Breeding of an elite <Emphasis Type="Italic">Laminaria</Emphasis> variety 90-1 through inter-specific gametophyte crossing

Laminaria longissima and Zaohoucheng No.1 (a commercial variety selected from Laminaria japonica) differ to a certain extent in their morphological characteristics and biological habits. It was assumed that varieties bred through their hybridization should exhibit high yield potential and tolerate relatively high seawater temperatures. Female gametophyte clones isolated from L. longissima were crossed with male clones isolated from Zaohoucheng No.1. Laminaria variety 90-1 was obtained after gametophyte crossing, continuous self-crossing and selection. This variety was genetically homozygous; the indices of variation of blade length, width and thickness of the final two selection cycles were 7–8%; i.e., not different significantly. Variety 90-1 grew faster, lost less tissue and had higher yield potential than two widely used commercial varieties of L. japonica (all commercial varieties currently used in China originate from this latter species). The blade of variety 90-1 increased 3.71 cm day⁻¹ on average during the whole period of cultivation, almost two-fold that of two controls, and growth was maintained even when seawater temperature was higher than 18°C–3°C higher than the temperature tolerated by other Laminaria varieties. Variety 90-1 increased yield by more than 70% over two controls and also synthesized desirable amounts of iodine, mannitol and algin. In blade length, variety 90-1 was more similar to L. longissima than to L. japonica, but more similar to L. japonica in blade width and thickness. Since the adoption of variety 90-1 in 1999, its culturing area has increased each year to reach its current area of 7,000 ha, i.e., almost one-third of the total cultivation acreage of Laminaria in China. Breeding of variety 90-1 has demonstrated that it is feasible to develop elite Laminaria varieties by crossing gametophytes from different Laminaria species in combination with successive self-crossing and selection.     

不同环境因子对珍稀观赏蕨类金毛狗配子体性别分化的影响

金毛狗[Cibotium barometz(L.)J.Sm.]是珍稀观赏蕨类的重要类群,为国家二级重点保护野生植物。该研究以金毛狗孢子为试验材料,探究培养密度、外源赤霉素以及光质等不同环境因子对金毛狗配子体性别分化的影响,为金毛狗人工繁育和蕨类植物配子体性别决定机制研究提供技术支持。结果表明:(1)低配子体培养密度(1个/cm2和5个/cm2)有利于颈卵器和雌配子体形成,随着配子体培养密度增加,颈卵器平均数量及雌配子体比率下降,精子器平均数量以及雄配子体和两性配子体比例增加,但配子体培养密度过高(80个/cm2)会导致大量无性配子体产生。(2)不同配子体培养密度下,随着培养时间延长,两性配子体比率均有增加,且增加幅度基本一致。(3)外源GA4显著抑制颈卵器和雌配子体形成,并显著促进精子器和雄配子体形成;外源GA3对金毛狗配子体性别分化没有显著影响。(4)白光、红光、蓝光等不同光质对金毛狗配子体性别分化未产生显著影响,但会影响配子体的发育和形态建成。     

Gametogenesis and chromosome number in Postelsia palmaeformis (Laminariales, Phaeophyceae)

Gametophytes of the ‘sea palm’, the kelp Postelsia palmaeformis Ruprecht, produced gametes whether or not chelated iron was supplied in the culture medium, in contrast to the inhibition of gametogenesis seen with the absence of iron in many other kelps. As gametogenesis proceeded, every cell of the gametophytes was converted into a gamete so that the gametophytes did not continue to grow vegetatively. The portion of the life history from spore release through germination, gametophyte growth, gametogenesis, fertilization and growth of the young sporophyte was completed in 9–10 days under laboratory conditions. Chromosome counts showed that sporophytes had a diploid number of 26–34 chromosomes while sporangia and gametophytes had a haploid number of 14–17 chromosomes, indicating a typical haplodiplophasic life history as seen in other Laminariales.     

Growth and reproductive characteristics in artificially formed clonal gametophytes ofDryopteris filix-mas (Dryopteridaceae)

Artificially formed clonal gametophytes ofDryopteris filix-mas (L.)Schott were able to grow and reproduce, although growth rates, proportions of hermaphrodites and reproductive efforts were low. Variable density and nutrient levels appeared to affect gametophytic life histories and they continued to influence the viability of the developing sporophyte generation. Differences between populations and sporophytes were discovered in life histories. Hermaphroditic gametophytes were considerably larger and possessed higher viability than did males. Clear differences between clones in the level of phenotypic plasticity in gametophyte size were observed. No neighbor effect on gametophyte sex expression was detected with the densities and nutrient levels used.     

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.     

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.     

Growth and gender in the gametophyte of Blechnum spicant L

The growth and gender in the gametophyte of Blechnum spicant L. were strongly affected by its origin, spore or mature homogenized gametophytes, and also by the addition of plant growth regulators to the culture medium. Spore-derived gametophytes cultured in Murashige and Skoog liquid medium were females and heart-shaped, whereas those derived from mature homogenized gametophytes were shorter and male or asexual (1:3) because of the release of antheridiogen to the culture medium. In the latter, maleness was especially increased by the addition of BA. This cytokinin is a strong inductor of maleness in homogenized cultures; however, even though BA influenced sexual organ formation in spore-derived gametophytes, it does not change the␣female sexual pattern that these gametophytes have. Separate male and female populations of gametophytes were obtained in this work.     

Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae)

Summary Protoplasts were isolated from sporophytes and from gametophyte cultures of several species in the order Laminariales. For each example, the isolation and culture procedures were investigated systematically, to identify conditions leading to plant regeneration. After dedifferentiation through a filamentous stage, protoplasts isolated from adultLaminaria saccharina sporophytes regenerated polystichous bladelets. In contrast, cells isolated fromLaminaria digitata sporophytes proved recalcitrant in culture, except when the donor plants were undifferentiated sporelings. The most critical factors for protoplast development were the origin of explants, the osmoticum used for cell isolation, cultivation in plain seawater, and the absence of stress during the first two weeks of culture. We also found that protoplast isolation from the sporophytes of members of the Laminariales results in the release of hydrogen peroxide, up to 5–120 μM final concentration in the macerating medium, a characteristic which may be related to protoplast recalcitrance. Protoplasts isolated from the gametophytic phase readily regenerated into normal gametophytes, capable of gametogenesis and producing sporophytes by fertilization.     

Size dependency of gametophytes decay inAthyrium brevifrons Nakai during spring desiccation

Gametophyte populations inAthyrium brevifrons were analysed with respect to population size and surviving area (%) of individual thalli in a transplant garden at Sapporo during 5–26 April 1983, to study the safe-microsite for gametophyte establishment in nature. Spores dispersed in August 1982 germinated and grew into thalli of various widths (<10 mm); 10.3% of the thalli matured by early October 1982. Maturation was attained by gametophytes of width 4–7 mm. The number of gametophytes gradually decreased with increasing width. By April 1983, 20.5% of total gametophytes were mature with a mode of 5–6 mm in width. The relative number of gametophytes with surviving area of 2–20% increased and that of 85–100% decreased in accordance with collection days delayed until after snow-melt. Surviving area (%) on gametophyte of all widths decreased with decreasing soil moisture contents. In particular, immature gametophytes of 2–4 mm width showed a significant correlation (P<0.01) between soil moisture content and relative number of gametophytes with 0–20% surviving area and mean surviving area (%) of every width of thalli. The spring desiccation might be a factor that reduces or limits gametophyte populations in nature.     

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。     

Comparative development of heavily asymmetric-cordate gametophytes of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> (Anemiaceae) focusing on meristem behavior

Development of heavily asymmetric cordate gametophytes of Anemia phyllitidis (Anemiaceae), one of the schizaeoid ferns, was examined using a sequential observation technique; epi-illuminated light micrographs of the same growing gametophytes were taken approximately every 24 h. The apical cell-like wedge-shaped cell was produced once from the terminal cell of a germ filament, but it stopped dividing soon after production of one or two derivative cells. Without a functional apical cell, the gametophyte developed by intercalary growth until the early stage of wing formation, and then the multicellular (pluricellular) meristem arose from the lower lateral side of the gametophyte. This was in sharp contrast to the observation that the multicellular meristem forms in place of the apical cell in typical cordate gametophytes. Loss of the functional apical cell probably caused a site-shift in the multicellular meristem of the Anemia phyllitidis gametophyte during evolution from apical to lateral. The results suggest that apical cell-based and multicellular meristems are primarily independent of each other. The multicellular meristem produced cells equally in the distal and proximal directions to form wings in both directions but proximally produced cells divided much less frequently. As a result, a heavily asymmetric gametophyte was formed.     

云贵水韭雌配子体发育及其濒危机制的研究

为了解云贵水韭(Isoetes yunguiensis)的生殖过程和濒危原因,采用人工培养获得其雌配子体,用半薄切片法观察雌配子体发育过程。结果表明,云贵水韭的大孢子萌发率约为26%;雌配子体异养,终生都在大孢子细胞壁内发育;颈卵器发育只有2枚颈沟细胞而无腹沟细胞。雌配子体发育停止在游离核时期、颈壁细胞数量偏少或排列紊乱、颈沟细胞异常、卵细胞凋亡等可能是导致生殖濒危的原因。同时,游离核是水韭生殖生物学研究和系统学研究的重要环节之一;缺失腹沟细胞可能是水韭类简化性进化的特征之一。     

Gametophyte contribution to sporophyte growth on the basis of carbon gain in the fern <Emphasis Type="Italic">Thelypteris palustris</Emphasis>: effect of gametophyte organic-matter production on sporophytes

At an early stage of growth gametophytes support the sporophytes of ferns. Young sporophytes become independent of gametophytes when the first leaves develop. Although large fern gametophytes produce multiple archegonia simultaneously, only one sporophyte is typically established on one gametophyte. The number of sporophytes is believed to be controlled in two possible directions, from gametophyte to sporophyte or from preceding sporophyte to another sporophyte. To investigate the effects of gametophytes on their sporophytes, we studied the relationship between organic matter production by gametophytes and the growth of young sporophytes of Thelypteris palustris. We cut gametophytes in half (CGs) to reduce the gametophytes’ production of matter. There was no significant difference between the growth of sporophytes on intact gametophytes (IGs) and that on CGs. According to our estimates, based on the rate of organic matter production, the large gametophyte was able to produce two or more sporophytes. The resources required for CGs to make similar-sized sporophytes was twice that for IGs. In polyembryony each of the multiple sporophytes was similar in size to the single sporophytes. Resource limitation does not seem to explain why fern gametophytes establish single sporophytes.     

Effects of daylength,irradiance and settlement density on the growth and reproduction of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> gametophytes

The effects of daylength, irradiance and spore settlement density on the growth, maturation and sporophyte production of Undaria pinnatifida (Harvey) Suringar gametophytes were examined using a factorial experimental design in culture. The growth of Undaria gametophytes increased with increasing daylength (8, 12 and 16 h), but the maximum fertility occurred at a daylength of 12 h followed by 8 and 16 h. Gametophytes grew better at the 16 h daylength under the same mean daily irradiance (MDI) of 20 μmol photons m⁻² s⁻¹. However, the fertility was higher at the short daylength (8 h), indicating that the maturation of U. pinnatifida gametophytes is influenced by daylength rather than by the MDI. Vegetative growth and sporophyte production of gametophytes were better at 60 μmol photons m⁻² s⁻¹ than at 30 μmol photons m⁻² s⁻¹ under a 8:16 h LD (Light: Dark) cycle, and their growth and maturation were density-dependant in 16 and 12 h daylength, respectively. These results suggest that the U. pinnatifida gametophytes require a certain amount of light for the growth and reproduction, and intraspecific competition occurred under the optimal growth and maturation conditions. However, the sporophyte recruits per unit has been enhanced with increasing spore settlement density at 8 and 12 h daylengths indicating that high settlement density gives a benefit for maintaining population, even though the sporophyte production of each female plant is inhibited. In conclusion, the vegetative growth, reproduction and sporophyte production of U. pinnatifida gametophytes are retarded at a low irradiance above growth saturation and a high settlement density, and are determined by daylength.     

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.     

Life-cycle of <Emphasis Type="Italic">Scinaia interrupta</Emphasis> (Nemaliales,Rhodophyta)

The life-cycle of Scinaia interrupta (A.P. de Candolle) M. J. Wynne was investigated in vitro using four irradiance regimes: 4, 8, 12 and 16 μmol photons m⁻² s⁻¹. A triphasic heteromorphic life-cycle was observed. Carpospores released by cystocarps of gametophytes collected in the field developed into filamentous tetrasporophytes, which produced tetrahedral tetrasporangia. Tetrasporangial development was accelerated under higher irradiance levels. Tetraspores germinated into filamentous protonemal gametophytes, initially identical to the tetrasporophyte. Filamentous gametophytes developed apical utricles and gave rise directly to the fleshy gametophyte. Further development of the fleshy gametophyte was not observed at the lowest irradiance regime (4 μmol photons m⁻² s⁻¹). The present study reports for the first time the influence of the irradiance regime on the initial tetrasporangial development and in the development of the fleshy gametophyte, and reinforces the importance of light intensity on Scinaia life-cycle. Production of apical utricles by the filamentous gametophyte is newly reported for the genus.     

Novel means for variety breeding and sporeling production in the brown seaweed Undaria pinnatifida (Phaeophyceae): Crossing female gametophytes from parthenosporophytes with male gametophyte clones

The unialgal haploid gametophyte clones are frequently used for variety breeding and sporeling production in the brown seaweed Undaria pinnatifida because a single crossing of a pair of the selected male and female gametophyte clones can generate sporophytic offspring with identical genotype and phenotype. As the seeds to be sprayed on the collectors, the detachment rate of the filamentous gametophytes is high in comparison to the seeded spores. In this investigation, we report the use of parthenogenesis to achieve the same goal in variety selection and sporeling production but with higher efficiency. The selected female unialgal gametophyte clone (Code: 06‐8‐1F) was induced to produce parthenosporophytes. These sporophytes were grown up in a controlled system and used to release zoospores. All zoospores generated into female gametophytes. These female gametophytes were allowed to go through parthenogenesis for the second year to confirm the applicability of this means. In the third year, the zoospores released from the parthenosporophytes were seeded on collectors over summer in female gametophyte form. In the early autumn, a selected male unialgal gametophyte clone (code: 5#F1‐2‐5M) was used to cross the seeded female gametophytes on the collectors. When the sporelings reached a mean length of 780 μm, they were transplanted to open sea on longlines for growing up. At harvest, the average length, width and wet weight of the adult sporophytes were 211 cm, 48.8 cm and 373 g, respectively. The sporophytic blades were uniformly smooth without wrinkles on both sides of the midrib, indicating top quality of the products. Amplified fragment length polymorphism analyses confirmed the identical genotypes of sporophytic offspring. These results suggested that this novel variety breeding and sporeling production method could serve as an efficient alternative to the traditional breeding technique for U. pinnatifida and possibly other commercial kelps that have identical life cycles.     

毛叶粉背蕨配子体的泌粉现象及其系统学意义

为探讨蕨类配子体泌粉现象的系统学意义,对5种粉背蕨属(Aleuritopteris)植物的配子体发育过程进行了观察。结果表明,毛叶粉背蕨(A.squamosa)的配子体(通常是雌配子体)也具有泌粉现象,而其他4种的配子体不具粉状分泌物。基于叶绿体rbc L序列的证据表明,毛叶粉背蕨与粉背蕨属其他植物、中国蕨属(Sinopteris)等构成一个具有强烈支持率的单系,即Hemionitids支,该支与美洲分布的隐囊蕨类互为姐妹群。因此,配子体泌粉现象在碎米蕨类中并非隐囊蕨类植物所特有,泌粉现象的产生,在系统发育上至少涉及2次独立的演化事件。毛叶粉背蕨的泌粉现象多发生在雌性配子体上,暗示配子体的泌粉可能与配子体的雌性分化和胚发生发育相关。     

Gametophyte development of Woodwardia radicans (L.) Sm.: Effect of population density and antheridiogen on sex expression

Abstract

Gametophyte development of Woorwardia radicans was studied in controlled culture conditions, as well as the effect of population density and antheridiogen on sex expression. The pattern of gametophyte development is not affected by temperature, while sex expression of gametophytes is affected by sowing density, by the presence in the culture medium of «antheridiogen» and by the nutritional conditions of gametophytes. The probable effects of antheridiogen and population density on the observed developmental pattern are considered.