Radiobiological damage: a new class identified in barley seeds stored after irradiation.

A new class of radiobiological damage has been identified in irradiated barley seeds. Consisting of both physiological and genetic damage, it appears to be independent of oxygen and long-lived free radicals, and develops very slowly during storage after irradiation. Increasing the storage temperature accelerates its development.

Direct embryogenesis from mesophyll cells of orchardgrass.

Segments taken from the basal 15 to 20 millimeters of the two innermost leaves of an orchardgrass (Dactylis glomerata L.) genotype produced somatic (nonzygotic) embryos directly from mesophyll cells without an intervening callus when cultured on an agar medium with 30 micromolar 3,6-dichloro-o-anisic acid (dicamba). This demonstration of high-frequency embryogenesis from mesophyll cells in Gramineae is strong evidence for totipotency of the cells.

Schedule dependence of vincristine lethality in Sarcoma 180 cells following partial synchronization with hydroxyurea.

The effects of exposure to 0.1, 0.5, or 2 microM vincristine for 4 hr were studied in Sarcoma 180 cells at various times after synchronization with 5 mM hydroxyurea for 1 hr. Maximum sensitivity to the lethal effects of vincristine was observed at 10 to 14 hr after hydroxyurea exposure at the higher vincristine concentrations, compared to a period of a maximum sensitivity to a second dose of hydroxyurea at 8 to 12 hr. Serial flow cytometry studies indicated that the apparent decrease in sensitivity to vincristine at 14 to 18 hr was due to the division of cells in the leading segment of the synchronized wave and their entry into the relatively resistant G1 phase prior to vincristine exposure. Synchronized cells that had not divided at the time of vincristine exposure were blocked transiently in G2. Serial metaphase index studies suggested that the G2 cells closest to the end of the cell cycle at the time of vincristine exposure were likely to exhibit the greatest degree of mitotic disorganization when they overcame the G2 block and entered metaphase. The present studies suggest that sensitivity to vincristine increases progressively as cells approach mitosis. The molecular mechanisms underlying this phenomenon are considered in relation to the increase in cell tubulin content during the course of cell cycle progression.

Effects of vincristine on cell survival, cell cycle progression, and mitotic accumulation in asynchronously growing Sarcoma 180 cells.

The effects of vincristine (VCR) on cell survival, cell cycle progression, DNA synthesis, and metaphase accumulation were studied in relation to drug concentration and drug exposure duration in Sarcoma 180 cells in vitro. VCR was found to affect cells in interphase, producing a transient G2 block at all drug concentrations and drug exposure durations studied. VCR did not affect DNA synthesis directly. Increases in the metaphase index were delayed and always peaked at approximately 8 hr after drug removal, regardless of the duration of drug exposure. Increases in the metaphase index of sufficient magnitude to be commensurate with VCR lethality were observed only with prolonged drug exposure. VCR produced both nuclear fragmentation and polyploidy. The proportion of cells undergoing polyploidy increased progressively with increasing drug exposure duration. Interference with cytokinesis during prolonged VCR exposure may represent a lethal effect of VCR that is separate from its short-term effects. This could serve as the basis for the clinical study of the antitumor effects of prolonged VCR infusions.

Effectiveness of physician antismoking advice.

The Dartmouth Primary Care Cooperative Information (COOP) Project conducted a controlled trial on the impact of antismoking advice from office-based physicians. Eighteen primary care medical practices were randomly assigned to be intervention or customary care practices. Medical personnel assigned to the intervention practices were to systematically identify cigarette smokers (among patients aged 35 to 59 years making an office visit), advise them to quit smoking, and provide educational materials. A random sample of 258 smokers was identified and followed-up four months later. Intervention-group smokers were more likely to report being advised to quit smoking (77 percent versus 47 percent) and to attempt quitting (39 percent versus 31 percent), but had success rates similar to those of the other group (6 percent versus 7 percent). We conclude that the medical office is an excellent place to identify large numbers of smokers and initiate attempts at quitting, but find that simple antismoking information and advice are not enough to improve cessation rates.

Mutagenic effectiveness and efficiency of sodium azide versus ethyl methanesulfonate in maize: induction of somatic mutations at the yg2 locus by treatment of seeds differing in metabolic state and cell population.

This study was conducted to compare the effectiveness and efficiency of sodium azide tNaN3) and ethyl methanesulfonate (EMS) for inducing somatic mutations at the yg2 locus in maize seeds of two different metabolic states and cell populations. Dormant or presoaked (72 h at 20 degrees C) seeds heterozygous for yg2 locus were treated with different concentrations of either EMS or NaN3. The cell populations with respect to the percentage of cells in G1, S, G2, and M were also determined for seeds of the two metabolic states. Dormant seeds possessed a higher percentage of cells in G1 and the presoaked seeds a higher percentage of cells in S, G2, and M. The frequency of yg2 sectors in leaves 4 and 5 increased with increasing concentration of both mutagens in both dormant and presoaked seeds. Both mutagens were more effective and efficient in the presoaked seeds. NaN3 was more effective than EMS in terms of number of sectors induced per unit of dose. However, EMS was more efficient as determined by sectors induced per unit of seedling injury and clearly had the ability to induce much higher sector frequencies (more than 10 times greater) than NaN3. The low ability of NaN3 (compared to EMS) to induce mutant sectors may be related to the cells not being treated at the optimum time during the cell cycle, but it is more likely due to its low effectiveness for inducing chromosome aberrations.

Construction of a GFP-BAR plasmid and its use for switchgrass transformation.

A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by replacing the uidA (ß-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l-1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red to yellow. Basta tolerance was observed in T1 plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene.

Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojecticle bombarded leaf cells.

Young leaf tissue of orchardgrass (Dactylis glomerata L.) was placed on Schenk and Hildebrandt medium containing 30 µM dicamba. Microprojectiles coated with DNA containing the selectable bar gene (Basta® tolerance) and the reporter gene uidA coding for ß-glucuronidase (GUS), both driven by the maize ubiquitin promoter (Ubi1), were propelled into the tissue with a particle inflow gun. Transient GUS expression was observed as blue spots of various sizes on leaf segments. Somatic embryos staining entirely blue were also produced, and embryos germinated on medium containing 3.0 mg 1-1 bialaphos. Leaves of 67 putative transformed plants were painted with 0.1% Basta. Ten showed no reaction, and 6 showed only a localized response. Cultured leaf segments from tolerant plants also produced somatic embryos that expressed GUS. The genetic transformation was confirmed by Southern blot hybridization and PCR analyses of T0 plants and by PCR analyses of somatic embryos produced from T0 plants.

Growth, proline accumulation and water relations of NaCl-selected and non-selected callus lines of Dactylis glomerata L.

Sodium chloride-tolerant calli were selected from leaf-derived embryogenic calli of Dactylis glomerata L. on agar solidified medium supplemented with 200 mM NaCl, a concentration lethal to non-selected calli. Growth characteristics, water relations and proline accumulation pattern were compared in selected and non-selected lines. The objective was to gain an understanding of the mechanism(s) of tolerance in the NaCl-tolerant line. Growth in the selected line, as expressed in terms of tolerance index (ratio of fresh wt. on NaCl medium:fresh wt. on NaCl free medium x 100), was greater than that of the non-selected line at all levels of NaCl between 50 and 300 mM. There was no significant difference in proline accumulation in the selected and non-selected lines. Maintenance of turgor by osmotic adjustment was observed in the non-selected line despite decreased growth. In contrast, the selected line lost either the need or the ability to adjust osmotically. There was little or no increase in symplastic osmolality in the selected line when exposed to NaCl. Presumably, selection was made for a salt-excluding tissue that has lost the ability to accumulate solutes and adjust turgor with NaCl stress.

Maximum r.b.e. of fission neutrons for induction of somatic mutations in maize.

This investigation was conducted to determine the maximum r.b.e. of fission neutrons for the induction of yellow-green sectors in maize. Seeds heterozygous for the yg2 locus were adjusted to 13 per cent water content, irradiated with varying doses for eitherfission neutrons or 60Co gamma-radiation, and soaked for 2 hours in nitrogen-bubbled water at ambient temperature before planting. Frequency of yg2 sectors, as scored in leaves 4 and 5, increase linearly for both radiations. The r.b.e. values were greater than1100 at 0.40 sectors per leaf 4 and greater than 600 at 0.20 sectors per leaf 5; however, at these low frequencies, the standard errors were 3 times greater than the r.b.e. At 6.0 sectors per leaf 4, the r.b.e. values were 111 plus or minus 9; and at 3.0sectors per leaf 5, 118 plus or minus 21. As measured by sectors per krad, mutagenic efficiency increased with increased dose of gamma-radiation; the opposite was true for fission neutrons. This trend was more evident for leaf 4 than for leaf 5.

Enhancement of somatic embryogenesis in orchardgrass leaf cultures by epinephrine.

Epinephrine at 10-100 µM stimulated somatic embryogenesis from orchardgrass (Dactylis glomerata L.) leaves cultured on SH medium with 30 µM of indole-3-acetic acid (IAA). Ethylene emanation was increased at epinephrine concentrations greater than 10 µM. Decarboxylation by the leaves of [1-(14)C]IAA included in the medium was decreased almost 3-fold by 10 µM epinephrine. Epinephrine at 10 µM enhanced the number of regenerated plants on SH medium with 30 µM dicamba (SH-30). Ethylene emanation was increased by epinephrine concentrations of 500 µM and greater included in SH-30 but somatic embryogenesis was decreased. Addition of 8 µM CoCl2, 6H2O (an ethylene biosynthesis inhibitor) to medium with 500 µM epinephrine decreased ethylene emanation to the control level but did not alleviate the decreased embryogenic response.

Embryoid and plantlet formation from leaf segments of Dactylis glomerata L.

The purpose of this investigation was to demonstrate callus induction and plantlet formation from cultured leaf segments of 12-15 week-old Dactylis glomerata L. (orchardgrass) plants. Flat half-leaf sections, approximately 2-3 mm square, from the three innermost (youngest) leaves were isolated and individually plated serially beginning at the leaf base on a solid SH medium containing 30 µM of 3,6-dichloro-oanisic acid (dicamba). Callus formed on leaf sections from all 50 plants used in the study. After transfer to SH medium with 1 µM dicamba, plantlets formed from leaf sections of 9 of the 50 plants. In most cases plantlets formed from embryogenic callus but in a few cases embryoids formed directly on the leaf surface without an intervening callus state. These developed into plantlets when transferred to low auxin medium. The response for both callus and plantlet formation decreased with increasing distance both spatially and temporally from the shoot apex. Histological examination of embryogenic callus revealed the presence of non-zygotic embryos in various stages of development. The results provide further support for compentency (if not totipotency) of Gramineae leaf cells.

Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.).

An embryogenic suspension culture of orchardgrass (Dactylis glomerata L.) consisting of small, embryogenic cell clusters was obtained from callus formed on basal sections of young leaves through a process of selective enrichment. These suspensions were used as a source of protoplasts. The isolated protoplasts divided at a frequency of 0.5-10% when plated in an agarose solidified culture medium. Conditioned medium, in which embryogenic Dactylis suspension cultures had been grown, was found to increase the rate of cell colony formation. Protoplast-derived colonies grew rapidly in a bead-type culture system of floating agarose slabs in liquid medium. New suspension cultures formed as the colonies grew out of the agarose. These cultures were embryogenic and formed green plantlets when plated on a solid medium lacking auxin. The plantlets were established in soil and grown to mature plants.

Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata.

Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

Micropropagation of switchgrass by node culture.

Switchgrass (Panicum virgatum L.) is naturally outcrossing; therefore, maintenance of genotype is difficult through sexual propagation. The objective of this study was to develop a micropropagation procedure for the multiplication of desired or selected genotypes. Nodal segments were surface sterilized, split longitudinally, and placed with the cut surface on solid MS medium containing 30 g L-1 maltose and different concentrations of 6-benzylaminopurine (BAP) as the only growth regulator. Best shoot proliferation was obtained with 12.5 micromoles BAP and culture at 29 degrees C. Shoots were easily rooted on MS medium without BAP. Under optimum conditions, it is possible to produce approximately 500 plantlets from one parent plant in 12 wk.

Inhibition of somatic embryogenesis in orchardgrass by endogenous cytokinins.

Endogenous indoleacetic acid (IAA) and cytokinin concentrations were measured by high performance liquid chromatography in leaf sections of an orchardgrass (Dactylis glomerata L.) genotype which exhibited a high capacity for somatic embryogenesis in vitro and in two genotypes that did not exhibit this capacity. The nonembryogenic genotypes contained 3- to 4-fold higher concentrations of zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, and total cytokinins than the embryogenic genotype. There were no significant differences in IAA concentrations between genotypes. Cytokinin concentrations between basal and distal sections of embryogenic genotype were not different, but the IAA concentration was significantly greater in basal sections. Somatic embryogenesis was inhibited in the embryogenic genotype by 0.001 micromolar exogenously added zeatin.

Transgenic plants of Orchardgrass (Dactylis glomerata L.) from protoplasts.

We have demonstrated the transfer and expression of a foreign chimeric gene in the grass species, Dactylis glomerata L. This species is a member of the Gramineae sub-family Pooideae, which includes the small grain cereals, from which transformed plants have not yet been obtained. A chimeric hygromycin-resistance gene was introduced into protoplasts isolated from an embryogenic suspension culture, using heat shock followed by electroporation or polyethylene glycol treatment. Cell colonies resistant to 20 µg/ml hygromycin were selected in liquid medium using an agarose bead type culture system. Transformed calli were identified by Southern hybridization. Embryogenic callus was induced to regenerate plants and transformed plants were shown to contain the hygromycin resistance gene.