Cryopreservation of Gametophytes of Laminaria japonica (Phaeophyta) Using Encapsulation-Dehydration with Two-Step Cooling Method

Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen using encapsulation-dehydration with two-step cooling method. Gametophytes cultured at 10℃ and under continuous irradiance of 30 μmol m^-2 s^-1 for 3 weeks were encapsulated in calcium alginate beads. The beads were dehydrated in 0.4 molLl sucrose prepared with seawater for 6 h, desiccated in an incubator set at 10℃ and 70% relative humidity for 4 h, pre-frozen at either -40℃ or -60℃ for 30 min, and stored in liquid nitrogen for 〉24 h. As high as 43% of survival rate was observed when gametophytes were thawed by placing the beads in 40℃ seawater and re-hydrated in 0.05 molL^-1 citrate sodium prepared using 30‰ NaCl 7 d later. More cells of male gametophytes survived the whole procedure in comparison with female gametophytes. The cells of gametophytes surviving the preservation were able to grow asexually and produce morphologically normal sporophytes.

Influence of Temperature and Salinity on Germination of Eelgrass (Zostera marina L.) Seeds

Seagrass restoration as part of ocean ecosystem protection has been launched for many years all over the world, but intensive research on this subject in China has just begun in recent years. Seed broadcasting has been widely accepted as the most potentially useful method for seagrass restoration over large areas. We examined the influence of key environmental factors on seed germination to help promote eelgrass bed restoration. Under anoxic conditions, the influence of temperature and salinity on the germination rate of eelgrass (Zostera marina L.) seeds was examined at different combinations of four temperatures (4, 9, 14, and 24℃) and nine salinities (5 to 45, increment of 5). The effect of significant interaction of temperature and salinity on germination rate was observed (ANOVA) (P<0.001). The highest germination rate (83.3 ± 3.5)% was reached in 8 weeks at 14℃ and salinity 5. Higher temperature significantly increased the germination rate at salinity 5 (P<0.001) during the whole observation period except for 24℃, while lower salinity significantly increased the germination rate at 14℃ (P<0.001). Although significant interaction was found between temperature and salinity (P<0.001), the influence of salinity was stronger than that of temperature for the germination of eelgrass seeds. These results provide useful information for the propagation of artificial seedlings for seagrass restoration in China.

海带配子体保存过程中污染菌的分离纯化及药敏试验

该论文通过对海带配子体保存中污染菌的分离纯化及药敏试验,分离获得一些主要的污染菌群并探索有效的抗生素及使用浓度。将污染的样本涂板,待长出菌落后,反复3区划线,分出单个菌落,得到纯培养。该试验分出2株真菌(粗丝和细丝)和1株细菌,然后再接种于液体培养基增菌,收集细菌或真菌用相应的方法提取DNA。真菌用18 s rDNA通用引物,细菌用16 s rDNA通用引物进行PCR反应,最后将PCR反应产物送基因公司测序,将测序结果在GeneBank中比对。结果表明:粗丝18 s rDNA序列与Fusarium proliferatum同源性最高,为100%;细丝18 s r DNA序列与Nectria mauritiicola同源性最高,为99%;细菌16 s rDNA序列与Halomonas sp.同源性最高,为99%。通过药敏试验观察发现粗丝的制霉菌素敏感抑菌圈直径为13~15 mm;细丝的先锋霉素敏感抑菌圈直径为14~16 mm;细菌的盐酸青霉素较敏感,其抑菌圈为15~17 mm。

温度和光照强度对多肋藻(Costaria costata)配子体生长影响研究

通过设置不同温度和光照强度,在实验室条件下检验了多肋藻雌、雄配子体在不同温度和光照强度条件下的生长情况。结果显示:多肋藻雌配子体在20℃和1 000 lx条件下生长最快,经过4周生长,配子体平均总长度由81. 27μm增加至10 234. 07μm,相对生长速率约16. 58%,生长速度显著大于其他试验组(P <0. 05);雄配子体在20℃和2 000 lx生长最快,配子体平均总长度由77. 63μm增加至17 215. 33μm,相对生长速率约18. 54%,生长速度与20℃和1 000 lx试验组差异不显著,但显著大于其他试验组(P <0. 05)。本研究为多肋藻配子体规模化扩增培养提供了数据参考。

铁盐对海带雌配子体生长发育的影响

为探明铁盐对不同品种(系)以及相同品种(系)在不同生长条件下海带雌配子体的影响,通过添加不同质量浓度的Fe^(3+),对培养在低温弱光[(10.0±1.0)℃、2μmol/(m^(2)·s),L]和高温强光[(15.0±1.0)℃、80μmol/(m^(2)·s),H]条件下的“901”、韩国海带和“连杂1号”3个品种(系)海带雌配子体的生长发育进行了观察。试验设置0、0.5、1.0、1.5 mg/L 4个Fe^(3+)质量浓度,光照周期为10 L∶14 D,试验持续1个月。结果表明,铁盐能够促使“901”雌配子体生长与成熟、排卵进行孤雌生殖,Fe^(3+)质量浓度越高,配子体排卵的比率越高,“901”(L)配子体排卵的比率低于“901”(H)。韩国海带和“连杂1号”很少有雌配子体细胞排卵,Fe^(3+)质量浓度越高,其营养生长越旺盛。韩国海带(L)的相对生长率低于韩国海带(H),“连杂1号”(L)的相对生长率略低于“连杂1号”(H)。试验中所生成的孢子体均为畸形,且最后均死亡。

海带配子体低温冷冻保存研究

设置3个不同的温度组A(-80℃)、B(-40℃)、C(-20℃)和1个对照组D(未冻存配子体细胞),对不同品种的海带配子体细胞段或细胞簇进行低温冷冻保存,15 d后取出,在适宜条件发育培养了6个月。结果表明,不同冻存温度、雌雄差异,对海带配子体细胞段或细胞簇存活率有显著的影响(P<0.05),A组和B组冻存的配子体细胞存活率显著高于C组。相同冻存条件下,细胞簇的成活率显著高于细胞段的存活率。A组和B组冻存的细胞簇成活率均为34%~43%,而A组和B组冻存的细胞段成活率均<30%,其中901雄配子体成活率>20%,其他均<10%。在复苏培养过程中,各组存活细胞段或细胞簇的生长状态均与对照组无显著差异。在发育培养过程中,A组和B组冻存的配子体细胞的发育进程及最终发育率无显著差异。

不同种类冷冻保护剂对海带配子体及孢子的毒性评价

为筛选用于海带超低温保存的冷冻保护剂,采用10%二甲基亚砜(DMSO)、10%甘油、10%蔗糖、10%乙二醇、10%DMSO+10%甘油、10%DMSO+8%葡萄糖、10%DMSO+10%蔗糖、10%DMSO+10%甘油+8%葡萄糖、10%DMSO+10%山梨醇等9种单一或复合冷冻保护剂,在0~4℃条件下分别对海带配子体和孢子进行处理,对配子体处理30 min、60 min、24 h,对孢子处理15、30、60 min,通过统计配子体细胞的存活率以及孢子萌发成配子体的比例,对不同种类的冷冻保护剂进行毒性评价。结果表明:9种冷冻保护剂均对海带配子体和孢子有一定的毒性作用,并且毒性随着处理时间的延长而增强。10%DMSO对海带配子体细胞的毒性最小,处理60 min后配子体细胞的存活率为100%;其次是10%乙二醇,处理30 min后配子体的存活率为100%;含10%甘油的单一或复合冷冻保护剂对配子体细胞毒性较大,处理后配子体的成活率显著低于DMSO和乙二醇组(P<0.05)。单一冷冻保护剂对海带孢子的毒性显著低于复合冷冻保护剂,除10%甘油组处理30、60 min外,其他单一冷冻保护剂试验组的配子体发生率均在60%~80%。

二甲基亚砜对海带配子体克隆低温冻存的影响

以海带配子体克隆为试验对象,采用4种体积分数二甲基亚砜(DMSO)冷冻保护液,对10个品种(系)20个克隆系进行了低温冷冻保存试验。结果表明,海带配子体克隆在-80℃冻存10 d,复苏后培养,DMSO各体积分数组均有海带配子体细胞存活,其中10%和15%体积分数组冻存的20个克隆系均有存活细胞,5%和20%体积分数组仅部分克隆系有存活细胞。对于相同克隆系,10%和15%体积分数组配子体存活率高于5%和20%体积分数组。指出,在海带配子体低温冻存中,10%和15%DMSO可作为冷冻保护液,为降低成本,优选10%DMSO作为冷冻保护液。提出,经-80℃冻存的海带配子体复苏后培养,进行繁育能力验证,能正常发育,形成孢子体,低温冻存不影响其繁育能力。