A novel strategy for identifying mutations that sensitize Drosophila eye development to caffeine and hydroxyurea.

This report describes a novel strategy for isolating Drosophila mutants with conditional eye phenotypes that should be generally applicable for identifying genes required for cellular responses to specific drugs. To test the strategy, we screened 3 of the 5 major chromosome arms for hydroxyurea- and (or) caffeine-sensitive (huc) mutants, and isolated mutations affecting 5 different complementation groups. Most of these were represented by single alleles; however, we also isolated multiple alleles of huc(29DE) gene, an essential gene that is also associated with a nonconditional pupal lethal phenotype. We also identified huc(95E) mutants, which are extremely sensitive to caffeine. Although huc(95E) is a nonessential gene, mutant imaginal disc cells undergo caffeine-dependent apoptosis, and huc(95E) gene function is required for the viability of the organism when mutant larvae are exposed to levels of caffeine that controls can easily tolerate. We have mapped the cytological positions of huc(29D) and huc(95E) as a first step toward molecularly characterizing the relevant genes.     

Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (<Emphasis Type="Italic">Pinus virginiana</Emphasis> Mill.)

Plant tissue necrosis and subsequent cell death are usually observed during in vitro regeneration in conifers, especially in plant regeneration via somatic organogenesis in pine species. Cell death is correlated with the elevated levels of peroxides. In this investigation, the effects of antioxidants on in vitro regeneration of Virginia pine (Pinus virginiana Mill.) were evaluated. Antioxidants, polyvinylpolypyrrolidone (PVPP) and 1,4-dithio-dl-threitol (DTT), were found to improve callus formation, shoot differentiation and growth, and shoot rooting by inhibiting tissue necrosis during the initiation of cultures and subculture of shoots. These treatments enabled the recovery and regeneration plants at high frequency through somatic organogenesis. Compared to the control, the frequencies of callus formation, shoot growth, and shoot rooting increased 15, 26, and 19%, respectively, by addition of 5 g/l PVPP and 2 g/l DTT. Higher peroxidase activity of tissue cultures during subculture from callus proliferation medium to shoot differentiation medium and to rooting medium was observed. The addition of antioxidants reduces and inhibits browning by reducing the accumulation of peroxidase.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - DTT 1,4-Dithio-dl-threitol - IBA Indole butyric acid - NAA -Naphthaleneacetic acid - PVPP Polyvinylpolypyrrolidone     

Exogenously added polyamines recover browning tissues into normal callus cultures and improve plant regeneration in pine

Polyamines are known to influence a variety of growth and developmental processes in higher plants. Tissue browning seriously reduces in vitro plant regeneration in pine species by decreasing the levels of antioxidant enzymes and polyamines in tissue cultures. In the present investigation, the effect of exogenously added polyamines on recovering browning tissues into normal callus cultures and on improving plant regeneration was examined in Virginia pine ( Pinus virginiana Mill.). Among the polyamines administered, 1.5 m M putrescine (Put), 1.5 m M spermidine (Spd), or 1.5 m M spermine (Spm) alone resulted in a 19.55%, 18.92%, and 1.45%, respectively, recovering of browning tissues into normal callus cultures in 5 weeks. A combination of Put with Spd or Spm did not result in an increase of recovering rate, compared to the Put or Spd alone. Exogenously added 1.5 m M Put, 1.5 m M Spd, 1.5 m M Put + 1.5 m M Spd, or 1.5 m M Put + 1.5 m M Spm recovers browning tissues into normal callus cultures by increasing the activity of antioxidant enzymes ascorbate peroxidase (APOX), glutathione reductase (GR) and superoxide dismutase (SOD) and by decreasing lipid peroxidation. Exogenously added 1.5 m M Put, 1.5 m M Spd, 1.5 m M Put + 1.5 m M Spd, or 1.5 m M Put + 1.5 m M Spm significantly improve the growth rate of callus cultures, shoot formation, and rooting adventitious shoots. These results demonstrated that exogenously added polyamines recover browning tissues into normal callus cultures by decreasing oxidative damage and improving plant regeneration by acting as plant growth substances.     

The Effect of Different Plant Growth Regulators on Adventitious Shoot Formation from Virginia Pine (Pinus virginiana) Zygotic Embryo Explants

The effects of different plant growth regulators on in vitro adventitious shoot formation in Virginia pine (Pinus virginiana Mill.) zygotic embryo explants were quantitatively evaluated. Using Tang and Ouyang (1999) (TE) basal medium supplemented with 11.4 μM indole-3-acetic acid (IAA) and 2.2 μM N⁶-benzyladenine (BA), callus was observed after 3–6 weeks of culture. Calluses were transferred to TE basal medium supplemented with 0.49 μM indole-3-butyric acid (IBA) and 8.8 μM BA for 6–9 weeks, where they produced numerous small shoot primordia. They were then transferred to TE basal medium supplemented with 0.49 μM IBA and 4.4 μM BA to promote growth and elongation of adventitious shoots. After elongated shoots were transferred to TE medium containing 0.05 μM α-naphthaleneacetic acid (NAA) for 6 weeks, adventitious roots were formed. Regenerated plantlets were established in soil in greenhouse.