紫菜叶状体细胞的酶法分离及其养殖研究

本文应用生物技术方法,进行紫菜细胞的菜苗和海上养殖已获得成功。用海螺酶将紫菜叶状体细胞分离成单细胞和原生质体,研究了叶状体的不同细胞类型的再生和发育。不同海水比重、不同温度对细胞的分离和培养的影响,并研究了单细胞和原生质体的附着及其海上的养殖。紫菜酶法采苗的成功,将会对传统的紫菜养殖产生根本的变革。     

同形鳞毛蕨成精子囊素系统的初步研究

研究了同形鳞毛蕨成精子囊素对该种和水蕨孢子萌发和配子体发育的影响,结果表明：同形鳞毛蕨配子体能产生成精子囊素,该成精子囊素能抑制同种孢子的萌发,抑制作用随配子体成熟度的增加而增强;同形鳞毛蕨成精子囊素还可促进同种孢子发育为雄配子体;光照条件下,同形鳞毛蕨成精子囊素对水蕨孢子萌发和配子体发育影响不大,黑暗条件下,同形鳞毛蕨成精子囊素能显著的促使水蕨孢子提早萌发,但都不影响其孢子最终萌发率和配子体的性别分化,表明同形鳞毛蕨和水蕨的成精子囊素不属于同一系统。     

鹧鸪菜生殖器官发育的研究

鹧鸪菜[Caloglossa leprieurii(Mont)J.Ag.]细胞含多核。在生殖季节,孢子体、雌、雄配子体在外形上有明显的差异:四分孢子体大而平展,分枝的上部肉眼可见有红色小斑点,即为四分孢子囊群。雌配子体顶部略蜷曲,分枝密,囊果呈球状,多位于分枝上部分枝点的腹面。雄配子体个体较小,枝顶蜷曲,颜色淡,特别生有精子囊的部分更淡。每种藻体的营养细胞的纹孔连结非常发达,每个细胞都具有4-5条,多者达7-8条。果胞枝由四个细胞组成,一般由围轴细胞分裂形成,果胞受精后,由支持细胞分裂形成辅助细胞,一个生育小枝上,可以连续形成5-7个甚至10个以上幼果胞系,但多数仅有一个果胞受精发育成囊果。当一个果胞受精时,其他果胞系就停止发育。精于囊位于分枝上部中肋的两侧,背腹面均有分布。一个营养细胞沿着叶面平行方向平周分裂成三个细胞,在两个外侧细胞各形成3-4个精子囊母细胞,每个精子囊母细胞斜壁分裂产生2-4个精子囊,放散后,余下的精子囊母细胞仍留在中间盘状细胞上。     

带形蜈蚣藻盘丝体孢子的形成及温度和光照强度对其放散的影响

在实验室条件下, 首次发现了带形蜈蚣藻(Grateloupia turuturu)盘状体产生的丝状体能够形成孢子, 暂命名为“盘丝体孢子”。研究详细观察了该盘丝体孢子的形成过程, 并探讨了不同温度6、12、16、20、24和30℃及不同光照强度10、30、45、60、90和120 μmol/(m2·s)对盘丝体孢子放散的影响。结果表明: (1) 带形蜈蚣藻雌配子体的囊果释放果孢子, 果孢子发育形成盘状体, 盘状体经过诱导再生出单列细胞的丝状体, 丝状体形成多室孢子囊, 并放散出大量盘丝体孢子; (2) 温度和光照强度均对丝状体中盘丝体孢子的放散产生显著影响。在温度为16℃、光照强度为60 μmol/(m2·s)时盘丝体孢子放散量有最大值; (3) 在温度低于12℃或高于24℃时, 盘丝体孢子的放散受到影响, 数量明显减少; (4) 在光照强度低于30 μmol/(m2·s)或高于90 μmol/(m2·s)时, 盘丝体孢子的放散明显受到抑制。研究结果补充了带形蜈蚣藻无性繁殖过程, 为其种质保存、人工育苗及养殖提供更为丰富的理论依据, 为探讨带形蜈蚣藻的起源与演化提供新思路。     

用细胞工程技术发展我国的紫菜养殖业

利用酶法将紫菜叶状体分离成单细胞和原生质体作为种苗用于紫菜养殖生产。此法与传统育苗法相比,能大幅度提高紫菜产量和质量,育苗周期由半年时间缩短到5天左右,经济效益可提高数倍。     

狭眼凤尾蕨配子体发育及其成精子囊素对水蕨配子体发育的影响

对狭眼凤尾蕨（Pteris biaurita）配子体发育特征及其外源成精子囊素对模式植物水蕨（Ceratopteris thalictroides）在黑暗和光照条件下孢子萌发和配子体发育的影响进行了研究。结果表明：（1）狭眼凤尾蕨孢子深褐色,三裂缝,孢子萌发为书带蕨型,原叶体发育为水蕨型,无毛状体产生;培养发现,其配子体能产生精子器,但不产生颈卵器,当接种密度适中时,可进行无配子生殖。（2）在光照和黑暗条件下狭眼凤尾蕨成精子囊素有促进和抑制水蕨孢子萌发的作用,但效果均不显著。（3）在光照条件下,狭眼凤尾蕨成精子囊素可以延迟水蕨心脏形配子体分生组织缺刻的形成,但对其配子体形态和性别分化无明显影响;而在黑暗条件下狭眼凤尾蕨成精囊素对水蕨长条形配子体的形态发育具有一定影响,与对照组相比其顶端分生组织发达,整体呈长楔形,对性别分化影响不显著。可见,狭眼凤尾蕨和水蕨不具有同种成精子囊素系统。     

昆明地区印度块菌子囊果的解剖结构研究

通过印度块菌子囊果石蜡切片的显微观察,对印度块菌子囊果包被及子实层的显微结构、子囊及子囊孢子的发育过程进行研究.结果表明:(1)印度块菌子囊果是由包被和子实层构成,子囊果的表面密被大量大小不一的疣状突起,包被由外皮层和内皮层构成;子实层由封闭的、大小不一的产孢组织构成,在产孢组织中包含有侧丝、产囊丝、子囊和子囊内的子囊孢子;(2)成熟印度块菌子囊果横切面上有明暗相间的迷宫状纹脉;(3)子囊卵形,由产囊丝顶端的细胞发育而来;(4)成熟子囊孢子红褐色,椭圆形至近球形不等,子囊孢子双层壁,外壁密布有刺状纹饰.子囊孢子在子囊内的发育过程中常出现败育现象,每个成熟子囊中含有1～5个子囊孢子(常见4个).     

蕨类植物性别分化对环境的响应

蕨类植物是维管植物中唯一的孢子体和配子体都能独立生活的类群.同型孢子蕨类配子体的性别分化受到激素和环境因子的影响.生理学研究表明,成精子囊素与赤霉素能诱导雄配子体发育,抑制雌配子体发育;脱落酸阻止成精子囊素诱导的精子器形成;乙烯合成前体ACC促进赤霉素诱导的精子器形成,而乙烯合成抑制因子AOA通过抑制细胞分化来抑制精子器形成.光照对不同种类蕨类配子体分化的影响存在差异.糖类能够促进雄配子体形成,并可加速成熟雌配子体向两性分化.钙离子、钴离子和甲硫氨酸等分别参与了蓝光和赤霉素对配子体性别分化的调控过程.培养密度影响配子体生长及性别表达,高密度下雄性和无性配子体居多,而低密度下两性和雌性配子体居多.近年来的突变体表型分析与分子生物学研究表明,成精子囊素通过影响ANI1、HER、TRA、FEM和MAN等基因的表达调控配子体性别分化.综述了蕨类植物性别分化对环境响应的研究进展.     

卡那霉菌原生质体的分离和再生

本文报道了卡那霉菌原生质体的分离和再生的条件。实验证明用玻璃纸平板培养法,培养卡那霉菌菌丝体可以代替常规的摇瓶培养法,而且操作简单,培养时间短。在培养菌丝体的琼脂培养基中加入甘氨酸后,对菌丝体的生长具有抑制作用,而这种菌丝体对溶菌酶的敏感性与不加甘氨酸培养基中长成的菌丝体对溶菌酶的敏感性基本相同。单独使用溶菌酶就可以分离原生质体,不用再加裂解酶。用蒸馏水处理原生质体,不能裂解原生质膜;0.1％的SDS能完全裂解原生质膜,而且能保留完整的菌丝细胞,从而可以准确计算出原生质体悬浮液中残存菌丝细胞数。原生质体的再生和生长,受再生培养基成份的影响,非高渗性的卡那霉菌产孢子培养基,可用作再生培养基,且能得到较高的再生频率,同时再生菌落的生长也较旺盛。原生质体在再生过程中,和分生孢子一样首先萌发芽管,未发现有如Okanishi所述的扩张现象。卡那霉菌原生质体的再生能力在4℃冰箱中能保存24小时。     

热带假丝酵母产生子囊孢子的条件及单倍体分离的研究

本文研究了诱导热带假丝酵母子囊孢子产生和子囊破壁的条件,以及单倍体分离和鉴别方法.结果表明:NaAc、KCl这两种成分即可满足热带假丝酵母的产孢营养要求,在含有NaAc 8.2 g/L、KCl 1.8 g/L的琼脂培养基中,28℃培养7 d,热带假丝酵母细胞的产孢率可达到47.5%;单纯使用蜗牛酶对破除热带假丝酵母子囊壁的效率甚低,酶解液加入适量的石英砂同时振荡处理4 h左右,可以显著提高对热带假丝酵母子囊破壁速率.用这种方法处理,绝大多数子囊壁均可破除.子囊破壁处理后再以52℃处理10 min杀死残余的营养体细胞,分离物的单倍体孢子比例可由灭活处理前的48%,提高到76%以上.     

DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal "seed" stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R-10 and 2% Macerozyme R-10. The highest number of protoplasts was obtained on a 50-rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as"seed" stock for algal mass culture.     

SPECIALIZED APPENDAGES ON SPERMATIA FROM THE RED ALGA AGLAOTHAMNION NEGLECTUM (CERAMIALES,CERAMIACEAE) SPECIFICALLY BIND WITH TRICHOGYNES1

Spermatia from Aglaothamnion neglectum Feldmann-Mazoyer specifically bind with trichogynes and hairs of female thalli, One of the functions of hairs on female thalli appears to be the catching of spermatia. Fertilization can occur if a spermatium binds first with a hair and then binds with a trichogyne. The binding of spermatia with trichogynes is not species specific, but only occurs beween closely related species. Spermatia have fimbriate coneshaped appendages projecting from each end that are responsible for initial binding with trichogynes.     

Detection of Epigenetic Variations in the Protoplast-Derived Germlings of Ulva reticulata Using Methylation Sensitive Amplification Polymorphism (MSAP)

Regeneration of protoplasts into de novo plants was reported for a large number of seaweed species. The regeneration of protoplasts into different morphotypes as a result of epigenetic variations was discussed for the first time in this study. The loci assessed for methylation modifications in normal filamentous thalli showed a frequency of 32.43% as unmethylated DNA, 24.32% as a hemimethylated, and 20.27% as a methylation of internal cytosine at both the strands. The corresponding methylation values for disk-type thalli were 27.02%, 32.43%, and 14.86%, respectively. The hypermethylation condition was apparent in the disk-type thalli with methylation ratio of 72.97% compared to that of normal filamentous thalli with 67.56%. The frequency of methylation polymorphic sites among the two morphotypes was 53%. The present study reveals the distinct expression of cytosine methylation and is thus correlated to differential morphogenesis of plants regenerated from cultured cells. The number of protoplasts regenerating into filamentous thalli declined with increasing temperature from 15°C, 20°C, 25°C, and 30°C. The disk-type variant had higher thermal stability at 30°C over normal filamentous thalli. Further, this variant could maintain itself for more than a year in the laboratory indicating its suitability for in vitro germplasm maintenance and propagation.     

Formation and regeneration of protoplasts and spheroplasts of gastrointestinal strains of lactobacilli.

Methods were developed for the formation of protoplasts and spheroplasts of gastrointestinal strains of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus salivarius. Attempts to regenerate vegetative cells from protoplasts were not successful, but spheroplasts could be regenerated consistently for five of six strains.     

Formation and regeneration of protoplasts and spheroplasts of gastrointestinal strains of lactobacilli

Methods were developed for the formation of protoplasts and spheroplasts of gastrointestinal strains of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus salivarius. Attempts to regenerate vegetative cells from protoplasts were not successful, but spheroplasts could be regenerated consistently for five of six strains.     

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     

Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae)

Protoplast were isolated from a filamentous brown alga, Sphacelaria sp. (Sphacelariales, Phaeophyta), using alginate-lyases extracted from marine molluscs, and commercial pectinase and cellulase. Yields were about 4000 protoplasts per gram of fresh tissue. Different types of protoplasts, originating from apical, subapical, nodal and internodal cells, could be readily identified based on their size and pigmentation. Apical cells produced a higher percentage of protoplasts (approx. 2%), compared with other cell types. All apical-cell protoplasts regenerated into new thalli and most other types of protoplasts divided at least once in culture, but did not develop further.     

Effects of nutrients,cell density and culture techniques on protoplast regeneration and early protonema development in a moss,Physcomitrella patens

To regenerate auxotrophic mutants of Physcomitrella patens, two media of increasing complexity were developed. The survival rate of protoplasts was around 30% higher on full medium when compared to standard minimal medium. Protoplast survival was higher in a medium containing 2.5 mmol/L ammonium tartrate compared to a medium with 5 mmol/L of this compound. Solid medium had a positive effect on protoplast survival compared to either liquid medium or solid medium overlaid with cellophane; the maximum survival rate being 31.6%. However, the number of surviving protoplasts without any cell division during the first ten days increased on solid medium. Density and survival rate of protoplasts were positively correlated, but the formation of long protonema filaments decreased markedly. The effect of different protoplast densities could be explained partly by physiologically active compounds excreted into the medium.     

DEVELOPMENT OF PROTOPLASTS OF ULVA FASCIATA (CHLOROPHYTA) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate protoplasts of the green alga Ulva fasciata Delile and subsequently induce them to form a microthallus suspension for algal seed stock. The protoplasts were covered with secreted mucilage following 6 h of culture when viewed with SEM. The mucilage fused to form thick layers during day 1 of culture. Microfibrillar cell walls were deposited into the thick layers of mucilage on the 5th day of culture. An average of about 10% of the freshly isolated protoplasts began to divide at 6–14 days. These protoplasts subsequently developed varied morphologies, depending on the time of collection during the year. Protoplasts isolated from U. fasciata collected in March to June developed frond thalli or microthalli when they were cultured in low or high densities (cells/area), respectively. The microthallus suspension was cultured for more than two years at 10–40 μ mol·m^{− 2} ·s^{− 1} . Frond thalli formed when the suspension was cultivated at 100–160 μ mol·m^{− 2} ·s^{− 1} . Therefore, microthallus suspension can serve as a seed stock of U. fasciata .     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.