Cybridization in Nicotiana tabacum L. using double inactivation of parental protoplasts and post-fusion selection based on nuclear-encoded and chloroplast-encoded marker genes

An effective selection system preceded by double inactivation of parental protoplasts was used to transfer Nicotiana suaveolens Leh. cytoplasmic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cultivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens plants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inactivated with iodoacetamide or rhodamine 6G, while those of the cytoplasm donor were inactivated by X-irradiation. The resultant microcalli were cultured on a shoot regeneration medium containing both kanamycin and spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants, out of a total of 44 regenerated plants transferred to the glasshouse, were obtained. Intraspecific somatic transfers of the CMS trait between N. tabacum cultivars with distinctlydifferent morphologies using single inactivation and nonselective shoot regeneration medium were demonstrated. The implications of the results for practical tobacco breeding as a means of circumventing lengthy backcrossing procedures are discussed.     

Revision and affinities ofGalium (Rubiaceae) in Madagascar

The only representatives ofRubiaceae-Rubieae in Madagascar are five species ofGalium. G. thunbergianum andG. chloroionanthum are essentially afromontane species, ± widely distributed in both the African mainland and Madagascar. The new speciesG. andringitrense andG. ankaratrense are endemic to Madagascar but show close affinities to two afromontane—afroalpine groups from the African mainland, theG. simense—G. ruwenzoriense andG. glaciale complex respectively.G. polyacanthum (new combination) exhibits certain primitive morphological traits and appears to be an old Madagascan endemic without close allies. New chromosome counts for populations from the African mainland demonstrate thatG. chloroionanthum andG. simense are 4x (x=11). Distribution patterns and origins of theGalium species in Madagascar and their relationships with African taxa are discussed.     

Suppression of nuclear factor-kappa B activity by nitric oxide and hyperoxia in oxygen-resistant cells

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO +/- O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-kappaB activity. Cells lacking functional NF-kappaB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-kappaB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-kappaB activity in cells exposed to NO +/- O2, IkappaBalpha was degraded, suggesting that pathways regulating the steady-state levels of IkappaB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-kappaB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-kappaB activity.     

A parametric study of acetabular cup design variables using finite element analysis and statistical design of experiments.

To isolate the primary variables influencing acetabular cup and interface stresses, we performed an evaluation of cup loading and cup support variables, using a Statistical Design of Experiments (SDOE) approach. We developed three-dimensional finite element (FEM) models of the pelvis and adjacent bone. Cup support variables included fixation mechanism (cemented or noncemented), amount of bone support, and presence of metal backing. Cup loading variables included head size and cup thickness, cup/head friction, and conformity between the cup and head. Interaction between and among variables was determined using SDOE techniques. Of the variables tested, conformity, head size, and backing emerged as significant influences on stresses. Since initially nonconforming surfaces would be expected to wear into conforming surfaces, conformity is not expected to be a clinically significant variable. This indicates that head size should be tightly toleranced during manufacturing, and that small changes in head size can have a disproportionate influence on the stress environment. In addition, attention should be paid to the use of nonmetal backed cups, in limiting cup/bone interface stresses. No combination of secondary variables could compensate for, or override the effect of, the primary variables. Based on the results using the SDOE approach, adaptive FEM models simulating the wear process may be able to limit their parameters to head size and cup backing.     

In vitro shoot culture and microtuber induction in the steroid yam Dioscorea composita Hemsl

The individual effects of sucrose, plant growth regulators and basal salt media formulations were investigated on microtuber induction and development in shoot cultures of the steroid yam Dioscorea composita. Sucrose at 8% (w/v) was the single most significant medium constituent for microtuber induction. Of the four cytokinins tested, 6-benzyladenine at 1.25 and 2.5 μM showed strong inhibitory effects on microtuber induction. By contrast, the auxins α-naphthaleneacetic acid and indole-3-butyric acid at 5.0 μM showed striking promotive effects on microtuber induction and growth. In the presence of either one of these auxins at 5.0 μM shoot cultures produced microtubers weighing 300–400 mg fresh weight whilst kinetin, 6-(γ,γ-dimethylallylamino)-purine, 6-benzyladenine and abscisic acid failed to promote microtuber growth (microtubers weighed generally <200 mg). Media formulations Lloyd and MacCown and White supported the lowest frequencies of microtuber induction when kinetin was present at 2.5 μM. Anderson Rhododendron was as effective as Murashige and Skoog overall in promoting both microtuber induction and growth. When removed from cultures and planted in sterilized moist sand, microtubers sprouted readily (60–87% within 2 weeks) and produced vigorous shoot growth and after 5–7 months minitubers of sizes (30–80 g) suitable for direct field planting. This revised version was published online in June 2006 with corrections to the Cover Date.