Differentiation of potato (Solanum tuberosum L.) plants from cultured leaf protoplasts

Through induction of totipotent stage in cultured cells plants were regenerated from protoplast derived callus colonies of potato. Growing of the plants in vitro with optimal aeration and pretreatment of the leaves in dark and cold prior to protoplast isolation has improved the protoplast yield and frequency of cell division. Protoplasts of four potato genotypes have started to divide within 2-5 days after plating them into culture medium containing 2,4-D (0.2 mg/l); ZEA (0.5 mg/l); and NAA (1 mg/l) as growth regulators. Embedding of the cells into agarose proved to be favourable to avoid cell browning and to increase colony formation. The series of hormone treatments based on complex action of NAA and BAP promoting colony growth and greening, ZEA and IAA inducing shoot redifferentiation, and GA3 plus NAA supporting shoot elongation and rooting, finally resulted in high frequency of plant regeneration from microcolonies.     

Scavenging behaviour and size-dependent carcass consumption of the black bullhead (Ameiurus melas)

This study examined the size-dependent scavenging behaviour of black bullheads Ameiurus melas under laboratory conditions, using common bleak Alburnus alburnus and pumpkinseed Lepomis gibbosus carcasses. Video camera observations showed that the activity of A. melas was higher at night, but substantial daytime activity was also recorded. Larger A. melas were more active than their smaller conspecifics, especially at night. All size classes exhibited a well-defined sequence of consuming different parts of the carcasses independent of size, but larger individuals tended to consume carcasses more efficiently. Carcasses of the softer-bodied A. alburnus were consumed more readily than those of the bonier L. gibbosus, independent of size. This scavenging behaviour of A. melas might facilitate the invasion success of the species.     

PEG-mediated transformation of leaf protoplasts of Solanum tuberosum L. cultivars

As an alternative to Agrobacterium-mediated gene transfer, direct transformation of potato (Solanum tuberosum L.) cultivars was achieved by using the Mg²⁺/PEG protocol (Negrutiu et al. 1987) to stimulate DNA uptake into leaf protoplasts. The frequency of kanamycin-resistant clones varied between 1–12% from experiment to experiment independently of the genotype used. A deleterious effect of heat shock treatment (45°C for 5 min.) has been found on colony formation during optimization of the transformation procedure. The Mg²⁺ ion concentration (15–35 mM), the denaturation (100°C, 10 min. right before the treatment) and the form of the plasmid DNA (linear or circular) had no significant effect on the efficacy of the transformation. Application of denatured calf thymus DNA as carrier molecules (at concentration of 50 g ml^-1, however, resulted in a significant decrease in the number of the resistant cell colonies). Sixty to 80 percent of the kanamycin-selected callus tissues regenerated shoots. The presence and expression of the introduced neomycin phosphotransferase neo gene in regenerants with normal morphology and tetraploid chromosome number was proved by biological tests based on rooting and callus formation in the presence of the antibiotic and by NPT enzyme activity assay. Southern analysis revealed the integration of the introduced DNA molecules carrying the neo marker gene, into the genome of potato.     

Modulation of the phosphoinositide 3-kinase pathway alters innate resistance to polymicrobial sepsis

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-beta, IL-2, IL-6, IL-10, IL-12, and TNF-alpha in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.     

Expression of His-tagged Shigella IpaC in Arabidopsis

Although the expression of histidine (His)-tagged proteins in bacteria is routine, few His-tagged proteins have been expressed in plants, and no His-tagged proteins from bacterial pathogens have been expressed in plants, to our knowledge. Here, we demonstrate expression of the Shigella flexneri invasion plasmid antigen, IpaC, in Arabidopsis thaliana. S. flexneri is the causitive trigger for bacillary dysentery, and IpaC is essential for bacterial entry into epithelial cells. IpaC, attached to a 5' leader containing six tandem His codons, was cloned into a pBI121 vector. This clone was introduced into Agrobacterium tumefaciens and Arabidopsis plants were then transformed. T1 and T2 plant generations were obtained. Total plant proteins were extracted from T2 leaves; the Bradford assay was used to determine protein concentrations. A nickel-coated ELISA plate method, using both anti-His and anti-IpaC 1 degrees antibodies, was used to detect and quantify IpaC in transgenic Arabidopsis plants. Between 1.9 and 2.3 microg IpaC/mg total plant protein was obtained; this equals 0.2% of total protein, an amount comparable to other recombinant protein estimates in plants. Expressing His-tagged proteins from bacterial pathogens, in plants, is important because plant material could ultimately be fed or applied intranasally to animals that are "at risk" for infection by such bacterial pathogens, thus causing them to raise antibodies against the pathogens--functioning as a vaccine.     

Effects of capture and video‐recording on the behavior and breeding success of Great Tits in urban and forest habitats

Behavioral research often involves capturing and video‐recording birds, but these procedures may have undesired effects on the behavior of birds that have rarely been quantified. In addition, birds in urban and more natural areas may differ in their sensitivity to disturbance. We examined the possible effects of both capturing, weighing and measuring, and taking a blood sample, and the presence of video‐cameras on the behavior of male and female Great Tits (Parus major) breeding in urban and forest habitats. Using a 2 × 2 block design, we compared the behavior and breeding success of parents that either were or were not captured on their nests a few days before behavioral observations, and of parents that either were or were not habituated to the presence of a concealed video‐recorder mounted on nest boxes. We found no significant effects of habituation to the camera on bird behavior, but males captured in their nest boxes were more vigilant and hesitated longer before entering nest boxes, and also had slightly lower provisioning rates than males that had not been captured. Captured females also tended to be more vigilant than females that had not been captured, but their provisioning rates were not affected. Capturing males also influenced the behavior of their non‐captured mates, but capturing females had no effect on the behavior of their non‐captured mates. We found no difference in the effects of capture on Great Tits in urban and forest habitats, and our treatments also had no effect on the mass, size, and survival of nestlings until fledging. Our results suggest that, for Great Tits, being captured results in sex‐dependent behavioral effects that can last for at least several days. As such, we suggest that the possibility of similar effects in other species of birds should be considered in behavioral studies where birds must be captured, and recommend either that behavioral data be collected before capturing birds or that all birds in a study should be captured and handled in a standardized way.     

Development of flooding-tolerant Arabidopsis thaliana by autoregulated cytokinin production

Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. This study was conducted to determine the effects of autoregulated cytokinin production on the flooding tolerance of Arabidopsis thaliana plants. A chimeric gene containing the senescence-specific SAG12 promoter and the ipt gene coding for isopentenyl transferase, a rate-limiting enzyme in the cytokinin biosynthesis pathway, was constructed. The chimeric gene was introduced into Arabidopsis plants by Agrobacterium-mediated vacuum infiltration. Four transgenic lines were chosen for flooding tolerance determinations. DNA hybridization analysis and PCR confirmed that all four of the transgenic lines carried the ipt gene. The segregation of kanamycin resistance in the T₂ generation indicated 1 to 3 integration events. GUS expression and RT-PCR of the ipt gene confirmed the senescence-specificity of the SAG12 promoter. Morphologically, the transgenic lines appeared healthy and normal. Transgenic plants began to flower at the same time as wild-type plants, but the period from flowering to senescence was lengthened by 7 to 12 days. Tolerance of the transgenic plants to waterlogging and complete submergence was assayed in three independent experiments. All four transgenic lines were consistently more tolerant to flooding than wild-type plants. The results indicated that endogenously produced cytokinin can regulate senescence caused by flooding stress, thereby, increasing plant tolerance to flooding. This study provides a novel mechanism to improve flooding tolerance in plants.     

Habitat use of invasive monkey goby (Neogobius fluviatilis) and pumpkinseed (Lepomis gibbosus) in Lake Balaton (Hungary): a comparison of electrofishing and fyke netting

Hydrobiologia - Detailed knowledge on the habitat preference of invasive fishes and the bias of different fishing methods in determining their population dynamic parameters are essential in...