Spermatophore cryopreservation and artificial insemination of black tiger shrimp, Penaeus monodon (Fabricius)

To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post‐thaw spermatophores. Spermatophores were collected during consecutive regenerations (15‐day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post‐thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen‐thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning.     

果树种质资源离体保存研究进展

综述了国内外果树种质离体保存方面的研究进展,简单介绍了种子保存,常规的细胞、组织和器官保存方法。超低温保存是一项长期稳定保存种质的方法,能减慢细胞代谢和完全抑制生长,概述了超低温保存的原理、技术要点和主要优点,以及影响超低温保存的因素:包括植物材料的性质、预处理、冰冻保护剂种类、降温方法、解冻方法以及解冻后处理等,并对今后果树种质保存提出了一些建议。     

香蕉茎尖的玻璃化法超低温保存及其植株再生

 以香蕉(Musa spp. ) 为试材, 对其离体培养茎尖玻璃化法超低温保存影响因素进行研究。结果表明, 不定芽在MS + 3.0～5.0 mg/L 6-BA + 0.1 mg/L NAA的培养基上分化较好。香蕉茎尖超低温保存较佳体系是: 2.0～3.0 cm的茎尖在含0.4 mol/L蔗糖培养基上预培养2 d, 剥取带1～2个叶原基的茎尖(长1.0～1.5 mm) , 室温(25℃) 下装载液(MS + 2 mol/L甘油+ 0.4 mol/L蔗糖) 装载20～30 min, 然后用玻璃化溶液( PVS2 ) 于0℃下处理40 min, 换1次PVS2后迅速投入液氮。保存至少1 h后, 在40℃水浴中化冻90 s, 用1.2 mol/L蔗糖培养液洗涤2次, 每次10 min, 然后转入含0.3 mol /L蔗糖的MS培养基上,暗培养10～15 h后转移到含0.5 mg/L 62BA的MS培养基中, 暗培养1周后转移到正常光下, 3个香蕉品种(巴西蕉、广东香蕉2 号、广东粉蕉1 号) 的成活率分别为75.9%、40.0%和69.6% , 再生率分别为63.4%、35.0%和63.4%。再生植株生长和分化正常, 生根后可移栽成活。     

香蕉茎尖滴冻法保存中细胞的超微结构变化

 应用透射电镜研究了香蕉茎尖滴冻法超低温保存中细胞超微结构的变化规律。结果表明: 装载后, 细胞液泡化程度降低, 出现质壁分离。脱水处理使质壁分离程度加重, 原生质体浓缩, 一部分细胞的细胞壁、膜系统发生了不可逆的损伤; 也有小部分位于分生组织区域的细胞结构虽然发生了变化, 但程度不深, 在恢复培养时会自动修复, 并再生出植株。细胞严重伤害主要发生在脱水处理过程中, 冷冻环节基本上不产生新的损伤。讨论了上述变化的可能机制。     

桃离体茎尖的超低温保存及植株再生

 以简单玻璃化法为基本方法, 研究了影响桃离体茎尖超低温保存后存活率的因子———低温驯化时间、蔗糖预培养时间、玻璃化液处理时间及化冻后植株再生条件; 建立了较为适宜的超低温保存技术程序———选择继代培养30 d的试管材料, 5℃低温驯化3～4周, 在含017 mol/L蔗糖的固体培养基预培养2 d, 再经玻璃化液PVS3处理100 min后浸入液氮, 化冻后茎尖存活率可达60%以上。     

Low temperature storage and in vitro germination of cherimoya (Annona cherimola Mill.) pollen

Due to the protogynous dichogamy of cherimoya and to the absence of proper pollinating vectors, hand-pollination with fresh pollen is a common practice for cherimoya commercial production. In order to optimize the process of hand-pollination, in this work we have studied the conservation of cherimoya pollen at −20, −80 and −196 °C for up to 3 months. In vitro pollen germination of fresh pollen was 57.1% and it was progressively reduced with conservation time at the three temperatures studied reaching a minimum after 3 months of storage of 10.4%, 14.2% and 13.6% at −20, −80 and −196 °C, respectively. Differences in germination among temperatures were only significant during the first 2 weeks of storage. Field pollinations with pollen stored for up to 3 months at the three temperatures show no yield differences compared to pollinations performed with fresh pollen. The results indicate that pollen collected and stored at sub-zero temperatures at the beginning of the cherimoya blooming season can be used along the whole blooming season avoiding the need of collecting fresh pollen daily.     

内皮生长晕细胞冻存和复苏的可行性

目的研究冻存对内皮生长晕细胞增殖能力和成血管能力的影响,探讨内皮生长晕细胞冻存和复苏的可行性。方法分离脐血中单个核细胞,采用贴壁培养法培养扩增内皮生长晕细胞,免疫组织化学染色和荧光染色法鉴定其内皮细胞特性。扩增后的细胞采用浓度为650μmol/L(体积比为50mL/L)和1040μmol/L(体积比为80mL/L)二甲基亚砜的培养基冻存至液氮中,24h后复苏并观察冻存细胞的复苏率、复苏后细胞的增殖能力和成血管能力的改变。结果采用贴壁法培养的细胞具有多种内皮细胞特性。细胞采用含不同浓度二甲基亚砜(50mL/L和80mL/L)的培养基冻存后,其复苏率差异无统计学意义,细胞的增殖能力在复苏后36h内50mL/L、80mL/L二甲基亚砜组均较未冻存组减弱(P<0.05),72h后三组间比较差异无统计学意义。而复苏后6h内成血管速率较未冻存组减弱(P<0.05),但30h后三组间比较差异无统计学意义。结论内皮生长晕细胞可以进行冻存和复苏。     

银杏愈伤组织超低温保存的研究

本文对银杏子叶愈伤组织进行了超低温保存研究。结果表明 ,冰冻保护剂、冷冻速度对解冻后材料相对存活率影响较大 ,冻前预培养和预冻至某一温度停留一段时间也较为重要。在含高浓度山梨醇或甘露醇的培养基上预培养 2d的愈伤组织 ,用 10 %二甲基亚砜 +0 .5mol·L- 1 山梨醇作为冰冻保护剂 ,以 1℃·min- 1 速度降温 ,在 - 15℃、- 35℃分别停留 10min和 30min后 ,投入液氮中保存 1d ,细胞相对存活率可达 6 0 %以上。再培养时 ,细胞能恢复生长。     

Somatic embryo culture for propagation,artificial seed production,and conservation of sawara cypress (<Emphasis Type="Italic">Chamaecyparis pisifera</Emphasis> Sieb. et Zucc.)

 Somatic embryogenesis in Chamaecyparis pisifera Sieb. et Zucc. was initiated from immature seeds collected from the end of June to early July. Mass propagation through adventitious shoot bud production from somatic embryo culture on Woody Plant (WP) medium and artificial seed production using sodium alginate was achieved. A high bud forming index value (25.8) was obtained on medium supplemented with 1 μM 6-benzylaminopurine. The conversion rates from artificial seeds under aseptic and nonaseptic conditions were 60%–100% and 10%–12%, respectively. For germplasm conservation, somatic embryos and embryogenic cells were successfully stored at 4°C (medium-term storage) and in liquid nitrogen for long-term storage. Received: December 21, 2001 / Accepted: August 1, 2002 Acknowledgments This work was supported in part by the Japan Science and Technology Corporation and in part by a Grant for Research for the Future Program from the Japan Society for the Promotion of Science. Correspondence to:E. Maruyama     

Trehalose in glycerol-free freezing extender enhances post-thaw survival of boar spermatozoa

Cryopreservation of boar semen is still considered suboptimal due to lower fertility as compared with fresh samples when glycerol, a permeating cryoprotectant, is used. Trehalose is a non-permeable cryoprotectant and nonreducing disaccharide known to stabilize proteins and biologic membranes. The aim of this study was to evaluate the cryosurvival and in vitro penetrability of boar spermatozoa when glycerol was replaced with trehalose in a freezing extender. Ejaculated Berkshire semen samples were diluted in egg yolk-based freezing extender containing glycerol (100 mM) or trehalose (0, 50, 100, 150, 200 and 250 mM) and cryopreserved using a straw freezing procedure. Thawed samples were analyzed for motility, viability, mitochondrial membrane potential (MMP), and acrosome integrity. In experiment 2, penetrability of spermatozoa cryopreserved with 100 mM glycerol or trehalose was examined. Replacement of cryoprotectant glycerol (100 mM) with trehalose had no effect on sperm viability, but replacing it with 100 mM trehalose improved motility, MMP and acrosome integrity significantly. Sperm motility and MMP were considerably higher in 100 mM trehalose, whereas the acrosome integrity was substantially higher in 100–250 mM trehalose. The in vitro penetration rate was also significantly higher in spermatozoa cryopreserved with trehalose (61.3%) than in those cryopreserved with glycerol (43.6%). In conclusion, 100 mM non-permeable trehalose can be used to replace glycerol, a permeating cryoprotectant, for maintenance of better post-thaw quality of boar spermatozoa.