in 33% the maize sperm nuclei were not released from the pollen tube; and (4) in 16% the sperm nuclei were released into the embryo sac but failed to move to either of the wheat gametes. In the remaining 29% sperm nuclei were more often found in the egg cell than at the polar nuclei. The results suggest that karyogamy occurs with very high efficiency when a sperm nucleus reaches the egg cell, but with only about 50% efficiency when a sperm nucleus reaches the polar nuclei.     

Polyspermy barriers in plants: from preventing to promoting fertilization

Barriers to polyspermy (fertilization of a female gamete by more than one sperm) are essential to successful reproduction in a wide range of organisms including mammals, echinoderms, fish, molluscs, and algae. In animals and fucoid algae, polyspermy results in early death of the zygote due to transmission of extra centrioles from the sperm and consequent disruptions to the mitotic spindle. Accordingly, a variety of mechanisms have evolved to prevent penetration of an egg by more than one sperm, or more than one sperm nucleus from fusing with an egg nucleus. The evolution of internal fertilization has also provided an opportunity to limit the number of sperm that gain access to each egg, as occurs in the mammalian female reproductive tract. Polyspermy and polyspermy barriers in plants have received much less attention. Plants lack centrioles and therefore, polyspermy would not be expected to cause lethal aberrant spindle organization. However, we find evidence from cytological, genetic and in vitro fertilization studies for polyspermy barriers in plants. Angiosperms, like mammals, are internally fertilized, and exert a high level of control over the number of sperm that have access to each female gamete. In particular, regulation of pollen tube growth ensures that in general only two sperm enter each embryo sac, where one fertilizes the egg and the other the central cell. Despite this 1:1 ratio of sperm to gametes within the embryo sac, angiosperms still require a mechanism to ensure that each female gamete is fertilized by one and only one sperm. Here, we present evidence suggesting that a polyspermy block on the egg may be part of the mechanism that promotes faithful double fertilization.     

The Fertilization and Early Development of Embryo and Endosperm in Stevia rebaudiana Bertani

The mature embryo sac is surrounded by endothelium tapetum. It is composed or an egg apparatus, one central cell with secondary nucleus, and 1–6 antipodal cells. About the 6th hour after pollination, female and male nuclei fuse with each other. The syngamy occurred almost simultaneously with the fusion of an other sperm nucleus and the secondary nucleus, but the velocity of the latter is faster than that of the syngamy. The fertilization of Stevia rebaudiana Bertani belongs to the premitotic type. About the 8th hour after pollination, primary endosperm nucleus is in mitosis, its dividing orientation may parallel or at right angle to the long axis of the embryo sac, and gives rise to two initial endosperm cells. The first five divisions of the endosperm cells are of synchronism. At the stage of heart-shaped embryo, the endosperm cells show the signs of digestion and absorbed. The endosperm development is of the cellular type. About the 10th hour after pollination, zygote divides for the first time. The division of the zygote is always transverse. The embryo development conforms to the Asterad type.     

Early Events in the Penetration of the Embryo Sac in Torenia fournieri(Lind.)

At maturity, Torenia fournieri(Lind.) has an embryo sac whichprotrudes through the micropyle placing the synergids, egg celland part of the central cell within the ovary locule adjacentto the placenta. The present study utilized this unique attributein combination with confocal and light microscopy to characterizethe timing and associated structural changes during pollinationevents leading to double fertilization. The observation of spermnuclei in living gametophyte tissue is an important advancein the identification, in real time, of stages leading to fertilizationin angiosperms. A continuum of fertilization occurred between12 and 16 h after pollination (hap), with peak frequency ofegg and sperm fusion at 14 hap (43%). Movement of the spermcells through the degenerated synergid took several hours andfusion between sperm and their respective female nuclei occurredsimultaneously. Changes in embryo sac structure were also documented.Cell walls in the region between the synergids and egg cellwere poorly developed prior to pollen tube penetration. Thickenedcell walls were observed around the periphery of the synergidsand egg cell following pollination, and in the central cellwhere it lay within the body of the ovule. Starch was observedin the cells of the embryo sac, although the number and distributionof granules varied before and after pollination. These temporaland spatial observations of the embryo sac inTorenia fournieriprovide a basis for further research to determine control mechanismsoperating during specific double fertilization events in angiosperms.Copyright 2000 Annals of Botany Company Double fertilization, embryo sac, sperm nuclei, Hoechst, Torenia fournieri     

Embryology of barley: Time course and analysis of controlled fertilization and early embryo formation based on serial sections

The aim of this work was to determine the sequences and their time schedule in fertilization, embryo and endosperm development in a barley strain grown under controlled conditions. This study involved controlled pollinations and serial sectioning of material embedded in glycol methacrylate.
In Hordeum vulgare cv. Bomi the pollen tube reaches the nucellus in the course of 40 minutes after pollination (m.a.P). In the next minutes the tube advances between the nucellar cells, through the filiform apparatus of the degenerating synergid and releases its contents, which is characterized by rod-shaped starch grains. One of the sperm nuclei is fused with the egg nucleus 45 m.a.P., the second is in contact with the two polar nuclei 50–60 m.a.P., but the real connection between one of the polar nuclei and the sperm nucleus does not occur before 10 hours after pollination (h.a.P.). Triple fusion between all three nuclei takes place about 13 h.a.P. The first division in the zygote takes place 22–24 h.a.P. The embryo has got 3 cells at 34 h.a.P., 4 cells at 38 h. and 52 h.a.P. the embryo is composed of 7 cells. Protoderm formation takes place from a 14-celled embryo stage at 72–80 h.a.P The primary endosperm nucleus divides 14 h.a.P. The next division takes place soon after the first; therefore the endosperm is 4-nucleated already 18. h.a.P. Further divisions are synchronized, and 30–38 h.a.P. there will be more than 32 nuclei. The synchronous divisions continue, and the cellularisation begins at about 70 h.a.P. There is a tight correlation between the enlargement of the ovule and the embryo sac in the first days of the embryo development.     

Disruption of endosperm development: an inbreeding effect in almond (Prunus dulcis)

A homozygous self-compatible almond, originated from self-fertilization of a self-compatible genotype and producing a reasonable yield following open pollination, exhibited a very high fruit drop rate when self-pollinated. To investigate whether fruit dropping in this individual is related to an abnormal development of the embryo sac following self-fertilization, histological sections of ovaries from self and cross-pollinated flowers were observed by light microscopy. Additionally, the presence of pollen tubes in the ovary and fruit set were determined for both types of pollination. Despite pollen tubes reached the ovary after both pollinations, differences in embryo sac and endosperm development after fertilization were found. Thus, while for cross-fertilized ovules a pro-embryo and an endosperm with abundant nuclei were generally observed, most self-fertilized ovules remained in a previous developmental stage in which the embryo sac was not elongated and endosperm nuclei were absent. Although 30 days after pollination fruit set was similar for both pollination types, at 60 days it was significantly reduced for self-pollination. These results provide evidence that the high fruit drop in this genotype is the consequence of a disrupted development of the endosperm, what could be an expression of its high level of inbreeding.     

小麦受精过程中酸性磷酸酶的超微细胞化学定位

小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）受精前成熟胚囊,除胚囊中央细胞的合点端细胞质中有酸性磷酸酶外,其余部位均未发现酸性磷酸酶。受精时期,以下部位存在酸性磷酸酶活性：卵细胞的细胞核内一部分染色质和细胞质中大部分线粒体;精、卵核融合时两核的核周腔内;退化助细胞合点端细胞质和一些液泡内;进入雌性细胞中的两个精核;胚囊各成员细胞的细胞壁及胚囊周围珠心细胞的细胞壁。二细胞原胚中未见有酸性磷酸酶。早期胚乳游离核染色质上有酸性磷酸酶。小麦受精过程酸性磷酸酶的分布特点可能与卵细胞生理状态的变化和细胞质中线粒体的改组、助细胞的退化、精核的生理状态以及精核与卵核的核膜融合等有关。     

The Ultracytochemical Localzation of Acid Phosphatase Activity in Wheat During Fertilization

No acid phosphatase activity was observed in the mature embryo sac of wheat (Triticum aestivum) except the chalazal cytoplasm Of the central cell before fertilization. During fertilization, acid phosphataseactivity was observed in the following loci: part of chromatin of the egg nucleus and most of the mitochondria in the egg cytoplasm; the perinuclear spaces of the egg and sperm nuclei at the fusion of the egg and sperm nuclei; the chalazal cytoplasm and some vacuoles of the degenerated synergid; two sperm nuclei within the cytoplasm of female cells; the cell wall of each cell of the embryo sac and that of the nucellar cells surrounding the embryo sac. No acid phosphatase was observed in the two-celled proembryo. Dense enzyme reaction product was localized in the chromatin of the free nuclei at early stage of the endosperm. The characteristic of acid phosphatase distribution during fertilization may be associated with the physiological change of the egg Cell, the reorganization of mitochondria in the egg cell cytoplasm, the degeneration of one of the two synergids, the physiological state of the sperm nuclei and the nuclear membrane fusion of the egg and sperm nuclei.     

黑节草双受精过程及胚胎发育的研究

黑节草从传粉到受精约需１３０ｄ,精子在花粉管中形成,胚囊发育属蓼型胚囊,因反足细胞较早退化,故受精前胚囊多只由卵器和中央细胞组成。精卵核融合时,精核染色质进入卵核后凝集成颗粒状,并在原位与卵核的染色质融合,雌、雄性核仁一直维持至合子的第一次分裂期前。双受精作用正常,属于有丝分裂前配子融合类型,初生胚乳核发生２－３次分裂后逐渐退化消失,胚的发育局限于球形胚阶段。     

Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indica/japonica Hybrids in Rice

Embryo sac abortion is one of the major reasons for sterility in indica/japonica hybrids in rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indica/japonica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagametogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.     

Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indicaljaponica Hybrids in Rice

Embryo sac abortion is one of the major masons for sterility in indicaljaponica hybrids In rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indicaljaponica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagamatogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucallus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.     

A one‐step rectification of sperm cell targeting ensures the success of double fertilization

Successful fertilization in animals depends on competition among millions of sperm cells, whereas double fertilization in flowering plants usually involves just one pollen tube releasing two immobile sperm cells. It is largely a mystery how the plant sperm cells fuse efficiently with their female targets within an embryo sac. We show that the initial positioning of sperm cells upon discharge from the pollen tube is usually inopportune for gamete fusions and that adjustment of sperm cell targeting occurs through release and re-adhesion of one sperm cell, while the other connected sperm cell remains in stagnation.This enables proper adhesion of each sperm cell to a female gamete and coordinates the gamete fusions. Our findings reveal inner embryo sac dynamics that ensure the reproductive success of flowering plants and suggest a requirement for sperm cell differentiation as the basis of double fertilization.     

Observations on Fertilization in Sugar Beet

The whole process of double fertilization in sugar beet has been observed, the main results are as follows: About 2 hours after pollination, the pollen grains germinate, the sperms in the pollen tube are long-oval. 15 hours after pollination, the pollen tube destroys a synergid and releases two sperms on one side or at the chalazal end of the egg cell. The sperms are spherical each having a cytoplasmic sheath. 17 hours after pollination, one sperm enters the egg cell, and the sperm nucleus fuses with the egg nucleus rapidly. 21 hours after pollination, the zygote is formed. In the meantime, the primary endosperm nucleus has divided into two free endosperm nuclei. 25 hours after pollination, the zygote begins to divide, forming a two-celled proembryo. The dormancy stage of the zygote is about 4 hours. In the meantime the endosperm is at the stage of four free nuclei. 17 hours after pollination, the sperm nucleus comes into contact and fuses with the secondary nucleus. The sperm nucleus fuses with the secondary nucleus, faster than the sperm with the egg. he first division of the primary endosperm nucleus is earlier than that of the zygote, it takes place about 20 hours after pollination, the dormancy stage of the primary endosperm is about 2 hours. The endosperm is free nuclear. The fertilization of sugar beet belongs to premitotic type of syngamy. From the stage of zygote to the two-celled proembryo, it can be seen that addition- al sperms enter the embryo sac, but polyspermy has not been observed yet.     

Ethylene Synthesis and Floral Senescence following Compatible and Incompatible Pollinations in Petunia inflata

Ethylene production and floral senescence following compatible and incompatible pollinations were studied in a self-incompatible species, Petunia inflata. Both compatible and incompatible pollinations resulted in a burst of ethylene synthesis that peaked 3 hours after pollination. P. inflata pollen was found to carry large amounts of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The amount of pollen-held ACC varied in different genetic backgrounds, and the magnitude of the peak correlated with the amount of ACC borne by the pollen. Aminooxyacetic acid (AOA), an inhibitor of ACC synthesis, had no inhibitory effect on this ethylene response, indicating that pollen-borne ACC was largely responsible for the early synthesis of ethylene. After compatible pollination, a second increase in ethylene synthesis began at 18 hours, and the first sign of senescence appeared at 36 hours. Upon treatment with AOA, the second phase of ethylene production was reduced by 95%, indicating that endogenous ACC synthesis was required for this phase of ethylene synthesis. AOA treatment also delayed senescence to 6 days after anthesis. After incompatible pollination, a second increase in ethylene production did not occur until 3 days, and the first sign of senescence occurred 12 hours later. Unpollinated flowers showed an increase in ethylene production 3 to 4 days after anthesis and displayed signs of senescence 1 day later. The significance of the early and late phases of pollination-induced ethylene synthesis is discussed.     

Disorganization of F-actin cytoskeleton precedes vacuolar disruption in pollen tubes during the in vivo self-incompatibility response in Nicotiana alata

Background and Aims The integrity of actin filaments (F-actin) is essential for pollen-tube growth. In S-RNase-based self-incompatibility (SI), incompatible pollen tubes are inhibited in the style. Consequently, research efforts have focused on the alterations of pollen F-actin cytoskeleton during the SI response. However, so far, these studies were carried out in in vitro-grown pollen tubes. This study aimed to assess the timing of in vivo changes of pollen F-actin cytoskeleton taking place after compatible and incompatible pollinations in Nicotiana alata. To our knowledge, this is the first report of the in vivo F-actin alterations occurring during pollen rejection in the S-RNase-based SI system. Methods The F-actin cytoskeleton and the vacuolar endomembrane system were fluorescently labelled in compatibly and incompatibly pollinated pistils at different times after pollination. The alterations induced by the SI reaction in pollen tubes were visualized by confocal laser scanning microscopy. Key Results Early after pollination, about 70 % of both compatible and incompatible pollen tubes showed an organized pattern of F-actin cables along the main axis of the cell. While in compatible pollinations this percentage was unchanged until pollen tubes reached the ovary, pollen tubes of incompatible pollinations underwent gradual and progressive F-actin disorganization. Colocalization of the F-actin cytoskeleton and the vacuolar endomembrane system, where S-RNases are compartmentalized, revealed that by day 6 after incompatible pollination, when the pollen-tube growth was already arrested, about 80 % of pollen tubes showed disrupted F-actin but a similar percentage had intact vacuolar compartments. Conclusions The results indicate that during the SI response in Nicotiana, disruption of the F-actin cytoskeleton precedes vacuolar membrane breakdown. Thus, incompatible pollen tubes undergo a sequential disorganization process of major subcellular structures. Results also suggest that the large pool of S-RNases released from vacuoles acts late in pollen rejection, after significant subcellular changes in incompatible pollen tubes.     

Fertilization and Histochemical Investigation on Pulsatilla chinensis (Bung) Regel

Fertilization and variation of protein and starch grains in Pulsatilla chinensis (Bung) Regel have been studied at light microscopic level with histochemical test. Based upon the observations, the main conclusions are summarized as follows: The mature pollen grains are two-celled in which the generative cell shows the stronger protein staining than the vegetative cell. And vegetative cells are full of starch garins. When the pollen tube enters into the embryo sac, one synergid is destroyed, or in a few cases synergids are intact. Occasionally two synergids are disorganized as pollen tube penetrates. However, most of the remaining syuergids break down during fertilization, only in a few cases it remains till early stage of embryo development. The contents discharged by the pollen tube consist of two sperms, which stain intensely blue with protein dyes, a great amount of protein and starch grains. Mature female gametophyte (embryo sac) consists of an egg apparatus, central cell, which has a huge secondary nucleus, and antipodal apparatus which retain in course of fertilization. A few of embryo sac contain two sets of egg apparatus, a central cell with two huge secondary nuclei and two sets of antipodal apparatus. In some nucleoli of the central cell the comb-like structure pattern may be detected clearly. There are 1–2 small nucleoli in some egg cells and central cells. All the cells in embryo sac show protein positive reaction. According to the different shades of the color in cells, its may be arranged in the following order: antipodal cells, synergids, central cell and egg cell. Only a few small starch grains are present near nuclei of central cell and egg cell before fertilization, but no starch grains remain in most of the central cell, the synergids and antipodal cells. The fertilization is of the premitotic type. The fusion of the sexual nuclei progresses in the following order: 1, sperms approach and lie on the egg nucleus and secondary nucleus; 2, sperm chromatin sinks themselves into female nucleus, and male nucleolus emerges with the sperm chromosome; and 3, male nucleoli fuse with the nucleoli of egg nucleus and central cell nucleus, and finally forming the zygote and the primary endosperm cells respectively. Nevertheless, as it is well known, the fertilization completes in central cell obviously earlier than that in egg cell. Though it has been explained in cereals and cotton, in Pulsatilla chinensis the main reason is that nucleolar fusion of the male and female nucleoli in egg nucleus is slower than that in secondary nucleus. And the dormancy of the primary endosperm nucleus is shorter than that of the zygote. In the process of fertilization, histochemical changes are considerably obvious in the following three parts: 1, from the begining of fusion of male and female nuclei to form zygote and primary endosperm cell, Protein staining around female nucleus appears to increase gradually; 2, no starch grains are detected in embryo sac. Though only starch grains are carried in by pollen tube, they are completely exhausted during this period; and 3, near completion of fertilization starch grains appear again in zygote, however, not yet in primary endosperm nucleus till its dividing for the first time. The present study reveals that antipodal cells and synergids seem to play a significant role in nutrition of the embryo sac during the fertilization.     

Invvestigations on Early Embryogenesis of Actinidia chinensis Planch var. chinensis

This paper deals with early embryogenesis of Actinidia chinensis var. chinensis. 1. Ovary superior consists of 34—45 carpels. Each carpel contains 11–45 ovules. The ovule is uni-integument and tenuinucellar. The ovule is anatropous. The archesporium is formed by a single cell, and directly develops into megaspore mother cell. Sometimes the archesporium consists of 2–3 cells, but only one of them develops into megaspore mother cell and the others are degenerated. 2. The mature pollen grain is two-celled and the embryo sac belongs to olygonum type. In most embryo sacs two polar nuclei are fused before fertilization. One of the synergids was destroyed as the pollen tube penetrated into embryo sac the other one disappeared after fertilization. In most cases the antipodal cells became degenerated in fertilization process, only some remained until the first division of primary endosperm nucleus. 3. In Beijing area the double fertilization of Actinidia chinensis occurred 30–72 hours after pollination. In the fertilization one sperm fused with egg nucleus and the other sperm fused with the secondary nucleus as usual. The fusion of the secondary nucleus with sperm was in advance of the fusion of the egg nudeus. 4. The endosperm is cellular type.     

Experimental insights on the function of ancillary pollen and stigma polymorphisms in plants with heteromorphic incompatibility

Most heterostylous plants possess a reciprocal arrangement of stigmas and anthers (reciprocal herkogamy), heteromorphic self‐incompatibility, and ancillary polymorphisms of pollen and stigmas. The topographical complementarity hypothesis proposes that ancillary polymorphisms function in the rejection of incompatible pollen thus promoting disassortative pollination. Here, we test this hypothesis by investigating patterns of pollen transfer and capture in populations of dimorphic Armeria maritima and A. pubigera and distylous Limonium vulgare (Plumbaginaceae), and by studying pollen adherence and germination patterns in A. maritima following controlled hand‐pollinations. Armeria lacks reciprocal herkogamy allowing the evaluation of the extent to which ancillary polymorphisms affect the composition of pollen loads. We compared the amounts of compatible and incompatible pollen on stigmas in natural populations and calculated the proficiencies of pollen transfer for each mating type. We detected disassortative pollination in each species, and mating types did not differ in compatible pollen capture, although cob stigmas captured more incompatible pollen. Controlled hand‐pollinations revealed the failure of incompatible pollen to adhere and germinate on stigmas. Our results provided evidence that, while structural in nature, pollen‐stigma dimorphisms are tightly associated with heteromorphic incompatibility and likely function to promote disassortative pollination, especially in the absence of reciprocal herkogamy.     

Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey

One major player known to be essential for successful gamete interactions during double fertilization in Arabidopsis thaliana is the recently identified family of egg cell-secreted EC1 proteins. Both gamete fusion events are affected in EC1-deficient female gametophytes. Here, we show that the number of ovules with unfused sperm cells is considerably higher than the number of undeveloped seeds in the same ec1-RNAi knockdown lines. We found that some sperm cells are able to fuse with the female gametes even 2 to 3 days after pollination, as reflected by delayed embryo and endosperm development, and by polytubey. We propose that the egg cell secretes EC1 proteins upon sperm arrival to promote rapid sperm activation, thereby accelerating gamete fusion and preventing polytubey.     

Nuclear behavior is defective in the maize (Zea mays L.) lethal ovule2 female gametophyte

It has long been known that the maize lethal ovule2 mutation results in ovule abortion but has a much smaller effect on pollen development or function. The behavior of the nuclei, the microtubular cytoskeleton and other events were examined in normal and lo2 mutant female gametophytes in order to obtain an understanding the role of this gene in embryo sac formation. The effect of the lo2 mutation is manifested following meiosis. When the surviving single megaspore carries the mutant lo2 allele, often both the megaspore and its nucleus greatly enlarge, but the nucleus either fails to divide or divides only once or twice. Micronuclei are frequently present, nuclei are often clustered and the abundance and patterns of microtubules are abnormal in the mutant embryo sacs. The mutant female gametophytes are blocked at the one-, two- or four-nucleate stage. Nearly all the embryo sacs containing the lo2 allele fail to function as evidenced by the failure of transmission of closely linked loci. When mutant female gametophyte development is arrested, the immature embryo sac degenerates. This mutation appears to identify a gene that is essential in the female gametophyte for normal nuclear division and migration and the normal accompanying tubulin cytoskeleton behavior.