甘蓝型油菜子叶和下胚轴再生植株无性系建立

以甘蓝型油菜(Brassica napus L.)豫油2号和6257的子叶和下胚轴为材料,在不同激素配比的MS培养基上诱导出了愈伤组织。将经过继代的部分愈伤转入分化培养基,分化结果表明:除基因型、外植体和分化培养基的激素配比对分化率有影响外,诱导愈伤培养基的激素配比对分化率也至关重要。豫油2号的子叶和下胚轴在最适诱导培养基(ZT 1+NAA0.5+2,4-D 0.2 mg/L)和最适分化培养基(ZT4+IAA 0.2 mg/L)组合中的愈伤分化率分别为12.5%和75%;6257的子叶和下胚轴在其最适诱导培养基(KT 2+NAA1+2,4-D 0.2 mg/L)和最适分化培养基(6-BA 4+IAA 0.02 mg/L)组合中的愈伤分化率分别为50%和37.5%。将其最适诱导培养基中的愈伤组织继代达8个月以上,建立了不同继代愈伤的再生植株无性系。     

Normal rat kidney proximal tubule cells in primary and multiple subcultures

Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.     

Synergism of thidiazuron and benzyladenine in axillary shoot formation depends on sequence of application in Miscanthus X ogiformis ‘Giganteus’

Shoots of Miscanthus X ogiformis Honda Giganteus transferred from medium with benzyladenine (BA) to thidiazuron (TDZ) formed significantly more axillary shoots than shoots grown continuously on either medium, or transferred from TDZ to BA. Shoot formation on axillary shoots transferred from BA-containing media to media with kinetin or isopentenyladenine (2iP) or transferred from media with TDZ, kinetin or 2iP to media with BA, corresponded to the number of shoots formed in the previous subculture. Shoot formation on shoots transferred from medium containing BA to media containing combinations of BA and/or TDZ increased with increasing concentrations of TDZ in the first subculture, whereas shoot formation in the second and third subculture depended on the BA concentration. When shoots were transferred from media with BA to media with TDZ, the time for shoot formation, as well as shoot size, indicate that the combined effect of BA and TDZ is expressed only during the early phase of the subculture. The results suggest that adenine- and phenylurea-type cytokinins have a common binding site in the plant cell, and a model is proposed.Abbreviations BA 6-benzyladenine - CBP cytokinin-binding protein - 2iP isopentenyladenine - KIN kinetin - MS Murashige & Skoog - TDZ thidiazuron     

The extended serial subculture of human diploid fibroblasts on microcarriers using a new medium supplement formulation

Human diploid fibroblasts serially passaged on microcarriers exhibit a decrease in their proliferative capacity with each transfer from microcarrier-to-microcarrier. This phenomenon, which does not occur in the same time scale with cells cultured in T-flasks, has been a serious barrier to the systematic utilization of microcarriers in the scale-up of anchorage-dependent human diploid cell cultures. This decreases in cell growth with each passage is shown to be related to the serum content of the medium, with high serum concentrations resulting in a more rapid decrease in cell growth with each serial transfer. As a result, methods for reducing the serum requirement of the cells were investigated. A new medium supplement mixture, PPRF92, has been developed, which allows the serial passaging of MRC5 cells on Cytodex 1 microcarriers through as many as 13 microcarrier-to-microcarrier tranfers, and at a serum levels as low as 1%, with no decrease in the proliferative capacity of the cells until they approach their reported population doubling limit. This new supplement mixture is a significant improvement to microcarrier technology in that it enables the use of microcarriers in the early stages of inocculum build-up for the production purposes. (c) 1992 John Wiley & Sons, Inc.     

石蒜愈伤组织的诱导及其继代培养

以石蒜鳞茎为外植体,研究了不同激素组合、鳞茎不同部位和不同生长时期等因素对石蒜愈伤组织诱导的影响及其继代培养。结果表明：MS＋2,4-D 1 mg·L~（-1）＋6-BA 1 mg·L~（-1）激素组合能较好的诱导出石蒜愈伤组织,诱导率达61.13%;外植体的选择是石蒜愈伤组织诱导的关键因素,内层鳞茎诱导愈伤组织的效果最好;在一个生长周期中,9、10月的鳞茎作为外植体诱导愈伤组织最佳;MS＋2,4-D 0.5 mg·L~（-1）＋KT 0.5 mg·L~（-1）是愈伤组织较好的继代培养基,继代周期为24～27 d。     

Plant development from microspore-derived embryos in oilseed rape as affected by chilling, desiccation and cotyledon excision

The present study evaluated the effects of chilling, partial desiccation, cotyledon excision and successive subculture of microspore-derived embryos on plant development in oilseed rape (Brassica napus L.). The results showed that out of the five media, all the genotypes showed the best response when the embryos were cultured on the half-strength Murashige and Skoog medium with 2.0 mg dm⁻³ benzylaminopurine. A cold treatment for 3 or 5 d further increased frequencies of embryo germination (90.0 %) and plantlet development (58.46 %). Desiccation for one day also increased the embryo germination and plantlet development in all genotypes tested. Cutting the cotyledons of the embryos at late cotyledonary stage significantly increased the frequency of plantlet development. The highest rate of plantlet development was obtained from cultures of embryos sampled with size of less than 4.0 mm. The successive subculture further improved the germination and development of plantlets from embryos. In the genotype ZJU452, the rate of plantlet development reached 99.78 % after the second subculture of embryos.     

继代十七年的玉米花粉胚性细胞系的异常核和异常分裂观察

对继代17年的玉米花粉胚性细胞系核形态和细胞分裂情况进行了观察分析,结果表明:随着继代培养时间的延长,异形核细胞比率增加,异形核类型和异常分裂增多。分析认为异形核和异常分裂现象出现和增多是导致胚性细胞系分化率降低、异形分化和非整倍体细胞产生的主要原因。并提出来自玉米农家种“八趟白”的部分胚性细胞系能长期保持倍性稳定和胚胎发生并再生,与不断挑选和继代培养中不加2,4-D有关。     

AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper

In order to further increase shoot regeneration frequency of Vigna mungo (L.) Hepper., the effects of AgNO₃ on this process was investigated in this study. The shoot tip and cotyledonary node explants were cultured on MS salts B5 Vitamins medium containing BA+TDZ+Ads+AgNO₃ for multiple shoot induction. AgNO₃ influenced the shoot bud formation and their subsequent proliferation. The best medium composition for multiple shoot induction was BA, TDZ combination with Ads and AgNO₃ in MSB5 medium. Maximum 39 shoots in cotyledonary node and 22 shoots in shoot tip were obtained per explants after 4 – 6 wk. of culture. Elongation and rooting were performed in GA₃ (0.6mg/l) and IBA (0.4mg/L) containing media respectively. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to the field with a survival rate of 78%.     

Instability of anthocyanin accumulation inVitis vinifera L. var. Gamay Fréaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, inVitis vinifera cell cultures. Therefore, four cell line suspensions ofVitis vinifera L. var. Gamay Fréaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73±0.15, 1.45±0.04, 0.77±0.024 and 0.27±0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% forV. vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities tol-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line showed greater potential in enhanced the anthocyanin production.     

植物激素对砀山酥梨脱病毒苗增殖生长的影响

砀山酥梨脱病毒苗培养基中添加外源激素能通过调节内源激素的含量,从而控制脱病毒苗的增殖和生长。苄基腺嘌呤(benzyladen ine,BA)处理可提高脱病毒苗内源玉米素核苷(zeatin riboside,ZR)含量,而脱病毒苗的有效增殖芽数与IAA/ZR比值呈显著负相关;1萘-乙酸(1-naphthalene acetic ac id,NAA)处理可显著提高内源吲哚乙酸(indole acetic ac id,IAA)含量,较高的内源IAA含量有利于芽梢的生长;继代组培苗体内含有一定量的内源赤霉素(G iberllic Ac id,GA1 3),适量的外源GA3处理,可提高内源GA1 3含量而显著降低脱落酸(absc isic ac id,ABA)含量,促进芽梢的伸长和叶面积的扩大。