Cytological Analyses on Development of Male and Female Gametophytes in an Interspecific Hybrid F1 from Cucumis hystrix Chakr. x Cucumis sativus L.

An interspecific hybrid F1 of Cucumis hystrix Chakr. x Cucumis sativus L. （NC4406） was used to establish the developmental sequence and to characterize the male and female gametophytes at cytological level for further understanding of the phylogenic relationship and the mechanism of fertility or sterility in the interspecific hybrid F1 The development of male and female gametophytes was studied through meiotic analysis and paraffin section observation technique, respectively. Meanwhile, the fertility level was assessed through hybrid F1 backcrossing to cultivated cucumber 4406. Variable chromosome configurations were observed in the pollen mother cells （PMCs） of hybrid F1 at metaphase Ⅰ , e.g., univalents, bivalents, trivalents, quadravalents, etc. At anaphase Ⅰ and Ⅱ, chromosome lagging and bridges were frequently observed as well, which led to the formation of polyads and only a partial number of microspores could develop into fertile pollen grains （about 23.3%）. Observations of the paraffin sections showed numerous degenerated and abnormal embryo sacs during the development of female gametophytes, and only 40% of the female gametophytes could develop into normal eight-nuclear megaspore. On an average, 22.8 and 6.3 seeds per fruit could be obtained from the reciprocal backcross. The interspecific hybrid F1 of C. hystrix x NC4406 was partially fertile; however, the meiotic behaviors of hybrid F1 showed a high level of intergenomic recombination between C. hystrix and C. sativus chromosomes, which indicated that it plays an important role for introgression of useful traits from C. hystrix into C. sativus.

EFFECTS OF ULTRASONIC TREATMENT ON FEMALE GAMETOPHYTES OF LAMINARIA JAPONICA (PHAEOPHYTA)

This study on the effects of ultrasonic treatment on female gametophytes of Laminariajaponica showed that:1. Ultrasonic treatment had shortening effect on filaments of female gametophytes. Within certainperiod of time, the average length of filamentous female gametophytes was shortened.2. Ultrasonic treatment had emptying effect on cells. The number of empty cells increased with timeof treatment. Ultrasonic treatment had harmful effect on cells.3. Ultrasonic treatment could break down cell walls. The combination of frequency of 20 kHz, out-put of 15 W, 40 s and 60 s of treatment was best for this purpose. After ultrasonic treatment, the regner-ation of female gametophytes into sporophytes was effected. Female gametophytes could not recover aftertoo long period of treatment.

Laminaria gametophyte clone culture and its application in sporeling cultivation

In China, the total annual production of cultured Laminaria japonica reached half million tons in dry weight there few years. The routine sporeling culture technique conducted in the greenhouse took at least three and half months. In such a case, sometimes the sporelings died within a few days due to destructive diseases. In Order to overcome the mentioned problems, a new sporeling culture technique, the clone technique, is developed. The method includes three stead: (1) Gametophyte clone culture. The spores and the gametophytes are cloned in flasks under favorable environments. (2) Sporeling cul ture. Male and female clones are crushed and spread onto a frame to allow the gametophytes to attach to the substrata. The frames are cultured in tanks, and the sporophytes reach 1 cm in length within one and a half months. (3) Outgrowing of the plant. The frames are put in the open sea when seawater temperature decreased to 20℃. After one month, the sporelings are large enough to be transplanted. It is concluded that the clone technique has the following advantages: (1)Large amount of clones can be produced in a short period of time. (2) Clone seeding method makes it free from the biological rhythm, one can seed the plant anytime all the year round. (3) It takes only one and a half months to complete the process of sporeling cultivation in the greenhouse. At present, this technique is used in the breeding of new strains of Laminaria.

Microsporogenesis and Male Gametophyte Development of Euphorbar pulcherrima

The microsporogenesis and male gametophyte development of Euphorbia pulcherrima were studied with paraffin section method. The results showed that the anther of Euphorbia pulcherrima had four chambers. The development of anther wall was dicotyledonous type; its wall consisted of epidermis, endothecium, middle layer and tapetum. The mature male gametophyte development experienced seven major stages as the followings： archesporial cells, aporgenous cells, microspore mother cells, tetrads of microspores （tetrahedral）, uninucleate microspores, 2-uncleate pollens and 2-celled pollen stage. The anther wall varied with the pollen development： the epidermis of anther ruptures at the anther mature stage; the middle layer disappears at tetrad stage; the endothelium became fibrous layer at the anther mature stage; the tapetum （glandular） was the most development at the microspore mother cells stage and disappeared at anther mature stage. The abnormal anther development was observed simultaneously.

Physiological differences in the growth and maturation of Eisenia bicyclis and Ecklonia cava gametophytes in Korea

The objective of this study was to examine the eff ects of temperature and light intensity on growth of female gametophytes of Eisenia bicyclis and Ecklonia cava and responses of these female gametophytes to Fe addition and daylength. Female gametophytes of each species were cultured at four temperatures (10, 15, 20, and 25°C) and under a combination of four light intensities (10, 20, 40, and 80 μmol photons/(m^2 ·s)) with two temperatures (15 and 20°C for E i . bicyclis;20 and 25°C for E c . cava ) to clarify their optimal growth conditions. Growth and maturation of female gametophytes of these two species under a combination of five Fe-EDTA concentrations (0, 1, 2, 4, and 8 μmol/L) and three daylengths (10, 12, and 14 h) were also examined. The growth of E i . bicyclis gametophyte was maximal at approximately 15–20°C, 20 μmol photons/(m^2 ·s), Fe-EDTA concentration of 8 μmol/L and daylengths of 12–14 h. While E c . c ava gametophytes showed optimal growth at approximately 20–25°C, 20 μmol photons/(m^2 ·s), Fe- EDTA concentration of 8 μmol/L and daylength of 14 h. Maturation of gametophytes was enhanced at Fe-EDTA concentration of 4 μmol/L for E i . bicyclis and at 2 μmol/L for E c . c ava . In conclusion, optimal growth temperatures and Fe-EDTA concentrations for maturation of E i . bicyclis and E c . c ava gametophytes were diff erent. Higher optimal growth temperature for E c . c ava gametophytes may contribute to its wider geographical distribution compared to E i . bicyclis which has restricted habitats in Korea. This suggests that addition of Fe ion could be used to recover beds of these two species in barren grounds of Korea.

Biological Features of Flowering and Development of Male Gametophyte in Anthurium andreanum

The aim of this study is to follow each development stage of inflorescence in order to understand the biological feature of flowering and the development of male gametophyte in Anthurium andreanum “Arizona' and to try to find the optimum conditions for its pollination. The methods of dissection and paraffin section were adopted to examine the structural characteristics of anthurium’s tiny floret and the development of the microspore. All the florets of the anthurium arrange on the rhachis helically sub- tended by a colorful bract. Each tiny floret has one gynoecium, four tepals and four stamina. The bract and the florets show different colors during the whole blooming period. The ovary is bicarpellary and has two locules, each of which has one anatropous ovule. The placenta is of a central placentation type. The stylar canal cells not only can produce the secretory mucilage but also can release their own cytoplasm caused by their self-disintegration before the pistil reaches its maturity. The wall of the anther is composed of four layers: epidermis, endothecium, middle layer and tapetum. The tapetal cells and the middle layers’ cells degenerated completely dur- ing meiosis of microsporocytes. The pollen grains were 2-celled at the time of anther dehiscence. Early morning, when the inflores- cences stay at their fifth development stage, is the optimum opportunity for pistil to get pollen grains. The pollen-collection should be done at the end of the seventh stage.

Florogenesis and Female Gametophyte Development in <i>Allium cepa</i>L. cv. Krishnapuram

Florogenesis is one of the most complicated and interesting processes in the nature. This process involves developmental, physiological and molecular events leading to transformation from vegetative to reproductive phase for optimal seed production and the continuation of species. The basic knowledge about flowering processes, male and female sexual systems support basic and applied research and breeding programs. Most of the onion varieties from India are short day varieties, more diverse than other exotic germplasm and useful as a source of new alleles for supporting breeding programs. The present investigation was focused to study for the first time florogenesis process by scanning electron study for the first time florogenesis process by scanning electron microscopy (SEM) and development of female gametophytes by light microscopy in order to acquire basic knowledge useful for optimizing in vitro process to produce gynogenic haploid to support and speed breeding program in short-day onion Allium cepa L. cv. Krishnapuram (KP) or Bangalore Rose. This study revealed that shoot primordium differentiated into inflorescence meristem in the month of December, while seeds were planted in the field in the September. The individual florets are preceded by a variying number of floral initials. The female gametophyte developed from chalazal side megaspore. The embryo sac development is a bisporic Allium type showing short-lived antipodals. The histological study suggests that the use of big or preanthesis flower buds with embryo sac for production of gynogenic haploids to support breeding program in onion cv. Krishnapuram (KP). However further studies are needed for confirmation of this observation.

Microsporogenesis and development of male gametophyte of B.rossica Fedtsch.et Flerov

There were four microsporanges in the mature anther of B. rossica Fedtsch. et Flerov. The development of the anther wall belonged to dicotyledonous type. The tapetum cell was dichotypic. Simultaneous cytocinesis in the microspore mother cells formed tetrad by reduction division and the array of microspore tetrads was different. There were tetrahedron type and bilateral symmetry type. The male gametophyte was 2-cell. The mature pollen was global and tricolporate. All those results were investigated under both light microscope （LM） and electron microscope （EM）

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales,Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been inte-grated into the genome of gametophytes of L. japonica, and the expression product showed the ex-pected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

GEX3, Expressed in the Male Gametophyte and in the Egg Cell of Arabidopsisthaliana, Is Essential for Micropylar Pollen Tube Guidance and Plays a Role during Early Embryogenesis

Double fertilization in flowering plants occurs when the two sperm cells, carried by the pollen tube, are released in a synergid cell of the embryo sac and then fertilize the egg and the central cell. Proteins on the surfaces of the sperm, egg, central, and synergid cells might be important for guidance and recognition/fusion of the gametes. Here, we present functional analyses ofArabidopsis GEX3, which encodes a plasma membrane-localized protein that has homologs in other plants. GEX3 is expressed in both the vegetative and sperm cells of the male gametophyte and in the egg cell of the female gametophyte. Transgenic lines in which GEX3 was down-regulated or overexpressed, using the Arabidopsis GEX2 promoter, had reduced seed set. Reciprocal crosses and imaging after pollination with a reporter line showed that, in both cases, the defect causing reduced seed set occurred in the female. In the antisense lines, micropylar pollen tube guidance failed, In the overexpression lines, fertilization of mutant ovules was mostly blocked because pollen tube guidance failed, although, occasionally, non-viable embryos were formed. We conclude that properly regulated expression of GEX3 in the egg cell of Arabidopsis is essential for pollen tube guidance.

Assessment of Genetic Diversities of Selected Laminaria （Laminariales, Phaeophyta） Gametophytes by Inter-Simple Sequence Repeat Analysis

Abstract: Inter-simple sequence repeat (ISSR) analysis was used to assess genetic diversity among 10 pairs of male and female Laminaria gametophytes. A total of 58 amplification loci was obtained from 10 selected ISSR primers, of which 34 revealed polymorphism among the gametophytes. Genetic distances were calculated with the Dice coefficient ranging from 0.006 to 0.223. A dendrogram based on the unweighted pair-group method arithmetic (UPGMA) average showed that most male and female gametophytes of the same species were clustered together and that 10 pairs of gametophytes were divided into four groups. This was generally consistent with the taxonomic categories. The main group consisted of six pairs of gametophytes, which were selected from Laminaria japonica Aresch. by intensive inbreeding through artificial hybridization. One specific marker was cloned, but was not converted successfully into a sequence characterized amplified region (SCAR) marker. Our results demonstrate the feasibility of applying ISSR markers to evaluate Laminaria germplasm diversities.

Inhibition of Ageratina adenophora on Spore Germination and Gametophyte Development of Macrothelypteris torresiana

Allelopathy of Ageratina adenophora plays an important role in its invasion. However, we have little knowledge of its allelpathic effects on ferns. In Petri dish bioassays, the inhibitory potential of aqueous leachates from roots, stems and leaves of A. adenophora was studied on the spore germination and gametophyte development of Macrothelypteris torresiana. All leachatss inhibited the spore germination and growth of the first rhizoid of M. torresiana and inhibitory effects increased with increasing leachate concentrations. Root leachates proved most inhibitory. Gametophyte rhizoids of M. torresiana treated with stem and leaf leachates of A. adenophora were erect, which was similar to those of the control. However, gametophyte rhizoids of M. torresiana treated with root leachates of A. adenophora were erect, but also curving or swollen. Moreover, curving and swollen rhizoids increased with increasing concentrations. As time went by, rhizoids treated with root leachates were not so curved and the swelling almost disappeared. Possible causes are discussed in the present study. The increasing concentrations of leaf leachates also delayed the stages of gametophyte development. With the treatment of root leachates, the delay was more obvious. Thus A. adenophora inhibited the spore germination and gametophyte development of M. torresiana and the root leachates were most inhibitory.

Effect of Heavy Metals （Cd, Cu） on the Gametophytes of Laminaria japonica Aresch

Effects of various concentrations of two heavy metals, namely Cd and Cu, on gametophytes of Laminariajaponica Aresch were determined by recording morphological changes of gametophytes, determining pH values and the heavy metal content of the culture solution, calculating the germination rate of sporophytes, and observing heavy metal （Cd） distribution using a fluorescence microscope. The results showed that heavy metals damaged the gametophytes, and were even lethal, and that the higher the concentration of heavy metal ions, the greater the injury to gametophytes. Gametophytes could not survive in culture solutions containing more than 100 mg/L Cd and 50 mg/L Cu and were only able to survive in culture solution containing a mixture of Cd and Cu up to a concentration of 10 mg/L, which indicates that gametophytes have a higher tolerance to Cd than Cu and that multiple heavy metal ions in solution markedly aggravate the damage to gametophytes compared with individual heavy metal ions. With increases in the concentration of the heavy metal, the burgeoning rate of sporophytes decreased acutely, and solutions containing multiple heavy metal ions caused even more marked harm to sporophytes than solutions containing a single heavy metal ion, because most sporophytes died in mixed solutions. The pH value of the culture medium dropped immediately at the beginning （the first day） of treatment, increased over the following days, and then decreased again. The pH of culture media containing multiple heavy metal ions showed greater variation than media containing a single heavy metal ion, with the extent of the decrease in pH of culture media containing multiple ions being greatest during the last period of the experiment. With increases in the concentration of heavy metals, the capacity of gametophytes to accumulate these ions increased. The blue fluorescent light emitted by the Cd-and Cd-binding protein complex existing in gametophytes in media containing different concentrations of Cd showed clearly the distribution of the ion in gametophytes and the results obtained were consistent with distribution determined using other methods. All results of the present study showed that gametophytes of L. japonica play a remarkable role as heavy metal decontaminators, especially with regard to Cd.

Epigenetic Repression of Male Gametophyte- Specific Genes in the Arabidopsis Sporophyte

Tissue formation, the identity of cells, and the functions they fulfill, are results of gene regulation. The male gametophyte of plants, pollen, is outstanding in this respect as several hundred genes expressed in pollen are not expressed in the sporophyte. How pollen-specific genes are down-regulated in the sporophyte has yet to be established. In this study, we have performed a bioinformatics analysis of publicly available genome-wide epigenetics data of sev- eral sporophytic tissues. By combining this analysis with DNase ! footprinting data, we assessed means by which the repression of pollen-specific genes in the Arabidopsis sporophyte is conferred. Our findings show that, in seedlings, the majority of pollen-specific genes are associated with histone-3 marked by mono- or trimethylation of Lys-27 （H3K27me1/ H3K27me3）, both of which are repressive markers for gene expression in the sporophyte. Analysis of DNase footprint profiles of pollen-specific genes in the sporophyte displayed closed chromatin proximal to the start codon. We describe a model of two-staged gene regulation in which a lack of nucleosome-free regions in promoters and histone modifications in open reading frames repress pollen-specific genes in the sporophyte.

Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana

The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase （NAGK）, which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium, nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine, nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Loss- of-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion, our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.

Programmed cell death may act as a surveillance mechanism to safeguard male gametophyte development in Arabidopsis

Programmed cell death(PCD)plays an important role in plant growth and development as well as in stress responses.During male gametophyte development,it has been proposed that PCD may act as a cellular surveillance mechanism to ensure successful progression of male gametogenesis,and this suicide protective machinery is repressed under favorable growth conditions.However,the regulatory mechanism of male gametophyte-specific PCD remains unknown.Here,we report the use of a TdT-mediated dUTP nick-end labelingbased strategy for genetic screening of Arabidopsis mutants that present PCD phenotype during male gametophyte development.By using this approach,we identified 12 mutants,designated as pcd in male gametogenesis(pig).pig mutants are defective at various stages of male gametophyte development,among which nine pig mutants show a microspore-specific PCD phenotype occurring mainly around pollen mitosis I or the bicellular stage.The PIG1 gene was identified by map-based cloning,and was found to be identical to ATAXIA TELANGIECTASIA MUTATED(ATM),a highly conserved gene in eukaryotes and a key regulator of the DNA damage response.Our results suggest that PCD may act as a general mechanism to safeguard the entire process of male gametophyte development.

Accumulation and Speciation of Arsenic in Pteris vittata Gametophytes and Sporophytes: Effects of Calcium and Phosphorus

Dear Editor, Environmental arsenic (As) contamination of both soil and water systems is a worldwide problem, and As can enter the human food chain through contaminated agricultural products and drinking water and contribute to the development of lung.

Effects of inorganic carbon concentration and pH on carbonic anhydrase activity of gametophytes of Saccharina japonica

Carbonic anhydrase(CA)was considered to be an important component of carbon concentrating mechanism(CCM)of algae.It was an inducible enzyme.Environmental factors,especially dissolved inorganic carbon and pH,were known to affect CA activity.Effects of inorganic carbon(CO_(2)and HCO_(3)^(-))and pH on CA activity of gametophytes of Saccharina japonica were evaluated in this study.Under high-CO_(2)condition(3%CO_(2)),the activity of external CA(C_(Aext))was significantly decreased(P<0.05)from 33.92 REA/g FW to 27.69 REA/g FW.In contrast,the internal CA(CA_(int))and total CA activities were elevated significantly(p<0.01)from 36.83 REA/g FW to 48.80 REA/g FW,and from 70.75 REA/g FW to 76.49 REA/g FW,respectively.Addition of an appropriate concentration of HCO_(3)^(-)to the medium,CA_(int)and total CA activities were promoted significantly,although the activity of C_(Aext)was inhibited significantly(P<0.05).In higher HCO_(3)^(-)concentration(up to 1680 mg/L),the activity of CA_(int)was promoted to 60.81REA/g FW,which was twice that of the control.Higher activities of CA_(int)and total CA were induced at low pH.At pH 6.0,the highest activities of CA_(int)(110.85 REA/g FW)and total CA(128.17 REA/g FW)were induced.However,at pH 9.0,the activities of CA_(int)and total CA were reduced to the lowest of 23.31REA/g FW and 42.19 REA/g FW,respectively.This is the first report about the successful detection of C_(Aext)activity of gametophytes of S.japonica.The results would provide data for the analysis of Ci acquisition and transport mechanism in S.japonica gametophytes.

Arabidopsis BIG1 and BIG5 are crucial for male gametophyte transmission

Arabidopsis contains five Brefeldin Ainhibited guanine nucleotide exchange factors(BIGs),which play a critical role in vesicle biogenesis for protein traffic from the Golgi to the plasma membrane.Biological processes regulated by BIG1-BIG4 are postulated to be distinct from those by BIG5. However, we show that the self-pollinated BIG1+/- big5 silique do not produce homozygous seeds, and some pollen tubes from BIG1+/- big5 anthers grew slowly in vitro and failed to target nearby ovules in vivo. We identified the big1 big5 homozygote from the progeny of BIG1+/- big5 plants transformed with BIG5, whose expression is driven by a pollen-specific promoter p Lat52, indicating that male gametophyte transmission is blocked in the double mutant. Confocal microscopy indicated that BIG1 and BIG5 are co-localized in trans Golgi network. Thus,our data indicate that BIG1 and BIG5 are crucial for male gametophyte transmission.

Arabidopsis thaliana NOP10 is required for gametophyte formation

The female gametophyte is crucial for sexual reproduction of higher plants, yet little is known about the molecular mechanisms underlying its development. Here, we report that Arabidopsis thaliana NOP10 （AtNOP10） is required for female gametophyte formation. AtNOP10 was expressed predominantly in the seedling and reproductive tissues, including anthers, pollen grains, and ovules. Mutations in AtNOP10 interrupted mitosis of the functional megaspore during early development and prevented polar nuclear fusion in the embryo sacs. AtNOP10 shares a high level of amino acid sequence similarity with Saccharornycescerevisiae （yeast） NOPIo （ScNOP1o）, an important compo- nent of the H/ACA small nucleolar ribonucleoprotein particles （H/ACA snoRNPs） implicated in 18S rRNA synthe- sis and rRNA pseudouridylation. Heterologous expression of ScNOP10 complemented the mutant phenotype of Atnop10. Thus, AtNOPIo influences functional megaspore mitosis and polar nuclear fusion during gametophyte formation in Arabidopsis.