小鼠单个植入前胚胎SSH方法的可行性

为建立一种能够一次性分离任意两个植入前胚胎之间全部的差别表达的基因的方法 ,在已有的单胚胎操作技术的基础之上 ,对单个植入前胚胎抑制性消减杂交 (singlepreimplantationembryosuppressionsubtractivehybridization ,SPE SSH)方法进行了初步的探索 ,分离到OM2和MⅡ d 2的基因片段 ,经GenBank和文献检索发现 ,这两个基因具有在MⅡ期和 2细胞期特异性表达的特点 .利用cDNA阵列所进行的鉴定也获得了同样的结果 ,而且所使用的材料极少 ,说明SPE SSH是一种强有力的分离和识别早期发育相关基因片段的实验技术 .若与单个卵裂球分离技术相结合 ,还可用于分离和识别人类早期分子诊断的标记性基因     

Study of the pre-implantation development of mice embryos marked with a green fluorescent protein (EGFP) gene

We present the data on the in vitro development of mouse embryos after injecting pronuclei with cloned DNA fragments with an enhanced green fluorescent protein (EGFP) gene under the control of different regulatory elements (promoters, enhancers, stop signals, etc). It was found that the microinjection procedure itself inhibits development independent of the genetic constructs applied. The development of transgenic embryos may be blocked at different stages of cleavage and morula or blastocyte stages. Most transgenic embryos proved to be mosaics. Transgenic cells are very frequently found in trophectoderm; i.e. they are not a part of the inner cell mass, which is a progenitor for the development of embryo tissues.     

Osteopontin improves in vitro development of porcine embryos and decreases apoptosis

An optimal environment for fertilization and early embryonic development is provided by the mammalian oviduct and uterus. The secretory cells lining the lumen of the oviduct and uterus synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. Western blotting in this study demonstrated that a 50-kDa secreted phosphoprotein 1 (SPP1) form was present in the uterus on Days 0, 3, and 5 in pregnant and nonbred gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5 in pregnant gilts, but in nonbred gilts the concentration of SPP1 on Day 0 was higher than Day 3, but not Day 5. In addition, we show that addition of 0.1 microg/ml SPP1 to the culture medium after fertilization increased the percent cleaved (24 hr: 23.6 +/- 1.29(a) vs. 18.7 +/- 0.65(b) (2-cell %)), and the percent blastocyst (37.2 +/- 1.12(a) vs. 30.9 +/- 0.56(b)) derived from IVF (P < 0.05). In parthenogenetic-derived embryos the percent cleaved was increased due to SPP1 at 24 hr (24.0 +/- 1.59(a) vs. 19.7 +/- 1.59(b) (>2-cell %)), and at 48 hr (72.9+/- 2.99(a) vs. 63.3 +/- 2.99(b)), but not the percent blastocyst. By TUNEL assay, SPP1 decreased both apoptosis (7.9 +/- 0.04(a) vs. 13.1 +/- 0.02(b)) and the percent fragmentation (45.2 +/- 0.07(a) vs. 58.8 +/- 0.03(b)). We conclude that SPP1 can improve development in vitro possibly by reducing the rate of apoptosis.     

Field performance of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L. plants propagated via somatic embryogenesis

Somatic embryogenesis is an in vitro clonal propagation method with potential to contribute to the improvement of cacao varieties. Before using this technology for commercial production, it is essential that somatic embryogenesis-derived plants be tested in field conditions. Therefore, we established a field test at Union Vale Estate, Saint Lucia. Thirty- to 50-yr-old trees were selected for clonal propagation as potentially high yielding based on local farmers observations. Clonal plants were propagated in vitro from immature flowers by embryogenesis and micropropagation. Multiple plants from nine genotypes were acclimated to greenhouse conditions then returned to Saint Lucia and planted in a field. Orthotropic rooted cuttings and locally propagated open pollinated seedlings were also planted for a total of 214 trees. Growth data were collected every 4–6 mo. including: stem diameter, stem height, length of the longest jorquette branch, number of jorquette branches, and dates of first flowering and fruiting. At 4.5 yr after planting in the field there were no major differences in all growth parameters among the propagation methods evaluated with exception of the orthotropic rooted cuttings. Trees grown from seeds were slightly taller then trees propagated by the other methods. Trees propagated as orthotropic rooted cuttings exhibited smaller average stem diameters, shorter stem heights to the jorquette, and shorter jorquette branches. We concluded that somatic embryo-derived plants demonstrated normal phenotypes in field conditions and have growth parameters similar to plants propagated by traditional methods.     

Patterns of somatic embryo formation in Siberian larch: Embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5–1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5–10 days, such embryos formed the embryogenic tissue including two cell types—elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Optimisation of a Selection Scheme using Kanamycin to Improve Transformation of Medicago truncatula cv. Jemalong

We developed an efficient method for in vitro selection of Medicago truncatula cv. Jemalong lines transformed with the nptII gene and subsequent confirmation of phenotype inheritance in these lines. For in vitro selection, the concentration of kanamycin inhibitory to embryogenic callus development and somatic embryo differentiation was identified by placing wounded leaves of non-transformed M. truncatula cv. Jemalong on Embryo Inducing Medium supplemented with 0, 85.8, 128.7, 171.6, 214.6, 257.5 and 343.3 μM of kanamycin. Differentiation of somatic embryos was inhibited with 171.6 μM of kanamycin but callus development was not altered. To confirm transgene inheritance, the kanamycin concentration to distinguish between resistant and non-resistant seedlings was identified by germinating non-transformed seeds of M. truncatula cv. Jemalong on 0.8% (w/v) water-agar plates containing 0, 171.6, 343.3, 514.9 and 686.6 μM of kanamycin. These concentrations did not impair seed germination since all the seedlings exhibited green cotyledons. The effect of kanamycin was only observed at 514.9 and 686.6 μM and on the first pair of leaves, which became white. Due to the high level of resistance to kanamycin of the seedlings the highest concentration is thought to be use to assure the selection efficiency. This optimised antibiotic selection scheme eliminates the regeneration of non-transformed escapes and discriminates between resistant and non-resistant seedlings, confirming the inheritance of the phenotype in transformed M. truncatula cv. Jemalong lines.     

Fertilization and Subsequent Development of Cattle Oocytes after Reduced Incubation with Spermatozoa

We studied the influence of the duration of the joint incubation of cattle oocytes and spermatozoa (18 versus 1 h) on fertilization, cleavage, and embryonic development in vitrountil the blastocyst stage. Spermatozoa of a bull of the Britanofrizskaya breed were used in the experiments. It was shown in the first experimental series that after a long-term incubation with the spermatozoa, the percentage of penetrated eggs increased 71.7 and 56.0% (p< 0.05) after 18-hour and 1-hour incubation, respectively. However, no differences were found in the number of normally fertilized eggs: 46.5 and 39.0%, respectively. In the second experimental series, no significant differences were found in either the number of cleaving embryos (41.2 and 32.2%, respectively) or the capacity of cleaving embryos to develop in vitrountil the blastocyst stage (21.7 and 15.8%, respectively). Thus, reduction in the time of the joint incubation of cattle gametes upon in vitrofertilization to 1 h did not reduce the number of normally fertilized eggs and did not affect their capacity for subsequent in vitrodevelopment.     

Genetic control of somatic embryogenesis induction in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill.

A reproducible protocol for somatic embryogenesis (SE) induction in Eucalyptus globulus from mature zygotic embryos is available since 2002. However, for the use of SE in tree breeding programs, the frequency of SE initiation needs to be improved and controlled, and this was investigated in 13 open-pollinated (OP) families over three consecutive years. A diallel mating design with five parent trees was used to study genetic control of SE induction. Results showed that SE induction varies across E. globulus families and over the years of seed production tested. Somatic embryogenesis was initiated on explants from 84% of the OP families tested in 2002 and 100% of the families tested in 2003 and 2004. The year 2003 gave best results for percentage of induction and total number of somatic embryos produced. Results concerning genetic control showed that SE induction is under the control of additive genetic effects, as 22.0% of variation in SE initiation was due to general combining ability (GCA) effect, whereas 6.4% was due to maternal effects. Neither specific combining ability (SCA) nor reciprocal effects were significant.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cultures and plant regeneration in <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. (muscadine grape)

A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l⁻¹ in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l⁻¹, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.