Localization of mitochondria in plant cells by vital staining with rhodamine 123

The positively-charged fluorescent dye rhodamine 123 (r-123) specifically stains mitochondria in living plant protoplasts, suspensionculture cells, and root hairs. This dye functions as a vital stain and permits visualization of the localization, distribution and movement of the mitochondria. Dehydration of root hairs caused mitochondria to aggregate into clumps. Mitochondria were either homogenous or heterogeneous and were frequently seen to accumulate in the perinuclear regions of suspension-culture cells but not in those of protoplasts or root-hair cells. Dinitrophenol and high concentrations of ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid and KCl immediately eliminated fluorescence in r-123-stained mitochondria whereas ionomycin enhanced it. Treatment of seedlings with r-123 resulted in differential brightness of fluorescence in different tissues. Meristematic tissues, such as root and shoot tips, exhibited the brightest fluorescence. The cytotoxicity of r-123 in both germinating seedlings and suspension-culture cells was low. The specificity, sensitivity and low toxicity of r-123 should make it a useful tool in experiments designed to examine agents and conditions which affect the location, the physiological status or the viability of mitochondria.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - DAPI 46-diamidino-2-phenylindole - r-123 rhodamine 123     

Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations Adh alcohol dehydrogenase - BAP benzylaminopurine - bp base pair(s) - GUS -glucuronidase - Kb kilobase(s) - MS Murashige and Skoog's medium - PCR polymerase chain reaction     

环形电极介导的小麦基因转化

用环形电极电激法有效地将外源DNA导入完整的小麦幼胚组织中。电激的物理参数采用770V／cm场强、800μF电容、100μg／mL质粒DNA(含有bar和GUS双标记基因),幼胚被电激3次。经PCR和Southern杂交分析表明,外源基因已稳定整合到小麦基因组中,转化频率为7．5％,高于相同处理条件下基因枪法的转化频率(4．2％)。     

The tumor suppressor p53 can reduce stable transfection in the presence of irradiation

The tumor suppressor p53 is believed to play an essential role in maintaining genome stability. Although it is currently unknown how p53 is involved in this important biological safeguard, several previous publications indicate that p53 can help to maintain genome integrity through the recombination-mediated DNA repair process. The integration of linearized plasmid DNA into the host chromosome utilizes the same repair process, and the frequency can be measured by clonogenic assays in which cells that were stably transfected by plasmid integration can be scored by their colony-forming abilities. To gain insight into whether p53 has a direct role in plasmid integration into the host chromosome, we determined the frequency of stable transfection with CHO cells expressing either wild-type or mutant p53 in the presence and absence of irradiation. We found that low-dose irradiation (50 to 100 cGy) increased stable transfection frequencies in CHO cells regardless of their p53 status. However, the increase of transfection frequency was significantly lower in CHO cells expressing wild-type p53. Our data thus suggest that wild-type p53 can suppress plasmid DNA integration into the host genome. This p53 function may play a direct and significant role in maintaining genome stability.     

Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues

A method of extracting proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from plant tissues with high protease activity was described. It resolved protein bands in highmolecular-weight regions of the gel and replaced commonly used procedures which showed severe degradation of proteins, even in the presence of protease inhibitors.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot induction and plant regeneration from flower buds in <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchids

Paphiopedilum species are recalcitrant in tissue culture, and no explant from mature plants has been successfully mass propagated in vitro. This study was aimed at inducing shoots and regenerating plants from the flowering plants of a sequentially flowering Paphiopedilum Deperle and a single floral Paphiopedilum Armeni White. By using cross-sectioned flower buds (FBs), we found that in both species, only sections that contained the base tissue of FBs were able to produce shoots and plants. We have also found that sections of FBs between 1.5 and 3.0 cm from Paphiopedilum Deperle were able to produce shoots, but only sections of FBs >2.5 cm from Paphiopedilum Armeni White were regenerable. Our microscopic observations revealed that the small bract at the FB base harbored a new miniature FB, which further harbored a primitive FB with dome-shaped meristem-like tissues that presumably led to the plant induction. The reiteration of this pattern resulted in a scorpioid cyme inflorescence architecture in the multifloral Paphiopedilum species, and its failure to reiterate resulted in a single flower. The induction rates were 57–75%, and all plants survived in a greenhouse. This method is potentially applicable for the micropropagation and conservation of slipper orchids.     

Cytopathology of soybean tissue culture cells infected with southern bean mosaic virus

Summary Virus distribution patterns and ultrastructural changes in soybean callus cells after infection with the type, or bean strain, of southern bean mosaic virus (SBMV) were observed. Calli grown in liquid Linsmaier and Skoog (LS) medium were inoculated with SBMV and incubated in fresh LS medium. Calli were sampled at 5, 10, 15, and 20 d after inoculation and examined by transmission electron microscopy. Five days after inoculation, viruslike particles (VLP) measuring 22 to 27 nm in d were observed in the cytoplasm. The particles formed loose aggregates with some tendency to associate in regular patterns. By the 10th d after infection, particles were observed in the vacuoles in similar loose arrangements. Viruslike particles were readily identified in vacuoles because of the absence of ribosomes. Crystalline aggregations of VLP were found from Day 10 to Day 20 in the cytoplasm only. Five days after inoculation particles similar to the VLP observed in the cytoplasm also were present in nuclei. Other cytopathic effects were noted, particularly several types of inclusion bodies. These observations differ considerably from reports of the type strain in intact bean plant tissues in the frequent occurrence of VLP in vacuoles and virus crystals in cytoplasm of soybean callus infected with the same strain of virus. Michigan Agricultural Experiment Station Journal Article Number 10020. We thank Dr. Karen Baker for useful suggestions.     

Freeze-dried microalgae of Nannochloropsis oculata improve soybean oil's oxidative stability

Marine microalga Nannochloropsis oculata possesses nutrients valuable for human health. In this study, we added freeze-dried N. oculata powder to soybean oil and observed a remarkable inhibition in oil oxidation. The amount of microalgae powder added was positively correlated to the increase in oil stability. The addition of 5.0 % (w/w) microalgae powder increased the oil stability index (OSI) values of soybean oil more than twofold at the tested temperatures 120 and 130 °C. N. oculata contains high levels of both phenolic compounds and α-tocopherols that could be the contributors to such an increase of the OSI. Two methods were conducted to assay the active ingredients released from microalgae: one employed three solvent systems to extract the microalgae and the other was the soybean oil added with microalgae. Analyses of free radical scavenging and reducing power suggested that the phenolic compounds dominated the antioxidation activities in soybean oil when it was infused with the microalgae powder. Our results suggest that N. oculata could potentially be used as an additive in cooking oil to increase the shelf life and nutritional value of the oil and to reduce the production of free radicals from lipid oxidation when the oil is used at high-temperature cooking processes.