The effect of cytokinins on growth and sexual organ development in the gametophyte of <Emphasis Type="Italic">Blechnum spicant</Emphasis> L.

In this study, we report the role of exogenous and endogenous cytokinins on growth and sexual organ development in the fern Blechnum spicant L. Spore-derived gametophytes (SG) were cultured in full-strength Murashige and Skoog (1962) liquid medium supplemented with (a) 4.44 μMN⁶-benzyladenine (BAP), (b) a crude extract from mature female gametophytes, and (c) 4.44 μM BAP in combination with the crude extract from mature gametophytes, respectively. Both BAP and the crude extract delayed the gametophyte development, and this effect was increased when they were added together. With respect to sexual organ development, BAP inhibited the sexual organ formation, while the crude extract favored antheridia formation; however, when added together, the percentage of antheridia decreased. The endogenous level of the cytokinins cis-zeatin (cZ), cis-zeatin-riboside (cZR), dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), isopentenyl-9-glucoside (iP9G), trans-zeatin (tZ), and trans-zeatin riboside (tZR) were analyzed in female and male gametophytes of B. spicant L. The endogenous levels of cytokinins tZ, cZ, DHZ, cZR, iP, and iPR were higher in female gametophytes than in male gametophytes, with the endogenous iP and iPR content being increased more than 300 and 400 times, respectively.     

Gibberellins and antheridiogen on sex in Blechnum spicant L.

The role of gibberellins (GAs) in determining sex in the gametophyte of the fern Blechnum spicant L. was studied through (a) the effect of exogenous GA₄₊₇ and GA₃ (b) quantitation of the endogenous levels of GA_1, GA₃, GA₄, GA₇, GA₉, and GA₂₀ in male and female gametophytes, and (c) the effect of flurprimidol, a GAs biosynthesis inhibitor of the steps of oxidation of ent-kaureno to ent-kaurenoic acid. Our results show that GA₄₊₇ had a slight effect of inducing either male or female sexual organs, antheridia and archegonia, respectively. The endogenous GAs content was not significantly different between sexes, but the GA₄, GA₇, and GA₂₀ levels were raised above those of the other GAs in both sexes. Neither antheridiogen biosynthesis nor antheridia formation was inhibited by flurprimidol. Gametophytes regenerated from homogenized mature gametophytes (HG) show a different physiological behavior than spore-derived gametophytes. In the first case, gametophytes are males and synthesize antheridiogen before they attain maturity, in contrast to what occurs in spore-derived gametophytes which are females and synthesize antheridiogen when mature.     

SEXUAL DEVELOPMENT OF ONOCLEA SENSIBILIS ON AGAR AND SOIL MEDIA WITHOUT THE ADDITION OF ANTHERIDIOGEN

Onoclea sensibilis gametophytes grown on agar, unsterilized soil or ashed soil without the addition of antheridiogen developed antheridia after the notch meristem was formed. There was a linear increase in maleness through time on all three media. Antheridial production on agar was delayed nearly 3 wk. Agar had a significantly higher proportion of females than ashed soil or unsterilized soil. It was concluded that agar is an inappropriate medium for experiments on the mechanism of sexual development in Onoclea gametophytes.     

麦秆蹄盖蕨（Athyrium fallaciosum）配子体的发育规律及播种密度对配子体性别分化的影响

在组织培养条件下,对麦秆蹄盖蕨（Athyrium fallaciosum）配子体发育的连续过程进行了详细观察。结果表明:麦秆蹄盖蕨孢子为四面体型; 孢子萌发为书带蕨型（Vittaria-type）;原叶体发育为铁线蕨型（Adiantum-type）,成熟原叶体为对称的心形;精子器近圆球形,成熟颈卵器细长,常向原叶体基部倾斜或弯曲。常规播种条件下,发现麦秆蹄盖蕨配子体有雌配子体、雄配子体、雌雄同体配子体和无性配子体类型。配子体的性别随密度不同而呈现一定的变化趋势,雄配子体随密度增大呈上升趋势;雌配子体随密度增大先上升后下降;雌雄同体配子体和无性配子体随密度变化不大。雌配子体和雌雄同体配子体具颈卵器数目一般为10～15个;精子器数目随密度的增大逐渐减少,雄配子体中具有约50个精子器,雌雄同体配子体具有约20个精子器。     

A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica

To determine a possible mechanism causing male and female sterility in Cryptomeria japonica male and female cones were collected from a C. japonica, tree, ShinDai2, that lacks pollen release and fertile seeds and specimens were processed to examine the development of pollen and female gametophytes using light microscopy and field emission scanning electron microscopy. Pre-meiotic development proceeded normally, but the formation of aberrant meiotic products was observed in cones of both sexes. In sterile microsporangia, heterogeneous microspore populations ranging from monads to polyads gave rise to mature pollen grains of non-uniform size. These pollen grains were covered with an amorphous layer and adhered to each other. In addition, they remained in the microsporangia and were not released even after the onset of pollen dissemination from fertile trees. In the ovules of sterile female cones, megaspores with abnormal shapes, numbers, and sizes formed, and the development of female gametophytes was arrested at the free nuclear or archegonium formation stages. These gametophytes collapsed, and no fertile embryo was generated. Results indicate that meiotic defects are important in the sterility mechanism.     

Effect of a cationic cellulosic polymer on gametophytes of Laminaria digitata (Laminariaceae, Phaeophyta): Improvement of dispersed `free-living' cultures

As filament aggregation is responsible for heterogeneity of Laminariales gametophyte cultures, a project was conducted to obtain stable homogeneous `free-living' cultures of Laminaria digitatagametophytes. The alga was cultivated at 15 ^° Cunder low light and in the presence of a cationic cellulosic polymer, JR125. With 0.1%polymer in the culture medium, the filaments were dispersed and did not adhere to the culture vessel. The absence of any effect of the closely related, but uncharged, polymer LR-250 Natrasol on filament aggregation indicated that the cationic nature of the JR125 molecule was involved in gametophyte dissociation. In the presence of JR125, the gametophytes showed active vegetative growth; the doubling time, measured as chlorophyll concentration, was 5 days. The outer surface of the cell wall was clearly modified by the polymer treatment, as observed by transmission electron microscopy, while neither the inner cell wall or cell organelles were affected. Physiological studies indicated that JR125 treatment did not disturb cell physiology, there being no effecton respiration, photosynthetic activity, sensitivity to high-light stress or modification of pigment or fluorescence characteristics. We have therefore established the conditions for maintaining a stable culture of mixed male and female Laminaria digitata gametophytes in active vegetative growth. The presence of JR 125 in the medium yields a homogeneous culture without cell physiology becoming modified. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effects of light on sex determination in gametophytes of the fern <Emphasis Type="Italic">Ceratopteris richardii</Emphasis>

The sexuality of homosporous fern gametophytes is usually determined by antheridiogen, a pheromone that promotes maleness. In this work the effect of photomorphogenically active light on antheridiogen-induced male development was examined for gametophytes of Ceratopteris richardii. Although blue light did not affect sensitivity to Ceratopteris antheridiogen (A_Ce) in wild-type gametophytes, it was found that the gametophytes of the her1 mutant, which are insensitive to A_Ce, developed into males when grown under blue light in the presence of A_Ce. Thus, we conclude that another A_Ce-signal transduction pathway activated by blue light exists latently in the gametophytes of C. richardii. Red light, on the other hand, suppressed male development. Because simultaneous red and blue light-irradiation did not promote male development in the her1 gametophytes, the action of red light seems to dominate that of blue light. The results of experiments with a photomorphogenic mutant also suggested that phytochrome may be involved in the action of red light.     

Do antheridiogens act via gametophyte size? A study of Woodwardia radicans (Blechnaceae)

For many plants, sex is not fixed by genotype but determined by environmental conditions during development. In homosporous pteridophytes, sex is environmentally determined by the presence or absence of antheridiogens, maleness-inducing pheromones. It has been proposed that antheridiogens primarily reduce growth rate, with small gametophyte size responsible for maleness. To test this hypothesis, the effects of antheridiogen and intergametophytic competition on gender expression and gametophyte size were studied in a culture experiment with Woodwardia radicans. We found that (1) antheridiogen inhibited growth of gametophytes; and (2) slow growth favored maleness, whereas fast growth favored femaleness, irrespective of the presence or absence of antheridiogen. Both conclusions are consistent with the hypothesis that, in W. radicans, antheridiogen effect is mediated by size. They also agree with the "size-advantage" hypothesis in which energetic limitations associated with relatively small individual size impose a less severe limitation for male reproductive success than for female reproductive success. The results are also discussed with regard to a genetic sex-determining pathway that has recently been identified.     

Isolation and culture of protoplasts from young sporophytes ofSalvinia natans aseptically obtained by co-culture of female and male gametophytes

Sporophytes were aseptically obtained by co-culture of female and male gametophytes derived from two types of spores (megaspores and microspores) of the heterosporous fernSalvinia natans All. Protoplasts isolated enzymatically from juvenile leaflets of sporophytes were cultured in a 1/10 Murashige and Skoog's medium containing 2.2 M naphthalene acetic acid, 2.2 M 6-benzyl-aminopurine, 0.35 M mannitol, and 0.05 M sucrose. Cell division took place within 6 days of culture, and cell-clusters composed of 9–10 cells were observed after 30 days of culture.Abbreviations BA 6-benzyl-aminopurine - MS Murashige and Skoog - NAA naphthaleneacetic acid     

THE DEVELOPMENT AND REPRODUCTIVE MORPHOLOGY OF HALOSACCION AMERICANUM I. K. LEE (RHODOPHYTA,PALMARIALES)1

Halosaccion americanum, a member of Palmariaceae, was grown in culture from spores and the life history was critically examined by the use of scanning EM and light microscopy. A mature tetrasporangium of H. americanum produces four spores that germinate to form two male and two female gametophytes. The male gametophytes grow to maturity in approximately eight months and macroscopically resemble the tetrasporophyte. Following the first division of the tetraspore, the two-celled female gametophyte consists of a vegetative cell and a carpogonium with trichogynes. Fertilization is accomplished by spermatia from male plants of the preceding generation, as male plants of the same season are immature. Spermatia are formed in a continuous layer over the surface of the mature male gametophytes and, when released, are entrained in long strands of mucous. Spermatia adhere to and fuse with trichogynes and, nuclear fusions presumably follow. The carposporophyte is absent; the new tetrasporophyte develops directly from the fertilized carpogonium. Growth of the sporophyte eventually obliterates the female gametophyte, and development into a mature tetrasporophyte proceeds over a period of approximately eight months. The development of H. americanum, with its extremely abbreviated female gametophyte stage and direct development of the tetrasporophyte from the zygote, indicates that this rhodophyte has the same life history as reported for other members of the Palmariales.     

Growth Promotion of Vegetative Gametophytes of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> by Blue Light

Through an acclimation period of 10 days, compared to white light, the maximal net photosynthetic rates were significantly higher for gametophytes of Undaria pinnatifida cultivated under blue light (400–500 nm), and were lower under red light (600–700 nm). Chlorophyll c and the carotenoid content of gametophytes were similar under blue light and red light but were much lower under white light. The growth rate of female gametophytes under blue light was higher than that under other lights, and the growth rate of male gametophytes showed little variation with respect to blue and white light. Male and female gametophytes were mixed together to form sporophytes under white, blue and red light. After approximately 5 days, 50% gametophytes became fertile under blue and white light, but remained vegetative under red light after 10 days.     

Plant regeneration and morphology during in-vitro organogenesis fromHeloniopsis orientalis (Thunb.) C. Tanaka

Heloniopsis orientalis (Liliaceae) is an important horticultural crop native to Korea. Under natural conditions, germination is poor and plant growth is delayed. Therefore, we have developed a vegetative propagation method to produce plants with vigorous growth characteristics via tissue culture. Leaf tissues were cultured on MS basal media supplemented with growth regulators 2,4-D, TDZ, BA, or zeatin. The regenerated shoots were then initiated directly from leaf expiants on an MS medium containing either 0.1 to 1.0 mg/L 2,4-D or 0.1 to 3.0 mg/L BA. Healthy plantlets with adventitious roots were formed on the medium supplemented with 0.1 mg/L BA. TDZ triggered callus initiation without caulogenesis or rhizogenesis, and callus formation was better on the half-strength MS medium than on the full-strength medium. After the plants were acclimatized for one month at 4°C, they were successfully transferred to soil. In addition, we used LM and SEM to investigate shoot morphogenesis at various stages of differentiation.     

Influence of some adjuvants on <Emphasis Type="Italic">in vitro</Emphasis> clonal propagation of male and female jojoba plants

Summary The influence of different adjuvants, activated charcoal (AC), casein hydrolyzate (CH), coconut water (CW), polyvinylpyrrolidone (PVP), and triiodobenzoic acid (TIBA), has been assessed on the shoot production potential of the nodal explants derived from in vitro-raised male and female jojoba (Simmondsia chinensis) shoots. Nodal explants of each sex were cultured separately on Murashige and Skoog medium supplemented with different levels of AC, CW, CH, PVP, and TIBA either alone or along with optimum levels of N ⁶-benzyladenine (BA; 10 μM for male, 20 μM for female). Some differences in response of the explants of both the sexes have been observed in terms of (1) percentage of explants developing shoots, (2) average shoot number, and (3) average shoot length. AC alone proved beneficial for elevating morphogenic response in male as well as female explants in comparison to basal medium or media containing AC and the optimum level of BA. When used alone, CH proved inhibitory for shoot differentiation in both sexes, especially in male explants. Addition of PVP to MS enhanced shoot proliferation in female explants only, but along with BA it increased the response of male explants. BA in combination with different levels of TIBA promoted shoot multiplication in female explants. Thus, explants of both male and female shoots exhibited differential morphogenic behavior under the influence of various adjuvants. However, BA alone proved to be the best for differentiation of shoots in both male (10 μM) as well as female (20 μM) explants.     

Gemmation in cultured gametophytes ofOsmunda regalis

Gametophytes ofOsmunda regalis were culturedin vitro to determine the optimal conditions for growth and development. Culture media with low ammonium concentration (Knop, Knudson or 1/4 Murashige and Skoog dilution) were the most effective for growth of this organism. Addition of sucrose and mannitol to the culture medium at the same osmoticum, inhibits or enhances, respectively, growth and development of the gametophytes, which show a strong autotrophyin vitro. In darkness, both two-dimensional growth and sexual organ formation took place. Gemmae formation is described for the first time in this species and this process does not occur without sucrose in the culture medium or in darkness.Abbreviations MS Murashige and Skoog medium (1962) - KD Knudson medium (1946) - KP Knop medium (1865)     

Microprojectile bombardment of <Emphasis Type="Italic">Laminaria japonica</Emphasis> gametophytes and rapid propagation of transgenic lines within a bubble-column bioreactor

A human acidic fibroblast growth factor gene, hafgf, was successfully transferred into Laminaria japonica (kelp) gametophytes via microprojectile bombardment using the biolistic PDS-1000/He gene gun. Following phosphinothricin screening, PCR detection and Southern blot analysis, transgenic L. japonica gametophytes were cultivated in an illuminated bubble-column bioreactor to optimize growth conditions. A maximal final dry cell density of 1,695 mg l⁻¹ was obtained in a batch culture having an initial dry cell density of 129.75 mg l⁻¹. This was achieved using an aeration rate of 1.08 l air min⁻¹ l⁻¹ culture in a medium containing 1.5 mM inorganic nitrate and 0.15 mM phosphate. In addition, the relationship between different nitrogen sources and growth of transgenic gametophytes indicated that both urea and sodium nitrate were effective nitrogen sources for cell growth, while ammonium ions inhibited growth of these gametophytes.     

Reproductive biology and population dynamics of wild gametophytes ofEquisetum

This is d study of the reproductive biology of wild gametophytes of Equisetum arvense L., E. fluviatile L., and E. palustre L., which form extensive populations during the summer months on previously inundated bare mud along the margins of reservoirs and lakes in Northern England and Wales. The garnetophytes have all the characteristics of pioneer species: they rapidly attain sexual maturity and are adversely affected by competition. Early in the summer mixtures of male and female gametophytes are produced. The former are smaller and their frequency (which is always less) diminishes throughout the season as a result of bryophytic competition. The rarity of bisexual, initially female, gametophytes, in wild populations is related to the absence of metabolite accumulation which mediates this change in rulture. Significantly different sex ratios between populations and species underlines the labile nature of the sex-determining mechanism in Equisetum. The high frequency of females bearing sporophytes indicates that intergarnetophytic fertilization is highly effective in nature. Absence of any correlation between the incidence of fertilized females and the proportions of males, in conjunction with a consideration of the male gamete dispersal distance, suggests that sporophyte formation is restricted by the availability of ripe archegonia. Sexual reproduction in Equisetum is probably limited by the narrow range of conditions under which gametophytes can become established rather than availability of water for fertilization. The majority of females, which bear one sporophyte, are smaller than unfertilized or polyembryonic females. Their small size results from nutrient demands and allelopathic compounds from the sporophytes which also probably prevent the establishment of gametophytes within mature stands of the parent species. Correlations between female diameters and frequencies of males suggest that gametophytes are more likely to produce archegonia under favourable conditions. The natural reproductive biology of Equisetum is in accord with predictions based on an understanding of the mechanism of sex determination in axenic culture. Several striking parallels between sexuality in Equisetum and dioecious angiosperms are revealed. The absence of winter flooding at two of the gametophyte sites led to the establishment of mature rtands of Equisetum, which produced cones after 3–4 years; two hybrids, E. fluviatile×E. arvense and E. fluviatile×E. palustre, were detected.     

Influence of tissue culture conditions on apogamy in Dryopteris affinis sp. affinis

The naturally-occurring apogamy of some ferns can be modified by culture conditions and growth regulators. Gametophytes of the apogamic fern Dryopteris affinis sp. affinis L., were cultured on Murashige and Skoog (MS) basal medium. Changes in concentration of MS medium components, sucrose, agar and different pH values were tested. The addition of benzyladenine (4.43 M) and naphthalene acetic acid (0.53 M) enhanced sporophyte proliferation on the gametophytes. After one month in culture, the gametophytes formed callus with a high morphogenic capacity. Culture of calli on medium without growth regulators yielded about 10,000 sporophytes per 1 g fresh weight of callus. This pattern of differentiation slowed with time to a point where only gametophyte regeneration was observed.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - F.W. fresh weight - MS Murashige & Skoog medium - NAA 1-naphthalene acetic acid - SE standard error     

HAPLOID PARTHENOGENETIC SPOROPHYTES OF LAMINARIA JAPONICA (PHAEOPHYCEAE)1

Parthenogenetic sporophytes were obtained from three strains of Laminaria japonica Areschoug. These sporophytes grew to maturity in the sea, producine spores that all grew into female gametophytes. These female gametophytes gave rise to another generation of parthenogenetic sporophytes during the next year, so that by the year 1990 parthenogenetic sporophytes had been cultivated for 12, 9, and 7 generations, respectively, for the three strains. When female gametophytes from parthenogenetic sporophytes were combined with normal male gametophytes, normal sporophytes that reproduced and gave rise to both female and male gametophytes were obtained. The parthenogenetic sporophytes were shorter and narrower than the normal sporophytes of the same strain. Chromosome counts on mature sporophytes showed that normal sporophytes (from fertilized eggs) were diploid (2n = approximately 40) and that the spores they produced were haploid (n = approximately 20), while nuclei from both somatic and sporangial cells in parthenogenetic sporophytes were haploid. All gametophytes were haploid. Young sporophytes derived from cultures with both female and male gametophytes were diploid, while young, sporophytes obtained from female gametophytes from parthenogenetic sporophytes had haploid, diploid, or polyploidy chromosome numbers. Polyploidy was associated with abnormal cell shapes. The presence of haploid parthenogenetic sporophytes should be use in breeding kelp strains with useful characteristics, since the sporophyte phenotype is expressed from a haploid genotype which can be more readily selected.     

Cultivation of the red alga Chondracanthus chamissoi: sexual reproduction and seedling production in culture under controlled conditions

The red alga Chondracanthus chamissoi (Gigartinales) is endemic to the southern-central region of South America. In the Pacific Ocean, it is distributed from north-central Peru to Chiloe Island. This species is of economic importance because it is edible and used for carrageenan production. The tetrasporophyte phase was grown in the laboratory, obtaining male and female gametophytes that were incubated under different photoperiod, pH, salinity and temperature conditions. These gametophytes developed and generated reproductive structures that led to in vitro maturation. Subsequently, fertilisation occurred and formation of cystocarps was observed. Finally, carpospores were released and the formation of sporophytes completed the life history of this species under laboratory conditions. Reproductive phase growth rates were recorded for each of the different culture conditions used. Sporophytes reached the highest daily growth rate (22%), while gametophyte’s daily growth rate was slower (9%). This research confirms, in vitro, the assumption that C. chamissoi has a sexual triphasic life history Polysiphonia type with isomorphic gametophytes and tetrasporophytes. The development of the complete life history took 20 months in the laboratory.     

GROWTH AND DEVELOPMENT OF THE MALE GAMETOPHYTE OF GINKGO BILOBA WITHIN THE OVULE (IN VIVO)

The mature male gametophyte of Ginkgo biloba can be divided into two regions: a large saccate structure that is suspended within the fertilization chamber above the archegonia, and a pervasive, highly branched haustorial system that ramifies through the intercellular air spaces of the apex of the nucellus. This morphology appears to differ in many ways from the simpler more typical male gametophytes of most other groups of seed plants. Growth and development of the male gametophyte of Ginkgo biloba were studied using computer reconstruction techniques to generate images of the gametophyte from data derived from serial sections through the ovule. These investigations reveal that morphological development of the male gametophyte of Ginkgo biloba is divided into three distinct phases: 1) Germination, characterized by an initial brief period of diffuse growth. This phenomenon has not been described for any other seed plant male gametophyte; 2) Initiation of tip growth and the formation of a tubular body, as typifies all seed plant male gametophytes. In Ginkgo, this is accompanied by a high degree of branching, giving rise to an extensively branched haustorial system; 3) Late swelling of the proximal unbranched portion of the gametophyte resulting in formation of the saccate structure that is characteristic of the mature gametophyte. This process appears to be very similar to late development in cycad male gametophytes. Thus, despite the seemingly anomalous morphology of the mature male gametophyte of Ginkgo biloba, specific patterns of growth and development are in many ways similar to growth processes expressed by the male gametophytes of some or all major groups of seed plants.