白檀未成熟胚的器官发生和植株再生

以白檀未成熟胚为试材,以改良MS为培养基,研究了白檀未成熟胚的器官发生和植株再生.结果表明:改良MS(mMS)能够很好的诱导愈伤组织,在添加了0.2 mg/L 6-BA+0.1 mg/L NAA的培养基中,诱导率高达92.5％;胚性愈伤组织分化最佳培养基为mMS+0.25 mg/L 6-BA+0.15 mg/L NAA,分化率为72.4％;分化出的胚性愈伤组织在空白mMS培养基上继代培养15 d,76.6％的组织分化出芽,再转入1/2mMS+ 1.5％蔗糖培养基上培养20 d,芽长至1.5 cm.     

有无透明带猪卵母细胞电激活参数的优化及体外培养方式的建立

[目的]摸索无透明带猪体外成熟卵母细胞(ZFOs)电激活的条件和无透明带孤雌激活胚胎(ZFEs)的体外培养方式,为手工克隆法生产广西巴马小型猪体细胞核移植奠定基础。[方法]用不同场强、脉冲时程和脉冲次数激活ZIOs、ZFOs,用不同培养方式(微滴法和微穴法)培养ZIEs和ZFEs,检查第7天的囊胚率及囊胚的总细胞数。[结果]有带猪体外成熟的卵母细胞电激活最佳场强和脉冲时程为125 V/(mm.80μs),微滴法培养的囊胚发育率为(30.54±11.06)%;无带猪体外成熟的卵母细胞电激活最佳场强和脉冲时程为85V/(mm.80μs),微穴法培养,其囊胚发育率为(12.77±6.98)%;单次脉冲的激活效果好于多次脉冲。[结论]对有无透明带胚胎的培养方式,微穴法培养无透明带胚胎,微滴法培养有带胚胎较合适,一次脉冲可以有效激活卵母细胞。     

显微注射胚胎干细胞对长爪沙鼠胚胎发育的影响

将不同数量的成系的小鼠胚胎干细胞(ES细胞)显微注射注入长爪沙鼠(Merionesunguiculatus)的不同胚期的胚胎中,研究其对胚胎发育的影响,以期初步探索嵌合胚胎制作的条件。试验结果表明,显微注射2～5、6～10和11～15个ES细胞数量之间对早囊/囊胚期胚胎的成活率和孵化率的影响均不显著,但显微注射11～15个ES细胞的成活率和孵化率较低,分别为82.14%和64.29%;显微注射2～5个ES细胞的成活率和孵化率均较高,分别为90.90%和78.79%。显微注射11～15个ES细胞和未经显微注射的胚胎成活率分别为95.12%和82.14%,差异极显著(P<0.01);孵化率分别为90.48%和64.29%,差异极显著(P<0.01)。不经显微注射的桑椹/囊胚、经显微注射的8～16-细胞期胚胎和早囊/囊胚,其成活率分别为94.92%、91.53%和87.50%,差异均不显著(P>0.05);从孵化率来看,未经显微注射的桑椹/囊胚和经显微注射的8～16-细胞期胚胎其孵化率分别为89.83%和61.02%,差异极显著(P<0.01)。     

Effects of Donor Cells Treated with Egg Extracts on the Development Competence of Cloned Embryos

Japanese Balck cattle fetal fibroblasts (JBCFF) were induced with Xenopus leavis egg extracts and somatic cell nuclear transfer (SCNT) was carried out with the reprogrammed JBCFF as donor cells in order to investigate their effects on SCNT efficiency.Three samples of egg extracts were acquired from different Xenopus laevis.The protein contents and kinds in extracts were evaluated with BCA Protein Quantification Kit and SDS-PAGE.Concentration of Digitonin to permeabilize JBCFF was optimized and assessed with PI staining.Reprogrammed cells treated with egg extract were used as donor in SCNT.Additionally the reconstructed embryos were activated with ionomycin+6-DMAP and A23187+6-DMAP to compare their effects on the development competence.The protein contents of extracts samples were 56.2255,64.6570 and 71.2158 μg/mL,respectively,the each extract had the same composition about 40-55 and 70-100 ku.The optimal concentration of Digitonin was 7 μg/mL and the permeabilization rate was 55.44%.After extracts treatment and continuous culture for 6-7 d,JBCFF formed well-defined colony structures.No significant composition difference was found in rates of fusion (92.83% vs 96.04%),cleavage (89.64% vs 89.78%) and blastocyst formation (24.06% vs 23.12%) of cloned embryos when the colony cells and JBCFF without extracts treatment were used as donor cells (P>0.05).Similarly,the two activation methods had no significant effect on the developmental competence of cloned embryos (cleavage rate 92.16% vs 92.28%,blastocyst rate 23.21% vs 24.18%).Conclusively,Xenopus leavis,egg extracts could induce JBCFF reprogramming to a low differentiated state.However donor cells with reprogramming partially could not improve the development of cloned embryos and its mechanism requires further research.     

水牛胚胎体外生产研究综述

近年来采用其它家畜上的程序来进行水牛体外胚胎生产（in vitro production,IVP）引起了越来越多的关注。水牛胚胎体外生产已获得了较高的卵母细胞成熟率、受精率和卵裂率,但囊胚发育率和体外胚的移植成功率仍较低,水牛IVP胚胎体外生产效率比黄牛低得多。在IVP技术应用于水牛生产育种之前,还有一些问题必须解决。本文对水牛胚胎体外生产的不同技术,包括未成熟卵母细胞的体外培养成熟和受精以及胚胎体外培养卵裂发育至囊胚等进行综述。文章还讨论了胚胎体外生产存在的问题及可能的解决方案。     

Effect of ABA,arginine and sucrose on protein content of date palm somatic embryos

Abscisic acid (ABA), arginine and sucrose were evaluated for their effects on the morphology, germination rates and protein content of date palm somatic embryos (SE). Different concentrations of these supplements in the culture medium were used. The comparative study of SE length and thickness between treated and untreated SE revealed no differences, except for ABA (20 μM), which increased thickness. A decrease of water content (WC) in favor of an increase in dry weight (DW) was observed in all treated SE, especially with sucrose (90 g l⁻¹) and ABA (20 μM). Only ABA (20 and 4 μM) caused a proliferation rate of the cultures higher than those in the control. Although all the tested compounds increased protein content, ABA (20 μM) was more effective in protein enrichment than arginine and sucrose treatments. The SDS-PAGE protein profiles showed a significant difference between treated and untreated SE. A protein band of 22 kDa, identified as glutelin in a previous work, was accumulated after treatment with 20 μM ABA or 3 mM arginine. These findings may contribute to further understanding of the mechanisms involved in the accumulation of specific storage proteins in several plants.     

牛体外受精卵的二步法培养体系的研究

以CR1aa为基础培养液,采用二步法对牛体外受精卵进行体外培养,完善牛体外受精卵的培养体系.实验一:对照组连续7 d均为CR1aa 50 mL/L FBS培养,处理组前3 d为CR1aa 3 mg/mLBSA培养,后4 d换为CR1aa 50 mL/L FBS.处理组的囊胚率显著高于对照组,但卵裂率和囊胚孵化率无显著差异.实验二:对照组连续7 d均为CR1aa 50 mL/L FBS培养,处理组前3 d为CR1aa培养,后4 d换为CR1aa 50 mL/L FBS.处理组的卵裂率显著高于对照组,但囊胚率和囊胚孵化率差异不显著.实验三:对照组连续7 d均为CR1aa 50 mL/L FBS 0.1mmol/L GSH培养,处理组前3 d为CR1aa 0.1 mmol/L GSH培养,后4 d换为CR1aa 50 mL/L FBS 0.1 mmol/L GSH.处理组的卵裂率显著高于对照组,囊胚率极显著高于对照组,但囊胚孵化率差异不显著.结果表明,GSH与二步法培养系统结合相对于传统的一步法培养系统更适于牛体外受精卵的体外培养.     

中药有效成分对小鼠2-细胞胚胎体外发育的作用

如何获得更多具有活力的可操作的桑椹胚、囊胚一直是胚胎体外培养的研究热点。近二十年来,虽经采用改进培养液成分、改善培养条件、建立体细胞与胚胎共培养体系和添加生长因子、细胞因子等手段,胚胎质量有所提高,但体外受精卵的囊胚发育率仍远低于体内受精,与体内受精囊胚相比,体外受精囊胚的细胞总数和内细胞团细胞数量明显减少,胚胎移植妊娠率和产仔率较低,这些问题均严重影响了胚胎工厂化生产和胚胎产业化的发展,以及育种工作的进程。因此,探索开辟胚胎体外培养生长发育的新途径,提高其发育率和移植妊娠率显得尤为必要。目前,国内外有关中草药有效成分对体外胚胎发育作用的研究甚少。本研究以ICR小鼠2-细胞胚胎为试验材料,将中药有效成分黄芩苷(Baicalin,Bai)、川芎嗪(Ligustrazine,Lig)和小檗碱(Berberine,BR)添加于mCZB基础液中,并以添加双抗的mCZB基础液为试验对照组,比较其对胚胎体外发育的影响。结果表明,体外培养120 h的胚胎孵化率,Bai组(80.6%)、Lig组(78.3%)及BR组(75.9%)极显著高于对照组(51.8%)(P<0.01)。孵化胚胎细胞计数,BR组、Lig组和Bai组(分别为87.2±8.6、83.9±7.7和81.9±6.2),与对照组(77.4±5.6)差异极显著(P<0.01)。说明3种中药有效成分均能显著促进小鼠早期胚胎的体外发育及胚胎细胞增殖。为进一步探讨Bai、Lig、BR对胚胎体外培养发育的效果和验证其胚胎质量,将不同组别培养至桑椹、囊胚期的胚胎进行两种程序常规冷冻,解冻后继续培养,观察其冷冻效果,并将各组解冻后发育至囊胚期胚胎移植。结果表明:冷冻解冻桑椹胚体外培养8~14 h囊胚发育率,程序一中Bai组(65.8%)和Lig组(81.1%)分别显著(P<0.05)和极显著(P<0.01)地高于对照组(48.6%)和BR组(46.7%);程序二中Bai组(80.6%)、Lig组(78.3%)极显著高于对照组(55.2%)和BR组(47.6%)(P<0.01)。冷冻解冻囊胚体外培养6~8 h囊胚发育率,程序一中各组间无显著差异(P>0.05);程序二中,中药有效成分各组间差异不显著(P>0.05),但均显著高于对照组(P<0.05)。两种冷冻程序效果差异不显著(P>0.05)。移植妊娠率和产仔率,Bai组(56.3%,63.3%)、Lig组(52.6%,67.3%)和BR组(55.0%,60.5%)均高于对照组(46.2%,52.8%)。说明3种中药有效成分对于提高小鼠2-细胞胚胎体外发育质量具有一定促进作用,并有利于提高冷冻解冻后移植胚胎的发育潜力。     

基因枪法转化小麦幼胚瞬时表达

自Vasil et al．（1996）利用基因枪将获得小麦对除草剂Basta具有抗性的再生植株以来，基因枪介导的小麦遗传转化研究发展很快（Altpeter et al．，1996）。在小麦基因枪转化中，优化基因枪转化因素，提高转化效率仍然是基因转化的关键问题。本实验研究了不同金粉用量、质粒DNA浓度及轰击枪数对小麦幼胚瞬时表达的影响，通过GUS染色和细胞压片，分析了gus基因的瞬间表达。     

农杆菌介导法将Bt基因转入大豆的研究

以6个品种大豆体细胞胚为受体,以Bt基因为目的基因,应用根癌农杆茵介导法对大豆进行了遗传转化,经体细胞胚萌发、生根及壮苗培养和卡那霉素选择培养,获得了完整的抗性再生苗,经PCR、PCR-Southern和点杂交分析鉴定,初步证明外源基因Bt已整合到大豆的基因组中。