预防组培中蓖麻子叶节玻璃化的研究

为了预防蓖麻子叶节在组织培养过程中的玻璃化现象,以建立其高效的遗传转化体系,本实验对影响子叶节生长状态的几个因素进行了初步研究。结果表明,在胚轴、子叶均为淡黄色的时期切取蓖麻子叶节,接种在1/8MS、内含10g/L琼脂粉、20g/L蔗糖、8.0mg/LZT、1.0mg/LNAA的培养基中进行培养,能有效地预防蓖麻子叶节玻璃化。     

油菜玻璃腋生枝的形态特征及影响其形成的因素

切除顶芽和叶片的油菜带节枝段,在含BA1mg/l和NAA0.1mg/l的MS培养基上,其腋生分生组织通常发育成三种类型腋生枝:正常的、半透明的或玻璃化的。顶芽和叶片的存在抑制玻璃腋生枝形成,叶片对腋生分生组织的正常发育也具有一定的作用。玻璃腋生枝的形成与培养基中的细胞分裂素有关。腋生分生组织发育成一定大小的正常腋芽后,不能形成玻璃腋生枝。     

青霉素在香石竹组织培养中的作用

青霉素10-80万单位/1对香石竹(Dianthus caryophyllus L.)愈伤组织芽的分化有促进作用,对愈伤组织苗的生长有抑制作用。在80万单位/1浓度时芽生长出现异常现象。青霉素对杆插苗的生长有抑制作用。随着苗的生长、浓度的提高,对发生根的抑制增强,对芽的抑制减弱,以至消失。在香石竹培养中对消除玻璃化苗、叶色彩化有明显效果。     

大蒜茎尖玻璃化法超低温保存技术研究

 用山东‘苍山大蒜’进行了茎尖玻璃化法超低温保存技术的研究。5～8 mm大蒜茎尖在MS +0.7 mol/L蔗糖的固体培养基上预培养7 d, 切取3.0～3.5 mm的茎尖, 在20℃下经60% PVS2 处理60 min,再于0℃下用PVS2 处理5～60 min后, 换适量新鲜PVS2 , 浸入液氮。保存2 d或1个月后取出, 在37℃水浴中解冻2 min, 用MS + 1.2 mol/L蔗糖液体培养基洗涤2次, 每次10 min, 经过恢复培养, 茎尖成活率最高可达到100%。     

蝴蝶兰类原球茎玻璃化产生的原因及恢复效果研究

对蝴蝶兰(Phalaenopsis)组培生产中引起类原球茎玻璃化的原因以及玻璃化类原球茎再利用的效果进行了研究.结果表明,随着植物生长调节剂浓度增高和继代培养的时间延长,类原球茎玻璃化程度加重.当NAA浓度为5 mg/L、BA浓度为10 mg/L,继代培养到第9代时,2种培养基中玻璃化率分别高达71%和65%.玻璃化类原球茎的平均增殖率仅为2.2,再生植株率为183株/瓶,明显低于正常类原球茎的5.3和1 297株/瓶.玻璃化类原球茎在无植物生长调节剂培养基中经2～3代恢复培养后,数量下降到0.84%,玻璃化现象可得到明显恢复,恢复后的类原球茎的分化能力和植株生长状态与正常类原球茎一致,无变异现象发生,可继续应用于组培生产.     

小鼠桑椹胚简易玻璃化冷冻技术再探讨

本试验继小鼠扩张囊胚玻璃化冷冻保存成功后，在室温（２５℃）下利用不同浓度的ＥＦＳ玻璃化溶液，对小鼠的桑椹胚简易玻璃化冷冻技术进行再探讨。结果是胚胎在１０％ＥＧ溶液中预先处理５分钟，再移入事先配置好含有ＥＦＳ３０的０．２５ｍｌ塑料细管中１分钟平衡后直接投入液氮中冷冻，解冻后获得的发育率最高（９４％）。冻胚移植后妊娠率和产仔率分别为５６％（９／１６）及４２％（４９／１１６）。与对照组相比差异不显著（Ｐ＞０．０５）     

转基因兔胚胎玻璃化冷冻保存的研究

在２５℃条件下,将兔体外受精精子载体转基因兔桑椹胚置于含有４０％乙二醇、１８％Ｆｉｃｏｌ、０．３ｍｏｌ蔗糖的ｍＰＢＳ溶液（ＥＦＳ４０）中平衡２分钟,然后直接投入液氮,成功地进行了玻璃化冷冻保存。解冻后桑椹胚发育至囊胚和孵化囊胚的比例分别为６５．８１％和３９．２４％,与未经冷冻的鲜胚发育比例（７１．０５％和４３．４２％）相比,没有明显的差异。７８枚经玻璃化冷冻和解冻的桑椹胚移植给５只受体,其中２只妊娠,共产下８只活仔兔。     

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.     

小鼠扩张囊胚低渗液处理试验

为研究解冻后的胚胎膨胀对其发育率的影响,本试验用２０～５０％的低渗ＰＢＳ溶液（即０．２０×、０．２５×、０．３０×及０．５０×）,分别将小鼠扩张囊胚在低渗液中处理３０分钟后培养,结果新鲜胚在０．５０×和０．３０×中处理后的发育率分别为１００％和９１％,０．２０×处理组的发育率降低到５３％。解冻后的胚胎直接用低渗液处理,０．５０×组只有８５％继续发育,低于０．２５×组完全不能发育。而解冻后经培养恢复到扩张囊胚后再做低渗处理,０．３０×组仍有９６％继续发育。     

Evaluation of apoptosis in long-term culture of vitrified mouse whole ovaries

The purpose of the present study was to investigate the development of follicles and incidence of apoptosis in vitrified neonatal mouse ovaries cultured in vitro in the presence of leukemia inhibitory factor (LIF). The vitrified and non-vitrified ovaries of 1-week-old mouse were cultured in the presence or absence of LIF for 7 days. At the beginning and at the end of culture period in each ovary of all groups of study the mean area and the development of ovarian follicles were analyzed; moreover, the incidence of apoptosis was assessed by transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method, DNA laddering and caspase-3/7 activity technique. The hormonal assay was done on the conditioned media collected during culture period. The proportion of preantral follicles and the levels of hormones increased in all cultured groups and it was significantly higher in LIF treated groups than in their control (P < 0.001). The ultrastructural characteristics of cell death, DNA fragmentation and TUNEL positive signals were prominent in vitrified cultured ovaries. The level of caspase-3/7 activity was higher in vitrified cultured ovaries.