Optimization of culture conditions for human corneal endothelial cells

Summary Long-term cultivation of human corneal endothelial cells (HCEC) was optimized with respect to different components of the culture system: 25 different nutrient media, different sera, 6 mitogens and various substrates were tested in their ability to influence clonal growth and morphology of HCEC. F99, a 1∶1 mixture of the two media M199 and Ham’s F12, was the most effective basal medium in promoting clonal growth of HCEC. Among various sera, human serum and fetal bovine serum showed optimal growth promoting activities in combination with F99, whereas newborn bovine serum (NBS) was by far superior for the development of a typically corneal endothelial morphology. Crude fibroblast growth factor (FGF), or alternatively endothelial cell growth supplement, was absolutely essential for clonal growth of HCEC at low serum concentrations, for example 5% NBS. Formation of a monolayer with a morphology similar to corneal endothelium in vivo was observed only on culture dishes coated with basal membrane components such as collagen type IV, laminin, or fibronectin. The most pronounced effect on morphologic appearance was obtained by culturing the cells on the extracellular matrix (ECM) produced by bovine corneal endothelial cells. Moreover, ECM could substitute for crude FGF in clonal growth assays.     

A simple,rapid protocol for adventitious shoot development from mature cotyledons of glycine max cv bragg

Mature soybean cotyledons (Maturity group VII) were cultured on modified MS containing 0–2.5 μM indole-butyric acid (IBA); 0–10 μM 6-benzylaminopurine (BAP) and 0.7% agar. Embryonic axes of explants were removed prior to culture initiation and cultures were incubated at 24°C with 45–50 μE. s⁻¹.M⁻² of mixed irradiance with 16 h photoperiod. Shoot proliferation occurred at 0–2.5 μM IBA and 5–10 μM BAP, while in the presence of 2.5 μM IBA alone, only roots developed. Abnormal shoots were produced with 2.5 μM IBA and 5–7.5 μM thidiazuron. Adventitious shoot development started 7–14 d after culture initiation in the region where the embryonic axis was previously attached to the cotyledon and shoots were visible within 28 days. The presence of the embryonic axis inhibited shoot morphogenesis. The shoots were rooted on half strength MS inorganic salts plus vitamins, 2% sucrose, 0.5 μM NAA acid or 2.5–5 μM IBA, or 5–10 μM IAA, and 0.7% agar. Rooted plants were acclimatized under a mist in the greenhouse. This simple, rapid,in vitro adventitious shoot development protocol could be adapted for transformation/regeneration studies in soybean. Trade and company names are used in the publication solely to provide specific information. Mention of a trade or company name does not constitute a warranty or an endorsement by the U.S. Department of Agriculture to the exclusion of other products or organizations not mentioned.     

聚乙二醇处理大豆种子子叶中几种酶活性和可溶性蛋白含量的变化

聚乙二醇处理的大豆种子的异柠檬酸裂解酶、苹果酸脱氢酶、过氧化氢酶、超氧物歧化酶、酸性磷酸酶、碱性磷酸酶的活性明显高于受低温吸胀冷害的种子子叶的活性,相关的酶活性协同地增长,而蛋白质的含量没有明显的变化。这些酶活性的提高可能是渗透调控处理对细胞膜系统修补的结果。     

Interactions Between Calonectria crotalariae and Heterodera glycines on Soybean

The interactions of Heterodera glycines at four egg inoculum levels (0, 100, 1,000, and 10,000 per pot) and three cyst levels (0, 100, and 200 per pot) and Calonectria crotalariae at 500, 5,000, and 50,000 microsclerotia per pot were evaluated on soybean. At the two lowest nematode egg levels, the presence of C. crotalariae did not affect nematode reproduction. At 10,000 eggs per pot, however, nematode reproduction was increased significantly at each microsclerotial level. The increase in nematode reproduction was stepwise at 500 and 5,000 microsclerotia per pot but declined at 50,000 microsclerotia per pot. Similar results were obtained when cysts rather than eggs were used as nematode inoculum. The nematode x fungus interaction significantly affected 60-day plant growth parameters of both Lee 74 and Centennial soybean. The nematode x fungus interaction was antagonistic to plant roots and significantly influenced root injury ratings. The presence of C. crotalariae in tissues of stock plants or plants used as race differentials did not alter the analysis of this population as race 3.     

Influence of Temperature and Soybean Phenology on Dormancy Induction of Heterodera glycines

Growth room and field experiments were conducted to determine the influence of soil temperature and soybean phenology on dormancy induction of a North Carolina population of Heterodera glycines race 1. Three temperature regimes and two photoperiods to regulate plant phenology were investigated in growth rooms. H. glycines hatch was greatest from the 26 and 22 C (day and night) temperature treatment, intermediate at 22 and 18 C, and least from the decreasing regime (26 and 22 C, 22 and 18 C, and 18 and 14 C). More eggs hatched and greater nematode reproduction occurred on pod-producing soybeans than on those that remained vegetative. In the field study, hatching patterns were not different between depodded and naturally senescing soybeans nor between the different maturity groups of soybean cultivars (groups V through VIII). Egg hatch (9-16%) was greatest in August and September when mean soil temperatures were between 25 and 29 C. Hatch declined to 1% in vitro and was not detectable in the bioassay in November. Greatest nematode numbers were observed on the latest maturing cultivar (group VIII) and fewest on the cultivar which matured earliest (group V). Decreasing temperature appears to be more important than soybean phenology in dormancy induction of H. glycines.     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Mechanical impedance increases aluminium tolerance of soybean (Glycine max) roots

The effect of aluminium (Al) on root elongation was studied in solution culture and sand culture. Compared to solution culture, in sand culture a ten times higher Al supply was necessary to inhibit root elongation to a comparable degree. This was due to a much lower Al uptake into the 5 mm root tips in sand culture. Fe concentrations in root tips were also lower in sand culture. Ca concentrations were higher and less depressed by Al, whereas Mg and K concentrations were not affected by the culture substrate. Regressions of Al concentrations in root tips versus inhibition of root elongation by Al revealed root damage at lower Al concentrations in sand culture. The effect of culture substrate on Al tolerance was independent of N source and could also be shown in flowing solution culture with and without sand. The results indicate that mechanical impedance in sand culture decreased Al uptake. This may be due to enhanced exudation of organic complexors thus reducing activites of monomeric Al species.     

Stomatal response to humidity in sugarcane and soybean: effect of vapour pressure difference on the kinetics of the blue light response

Abstract. The effect of atmospheric humidity on the kinetics of stomatal responses was quantified in gas exchange experiments using sugarcane ( Saccharum spp. hybrid) and soybean ( Glycine max ). Pulses of blue light were used to elicit pulses of stomatal conductance that were mediated by the specific blue light response of guard cells. Kinetic parameters of the conductance response were more closely related to leaf-air vapour pressure difference (VPD) than to relative humidity or transpiration. Increasing VPD significantly accelerated stomatal opening in both sugarcane and soybean, despite an approximately five-fold faster response in sugarcane. In contrast, the kinetics of stomatal recovery (closure) following the pulse were similar in the two species. Acceleration of opening by high VPD was observed even under conditions where soybean exhibited a feedforward response of decreasing transpiration (E) with increasing evaporative demand (VPD). This result suggests that epidermal, rather than bulk leaf, water status mediates the VPD effect on stomatal kinetics. The data are consistent with the hypothesis that increased cpidermal water loss at high VPD decreases the backpressure exerted by neighbouring cells on guard cells. allowing more rapid stomatal opening per unit of guard cell metabolic response to blue light.     

长期低浓度SO2对大豆生长和产量的影响

随着化石燃料消耗量不断增加,由此产生的主要大气污染物之一SO_2的浓度和影响范围也日趋增大。SO_2对植物,特别是对农作物的影响已受到普遍重视。本研究选择我国北方种植面积大、分布广的大豆为供试作物,在野外开顶式熏气装置中进行低浓度SO_2长期暴露试验,观察SO_2对大豆生长发育及产量的影响,以期为制订农田大气环境质量标准提供有一定参考价值的生态学基准。     

Influence of single amino acids on the development of hamster one-cell embryos in vitro

One-cell hamster embryos placed in culture have always shown a complete block to development at the two-cell stage. In a preliminary study using a chemically defined culture medium containing 20 amino acids (HECM-1), many one-cell embryos were able to escape the "two-cell block" and develop to the four-cell stage. Use of a simpler formulation containing only the amino acids hypotaurine and glutamine revealed marked inhibitory and stimulatory effects of adding the other amino acids. In the first experiment, 19 amino acids were separately examined for effects on one-cell embryo development. Six amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited embryo development (reduced mean cell number; MCN), and three others (glycine, cystine, and lysine) stimulated development (increased MCN), compared with basic medium containing only glutamine and hypotaurine (low control). When the responses with the six inhibitory amino acids were totalled, only 3 of 185 (2%) one-cell embryos reached the six-or seven-cell stage compared to a total of 15 of 76 (20%) embryos that developed to these stages using the three stimulatory amino acids. When tested together in a second experiment, the six inhibitory amino acids significantly reduced the MCN, from 4.28 +/- 0.44 (low control) to 3.71 +/- 0.55. In this group, 17 of 117 (15%) of one-cell embryos reached more than four-cell and only 4 of 117 (3%) reached six- or 7-cell stages, compared with 39 of 117 (33%) and 12 of 117 (10%), respectively, for the basal medium group.(ABSTRACT TRUNCATED AT 250 WORDS)