虎雪兰玻璃化超低温法脱毒技术的研究

以兰属花卉虎雪兰组培原球茎为试材,采用玻璃化超低温法对兰花病毒脱除进行了研究,以期为虎雪兰玻璃化超低温法脱毒体系的建立提供参考依据。结果表明:在蔗糖浓度0.5 mol·L-1预培养4 d,然后在蔗糖0.6 mol·L-1加载液冰上处理50 min,之后转入PVS2溶液冰上玻璃化处理120 min,再液氮冷冻40 min,37℃水浴解冻3 min,最后卸载液(1/2MS+1.2 mol·L-1蔗糖)卸载20 min,待恢复培养后,原球茎成活率可达到65%以上,随机检测不同处理样品的脱毒率可达97%。     

Vitrification of mouse two-cell and blastocyst stage embryos in simplified closed system using either a hemi-straw or a hollow fiber device

Two-cell stage and blastocyst stage mouse embryos were equilibrated in a medium containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 8–15 min. Vitrification was performed in a medium containing 0.5 M sucrose and either 15% EG + 15% DMSO, 17.5% EG + 17.5% DMSO, or 20% EG + 20% DMSO for 30 s. They were then placed either on a hemi-straw (HS) or a hollow fiber vitrification (HFV) device and vitrified by cooled air inside a 0.5-ml straw. In two-cell embryos, a 100% survival rate was obtained from all groups except the 20% HS group (P > .05). All vitrified two-cell groups showed similar rates of blastocyst development to that of fresh control group (P > .05), except 17.5% and 20% HFV groups, which were significantly lower than the other groups (P < .05). In the blastocyst embryos, the HFV groups were divided into two subgroups (non-collapsed; HFV-NC and collapsed; HFV-C blastocyst). Re-expansion rate in 15% HFV-NC, 17.5% HFV-NC, and 15% HFV-C groups was reduced (P < .05), whereas the rest were similar to control. In conclusion, we established a simplified, reliable, and closed system for HFV vitrification applying hemi-straw, which does not require skilled practitioners.     

胡杨组织培养研究概述与问题讨论

胡杨插条繁殖生根极难，因而进行胡杨试管繁殖研究具有重要意义。对胡杨试管苗再生的途径及影响因子进行了综述，对胡杨组织培养中存在的黄萎、玻璃化等主要问题进行了讨论。试管苗黄萎问题没有解决，有必要进一步开展黄萎发生机理和高效再生体系的研究。     

驱蚊香草的组织培养技术

利用驱蚊香草茎段进行组织培养,研究了添加在培养基中不同激素组成、浓度、培养条件等因素对不定芽诱导、继代增殖、防止玻璃化以及生根的影响.结果表明:适于驱蚊香草不定芽诱导的培养基为MS 6-BA1.5 mg/L NAA0.05 mg/L,适于继代增值的培养基为MS 6-BA1.5 mg/L NAA0.05mg/L,适于生根的培养基为配方为1/2MS IBA0.5mg/L NAA0.1 mg/L;用透气设施的封口材料明显地降低驱蚊香草的玻璃化;基质培养基生根优于琼脂培养基.     

Comparative study on antioxidative system in normal and vitrified shoots of Populus suaveolens in tissue culture

To explore the physiological and biochemical mechanism of the occurrence of vitrified shoots of Populus suaveolens in tissue culture, the changes in water, chlorphyll, lignin, H₂O₂, phenylalanine ammonialyase (PAL), malonaldehyde (MDA), protective enzymatic systems, and some key enzymes involved in the ascorbate- glutathione cycle were comparatively studied in both normal and vitrified shoots of P. suaveolens. The results show that the lower activities of peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and PAL, and the less contents of chlorphyll, lignin, ascorbate (ASA) and reduced glutathione (GSH) as well as the lower ratios of ASA / DHA and GSH / GSSG are observed in vitrified shoots than in normal ones during the whole culture period. While in comparison with normal shoots, the higher activity of superoxide dismutase (SOD) and the more concentrations of water, H₂O₂, MDA, dehydroascorbate (DHA) and oxidized glutathione (GSSG) are found in vitrified shoots. Statistical analysis indicates that the enhanced activity of SOD and the decreased activities of CAT and POD as well as some enzymes involved in the ascorbate-glutathione cycle might be closely correlated to the accumulation of H₂O₂. The less regeneration of ASA and GSH and the lower capacity of the ascorbate-glutathione cycle observed in vitrified shoots might be due to a significant decrease in APX, MDAR, DHAR and GR activities and a decline in redox status of ASA and GSH. The decreases in chlorphyll content might result in a decline in photosynthesis. The lower activities of POD and PAL could result in the decrease of lignin synthesis and cell wall ligination, which might be the key factor leading to the increase in water content. It is concluded that the deficiency of detoxification capacity caused by the lower capacity of the ascorbate-glutathione pathway and the decreased activity of protective enzymatic system might lead to the large accumulation of H₂O₂ and the enhancement of membrane lipid peroxidation, which might be the main cause leading to the occurrence of vitrifying shoots of P. suaveolens in tissue culture. [Supported by National Natural Science Foundation of China (Grant No. 30271093) and the Foundation of State-designated Base for Biology Researching and Teaching in Beijing Forestry University]     

心叶球兰组织培养与快速繁殖试验

以心叶球兰叶片为外植体,愈伤组织诱导和继代增殖培养基MS+6-BA2.0mg.L-1+NAA0.5mg.L-1+2,4-D1.0mg.L-1+GA1.0mg.L-1+白糖3%,诱导的愈伤组织多,生长旺盛,增殖倍数为2～3倍;丛芽诱导培养基MS+KT0.5mg.L-1+NAA0.5mg.L-1+GA1.0mg.L-1+白糖3%,芽的诱导率为79.3%;在丛芽增殖及复壮培养基1/2MS+IBA1.0mg.L-1+白糖2%上,有20%～30%玻璃化丛芽转为绿色苗,玻璃化单芽经复壮培养长成绿色苗;诱导生根培养基为1/2MS+IBA0.5mg.L-1+白糖2%,玻璃化生根率为56.4%,淡绿色苗生根率达91.1%。瓶苗移栽基质泥炭土∶珍珠岩∶甘蔗渣(1∶1∶1)的成活率最高,达91.4%。     

6种草本药用植物种子超低温保存技术研究

以草本药用植物杜若、过江藤、夏枯草、皱果苋、罗勒和山香的成熟种子为材料,探讨了含水量和冷冻方法对种子超低温保存的影响。结果表明,经液氮超低温冷冻后,6种草本药用植物种子发芽率较对照组均有显著差异(p0.05);适宜的含水量下,种子经过超低温冷冻后其发芽率甚至高于对照组。3种冷冻方法中,玻璃化冷冻法更适合过江藤和山香种子的超低温保存,缓慢冷冻法更适合皱果苋、杜若和夏枯草种子的超低温保存,直接冷冻法适合于杜若和罗勒种子的超低温保存。所以,液氮超低温冷冻法保存杜若等6种草本药用植物种子是可行的;含水量对超低温保存前后夏枯草、山香和罗勒种子的发芽率影响显著。     

胎牛血清在猪孤雌囊胚玻璃化冷冻后恢复培养中的作用研究

【目的】研究胎牛血清(fetal bovine serum,FBS)在猪孤雌囊胚玻璃化冷冻后恢复培养中的作用。【方法】本试验以体外培养第5天的猪孤雌激活囊胚为材料,将新鲜和冷冻囊胚分别在含10% FBS(V/V)的胚胎培养液中继续培养48 h,即分为新鲜组(Fresh)、新鲜+FBS组(Fresh+FBS)、冷冻组(Vitrified)、冷冻+FBS组(Vitrified+FBS)。观察各组囊胚的扩张和孵化能力,检测胚胎的细胞膜损伤、凋亡细胞数目、总细胞数目、胞内活性氧(ROS)水平、线粒体活性以及发育相关基因的表达水平。【结果】与Fresh和Vitrified组相比,Fresh+FBS和Vitrified+FBS组的完全扩张率、孵化率和囊胚细胞总数均显著提高(P＜0.05),细胞膜损伤率和细胞凋亡率均显著降低(P＜0.05)。与Fresh组相比,Vitrified组ROS水平显著升高(P<0.05),Fresh+FBS和Vitrified+FBS组ROS水平均显著降低(P<0.05)。Vitrified+FBS组的线粒体活性显著高于Vitrified组(P<0.05)、显著低于Fresh+FBS组(P<0.05),与Fresh组无显著差异(P＞0.05)。相比于Fresh组,Vitrified组POU结构域与类转录因子1(POU5F1)表达水平显著上升(P＜0.05)、过氧化氢酶(CAT)表达水平显著下降(P＜0.05)。增殖细胞核抗原(PCNA)、DNA甲基转移酶3A(DNMT3A)、超氧化物歧化酶1(SOD1)、BCL2相关X蛋白(BAX)/BCL2L1的表达水平在Fresh和Vitrified组之间均无显著性差异(P＞0.05)。Fresh+FBS和Vitrified+FBS组中PCNA、SOD1和CAT的表达水平显著高于Fresh和Vitrified组(P<0.05)。【结论】体外培养第5天的新鲜和冷冻猪孤雌囊胚在10% FBS中继续培养,其发育能力及胚胎质量均得到明显改善。     

牛IVF性控切割胚胎和整胚的冷冻保存研究

本研究分3个实验对比了采用不同处理的常规冷冻和玻璃化冷冻保存358枚体外受精(IVF)性控切割囊胚和326枚整胚的保存效果。结果表明：(1)用于冷冻整胚的常规方法对冷冻性控切割胚胎同样有效；(2)利用含10％EG 10％FBS的DPBS冷冻牛IVF控切割胚胎或整胚的效果明显优于用含10％甘油 0.4%BSA或1．4mol／LEG 0．1％PVOH的DMPBS；(3)采用微玻管玻璃化冷冻保存牛IVF性控切割胚胎的效果显著优于常规冷冻法。     

细胞松弛素B对牛GV期卵母细胞玻璃化冷冻效果的影响

本试验通过玻璃化冷冻前不同浓度细胞松弛素B(cytochalasin-B,CB)预处理来分析CB对牛GV期卵母细胞固体表面玻璃化(SSV)冷冻后发育潜力的影响。试验结果表明,玻璃化冷冻后,CB处理组和未处理组之间卵母细胞成熟率差异不显著(P>0.05),但均显著低于体外培养组。说明试验中所用CB浓度 (2.5、7.5、15、20和30 μg/mL)对牛GV期卵母细胞玻璃化冷冻效果的改善作用不明显。