Lipid-based transfection as a method for gene delivery in zebrafish (Danio rerio) embryos

A major challenge to the widespread production of transgenic, knockout and knockdown zebrafish has been the absence of a simple and effective procedure for introducing macromolecules into the fertilized egg. None of the existing techniques for gene transfer in fish embryos has proven to be a major advance over cytoplasm microinjection, which is a technically demanding and time‐consuming procedure. This report addresses this need, considering that the development of protocols for lipid‐based transfection with fish embryos would considerably simplify gene transfer in this complex biological model. In this study, lipid‐based transfection with two different reporter vectors was carried out in zebrafish embryos at different developmental stages. The parameters tested included different plasmid/transfection reagent ratios as well as the influence of an added transfection enhancer reagent. When embryos were transfected in the blastula stage with a pEGFP‐N1 vector, more than 35% successfully incorporated the plasmid and expressed the fluorescent protein 24 h after transfection. The transfection enhancer did not show any significant effect in our experiments. This work presents an approach to implement this technique as a faster, cheaper and more practical alternative than microinjection.     

糖水乳蟠桃罐头加工技术研究

乳蟠桃含有丰富的营养价值,作为我国药食兼用的食品之一,具有很好的保健功能,受到人们的青睐。本实验以新疆乳蟠桃为材料,通过对蟠桃罐头的单因素、正交实验分析得到蟠桃罐头生产加工的最佳配方：糖26%（m/v）、柠檬酸0.20%（m/v）、抗坏血酸0.15%（m/v）。     

普通小麦闭颖特性遗传分析及离体培养特性研究

为了给闭颖小麦的分子育种提供理论依据,进行了闭颖小麦闭颖授粉特性的遗传分析,同时对6个闭颖小麦系的花药、幼胚及成熟胚进行了离体培养特性的比较研究。结果表明,5组开、闭颖小麦杂交F1代个体全表现为开颖授粉,F2代个体开、闭颖分离,经卡平方适合性测验,开、闭颖株数均符合3∶1分离比例,表明小麦闭颖变异材料的闭颖授粉特性受一对隐性基因控制;不同闭颖小麦系离体愈伤组织诱导与再生特性有一定差异,供试的6个闭颖系中,133-1和133-8的离体培养特性优良,其中,133-1的成熟胚愈伤组织分化率最高,达到27.93%,133-8的幼胚分化率最高,达到40.70%,两材料的花药分化率也最好,都为4.48%,与对照材料小偃22处于同一水平;133-A的离体组织培养特性最差;3种外植体离体培养中,幼胚的培养再生效率最高,成熟胚次之,花药最差,供试材料的出愈率与分化率之间不具备明显的相关关系。     

紫薇幼胚培养及植株再生初探

[目的]对紫薇幼胚培养进行尝试性研究,探讨其适宜的生长分化条件。[方法]以紫薇(Lagerstroemia indica)幼胚为外植体,诱导其萌发并建立无菌系。在此基础上探讨了不同激素水平和培养方式对紫薇幼胚培养的影响。[结果]紫薇幼胚去种皮后易于萌发,其萌发率可达100%;启动培养基为MS+BA0.5+NAA0.5+sucrose3.0%+agar0.7%;丛生芽诱导培养基为MS+BA0.5+NAA0.1+sucrose3.0%+agar0.7%+椰乳10%;生根培养基为MS+BA0.5+IBA0.1+sucrose3.0%+agar0.7%+椰乳10%。[结论]该研究可为紫薇的后续优化育种提供技术参考。     

AgNO3对银杏胚愈伤组织诱导及分化的影响

为探讨AgNO3对不同发育期银杏胚愈伤组织诱导及分化的影响,以银杏3个不同发育程度合子胚为外植体,添加0～5 mg/L AgNO3做处理。结果表明：AgNO3能显著提高胚的愈伤诱导率：早期心形胚鱼雷形胚,早期子叶胚,成熟胚子叶分别在浓度1,0．5,0．1 mg/L时达到最大诱导率,为87％,77％,98％。隔代添加AgNO3促进了早期子叶胚愈伤的分化：1．0 mg/L处理在第90天分化出愈伤化银杏叶和胚类似物,3．0 mg/L处理分化出短小叶片;常规添加AgNO3的处理在0．1 mg/L分化出芽点。遮光处理表明：黑暗培养对愈伤诱导率影响不明显;添加活性炭时,AgNO3促进胚轴愈伤而抑制子叶愈伤,胚轴愈伤组织白色透明。综合分析表明：AgNO3能提高银杏胚的愈伤组织诱导率并促进其分化。     

杂交鹅掌楸体细胞胚萌发培养的研究

以杂交鹅掌楸体细胞胚为材料,研究体胚不同发育阶段、大量元素和维生素C、基因型、激素组合以及移栽基质对体胚苗萌发和移栽存活的影响。结果表明：鱼雷型胚萌发效果最好;3/4 MS培养基较适合体胚苗的萌发;Vc有利用保持体胚苗维持正常形态。不同基因型体胚苗萌发差异较大,基因型1×5002萌发效果最好,成苗率为84.26%;而1×4088仅有13.24%。KT 0.1 mg/L＋IBA 0.1 mg/L有利于促进体细胞胚的萌发。不同基因型体胚苗移栽存活率有差异,黄心土较混合泥炭土更适合体胚苗移栽。     

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L. ''Moldova''

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape 'Moldova' (Vitis vinifera L. 'Moldova'). Primary calli were initiated on Nitsch and Nitsch (NN) medium supplemented with 1.0mg'L-1 2,4-D and 0.5 mg'L-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg-L-1 6-BA and 2 mg.L-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg.L-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dy-namic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic em-bryos, including typical epiderma, cotyledon primordium and vascular tissue.     

Bovine Immunodeficiency Virus in Relation to Embryos Fertilized In Vitro

The association of bovine immunodeficiency virus (BIV) with embryos derived by in vitro fertilization from oocytes of experimentally infected heifers or oocytes/embryos exposed to the virus in vitro was investigated. Using a nested-PCR assay, proviral DNA of BIV was not detected in follicular fluid or in embryos derived from BIV-infected donors. In vitro exposure of oocytes to BIV during maturation or insemination with BIV-infected semen resulted in zona pellucida-intact embryos testing negative for BIV provirus. However, exposure of zona pellucida-free day-7 embryos to the virus resulted in a positive BIV assay for 28% of the batches of embryos, suggesting that the zona pellucida has a role in protecting against BIV infection. The presence of BIV in the IVF system had no apparent effect on the development of bovine embryos to the blastocyst stage.     

In vitro development of bovine nuclear transfer embryos reconstructed with mammary gland epithelial cells at different passages

In the present study, the effect of passage of nuclear donor cells on the in vitro development of nuclear transfer (NT) embryos was investigated using colostrum‐derived mammary gland epithelial (MGE) cells at different passages (3–30 passages) to find reliable passages for the efficiency of cloning. Development of NT embryos to the blastocyst stage was affected by the number of passages of MGE cells (P < 0.05). Nuclear transfer embryos reconstructed with MGE cells at 3–7 passages showed a significantly higher blastocyst development (31.3–48.5%) than those with the cells at 10–30 passages (2.5–12.5%, P < 0.05). No difference in the proportion of the MGE cells with normal diploid was observed among passage of 3, 15 and 30 (P > 0.05). The use of MGE cells at early passages for nuclear donor cells may be advantageous for the production of NT embryos.     

Vernalization of immature embryos of winter wheat genotypes

Summary The effect of direct vernalization of immature embryos on flowering was studied in six winter wheat genotypes. Fourteen-, 17-, and 20-day-old embryos were excised and vernalized for 0–6 weeks on synthetic medium during a conditioning period. Percent germination of embryos was high (overall 96.1%), and free from genotypic effects. Genotypes differed for flowering in response to cold treatment of excised embryos. Embryo vernalization was as effective as or more than conventional vernalization (control, seedling vernalization for 6 weeks). Seventeen-day-old embryos were the most responsive to vernalization. With a 5-week vernalization of 17-day-old embryos, the percentage of plants anthesed was higher than those from 14-and 20-day-old embryos. For 17-day-old embryos vernalized for 5 weeks, the mean number of days from culture to anthesis was less than that of 6 week vernalization, less than that of 14- and 20-day-old embryos, and less than controls.Purdue Univ., Agronomy Dept., W. Lafayette, IN 47907, USA.