Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation, colchicine treatment and gene transfection. Results are as follows: (Ⅰ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3% vs. 4.9%, P<0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25μm) as donor nuclei was higher than that from large cells (25-33μm) and small cells (8-15μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS(11.8% vs. 18.6%, P>0.05). (Ⅳ) Fetal fibroblasts treated with 0.05μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P>0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P<0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05μmol/L colchicine could facilitate the development of reconstructed embryos. Additionally, as cells transfected with GFP gene were used as donor nuclei, adverse effect on the development of reconstructed embryos was observed. Therefore, the developmental efficiency of reconstructed embryos could be improved if proper treatments to donor cells were used.     

Modification of cell development in vitro: The effect of colchicine on anther and isolated microspore culture in Brassica napus

In an attempt to discover the biological basis of microspore derived embryogenesis, the effect of the antimicrotubule agent colchicine on anther and free microspore embryogenesis was investigated. The microtubule inhibitor colchicine promoted embryogenesis from cultured anthers, both with regard to the number of anthers responding and the number of embryos being produced per anther. A similar promotional response was also observed with cultured microspores. Although the parameters for cultured anthers and free microspores differed, administration of the drug for a short period immediately prior to pollen mitosis I seems to exert the maximum promotional effect. Of the five cultivars of Brassica napus studied, all responded to colchicine treatment. However, the drug did release more embryogenic potential in poor-responding varieties (i.e. Lirawell and Optima) than in the highest responding variety (Topas). Colchicine also resulted in increased embryogenic response in microspores cultured at lower temperatures.These results are considered in terms of models proposed to explain the switch in microspore development from a gametophytic to a sporophytic pathway. The use ofcolchicine as agent to promote embryogenesis in previously recalcitrant species other than Brassica is also discussed.     

Ciliary Proteins and Cytodifferentiation in Stentor coeruleus*

To elucidate further the molecular events required for cytodifferentiation in Stentor coeruleus, the effects of several chemical metabolic inhibitors were tested on the outgrowth in situ of the membranellar cilia of the oral feeding organelle. The chemicals used included several inhibitors of cytoplasmic and mitochondrial protein synthesis (cycloheximide, emetine, and chloramphenicol), and an antimitotic agent (colchicine). Ciliary growth was affected only by colchicine, suggesting that a pool of “ciliary proteins” exists in interphase Stentor of sufficient size to permit complete reformation of the membranellar cilia. The implication of these observations to an understanding of the more complicated process of oral regeneration is discussed.     

Noradrenaline induced secretion of nonelectrolytes through frog skin

Summary Addition of noradrenaline (4×10^–5 m) to the inner bathing fluid in the skin of the frogRana esculenta results in increased unidirectional fluxes of urea, thiourea, N-methyl-thiourea, N-N-dimethylthiourea and mannitol. Fluxes towards the external medium ( ₀) undergo a much greater increase than those moving in the opposite direction ( _i ). The effect of noradrenaline on ( ₀) is higher for urea and thiourea than mannitol, while its effect on ( ₀) thiourea derivatives is related to lipid solubility. This phenomenon does not occur for ( _i ) of the same molecules.FCCP (10^–6 m) pretreatment strongly inhibits the noradrenaline effect on ( ₀). In skin pretreated whith colchicine (2×10^–5 m) both urea fluxes are increased to the same extent by noradrenaline. Noradrenaline is concluded to exert two separate effects: (1) a change in permeability in both directions; (2) a secretion of nonelectrolytes towards the external fluid. Such secretion is most probably associated with the hormone-induced secretion of fluid and electrolytes, perhaps mediated by an exocytotic mechanism.     

FLAGELLAR DEVELOPMENT AND REGENERATION IN VOLVOX CARTERI (CHLOROPHYTA)1

The biflagellate somatic cells of Volvox carteri f. nagariensis lyengar exhibit an asymmetric pattern of flagellar development. Initiallt each somatic cell has two short (4 μm) flagella but after several hours one flagellum on each cell elongates unitl it reaches a length of 12 μm. Due to the regular arrangement of somatic cells in the Volvox spheroid it is apparent that the same flagellum on each somatic is the first to elongale. The asymmetric flagellar length is maintained for about 8 h after which the second flagellum on each somatic cell elongates. When the second flagellum attains the same length (12 μm) as the first flagellum, both flagella elongale at the same rate until reaching a final length of 22 μm. Experimental removal of somatic cell flagella results in their regeneration. Somatis cells regenerate both flagella simultaneously and full length flagella are produced in about 2 h. The intial rate of flagellar regeneration is about ten times faster than the intial rate of flagllar growth in development. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial rate of flagellar regeneration but the flagella produced in the presence of the drug are half the length of flagella produced in its absence. Somatic cells are able to regenerate flagella up to the time of α and β tubulin, the major structural proteins of the flagellar axoneme, and other cellular proteins.     

八倍体小偃麦与普通小麦杂交后代花药培养的研究

本试验研究了八倍体小偃麦与普通小麦杂交后代愈伤组织的诱导频率、雄核发育和秋水仙碱诱导小孢子染色体加倍的效果。结果表明:八倍体小偃麦与普通小麦杂种F_1愈伤组织的产量具有明显的杂种优势;其雄核发育存在均等分裂和不均等分裂等类型,这与小麦中的观察结果相似;在培养基中加入秋水仙碱可以有效地诱导小孢子第一次有丝分裂时染色体加倍。     

西南桦染色体加倍的影响因子初步研究

以西南桦种子无菌萌发小苗的顶芽为外植体,探讨秋水仙素的浓度、处理时间及预培养时间对西南桦染色体加倍的影响,初步建立西南桦染色体加倍的方法。结果表明,使西南桦染色体加倍的秋水仙素浓度以120 mg·L-1为宜;在秋水仙素加倍处理前,预培养10 d后再加倍15 d效果较好,此时得到的西南桦嵌合体较多。     

组织培养与秋水仙碱诱导相结合培育植物多倍体的应用(综述)

本文概述组织培养与秋水仙碱诱导相结合培育植物多倍体的优点、诱导方法、最佳药液处理浓度与处理时间组合的选择及诱导材料的选取,并展望其应用前景。     

小麦花培苗染色体加倍技术研究

为了提高冬小麦花培苗染色体加倍效率,分别用不同浓度的秋水仙碱对参试的冬小麦材料的花药愈伤组织、再生植株根系和花培苗分蘖节进行了加倍处理。结果表明,用0.02‰和0.05‰秋水仙碱浓度处理的愈伤组织再生植株结实率达33.3%～61.5%。用0.2％的秋水仙碱浸根处理5 h,结实株率平均高达37.5％。用0.04％的秋水仙碱1%的二甲亚砜溶液浸泡分蘖节的时间应在5～10 h之间较为适宜,结实株率平均可达50％以上。