In Vitro Cellular & Developmental Biology - Plant

A reproducible system for gene transfer in lentil through particle bombardment is presented. Lentil cotyledonary nodes excised from germinated seedlings were bombarded with a plasmid containing a mutant acetolactate synthase gene (ALS) from tobacco conferring resistance to sulfonylurea herbicides. Putative transgenic shoots regenerated on Murashige and Skoog medium supplemented with 6-benzylaminopurine (BA) and chlorsulfuron (5 nM for first 4 wk followed by 2.5 nM for the remainder of the culture period) were micrografted and successfully transferred to soil. T0 and selfed progeny plants were screened using metsulfuron herbicide leaflet painting. The non-transformed escapes died and transformed plants survived the test. The surviving plants were phenotypically normal and produced viable seeds. The presence and stable transmission of the transgene into genomic DNA of screened T1 transformants was confirmed by PCR and Southern hybridization. This method for producing transformed plants will allow new opportunities for lentil breeding to produce improved cultivars.

Populations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy.

Spent media from five different insect cell lines when inoculated intoTrichoplusia ni (TN-368) cultures produced cytotoxicity resulting in rounding and detachment of cells. The substance in spent medium from the established cell lineCarpocapsa pomonella (CP-169) is believed to be a toxin, based on the failure to serially passage the agent, the early appearance of the cytotoxic effect, and the inability to detect microbes by culturing techniques as well as by electron microscopy. The ability to extract the toxic substance from CP-169 cells indicates that it is cell associated. Biophysical and biochemical properties of the CP-169 cytotoxin are presented.

Viral infection is one of the most serious biotic stresses, which disturbs the growth and productivity of many horticultural crops, including that of fig (Ficus carica L.). The production of plants free of viruses, such as fig mosaic virus (FMV), has become a priority in many plant breeding programs. In this study, leaves from plants of two fig cultivars, Kodato and Dattora, infected with FMV were collected from both Mecca and Al-Taif, Saudi Arabia. Transmission electron microscopy of ultrathin leaf sections showed double membrane bodies, characteristic of FMV particles, only in the mesophyll cells of infected samples. Protein analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a protein band with a molecular weight of 35 kDa, which corresponded to the viral coat protein; and FMV was confirmed by Western blot and enzyme-linked immunosorbent assay (ELISA) tests. To obtain virus-free plants, apical shoot culture was applied. A comparison of various artificial media with different concentrations of growth regulators was evaluated to optimize shoot formation, shoot multiplication, and root formation, and was followed by plant acclimation ex vitro. Direct ELISA analysis of shoots micropropagated from meristem tip explants indicated that there were virus-free shoots, when compared to infected plants (positive control), while there were no significant differences between these explants and healthy samples (negative control). This study demonstrated that in vitro micropropagation of Saudi F. carica infected with FMV virus led to the successful elimination of the virus.

Mature acini with attached segments of intercalated ducts were dissociated from the submandibular glands of rats and grown in primary culture on gels of reconstituted rat tail collagen. Screening evaluations indicated that the following new conditions promoted a substantial improvement in the survival of the cells as compared with our previously reported results: a) adding dexamethasone, epidermal growth factor, and retinoic acid to the medium, b) decreasing the fetal bovine serum in the medium to 1%; and c) adjusting the oxygen in the gas phase to 50%. A detailed evaluation, including light and electron microscopy and biochemical analysis, then provided the following observations. The acinar-ductal complexes enlarged throughout the 22-d culture period, and after 4d sheets comprised of a one- to two-cell thick layer of acinar cells spread among the complexes. Synthesis of mucin, and its secretion in response to norepinephrine or cAMP, dropped precipitously to very low levels after 2 d. However, synthesis of DNA, general proteins, and glycoproteins dropped only transiently after 2 d, rising to levels approaching those of freshly dissociated complexes by 22 d. These data indicate that a shift occurred from the synthesis of large quantities of secretory proteins and glycoproteins, especially mucins, during the first 2d in culture, to other materials thereafter. Overall, the new culture conditions resulted in substantial growth and survival of acinar cells through 22 d in primary culture, but the important acinar characteristic of the synthesis and secretion of mucins was essentially lost after 4 d.

Shoot tip cultures from 2- to 3-d-old seedlings ofSorghum bicolor (L.) Moench cv. IS3620C develop highly embryogenic callus from which plants can be regenerated when transferred to plant growth regulator-free medium. Isolated shoot tips were cultured on Murashige and Skoog medium supplemented with 2.5 mg/liter 2,4-dichloro-phenoxyacetic acid and 0.05 mg/liter kinetin. Purple pigmentation characteristic of sorghum cultures on growth regulator-free medium is virtually eliminated with the shoot tip culture. Embryogenic callus is white and hard with an undulating appearance but can be separated into multiple bipolar structures by application of gentle pressure. The well-developed embryos have a cup-shaped scutellum. These germinate like zygotic embryos and develop root-shoot axis. Lack of vascular connections to the parent tissue and the synchronous development of the plumule and radicle suggest that these embryos may be of unicellular origin. In contrast, when the entire seedling serves as the explant, all meristematic centers in the shoot, including the coleoptile sheath close to the apical meristem respond to plant growth regulators in the medium by callus formation. Upon subsequent reculture onto growth regulator-free medium several modes of development occur. The differential response of these tissues to identical culture conditions indicate the presence of different population of cells that respond differently to exogenous plant growth regulators.

The first continuous coleopteran cell line, designated DSIR-HA-1179, was derived from the scarab beetleHeteronychus arator. Most cells are spindle-shaped, 25 to 40 µm long and 15 to 20 µm wide, adhere strongly to plastic surfaces, and are highly resistant to trypsin and collagenase treatment. The population doubling time of 6 d, at 27° C, is slow compared with most dipteran and lepidopteran cell lines. The cells cross-react in comparative radioimmunoassays withH. arator larval tissue but not other insect cell lines and support the replication ofOryctes baculovirus and at least two nodaviruses, black beetle virus, and Flock House virus.

The concentration of each of 10 pesticides (azinphosmethyl, captan, carbaryl, chlordimedorm, dichlorvos, dimenthoate, fenvalerate, methomyl, methyl parathion, trichlorfon) causing a 50% inhibition (ID50) in cell number relative to an untreated culture for a time period equal to four cell doublings was determined for the TN368 and IPLB-HZ1075 cell lines. The range of ID50 values with either of the cell lines was similar, with captan being most toxic within an ID50 range of 5 to 6 μM/2×105 cells/ml, and methomyl least toxic within a range of 2900 to 3200 μM/2×105 cells/ml. Yet there were significant differences between cell lines in pesticide susceptibility. Fenvalerate, dichlorvos, and chlordimeform were 16, 3, and 1.5 times more toxic, respectively, for TN368 cells than HZ1075 cells, whereas dimethoate and carbaryl were each 2 times more toxic for HZ1075 cells. In general, increasing toxicity paralleled decreasing water solubility, although the order of the pesticides varied somewhat according to the particular cell line and medium. Moreover, there was little aberrant cell morphology in either of the cell cultures during incubation with most of the pesticides at their ID50 levels. Preincubation of TN368 cells with any one of seven different pesticideis before inoculation withAutographa californica MNPV, and subsequent incubation of infected cells in medium plus pesticide, did not significantly suppress polyhedra development except for trichlorfon-incubated cells. In addition, there was a small but consistent variation from control cells in extracellular virus titers assayed from two of five of the pesticide incubations. The titer was consistently depressed with trichlorfon and elevated with fenvalerate, however, further work is required to determine the biological significance of these differences.