Bernard J. Mulligan

Direct DNA transfer to plant cells.

A range of somatic cell and molecular techniques are now available to supplement conventional plant breeding. The introduction and expression of foreign DNA has been used to modify basic aspects of physiology and development, to introduce commercially important characteristics such as herbicide and insect resistance into plants and to insert genes suitable as dominant selectable markers for somatic hybridisation. Several techniques for direct DNA delivery are available, ranging from uptake of DNA into isolated protoplasts mediated by chemical procedures or electroporation, to injection and the use of high-velocity particles to introduce DNA into intact tissues. Direct DNA uptake is applicable to both stable and transient gene expression studies and utilises a range of vectors, including those employed for gene cloning. Although the frequency of stable transformation is low, direct DNA uptake is applicable to those plants not amenable to Agrobacterium transformation, particularly monocotyledons.

Surfactant effects on yeast cells

Abstract The nonionic surfactant, Pluronic F-68, is finding increasingly wide spread use in eukaryotic cell cultures, although its mechanism of action in these different systems is obscure. Here we demonstrate that surfactants can alter the rate of fluorescein diacetate uptake into yeast cells and propose that this involves changes in membrane permeability. This effect occurs independently of changes in both membrane and soluble cell protein profiles and is not accompanied by any obvious alterations in yeast cell or membrane structure. Comparison by patch-clamp analysis revealed that both Pluronic and Triton X-100 altered the permeability of artificial lipid bilayers, but the magnitude of response differed according to surfactant used. Possible mechanisms of surfactant-mediated effects are discussed, including the formation of membrane pores.

Yeast responses to nonionic surfactants

The effects of the nonionic surfactant Pluronic F-68 in batch cultures of Saccharomyces cerevisiae (X 2180 1B) on 1) culture growth and cell viability; 2) whole-cell polypeptide profiles; 3) membrane permeabilization, as measured by in situ alcohol dehydrogenase activity; 4) the action of the antibiotics, cycloheximide, and amphotericin B; and 5) flocculation ability have been studied. The effects of Pluronic F-68 have been compared with those of Triton X100. Consistent with its recognized biocompatibility, Pluronic F-68 (0.1–1.0% w/v) did not affect culture growth kinetics, whereas Triton X100 initially inhibited the onset of the logarithmic phase and slowed cell senescence. These changes were not reflected by changes in the profiles of cell-associated polypeptides. Cell-permeabilizing effects of both surfactants were demonstrated by the in situ alcohol dehydrogenase assay and by enhancement of growth-inhibiting effects of sublethal doses of cycloheximide (0.1 μg ml−1) or amphotericin (50 μg ml−1). No evidence of surfactant-induced flocculation was observed with this strain of yeast, which has minimal inherent flocculating ability. The results are discussed in relation to the principal physicochemical properties of the surfactants (e.g., critical micelle concentration) and their effects on cell permeability.

EFFECTS OF EMULSIFIED PERFLUOROCHEMICALS ON GROWTH AND ULTRASTRUCTURE OF MICROBIAL CELLS IN CULTURE

SummaryThe effects of emulsified perfluorochemicals (PFCs) and some of their constituents on growth and ultrastructure of microbial cells in culture have been studied. Growth rate ofE. coli was inhibited by stem emulsion from the proprietary formulation, Fluosol-DA 20%, and also by a combination of the Pluronic F-68 surfactant and yolk phospholipid emulsion stabilizer. Yeast growth was also inhibited by Fluosol stem emulsion and by pluronic alone. Electron microscopical examination of thin sections of yeast cells following culture in perfluorodecalin emulsion revealed cytoplasmic vacuolation and other ultrastructural perturbations resembling those reported previously in mammalian cells cultured with emulsified PFCs; no comparable structural changes inE. coli were observed.

Characterization of gamma irradiation-induced deletion mutations at a selectable locus in Arabidopsis.

Seeds of transgenic Arabidopsis, containing a negatively selectable suicide marker, a 35Stms2 construct introduced as a transgene, were gamma-irradiated at a range of doses from 20-120 krad. Batches of M2 seeds, from M1 plants irradiated at doses of 40, 45 and 60 krad, were screened by germinating them on medium containing NAM under conditions that selectively inhibited growth of plants expressing the tms2 gene product. Nine candidate loss-of-transgene mutants were isolated. The frequency of such mutations (0.0125 to 0.025%) did not vary significantly with irradiation dose or M1 pool size. DNA from the mutants and the parent was hybridized in Southern blots, using probes complementary to various regions of the transgene. All nine mutants were null for both the tms2 coding sequence and the 35S promoter. Six of the nine mutants were null for the entire transgene construct of 9 kbp. DNA from one mutant contained one of the T-DNA borders and gave a hybridization pattern consistent with a deletion at least 5 kbp. The two remaining mutant lines gave identical patterns of hybridization, consistent with a 5.6-kbp internal deletion within the transgene. From the Southern blots, and on the basis of lineage, the nine lines represent the progeny of either seven or eight independent mutations. We have established conditions capable of producing deletion mutations of at least 5 kbp, but without apparently introducing small deletions or rearrangements. Such deletion mutations are ideally suited for cloning by subtractive hybridization, and should also be readily detectable by RFLP analysis, facilitating map-based cloning procedures.

Factors affecting single strand-preferring nuclease activity during leaf aging and dark-induced senescence in barley (Hordeum vulgare L.)

Abstract Nucleases (SSNs) which preferentially degrade single-stranded nucleic acids have been implicated in programmed cell death pathways, including senescence, in higher plants, though the biological roles of such enzymes are obscure. An assessment has been made of changes in SSN activity that occur during dark-induced senescence of the primary leaf of barley. Specifically, changes have been studied of SSN activity in differently aged sections of primary leaf of seedlings grown either under a 16 h light/8 h dark cycle or following transfer to continuous darkness. Prolonged darkness caused an increase in SSN activity, due to four different SSN isoforms, a major activity of ca. 37 kD and minor activities of ca. 36.5, 40.1 and 43 kD. The increase occurred most rapidly and to a greater extent, in older cells towards the leaf tip, with a progressively smaller increase towards the leaf base. Increased SSN activity was apparent within 8–12 h of darkness, but did not persist or accumulate in plants grown under a light/ dark cycle. The results show that, within the primary leaf, in addition to light/dark control, factors related to leaf tissue age also participate in the control of enzyme activity. Possible roles of SSNs in leaf senescence and cell death are discussed.

Effects of Pluronic F-68 on shoot regeneration from cultured jute cotyledons and on growth of transformed roots

The effects have been studied of the non-ionic surfactant, Pluronic F-68, on the growth in culture of jute (Corchorus capsularis L.) cotyledons with attached petioles, cotyledon explants and transformed roots. Supplementation of culture medium with 0.001–0.5% (w/v) of either commercial grade Pluronic F-68 or a purified fraction prepared by passage through silica gel, stimulated shoot production from the petioles of C. capsularis var. D154 and C134 cotyledons. This effect was most marked in C134, because of the failure of control cotyledons to produce shoots in the absence of Pluronic. Plants regenerated from Pluronic-treated cotyledons were morphologically normal. Growth of transformed roots of C. capsularis var. D154 was stimulated in medium supplemented with commercial grade or purified Pluronic F-68, with maximum increases in both fresh and dry weights with 0.1% (w/v) of the surfactant. Roots cultured in the presence of Pluronic F-68 could be maintained without sub-culture for up to 70 days, whereas roots cultured in the absence of Pluronic required subculture every 7 days, to prevent necrosis. Transformed roots also produced callus in the presence of 0.001–1.0% (w/v) of either commercial grade or purified Pluronic. The biotechnological implications of these results are discussed in relation to the potential value of non-ionic surfactants as growth-stimulating additives to plant culture media.

Surfactant stimulation of growth in cultured plant cells, tissues and organs

The culture of plant cell, issues and organs under, defined laborato, onditions both pu, ndapplied applications. A unique feature of plant cells is that they exhibit totipotency. This enables intact, fertile plants to be regenerated from tissues which are given the appropriate stimuli from exogenously supplied growth regulators, such as auxins and cytokinins, in the culture medium. The ability to regenerate plants is fundamental to the multiplication of elite individuals by micropropagation. Additionally, the genetic engineering of plants, through exposure of somaclonal variation, somatic hybridization by protoplast fusion and transformation involving Agrobacterium-mediated gene delivery or direct DNA uptake, also neccessitates reproducible plant regeneration.

Microbial cell responses to a non-ionic surfactant

SummaryThe effects of a non-ionic surfactant, Pluronic F-68, on growth of microbial cell cultures have been studied. Growth ofSaccharomyces cerevisiae at 30°C or 37°C as measured by viable cell counts was unaffected by culture with pluronic. However, corresponding absorbance measurements forS. cerevisiae incubated with 5–10% pluronic were lower than controls at both temperatures. Absorbance ofE. coli cultures was also significantly reduced by incubation with 5.0–10.0% pluronic at both temperatures although viable counts again revealed no significant inhibition of growth.

Application of rice (Oryza sativa L.) suspension culture in studying senescence in vitro (I).: Single strand preferring nuclease activity

Single Strand-Preferring Nucleases (SSPNs) have been implicated in the triggering and progress of cell death pathways, including senescence in higher plants, though the biological roles of such enzymes are still obscure. In the present study, heterotrophic cell suspension cultures of Oryza sativa L. (rice) cv Taipei 309 were used to investigate changes in Single Strand-Preferring Nuclease activity associated with cell death in vitro . An acid nuclease activity (pH 5.5) was found which was strongly stimulated in the presence of 10 mM Zn 2+ and inhibited by 10 mM EDTA and EGTA. An increase in SSPN activity was concomitant with a loss of cell viability, total protein and the onset of stationary phase of growth in the cell cultures. Using DNA-SDS-PAGE two major SSPNs were detected with the molecular weights of 26 KDa and 53.5 KDa. There are possibilities that these two enzymes represent monomeric and dimeric forms of the same nuclease or they are two different enzymes. The 26 KDa nuclease was partially purified using heparin-Sepharose column chromatography. The results confirm the value of plant cell suspension cultures for the investigation of the molecular process underlying plant cell ageing, senescence processes and programmed cell death (PCD). Possible senescence-associated roles of SSPNs are discussed.