【作者】 刘建新；

【导师】 董良峰； 吴超元；

【作者基本信息】 中国科学院海洋研究所 ， 海洋生物， 2001， 硕士

【摘要】 为了进一步完善裙带菜单倍体克隆（简称单克隆，以下同）育苗技术，实现该技术的产业化，本文对单克隆的快速扩增以及育苗条件进行了优化研究。获得如下结果：一、光对裙带菜排卵的干扰现象： 一般附苗后4-5天雌雄配子即已形成。接近成熟的卵囊细胞翘起，长度达到50-55μm，直径为19-20μm，一到两天后即可排卵。精子囊略透明，颜色微绿。在11:13（L:D）的光周期下，排卵一般在黑暗开始后5分钟即已开始，5-15分钟后达到高峰。排卵的过程只有几秒钟。卵排出后停留在卵囊开口处，仍与卵囊牢固相连。 虽然排卵过程只有几秒钟，一旦这个过程受到光的干扰，就会诱导卵脱离卵囊。实验证实，5-6μmol m^-2s^-1的微弱光强（显微镜下镜检的光强）即能引起95％的卵从卵囊脱落。根据我们的观察，一旦卵已排出并与卵囊相连，将不再受光的干扰而脱落。脱落的卵很快死亡分解。将配子已经形成但尚未释放的培养转移至连续光下以后，卵仍能排放，但排出的卵大部分脱落（75％），进一步说明了光对排卵的干扰作用。 在育苗实践中，为了方便工作，育苗车间内在天黑后的2-3小时内仍有照明，而在第二天早晨取样观察，就会发现许多空的卵囊。我们的实验证实了这是由于排卵过程受到光的干扰引起的。因此，在育苗实践中，为了提高出苗率，在出苗期间，天黑后2-3小时内育苗车间内应避免照明，以降低光导卵的脱落。二、单克隆快速扩增以及育苗条件的优化： （一）、单克隆快速扩增的优化。 实验结果表明： 1．单克隆快速生长的适宜光照强度为80-120μmol m^-2s（-1）。单克隆的培养密度宜 控制在1-10g/L。接种密度以1g/L为宜。 2．单克隆藻体内能贮藏氮，因此在单克隆快速扩增过程中，可采用间歇施肥法补 充氮源。实验证明，培养液中氨态氮的适宜浓度为20μmol/L，可维持单克隆 6天的生长之用（单克隆培养密度1-10g/L）。 3．实验证明，在65天内，“老水”对单克隆的生长没有明显的抑制作用。而且， PES培养基除去氮的其它成分也可满足单克隆65天的生长需求。 4．单克隆快速生长的适宜温度为22℃。 另外，单克隆密度与消光值之间存在着线形关系，可以通过测量消光值来计 算单克隆的培养密度和鲜重，以代替称量法测鲜重。 综上，可提出如下单克隆快速扩增的模式：温度为22℃；初始光强控制在 80-120μmol m^-2s^-1；单克隆的接种密度为1g/L；PES培养基（除去氮的成分） 刘建新 中国科学院海洋研究所硕士论文 可供单克隆65天的培养之用，在这65天内，每隔六天左右将初始氨态氮补充至 20 pmol几 单克隆的培养密度控制在110g儿：单克隆培养密度和鲜重可通过测 量消光值计算得到。 依以上模式，并及时进行扩养，单克隆的鲜重平均日增长率可达25％，以此增 长率计算，109单克隆经一个月培养后鲜重可达8070克，可培育苗帘2000多个，：实现海面养殖1000多亩，达到了生产性育苗的要求。 （二）、单克隆育苗条件的优化 根据实验结果，可采取如下的育苗优化措施： 1．光对排卵有干扰作用。因此，在出苗期间，在天黑后2－3小时内，育苗车间 内应避免照明，以降低光导的卵子脱落。 2．in mol／L磷（PO。’－－P）和 70 u mol／L氮（NH。”－N）有利于加速出苗。 3．铁对配子体的发育有明显的促进作用。2．5 11 mol儿的 Fe（Ill）能使出苗时间 提前卜 天，出苗率增加 15％左右。 4．单克隆育苗较适宜的温度为 17’C。 依以上条件，个5天左右即可出苗，比一般出苗时间提前1－2天。出苗率也 有所增加，例如在实验室条件下，出苗率由原来的50－60％增至80－90％，增加了30％ 左右。 本项研究进一步完善了裙带菜单克隆育苗技术，将大大有利于该技术的进一步推 广。更多还原

【Abstract】 A new sporeling culture method using gametophyte clones was developed in the early nineties. In the course of extension, there exits two problems: commercial scale of gametophyte clones culture and high outgrowth rate in sporeling raising. The aim of this study, therefore, is to solve these two problems: I . Light etachment of egg from the tip of oogonium: In general, female and male gametes formed five to six days after the attachment of gametophyte fragments to the slides. In female gametophyte, the sign of approaching maturity was the erection of the swollen tip cell which is about 50?5 11 m in length and 19 20 l.A m in diameter, in male gametophyte, a transparent, greenish and papilla-like protrusion formed, through which sperms were eventually released. It was observed that under normal 11:13 LD photoperiod regime, egg discharge occurred five minutes after the beginning of the dark period and peaked 5-15 minutes later. The course of egg discharge took only a few seconds. The egg extruded out was firmly attached to the oogonium apex. The course of egg discharge took only a few seconds, but once it was disturbed by irradiance of as low as 5-6 i mol m2s necessary for microscopic observation, 95% of the discharged eggs separated from the oogonia. However, egg can not be detached by light once it has been discharged and attached to the oogonium apex. Even the detached eggs could be fertilized, but they died and decomposed finally. It was found that when the slides were transferred from the normal 11:13 photoperiod regime to continuous light, egg discharge occurred five minutes after the ending of the ormal light cycle and peaked 5-15 minutes later the first day after transfer. However, further observation revealed that 75% of the eggs separated from oogonia in a few seconds. The extension of the clone technique in Undaria pinnat fida sporeling culture carried out in Shandong Province since 1997 led to large-scale production of healthy sporelings for commercial cultivation. However, the following three years?practice showed that the sporeling outgrowth rates were uneven, in some cases, very low, even 50% lower than the usual rates. In sporeling culture practice, lights in the greenhouse were sometimes still on in the period 2-3 hours after dusk; consequently many empty oogonia were found the next morning. As loss of eggs was proved to be caused by the disturbance of light, darkness control in the period 2-3 hours after dusk was suggested as a means to enhance the outgrowth rate. II . Optimization of gametophyte clone mass culture and sporeling raising techniques 1. Fast propagation of gaznetophyte clones: (1). Clones grew best under irradiance of 80?20 l.mol m2s Culture density be better controlled between log/L. lg/L was considered as the suitable initial culture density. (2).Fast growth of clone consumes a lot of nitrogen. As clone can store nitrogen inside the cell, ammonium and nitrate nitrogen can be supplemented periodically to ensure fast growth of clone. Favorable ammonium itrogen concentration in culture medium is 20 .tmol/L which meets the demand of growth of clone in six days (clone density is 1 lOg/L). (3).Old culture medium had no apparent inhibitory effect on the growth of clone in 65 days. Other elements in PES medium exclude N were proved to be sufficient for growth of clone within this period. (4). Favorable temperature for growth of clone is 220C.更多还原