Assessing the Germplasm of Laminaria （Phaeophyceae） with Random Amplified Polymorphic DNA （RAPD） Method

Eighteen gametophytes including L.japonica ,L.ochotensis and L.longissima,were verified with random amplified polymorphic DNA (RAPD) technique.Eighteen ten-base primers were chosen from 100 primers selected for final amplification test.Among the total of 205 bands amplified ,181(88.3%) were polymorphic ,The genetic distance among different strains ranged from 0.072 to 0.391,The dendrogram constructed by unweighted pair -group method with arithmetic(UPGMA) method showed that the female and male gamtophytes of the same cell lines could be grouped in pairs respectively,It indicated that RAPD analysis could be used not only to distin-gusih different strains of Laminariz ,but also to distinguish male and female gametophyte within the same cell lines,There is ambiguous systematic relationship if judged mereely by the present data It seems that the use of RAPD marker is limited to elucidstion of the phylogenetic relationship among the species of Laminaria.     

海带雌性原生质体的分离与培养

于1993年1月 - 1993年4月，为了制备遗传性纯一的基因工程受体，用酶法从海带雌配子体单性生殖系分离雌性单倍体原生质体。从皱纹盘鲍消化腺及消化器官提取出鲍酶，在25°C下、1％（W／V）的鲍酶与2％（W／V）的纤维素酶（Onozuka R－10）合用能有效解离单细胞和原生质体。海带雌性原生质体经2d培养再生了细胞壁。     

雌雄比对海带雌配子体发育的影响

本实验进行了不同雌雄比对海带雌配子体发育的影响。结果表明：雌雄比(鲜重)为1：1及2：1，实验组出现排卵时间最早，且发育率高，实验第20天，发育率超过70％，孢子体正常。5：1，10：1，20：1实验组随着雄性比例的减少，发育率逐渐降低，随着培养天数的增加，三个实验组均出现未受精卵及畸形孢子体，雌雄比越大，未受精卵数目越多。     

海带配子体无性繁殖(克隆)技术研究与应用进展

海带具有异型世代交替,其配子体在单独分离培养时可进行营养生长,形成无性繁殖系(克隆)。本文简要回顾了海带配子体克隆技术研究与应用的历程,综述海带配子体克隆在海带种质资源保存、海带生理和遗传学研究及海带育种和苗种繁育中的应用现状。     

Intraspecific crossings ofUndaria pinnatifida (Harv.) Sur.—A possible time-saving way of strain selection

Unicellular gametophytes ofUndaria pinnatifida (Harv.) Sur. were isolated in Qingdao, P. R. China in April 1993 and in Tokushima, southern Japan in March 1995. Different intraspecific crossings by using unicellular male and female gametophytes were successfully undertaken in Sept. of 1995 in Qingdao. Sporophytes were transplanted to two different locations for open sea cultivation. One was at Zhanqiao (ZQ) Bay where the water current was slower than that of another location—Taipingjiao(TPJ). A total of 218 adult sporophytes were harvested on January 12 and 18 from TPJ and ZQ respectively. For eacn combination, 10 sporophytes were cultivated. Analysis of the morphological characteristics of adult sporophytes indicated that the longest length between two bases of the serration of pinnate blades (W2) is a morphological characteristic that can be transferred from the parent plant to the next generations regardless of environmental variations. There was evidence that W2 was apparently determined by sex-linked factors, i.e., by male parental gametophyte. Sporophytes from certain crossing combinations showed more vigorous growth than those from other crossing combinations. It is therefore possible to select gametophyte strains which can be used as parental gametophytes for the seedling production of sporophytes with more vigorous growth within shorter cultivation period. The morphology of hybrids from a Qingdao strain and a Tokushima cultivated strain resembled that of both parental plants in frond features (wrinkled or smooth) and W2. Sporophyll formation also varied with strains. The fact that adult sporophytes resulting from the same crossing combinations have identical morphological characteristics under the same environmental conditions indicates the possibility of a new way to select strains which are expected to be ideal for commerical production by purposely selecting, propagating, and seeding unicellular gametophytes for sporeling production through freeliving techniques of gametophytes. Project 39400102 supported by NSFC, also supported by National Climbing Plan B (PD-B642); and Bioengineering Center, SSTC.     

Identification of a Putative Tetrasporophyte-Specific Gene in Gracilaria lemaneiformis(Gracilariales, Rhodophyte)

A putative tetrasporophyte-specific gene, designated as SSH466 (GenBank accession No. DQ019223), was one of the genes identified in this work using suppression subtractive hybridization (SSH) method in Gracilaria lemaneiformis. The full length of the gene was obtained using SMART RACE strategy. Sequence analysis revealed that the gene had 1 019 nucleotides, including an open reading frame of 498 nucleotides encoding 166 amino acid residues, 158 nucleotides of 5' untranslated region and 363 nucleo- tides of 3' non-coding region. Protein motif and secondary structure prediction showed that there existed a transmembrane domain with a unique β-sheet. Thus, SSH466 protein might be a cross-membrane protein. Sequence homology search in the public GenBank databases did not reveal any significant match with SSH466. Virtual Northern blot analysis confirmed that it was a tetrasporo- phyte-specific gene.     

Zoospore-derived monoecious gametophytes in Undaria pinnatifida (Phaeophyceae)

The annual life cycle of the brown seaweed Undaria pinnatifi da(Harvey)Suringer comprises a macroscopic diploid sporophyte stage and a microscopic haploid gametophyte stage.In 2011,an unusual zoospore-derived monoecious gametophyte isolate(designated as line 10-5-3)of U.pinnatifi da was observed.To understand this phenomenon,a comprehensive screening of eighty-two previously identifi ed male gametophyte cultures,isolated from three randomly selected cultivars(lines 10,7,and 5)was performed.Thirty-six of the isolates developed both antheridia and oogonia on the same fi lamentous fragment in a standard gametogenesis test(SGT: 18°C,60 μmol photons/(m 2·s)).Selfi ng of the monoecious gametophyte or crossing it with a normal male gametophyte both gave rise to morphologically normal sporophytic offspring.However,crossing resulted in a much higher fertilization rate(89.7%).The hybrid and selfed sporophytic offspring were grown to maturity in fl ow tanks at an ambient temperature of 10–18°C over a period of 69 days.Active zoospores were released from both types of mature sporophylls.The majority of these developed into male gametophytes,while 15%–20% developed into the observed monoecious structures on the same fi lament.Using PCR amplifi cation it was found that all the monoecious gametophyte isolates and the sporophytic offspring resulting from the selfi ng and crossing lacked the femalelinked microsatellite sequence(a part of the locus Up-AC-2A8,GenBank accession No.AY738602.1),indicating their male nature.U.pinnatifi da is an invasive species in some regions and the implications of the above fi ndings for this species in nature are briefl y discussed.     

海带杂种优势苗种繁育技术研究

用生物反应技术进行海带配子体克隆的连续培养,将丝状体克隆细胞粉碎后,用两系法培育杂种优势苗种.实验结果表明,雌、雄海带配子体克隆细胞的日增重速率可达14.03%和13.87%,经42d培养增重6.9倍和6.8倍;雌、雄配子体克隆细胞杂交16d,排卵率为100%;培养40d后幼孢子体长度为2~3cm,经海上养殖实验,初步筛选出了一个较好的杂交组合.     

节能型海带育苗新技术——黑暗流水储存配子体

目前采用的海带苗培育方法已经有50多年的历史,它将21~28℃的自然海水降温至6~8℃用于育苗。缺点是海水降温成本高、管理需要人工多。而本技术在育苗过程中的一定时期,将苗帘集中堆放,在流水黑暗条件下储存配子体14 d,储存期间无需人工管理。以此配子体储存技术,可以将海带育苗平均水温从目前的7℃左右提高到10℃左右。     

海带配子体克隆细胞附着的初步研究

海带是我国海水养殖主要品种之一,其综合应用价值较高[1].20世纪90年代末期,海带配子体克隆在海带苗种繁育中的应用,为海带育苗生产开辟了新途径[2].主要运用生长发育调控技术实现海带配子体克隆细胞大量扩增和同步成熟来繁育海带优良苗种,这样可大幅度降低育苗成本,缩短育苗周期,该项技术具有良好的应用前景.目前海带配子体克隆育苗技术尚未在海带育苗生产中得到规模化应用,其作为一项高新技术,部分关键技术仍有待进一步解决.在与有关生产单位进行海带配子体克隆育苗实验的工作中,笔者认为克隆细胞的附着是影响海带商品苗质量(出苗密度及出苗均匀程度)的关键问题之一.