Use of diathermy for weeding heterogeneous tissue cultures

Summary Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelail cells. Primary cell cultures were observed at ×100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture. Dr. Marks receives a Medical Postgraduate Research Scholarship from the National Health and Medical Research Council of Australia. Financial support was received from the Leo Leukaemia and Cancer Research Trust and the Scleroderma Association of New South Wales.     

利用组织培养选择烟草耐盐愈伤组织变异体并分化出再生植株

烟草(品种革新一号)叶片为外植体,直接置入含0.5%NaCl的修改MS培养基中,诱发产生耐盐的愈伤组织。然后采取逐步提高NaCl浓度的措施,分别获得耐0.5%、1.0%、1.5%及2.0%NaCl细胞系。耐盐细胞系在无盐条件下,生长9—11代后仍保持其耐盐性。从各个耐盐细胞系均分别获得再生苗。耐2.0%NaCl的04—9细胞系共得到15株再生植株,叶片狭长、多锯齿、并具有较多的花茎,多数花粉粒畸形,经过人工授粉获得少量种子。04-9变异型再生植株水培于含有1.0—2.0%NaCl的Hogland溶液中生长85天,仍然存活。原始型愈伤组织的细胞呈不规则椭圆形,耐盐细胞系的细胞均近似圆形;耐盐浓度愈高则细胞愈小。耐盐细胞系愈伤组织的叶绿素含量随耐盐浓度增高而增加;渗透势则随耐盐水平提高而降低。耐2.0%NaCl细胞系04—9愈伤组织内脯氨酸含量高40.7倍,其再生植株叶片内的脯氨酸含量亦较原始型增加两倍。耐2.0%NaCl细胞系再生植株的幼年与成年叶片的过氧化物同工酶的酶谱与原始型均有显著差别。以上试验结果均说明耐2.0%NaCl细胞系04—9及其再生植株是一个耐盐变异体。     

Defense reactions of mosquitoes to filarial worms: potential mechanism for avoidance of the response by Brugia pahangi microfilariae

The melanization response of Aedes trivittatus and the black-eyed Liverpool (LVP) and Rocke-feller (RKF) strains of Aedes aegypti against intrathoracically inoculated Brugia pahangi microfilariae (mff) that previously had penetrated LVP, RKF, or A. trivittatus midguts in vitro was assessed at 1, 3, and 5 days postinoculation (PI). LVP and RKF midgut-derived mff almost totally avoided the melanization response and developed normally in LVP strain A. aegypti, and although over 90% of these mff died by 5 days PI in RKF mosquitoes, the majority of these were not melanized. A. aegypti midgut-derived mff also were able to avoid the response of A. trivittatus in 33–43% of the cases. Penetration through A. trivittatus midguts, however, did not significantly affect the ability of mff to avoid the melanization response in any of the mosquitoes examined. Allogeneic and xenogeneic tissue inplants were accepted by all three mosquito species examined. Data presented support the hypothesis that mff avoid immune recognition in compatible mosquitoes by coating themselves with midgut material(s) during penetration of the midgut in their migration to the hemocoel.     

Differential response of Trifolium species to 4-(2,4-dichlorophenoxy)butyric acid treatments

The differential response of white clover ( Trifolium repens L. cv. Regal Ladino) and berseem clover ( Trifolium alexandrinum L. cv. Mississippi ecotype) was investigated by treating greenhouse cultured plants with 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). Berseem clover plants were significantly injured by a treatment concentration of 0.6 kg ha^-1 of 2,4-DB, whereas white clover plants were not injured by treatment levels below 2.4 kg ha^-1. The metabolism of 2,4-DB in cell suspension cultures of white clover and berseem clover was investigated using [ring-¹⁴C]-2,4-DB and non-labeled 2,4-DB. White clover cell cultures metabolized ca 4-fold more 2,4-DB than berseem cultures over a 44-h treatment period. The decrease in berseem cell population was 4-fold greater than the decrease in white clover cell population in response to the 8 μ M 2,4-DB treatment. The herbicide and its [ring-¹⁴C]-labeled metabolites were isolated from treated cells and medium after 44 h by partition and thin-layer chromatography. White clover cells metabolized 90% of the [¹⁴C]-2,4-DB and berseem clover cells metabolized 22% of the herbicide. The major portion of the radiolabel was in the glycoside fractions from extracts of both species. The differential response of Trifolium species to 2,4-DB is implied to be due to the differential rate of 2,4-DB metabolism to a glycoside by the clover plants.     

The characteristics of light in floral induction in vitro of Cichorium intybus. The possible role of phytochrome

The action of light in the initiation of floral buds in vitro has been studied using monochromatic light qualities on root explants of a long day plant, Cichorium intybus L. cv. Witloof. Red light (660 nm, 0.30 W m^-2) promotes flowering, while far-red (730 nm, 0.31 W m^-2) and irradiation with combined red + far-red (0.20 + 0.41 W m^-2) have no effect. In short day conditions floral response can be obtained in two ways: 1) by interrupting the dark period with 5 brief irradiations of red light (0.45 W m^-2, 12 min) at regular intervals, although these are counteracted by far-red irradiations of equal intensity and duration; 2) by interrupting the long night with 5 h red light applied during the second third of the night, while at the beginning or at the end it is ineffective. Red light efficiency appears to depend on the photosynthetic activity of the tissues, so that flowering increases with increasing intensity of white light and is suppressed if no white light is supplied. The reproductive development is determined by the coordination of proper irradiation conditions with sufficient sensitivity of the perceiving meristematic cells. The period of highest sensitivity to environmental light conditions in the life cycle of a Cichorium root explant occurs between the 8th and the 16th day after the start of the culture. The data strongly suggest that phytochrome is involved in flower induction of Cichorium in vitro.     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Glutamate synthase in greening callus of Bouvardia ternifolia Schlecht

The distribution of the two glutamate-synthase (GOGAT) activities known to exist in higher plants (NADH dependent, EC 2.6.1.53; and ferredoxin dependent, EC 1.4.7.1) was studied in non-chlorophyllous and chlorophyllous cultured tissue as well as in young leaves of Bouvardia ternifolia. The NADH-GOGAT was present in all three tissues. Using a sucrose gradient we found it in both the soluble and the plastid fraction of non-chlorophyllous and chlorophyllous tissue, but exclusively in the chloroplast fraction of the leaves. Ferredoxin-GOGAT was found only in green tissues and was confined to the chloroplasts. Ferredoxin-GOGAT activity increased in parallel with the chlorophyll content of the callus during the greening process in Murashige-Skoog medium (nitrate and ammonium as the nitrogen sources), while NADH-GOGAT was not affected by the greening process in this medium. Furthermore, both activities were differentially affected by either nitrate or ammonium as the sole nitrogen source in the medium during this process. It is suggested that each GOGAT activity is a different entity or is differently regulated.Abbreviations GOGAT glutamate synthase - MS Murashige-Skoog (1962) medium - PMSF phenylmethylsulfonyl fluoride     

Lipoxygenase Metabolism of Arachidonic Acid in Brain

When blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, gas chromatography/mass spectrometry confirmed that the major radioactive lipoxygenase enzyme product of arachidonic acid was 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), with lesser amounts of 5-hydroxy-5,6,8,11,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. When 12-[2H]HETE was used to measure endogenous 12-HETE in brain tissue frozen with liquid nitrogen, the level of 12-HETE was 41 +/- 6 ng/g of wet weight tissue. This frozen tissue level was not due to the presence of blood. When brain slices were incubated in vitro for 20 min, the 12-HETE level increased to 964 +/- 35 ng/g of wet weight tissue. Elimination of residual intravascular blood before tissue incubation reduced the brain slice 12-HETE concentration by one-half.     

UV-microscopic studies on the vacuolar changes caused by the flavone “aglycone” isovitexin inSilene pratensis plants

Summary UV-microscopic and chromatographic studies have been performed on the variation in contents and configuration of the flavones present in epidermal cells of the petals, stem leaves, rosette leaves and cotyledons ofSilene pratensis plants. Most of the flavone contents is located in the vacuole of the upper epidermis cells, the concentration depending on the light intensity at which the plants were grown. In plants able to glycosylate isovitexin in the petals (genotypegG/. gl/gl fg/fg, accumulating isovitexin 7-O-glucoside) the vacuole is completely filled with the UV absorbing flavone. In plants which are unable to glycosylate isovitexin in their petals (genotypeg/g gl/gl fg/fg, accumulating only isovitexin) the upper epidermal cells of stem leaves and petals contain droplet like structures in their vacuoles. At high light intensities these structures increase in mass and become detectable in the visible light. These denser structures often condense to structures with radiating threads.As compared with the accumulation of isovitexin in upper epidermal cells of stem leaves and petals in genotypeg/g gl/gl fg/fg, the cotyledons and the rosette leaves contain two isovitexin glycosides. In the latter organs the upper epidermal cells are very similar to the upper epidermal cells fromgG/. gl/gl fg/fg plants, having a vacuole filled with UV absorbing material. It appears therefore that isovitexin itself causes the formation of the structurés in the cells. It was shown by varying the light intensity that a relative high concentration of isovitexin is necessary for the droplet like structures to appear. Still higher concentrations are needed for the formation of the structures with radiating threads. It is hypothesized that isovitexin interferes with the energy supply of the cells, which therefore are not able to maintain their turgor.     

Callus initiation and plant regeneration in ragi (Eleusine coracana Gaertn.)

Callus was successfully initiated on root, mesocotyl and leaf base segments of 3- to 4-day-old seedlings of ragi (Eleusine coracana Gaertn.). 2,4-D along with casein hydrolysate for Murashige and Skoog's basal medium was found to be most effective for callus initiation and maintenance. Mesocotyl and leaf base tissue derived calli gave shoot buds in medium in which the 2,4-D concentration was lowered.