Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system

A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-μm pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal. Received: 21 April 1997 / Revision received: 27 June 1997 / Accepted: 5 July 1997     

海带(Laminaria japonica)丝状体遗传多样性的比较研究

采用垂直板式聚丙烯酰胺凝胶电泳技术对分别来自中国青岛与日本北海道海带(Laminaria japonica)丝状体的6种酶系统（乙醇脱氢酶、谷氨酸脱氢酶、异柠檬酸脱氢酶、乳酸脱氢酶、山梨醇脱氢酶和超氧化物歧化酶）进行了检测,发现存在22个基因位点,分析了不同来源海带丝状体的遗传多样性与遗传分化程度。结果表明：日本北海道海带丝状体的预期杂合度、每个位点有效等位基因数等遗传参数均大于中国青岛海带丝状体。对日本北海道海带丝状体与中国青岛海带丝状体的遗传相似系数和遗传距离进行了计算分析,显示两者发生了遗传分化。日本北海道海带丝状体的遗传多样性高于中国青岛海带丝状体。研究结果还发现在中国青岛海带丝状体的Idh、Ldh 和Sdh中,以及日本北海道海带丝状体的Adh、Gdh、Idh、Ldh、Sdh和Sod中存在着不同迁移率的不等距双带酶,可能由特殊基因控制,并集中在第3个基因位点上。     

海带喇叭丝对接的新类型

在对海带髓部组织进行显微观察时,发现了两种海带喇叭丝对接的新类型,即三细胞对接型和四细胞对接型。这两种类型的数量较少,只存在于带片与带柄的交接处,其功能可能与提高物质转运效率和横向运输有关。     

海带无性繁殖系的形成及孢子体诱导

海带无性繁殖系的形成及孢子体诱导周志刚吴超元（中国科学院海洋研究所青岛２６６０７１）海带（Ｌａｍｉｎａｒｉａ）是具异型世代交替的大型经济褐藻,在生活史中出现一至数个细胞组成的雌、雄配子体阶段,再由雌配子体的卵囊受精长成孢子体［１］。现行的育苗法是于初...     

温度对海带幼孢子体生长和光合作用的影响

根据欧盟项目的总体研究计划 ,在室内和围隔实验中研究了海带幼孢子体在不同温度条件下的生长情况和光合作用曲线 ,得出幼孢子体在 15°C下生长情况最好 ,而且其长度和重量的日增加量均和温度有线性相关关系。 Pm 值在 15℃时为 5 79mol O2 / (g DW· h) ,围隔实验中为 6 4 2 (mol O2 / (g DW· h)。由于围隔实验海区的光照强于室内 ,使得围隔中海带的光合作用曲线其光饱和点高于室内实验海带的光饱和点。海带光饱和参数 (Ik)与温度有线性相关关系 (R2 =0 .736 7,p<0 .0 5 )。5℃时 ,Ik 值平均为 96μE/ (m2 · s) ,10℃时为 71μE/ (m2 · s) ,15℃时为 31μE/ (m2 · s)。呼吸速率 R值在 5℃时最低 ,为 5 4μmol O2 / (g DW·h) ,10℃、15℃和围隔中均高于 5℃时的呼吸速率 ,并且在 10℃达到最高。本次实验研究得出的生长和光合作用参数将有助于合理确定桑沟湾中海带的养殖容量。     

不同海带品系抗氧化系统活性与耐热性的相关性研究

以山东沿海广泛培育的两个海带品系耐高温海带901(简称901)和热敏感海带荣成1号(简称RC)为实验材料,研究不同温度条件下其抗氧化系统活性与耐热性状的相关性.结果表明,常温条件下(10℃)901与RC抗氧化系统的基础活性与耐热性状间无显著相关.高温胁迫条件下(18℃)RC体内的活性氧(reactive oxygen species,ROS)含量快速升高,而901的ROS在整个胁迫过程中变化不大,说明ROS可能对高温氧化胁迫具有重要意义.RC的抗氧化系统对高温胁迫比901敏感,表现为出现活性有效抑制的时间和幅度都强于901.超氧化物歧化酶、谷胱甘肽过氧化物酶和抗坏血酸和维生素E可能在保护海带细胞免受高温胁迫方面具有重要作用.901的叶绿素对高温胁迫的响应明显滞后于RC,叶绿素含量的半伤害时间可作为判定幼苗期901和RC耐热性的灵敏指标.导致不同品系海带耐热性差异的并非其抗氧化系统基础活性的高低,而是该系统对高温胁迫的应激性和敏感性差异.     

The response of the early developmental stages of Laminaria japonica to enhanced ultraviolet-B radiation

The responses of the early development of Laminaria japonica collected from Kiaochow Bay in China to enhanced ultraviolet-B radiation (UV-B, 280—320 nm) were studied in the laboratory. The low UV-B ra-diations (11.7—23.4 J·m-2·d-1) had no significant effects on zoospores attachment, but when the UV-B dose > 35.1 J·m-2·d-1 the attachment decreased significantly compared with the control. Germination of embryospores was >93% under the low (11.7—35.1 J·m-2·d-1) doses, and in the range of 78.5%—88.5% under the high (46.8—70.2 J·m-2·d-1) UV-B doses, indicating a significant radiation effect. Under the higher UV-B exposure (35.1—70.2 J·m-2·d-1), all of the few gametophytes formed from embryospores died 120 h post-release. After exposure to the low UV-B radiation (11.7—23.4 J·m-2·d-1), the formation of sporophytes decreased and the female gametophyte clones increased compared with the control. However, the sex ratio and the relative growth of female gametophytes/sporophytes had not signifi-cantly changed. According to the results, enhanced UV-B radiation has a significant effect on the early development of L. japonica under laboratory conditions, suggesting that the UV-B radiation could not be overlooked as one of the important environmental factors influencing the ontogeny of macroalgae living in marine ecosystems.     

Development of 18 polymorphic microsatellite DNA markers of Laminaria japonica (Phaeophyceae)

Eighteen polymorphic microsatellite DNA markers were developed for Laminaria japonica, a brown alga cultured intensively in China. These markers are independent from each other and are moderately variable in L. japonica. The number of alleles and gene diversity detected in 53 gametophyte clones representing the varieties of L. japonica cultured once or currently in China range from two to nine and from 0.355 to 0.768, respectively. These markers will certainly facilitate the management and exploitation of the germplasm resource of L. japonica conserved indoor as gametophyte clones and the determination of the genetic diversity of L. japonica naturally distributed.     

Aerobic microbial degradation of alginate in Laminaria hyperborea stipes containing different levels of polyphenols

The polyphenols present in brown seaweed tissue may seriously affect aerobic microbial degradation, particularly the alginate present. Laminaria hyperborea stipes, harvested at 59 °N off the Norwegian coast in autumn, were degraded at different levels of polyphenols in aerated batch reactors at 35 °C and pH 7. This was achieved by manipulating the relative amounts of peripheral tissue, by removing or adding the mechanically peeled outer phenolic layer, using standardized inocula already adapted to L. hyperborea degradation. The degradation of organic matter was clearly depressed by increasing the amount of peripheral tissue. Alginate lyase activity was also negatively correlated to the amount of peripheral tissue loaded, presumably due to the release of reactive polyphenols. The total digestion rates of alginate were reduced by more than a factor of two at enhanced amounts of peripheral tissue. The guluronic content of extracted Na-alginate increased during the degradation, despite the presence of significant amounts of guluronate specific alginate lyase activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mariculture of Laminaria japonica (Laminariales, Phaeophyceae) using protoplast regeneration

The conditions for culture of viable protoplasts from Laminaria japonica were investigated and the regenerative processes were observed in detail. As a result of culturing at four water temperatures (5, 10, 15, and 18°C), we found that low water temperature was better for survival, division and rhizoidal formation of protoplast‐derived cells. Only epidermis‐derived protoplasts developed into normal sporophytes through a direct developmental process. Protoplast‐derived cells divided after 5 days and 2–10 celled germlings formed the first rhizoids after 15 days. Only initial sporophytes with the first rhizoids grew to normal sporophytes with multilayered blades, stipes and holdfasts. When these young sporophytes were transplanted into the sea, they grew to normal fertile sporophytes.     

搅拌式光生物反应器培养海带配子体细胞：不同脉冲补料方式的研究

本文以批次培养为对照,研究了七种脉冲补料方式对搅拌式光生物反应器中培养大型褐藻海带配子体细胞生长和培养液内氮磷营养盐消耗的影响,并首次探讨了脉冲补料方式下不同补料时间点和补料量的影响作用。培养条件设定为50 mg DCW (细胞干重) L-1接种密度、培养液为改良的APSW人工海水、光强60 µE m-2 s-1、光周期16/8 h L/D、通气速率和搅拌速率分别为50 mL min-1和100 rpm。结果表明少量补料利于细胞对氮磷的协同吸收,进而利于生物量扩增。当培养液内氮磷富足或耗尽时补料对于生物量大量生产效果甚微,可能由于氮磷吸收变缓、其储存现象显著,或是其吸收协同性降低。文中当细胞生长至对数中期开始频繁补加少量氮磷营养盐,即维持培养液内氮磷浓度在各自起始浓度的1/3至1/2之间,对生物量生产最有效,生物量增长倍数高达12.270倍。     

Preparation of protoplasts from Laminaria japonica using native and recombinant abalone alginate lyases

Laminaria japonica protoplasts were released with high yields using the abalone alginate lyase HdAly in combination with a cellulase and chelating agents. Addition of EDTA at concentrations higher than 10 mM to Laminaria thalli which had been preincubated with HdAly and Cellulase Onozuka, dramatically improved the yield of protoplasts. EDTA was far more effective than EGTA, indicating that chelating divalent metal ions such as Mg²⁺ and Sr²⁺ in addition to Ca²⁺ is a key factor for high-yield production of Laminaria protoplasts. Protoplasts had a mean diameter of 27 μm, suggesting that most protoplasts were derived from cortical cells rather than epidermal layer cells. Recombinant HdAly (rHdAly) was produced from a cDNA clone in the Sf9 insect cell expression system. rHdAly had substantially the same enzymatic properties and protoplast-producing ability as did native HdAly. The optimal conditions for high yield production of protoplasts from Laminaria using native and recombinant HdAlys were investigated.     

活性氧在海带抗褐藻酸降解菌感染中的作用

海带是我国大型经济海藻养殖的主要对象 ,养殖历史长 ,用途广泛。海带人工育苗是发展我国海带养殖事业的一个重要环节。海带病烂 (烂柄病 ) ,尤其在育苗期 ,将造成很大的经济损失。研究指出 ,海带的病烂与褐藻酸降解菌的大量繁殖有关[1- 3 ] 。褐藻酸降解菌是海带藻体上主要附生细菌 ,正常情况下并不引起病烂 ,但若养殖环境恶化 ,如过于密植、损伤、温度过高等 ,使海带的抵抗力下降 ,细菌易于侵入并大量繁殖而导致病害。本文以褐藻酸降解菌为病原细菌 ,研究海带活性氧的产生及其在抵抗褐藻酸降解菌感染过程中所起的作用 ,以期为揭示海带活性…     

Direct determination of alginate content in brown algae by near infra-red (NIR) spectroscopy

The methods used to quantify total alginate in brown algal tissue are time-consuming and may also be misleading, so faster and simpler methods for measuring alginate content would be beneficial in a variety of applications. This study reports on the use of near infra-red (NIR) analysis to monitor the alginate content of Laminaria hyperborea stipe during biodegradation. NIR reflectance spectra were recorded for 78 different freeze-dried samples of its stipe. The samples were collected during several biological degradation experiments and the total alginate content varied from 2.2 to 40.8% Na-alginate (w/w), determined by established methods based on ion exchange. Data analysis was performed using multivariate calibration methods in order to relate the spectral data to the alginate content. PLS2 analysis revealed some dependence on material type, probably reflecting differences in polyphenol content. In the end, a PLS1 model with 9 components was selected. The calculated model was validated both with internal data and with an external test set. Internal full cross validation explained 96.6% of the variance in alginate content. The external validation showed that the PLS1 model was able to predict the alginate concentration with a root mean square prediction accuracy of 2.1%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microsatellite DNA Variation of the Gametophyte Clones Isolated from Introduced Laminaria japonica （Phaeophyta） and L. Iongissima of China and Varieties Derived from them

The variation of 90 Laminaria gametophyte clones representing the introduced Laminaria japonica （Group 1） and Laminaria Iongissima （Group 2）, the varieties of L. japonica （Group 3） and the varieties derived from interspecific hybrids （Group 4） was determined with 18 microsatellite markers. The allelic diversity and Nei＇s gene diversity of Group 1 were significantly higher than those of Group 2 （2.9 vs. 1.8 and 0.414 vs. 0.161, respectively）, demonstrating that the variation of the introduced L. japonica is richer than that of L. Iongissima. Both allelic diversity and Nei＇s gene diversity of Group 3 were lower than those of Group 1, indicating that only a portion of variation of L. japonica was incorporated into the varieties of L. japonica. Significant genetic differentiation was detected between four groups and between female （Population 1 ） and male （Population 2） gametophyte clones in each group. The variation among groups accounted for 39.95%, while that among populations accounted for 21.65% of the total. The genetic distance between Group 1 and Group 4 was obviously longer than that between Group 2 and Group 4 （0.686 vs. 0.291）, indicating that maternal gametophyte clone contributed more variation to the hybrids than the paternal gametophyte clone did.     

Genetic Mapping of Laminaria japonica and L. Iongissima Using Amplified Fragment Length Polymorphism Markers in a ＂Two-Way Pseudo- Testcross＂ Strategy

With a ＂two-way pseudo-testcross＂ mapping strategy, we applied the amplified fragment length polymorphism （AFLP） markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family （Laminaria iongissima Aresch. × L. Japonica Miyabe） with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1：1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3：1 Mendelian segregation ratio. Among the loci with 1：1 segregating ratios, 79 loci were ordered in 14 linkage groups （648.6 cM） of the paternal map, and 72 loci were ordered in 14 linkage groups （601.9 cM） of the maternal map. The average density of loci was approximately 1 per 8 cM. To Investigate the homologies between two parental maps, we used 58 loci segregated 3：1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.     

虎杖的组织培养与快速繁殖

以虎杖茎段、叶柄、叶片为外植体探讨了愈伤组织诱导、分化和植株再生的条件,筛选出茎段生长培养基为1/2MS+BA1.0mg·L-1+KT0.5mg·L-1+NAA0.2mg·L-1,茎段、叶柄和叶片外植体愈伤组织诱导培养基为MS+BA1.0～2.0mg·L-1+KT0.2～0.5mg·L-1+NAA0.2～0.5mg·L-1或MS+BA2.0～3.0mg·L-1+KT0.2～0.5mg·L-1+2,4-D0.5mg·L-1;丛生芽诱导培养基为MS+BA2.0mg·L-1+KT0.5mg·L-1+IBA0.2mg·L-1+LH1000;不定根及根状茎诱导培养基为1/2MS+IBA0.2mg·L-1.