Using Isolated Embryo Sacs and Early Proembryos for Localization of Calmodulin mRNA Before and After Fertilization in Nicotiana

An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to small amounts of materials in which a whole view of CaM mRNA distribution can be obtained. The authors revealed that CaM mRNA expression changes dramatically before and after fertilization. Especially interesting is that a prominent CaM mRNA band appears between the egg apparatus and polar nuclei temporarily during the period of pollination and fertilization. The band disappears just prior to fertilization and expands to a fan_shaped region that occupies the micropylar portion of the embryo sac. After fertilization, CaM mRNA accumulates in the elongated zygotes with higher concentration in their chalazal portion than in the micropylar portion. Such an asymmetrical pattern continues to manifest in the early proembryos. It is supposed that CaM mRNA may be involved in the early events and signaling steps associated with double fertilization and zygote polarization in higher plants.

Embryogenesis of Polyembryonic Rice ApⅢ: Structural and Histochemical Studies of Egg Apparatus Around Fertilization

The structural and histochemical changes of the egg apparatus in the polyembryonic rice ( Oryza sativa L.), ApⅢ with the highest frequence of additional embryos among the polyembryonic rice investigated, before and after fertilization were studied and compared with those of normal and other polyembryonic rices in a similar developmental period. A total of 2 932 ovules were observed and each of them contained only a single embryo sac with a set of egg apparatus. Among 1 655 embryo sacs, there were 1 643 embryo sacs (99.27%) with one normal egg apparatus in each embryo sac, and only 12 embryo sacs (0.73%) from the remainder with 4_celled egg apparatus, i.e. two eggs and two synergids. Neither the numerous poly_egg apparatus and egg_like cells, nor the double set of embryo sacs each containing one egg apparatus and other abnormal egg apparatus in single ovary, which were reported by earlier investigators to have high frequency of embryo production in SB_1 and ApⅣ, were observed. The egg cell was located at the subterminal site of the micropylar end of embryo sac. The cytoplasm of egg cell was rich in protein materials and polysaccharide grains, which did not disappear until the division of zygote. The prominent nucleus was closely surrounded by protein and polysaccharide grains, which did not disappear until the division of zygote. No cytological difference was found between egg cells from the normal and abnormal egg apparatus. The two synergids were fully developed and situated at the upper most part of the micropylar end of the mature embryo sac. In most embryo sacs, the synergids were flask_shaped with longer necks, and a widened cap_shaped top, in close contact with the micropyle. The synergids had a well developed filiform apparatus. The characteristic appearance of the filiform apparatus as well as the cap_neck region of synergids before and after pollen tube penetration were easily distinguishable from the egg cell. The structure, the stainability with Coomassie Brilliant Blue and PAS reaction, the process of accumulation, distribution and disapperance of the cytoplasmic protein materials and polysaccharide grains of the two synergids, the persistent and rarely the receptive synergids before and after pollen tube penetration, were closely similar to those of egg cell of the same developmental stage. In comparison with normal and other polyembryonic rice reported, the size of nucleus and nucleolus and their stainability also strongly resembled those of egg cell. Based on the results observed, the main conclusions are summarized as follows: (1) the additional embryos very frequently developed in the young and mature seed of polyembryonic rice ApⅢ were produced by one or two synergids of normal egg apparatus, rarely by 4_celled egg apparatus; (2) during fertilization, the synergids, in addition to the natural specific function of introducing pollen tube and transferring sperms to egg cell and central cell, could be closely associated with the potentiality to breed one or two additional embryos; and (3) as compared with that of normal or other polyembryonic rice it is firstly disclosed that in a few embryo sacs of ApⅢ, the cytoplasmic and nuclear structure, the active anabolism and catabolism of protein and polysaccharide materials and the delayed disorganization at the mid_basal region of the receptive and persistent synergid still remained unchanged before the division of zygote. Such salient features could be the predisposition for the origin of additional embryos in ApⅢ.

CHANGES OF MICROTUBULAR ORGANIZATION DURING THE EMBRYO SAC DEVELOPMENT IN RICE

Changes in the patterns of microtubular distributions and organization in various stages of development of the embryo sac in rice ( Oryza saliva L.) were different. In the megasporocyte, most microtubules were radially distributed but some were longitudinally oriented. Similar distributional patterns were seen in the dyad cells and the functional megaspore. Microtubules in the uni-nucleate embryo sac were mostly randomly distributed, although some radiated type was also present. The pattern of distribution of the microtubules in the two and four-nucleate embryo sac was quite similar and the microtubules were mainly of the perinuclear type. Microtubules in the cells of the eight-nucleate embryo sac were more complex. In the egg cell, the microtubules were mostly randomly distributed whereas in the synergids they were predominantly in longitudinal alignment. Those in the central cell were transversely aligned. The antipodal mass had very few microtubules and few longitudinally aligned ones were present in the cytoplasm.

Further Studies on Microtubule Organizational Changes During Megagametogenesis in Rice Embryo Sac

Changes in the pattern of microtubule distribution and organization during megagametogenesis in the embryo sac of rice (Oryza sativa L. cv. IR36) were re-examined using a modified polyethylene glycol sectioning technique before immuno-fluorescence staining of microtubules. In the sectioned materials the pattern of distribution and structural organization of the microtubule cytoskeleton were quite well preserved. Fine details of the patterns of structural changes and re-organization of the microtubule cytoskeleton in the major stages of development during embryo sac megagametogenesis (viz. functional megaspore, uni-nucleate, 2-nucleate, 4-nucleate, 8-nucleate and mature stage) could be clearly observed and easily followed. Some new organizational patterns of microtubules associated with the probable movement and positioning of the polar nuclei were observed.

A New Type of Embryo Sac in Cardiopteris and Its Systematic Implication

报道心翼果属 (CardiopterisWall.exRoyle)一种新的胚囊类型 :胚珠无珠被、直生、薄珠心。成熟胚囊中 ,卵器位于合点端 ,反足细胞位于珠孔端。这样的胚囊在所有进行过胚胎学研究的被子植物中系首次发现。合子和胚胎也位于合点端。心翼果属的这一新特征无疑是特异性的 。

Changes of Calmodulin Distribution in the Embryo Sac of Oryza sativa Before and After Fertilization: an Immunogold Electron Microscope Study

Changes of calmodulin (CaM) distribution in the embryo sac of rice (Oryza sativa subsp. Japonica) at various stages before and after fertilization have been investigated by using immunogold electron microscopy. Before pollination, both cytoplasm and vacuoles of the egg cell, synergids and central cell were labeled by gold particles. A small amount of gold particles were localized in the nucleus, endoplasmic reticulum, mitochondria and dictyosomes. From pollination to fertilization, CaM amount increased in these cells, especially rich in the starch of amyloplasts. Increase of gold particles in the central cell began about 2 h earlier than that in the egg cell. There was no distinct difference of CaM amount between the degenerated and the persistent synergids. It is interesting to observe an obvious change of CaM distribution form during pollination and fertilization from scattered single particles to clustered particles, and back again to single particles after the fertilization finished. CaM was also localized extracellularly in the embryo sac wall as well as in the wall and intercellular space of nucellus cells. The extracellular CaM also changes in its amount and form after pollination. These results suggest that CaM, either intra- or extra-cellular, may play important roles in fertilization and zygote formation.

Abnormal Structure of Embryo Sac in Autotetraploid Rice

The structures of mature embryo sacs in 13 genetic stock lines of autotetraploid rice （Oryza sativa L.）, including indica, japonica and javanica, were studied by using the whole-mount stain-clearing laser scanning confocal microscopy （WCLSM）. Among the 13 autotetraploid rice, the majority of ovaries possess normal polygonum-type embryo sacs, while a few ovaries were characterized by abnormal embryo sacs. The abnormalities of embryo sacs could be classified into six categories, i. e. no female germ unit, abnormal polar nuclei, embryo sac degeneration, no egg apparatus, small embryo sac and ＇double set＇ of embryo sacs. The frequency of abnormal embryo sac in japonica （26.6%） was higher than that in indica （19.34%）. In addition, the major abnormalities in each autotetraploid line varied, suggesting that the abnormalities may be related to the genotypes of the varieties.

Production of Partial New-typed Brassica Napus by Introgression of Genomic Components from B. rapa and B. carinata

A breeding strategy for widening the germplasm of Brassica napus was proposed by introgression of the A^r subgenome of B. rapa （A^rA^r） and C^c of B. carinata （B^cB^cC^cC^c） into natural B. napus （AnAncncn）. The progenies with 38 chromosomes that were derived from the self-pollinated seeds of pentaploid hybrids （A^rA^nB^cC^cC^n） were used for further research. Some of the partial new-typed B. napus showed normal meiotic behavior, high portion of germinated pollen and normal embryological development. This indicates that the selected new-typed B. napus had a balanced genetic base. Molecular analysis showed that about 50% of the genome in the new-typed B. napus was replaced by A^r and C^c subgenome from B. rapa and B. carinata. Considering the genetic diversity among different lines of new-typed B. napus, it was deduced that the introgression of the genomic components from B, rapa and B. carinata could widen the genetic diversity of rapeseed.

Haploid Induction via In vitro Gynogenesis in Tomato(Solanum lycopersicum L.)

In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method of ovule isolation was established in this study. Using this method, 100-150 ovules could be isolated from one ovary. Isolated ovules were cultured on three induction media to induce gynogenesis in vitro. During culture, ovules were enlarged markedly, with opaque white color. When observed microscopically, there were cell divisions and cell clumps in embryo sacs. Subsequently, the cell clumps in embryo sacs ceased growth, likely because the integument grew faster than embryo sacs did and hindered the fiarther development of embryo sacs. Therefore, subsequent callus morphogenesis might be originated from the integument. Thousands ofcalli from the two tomato varieties were obtained. Five diploid plants were regenerated after 15 months of subculturing. To eliminate the hindering effect of integument on embryo sac cells, the protoplasts of embryo sacs were prepared and cultured. After 48 hours of culture, the protoplasts of embryo sacs doubled in size and gradually formed clusters of cells. These results suggested that gynogenesis might be a potential way for haploid induction in tomato.

Pseudogamous Apomixis in Maize and Sorghum in Diploid-Tetraploid Crosses

Apomictic seed development is a complex process including formation of unreduced embryo sac,parthenogenetic embryo development from the egg cell,and endosperm formation either autonomously,or due to fertilization of polar nuclei by the sperm(under pseudogamous form of apomixis).In the latter case,an obstacle to the normal endosperm development is disturbance of maternal(m)-to-paternal(p)genomic ratio 2m:1p that occurs in the cases of pollination of unreduced embryo sac with haploid sperms.Usage of tetraploid pollinators can overcome this problem because in such crosses maternal-to-paternal genomic ratio is 4m:2p that provides formation of kernels with plump endosperm.Using tetraploid lines as pollen parents we observed formation of plump kernels on the ears and panicles of diploid maize and sorghum accessions.These kernels had hybrid endosperm and diploid maternal-type embryo or hybrid embryo with different ploidy level(2n,3n,4n).The frequencies of plump kernels on the ear ranged from 0.2-0.3%to 5.7-6.2%counting from the number of ovaries.Maternal-type plants were found in two maize lines,their frequency varying from 10.7 to 37.5%of the progeny plants.In CMS-lines of sorghum pollinated with tetraploid sorghum accessions,the frequency of plump kernels ranged from 0.6 to 14.0%counting from the number of ovaries;the frequency of maternal-type plants varied from 33.0 up to 96.1%.The hybrid nature of endosperm of the kernels that gave rise to maternal-type plants has been proved by marker gene expression and by SDS-electrophoresis of endosperm proteins.These data testify to variable modes of seed formation under diploid×tetraploid crosses in maize and sorghum both by amphi-and by apomixis.Therefore,usage of tetraploid pollinators might be a promising approach for isolation of apomixis in maize and sorghum accessions.

Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid

Autotetraploid indic, a/japonica rice hybrid combines both the advantages of polyploidy and the heterosis between indica and japonica rice. Embryo sac abortion is an important factor influencing spikelet fertility in autotetraploid dce hybrid. To clarify the cytological mechanism of embryo sac abortion, the megasporogenesis and megagametogenesis in an autotetraploid japonicaAndica hybrid were examined by the whole-mount eosin B-staining confocal laser scanning microscopy （WE-CLSM） technique. Abnormalities were observed from the megasporocyte stage to the mature embryo sac stage. The degeneration of the tetrad cells and the functional megaspore was the characteristic of abnormalities during megasporogenesis. Abnormal small embryo sacs and disordered number of nuclei were frequently observed during embryo sac development. Some interesting phenomena, such as two functional megaspores, the diplospory-like megasporocyte, and five-nucleate embryo sac were found. The abnormalities that occurred during female gametophyte development resulted in more than five types of abnormal embryo sacs （i.e. embryo sac degeneration, embryo sac without female germ unit, embryo sac without egg apparatus, embryo sac with abnormal polar nuclei and abnormal small embryo sac） in autotetraploid japonica/ndica hybdd. Embryo sac fertility was lower in diploid japonica/ndica hybdd than in autotetraploid japonicaAndica hybrid although many abnormal phenomena were observed in autotetraploid hybrid.

ABORTED GAMETOPHYTE 1 is required for gametogenesis in Arabidopsis

In flowering plants, the male and female gameto- genesis is a crucial step of sexual reproduction. Although many genes have been identified as being involved in the gametogenesis process, the genetic mechanisms underlying gametogenesis remains poorly understood. We reported here characterization of the gene, ABORTED GAMETOPHYTE 1 （AOGI） that is newly identified as essential for gametogenesis in Arabidopsis thaliana. AOG1 is expressed predominantly in reproductive tissues including the developing pollen grains and ovules. The AOG1 protein shares no significant amino acid sequence similarity with other documented proteins and is located mainly in nuclei of the cells. Mutation in AOG~ caused degeneration of pollen at the uninucleate microspore stage and severe defect in embryo sacs, leading to a significant reduction in male and female fertility.Furthermore, the molecular analyses showed that the aogl mutant significantly affected the expression of several genes, which are required for gametogenesis. Our results suggest that AOG1 plays important roles in gameto- genesis at the stage prior to pollen mitosis 1 （PMI） in Arabidopsis, possibly through collaboration with other genes.

Two Tightly Linked Genes at the hsal Locus Cause Both F1 and F2 Hybrid Sterility in Rice

Molecular mechanisms of hybrid breakdown associated with sterility (F2 sterility) are poorly understood as compared with those of F1 hybrid sterility. Previously, we characterized three unlinked epistatic loci, hybrid sterility-a1 (hsa1), hsa2, and hsa3, responsible for the F2 sterility in a cross between Oryza sativa ssp. indica and japonica. In this study, we identified that the hsa1 locus contains two interacting genes, HSA1a and HSA1b, within a 30-kb region. HSA1a-j (japonica allele) encodes a highly conserved plant-specific domain of unknown function protein (DUF1618), whereasthe indica allele (HSA1a-is) has two deletion mutations that cause disruption of domain structure. The second gene, HSA1b-is, encodes an uncharacterized proteinwith some similarity to a nucleotide-binding protein. Homozygous introgression of indica HSA1a-is-HSA1b-is alleles into japonica showed female gamete abortion at an early mitotic stage. The fact that the recombinant haplotype HSA1a-j-HSA1b-is caused semi-sterility in the heterozygous state with the HSA1a-is-HSA1b-is haplotype suggests that variation in the hsa1 locus is a possible cause of the wide-spectrum sterility barriers seen in F1 hybrids and successive generations in rice. We propose a simple genetic model to explain how a single causal mechanism can drive both F1 and F2 hybrid sterility.

Phenotypic Characterization of a Female Sterile Mutant in Rice

A female sterile mutant, derived from a spontaneous mutation, was first discovered In rlce （Oryzs satlvs L. sep. Indlca） restorer llne 202R. Wlth normal fiowerlng, the mutant exhlblts an extremely low seed-sattlng rate. When the mutant Is crossed as a pollen donor, the seeds set normally; whereas when It Is used as a pollen recelver, no seeds are obtalned even wlth mlxed pollen gralns of dlfferent varletles sprlnkled over the atlgmas. The fioret of the mutant, conslatlng of slx stamens and one platll, looks the same as that of the wlld type In the malefemale organs, except that less than 10% of the mutant florets have three atlgmas on the ovary. Although the mutant has a low seed-setting rate, Its pollen fertility Is approximately 87.1%, which Is equal to that of the wild type. In addition, more than 90% of the mature embryo sacs of the mutant have complete Inner structures. At every stage after pollination, the sperm, embryo, and endosperm are not found In the mutant embryo sac, whereas the disintegration of the egg cell that does not accomplish fertilization Is visible. Through observetlons with a fluorescence microscope, we have found that the pollen grains germinate normally, whereas the pollen tube abnormally elongates in the style-transmitting tissue. The mutant pollen tubes display various defects In the style, such as slower elongation, conversed elongation, distorted elongation, swollen tips, or branched tips. As a result, the growth of the pollen tubes ceases In the style, and, therefore, the pollen tubes cannot reach the embryo sac and the process of double fertilization Is blocked. Based on these observations, we conclude that this mutant, designated as fs-202R, Is a novel type of female sterile mutation In rice, which causes the arrest of the elongation of the pollen tube.

The Arabidopsis R-SNARE protein YKT61 is essential for gametophyte development

Gametophyte development is a pre-requisite for plant reproduction and seed yield;therefore,studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture.The biogenesis and dynamics of endomembrane compartments are critical for cell survival,and their regulatory mechanisms are just beginning to be revealed.Here,we report that the Arabidopsis thaliana SNARE(soluble N-ethylmaleimide sensitive factor attachment protein receptor)protein YKT61 is essential for both male and female gametogenesis.By using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based genome editing,we demonstrated that male and female gametophytes carrying YKT61 loss-offunction alleles do not survive.Specifically,loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes.A three-base-pair deletion in YKT61 in the ykt61-3 mutant resulted in a single-amino acid deletion in the longin domain of YKT61;the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association,suggesting that the N-terminal longin domain of YKT61 plays multiple roles in its function.This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9-mediated editing and expression from a Cas9-resistant transgene.