Engineering gibberellin metabolism in Solanum nigrum L. by ectopic expression of gibberellin oxidase genes.

Gibberellins (GAs) control many aspects of plant development, including seed germination, shoot growth, flower induction and growth and fruit expansion. Leaf explants of Solanum nigrum (Black Nightshade; Solanaceae) were used for Agrobacterium-mediated delivery of GA-biosynthetic genes to determine the influence of their encoded enzymes on the production of bioactive GAs and plant stature in this species. Constructs were prepared containing the neomycin phosphotransferase (nptII) gene for kanamycin resistance as a selectable marker, and the GA-biosynthetic genes, their expression under the control of the CaMV 35S promoter. The GA-biosynthetic genes comprised AtGA20ox1, isolated from Arabidopsis thaliana, the product from which catalyses the formation of C(19)-GAs, and MmGA3ox1 and MmGA3ox2, isolated from Marah macrocarpus, which encode functionally different GA 3-oxidases that convert C(19)-GAs to biologically active forms. Increase in stature was observed in plants transformed with AtGA20ox1, MmGA3ox2 and MmGA3ox1 + MmGA3ox2, their presence and expression being confirmed by PCR and RT-PCR, respectively, accompanied by an increase in GA(1) content. Interestingly, MmGA3ox1 alone did not induce a sustained increase in plant height, probably because of only a marginal increase in bioactive GA(1) content in the transformed plants. The results are discussed in the context of regulating plant stature, since this strategy would decrease the use of chemicals to promote plant growth.

Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species.

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C(19)-GAs, including the bioactive GAs GA(1 )and GA(4), by 2beta-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA(1) and GA(4). Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds.

Drosophila melanogaster, a model system for comparative studies on the responses to real and simulated microgravity.

A key requirement to enhance our understanding of the response of biological organisms to different levels of gravity is the availability of experimental systems that can simulate microgravity and hypergravity in ground-based laboratories. This paper compares the results obtained from analysing gene expression profiles of Drosophila in space versus those obtained in a random position machine (RPM) and by centrifugation. The correlation found validates the use of the RPM simulation technique to establish the effects of real microgravity on biological systems. This work is being extended to investigate Drosophila development in another gravity modifying instrument, the levitation magnet.

Hemoglobin promotes somatic embryogenesis in peanut cultures.

Critical parameters influencing somatic embryogenesis include growth regulators and oxygen supply. Consequently, the present investigation has focused on optimization of a somatic embryogenic system for peanut (Arachis hypogaea L.) through media supplementation with the auxin, picloram. The latter at 30 mg L(-1) was optimal for inducing regeneration of somatic embryos from cultured explants of zygotic embryos. In contrast, somatic embryogenesis did not occur in the absence of this growth regulator. An assessment has also been made of the beneficial effect on somatic embryogenesis and plant regeneration of the commercial hemoglobin (Hb) solution, Erythrogen. Hemoglobin at 1:50 and 1:100 (v:v) stimulated increases in mean fresh weight (up to a maximum of 57% over control), mean number of explants producing somatic embryos (15%) and mean number of somatic embryos per explant (29%).

Metabolic responses of cultured cells to oxygenated perfluorocarbon.

Protoplast-derived cells of albino Petunia hybrida cv. Comanche were used as a model, nonphotosynthetic, eukaryotic plant system to study changes in (1) the rate of oxygen consumption as measured by a Clark-type oxygen microelectrode, (2) mitochondrial membrane potential (MMP) as assessed by Rhodamine 123 fluorescence, and (3) intracellular activities of superoxide dismutases (SOD, EC 1.15.1.1) and catalases (CAT, EC 1.11.1.6), following culture for up to 14 d in aqueous nutrient medium overlaying oxygen-gassed perfluorodecalin (Flutec PP5; F2 Chemicals, Preston, UK). The mean (+/- s.e.m., n = 7) rate of oxygen consumption of Petunia cells after 24 h of culture in the presence of oxygenated PFC was 14.3 +/- 1.6 mol O2 ml(-1) min(-1), compared to 9.7 +/- 0.8 micromol O2 ml(-1) min(-1) for untreated (control) cells (P < 0.05). Similarly, the culture of cells with oxygenated PFC for 24 h resulted in a significant (P < 0.05) increase of over 50% in the mean MMP, compared to the control. Culture of protoplasts with oxygenated PFC also produced significant (P < 0.05) increases in both mean SOD and CAT activities after 3-7 d of culture, the former comparable to that reported previously for protoplasts of Salpiglossis sinuata cultured with oxygenated PFC.

Ultrasound-induced physiological changes in cultured cells of Petunia hybrida.

Petunia hybrida cell suspension cultures were exposed to ultrasonic standing wave fields at 2.43 MHz for 40 min with mean sound pressures (within homogenous sound fields) varying from 0 (control) to ca. 1.1 MPa. Mean (+/- s.d.; n =6-9) cell viability was reduced to 87+/-10% at 0.6 MPa and to 59 +/- 23% at 1.1 MPa, compared to an initial control value of 92 +/- 6% (P <0.05). Mean (n = 3) cell alkaline phosphatase concentration increased linearly with sound pressure from a control value of 0.006+/-0.001 to 0.02+/-0.01 Sigma-Units microg(-1) protein at 1.1 MPa (P<0.05). Similarly, mean cell catalase activity increased from a control value of 0.020 +/- 0.003 to 0.026 +/- 0.008 arbitrary units at 1.1 MPa. In contrast, mean cellular lactate dehydrogenase concentration was unchanged. These observations indicate that cellular repair processes associated with increased alkaline phosphatase activity might be triggered by physical cell damage caused by ultrasound. The observed increase in catalase activity suggests increasing production of free radicals and other sonochemicals, which warrants further study. The absence of changes in lactate dehydrogenase indicates that there was no major damage to respiratory pathways or to overall cellular integrity.

Hemoglobin-stimulated growth and antioxidant activities in cultured cotton cells.

Changes in cellular reactive oxygen scavenging enzymes were assessed in suspension-derived cells of cotton (Gossypium herbaceum) cv. Dhumad following culture with a commercial bovine hemoglobin (Hb) solution (Erythrogen) at 1:100-1:1000 (v:v). Mean (+/- SEM) fresh (f.wt.) and dry weights (d.wt.) of cells after 25 d of culture were significantly (p <.05) greater in medium supplemented with 1:750 and 1:1000 (v:v) Erythrogen, compared to controls lacking Erythrogen. For example, with 1:750 (v:v) Erythrogen, mean cell f.wt. and d.wt. were increased by 45 and 31%, respectively. Total soluble cellular protein increased by 141, 176, and 191% with Erythrogen at 1:50, 1:750, and 1:1000 (v:v), respectively. Cellular catalase and glutathione reductase activities decreased significantly (p <.05) following addition of low concentrations (1:1000 and 1:750 v:v) of Erythrogen to culture medium. However, increasing the concentration of Erythrogen to a maximum of 1:100 (v:v), caused a concomitant increase in catalase to a maximum of 62% over control. Mean total superoxide dismutase activity increased linearly with increasing Erythrogen concentration, reaching a maximum mean value over 2-fold greater than control with 1:100 (v:v) Erythrogen. A similar trend was observed in cellular H2O2 content, which reached a maximum of 98% over control with 1:250 (v:v) Erythrogen. These results demonstrate that culture of cotton cells with Hb solution causes changes in cellular oxygenation sufficient to modify cellular antioxidant status.

Strategies for signal amplification in nucleic acid detection.

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.

Haemoglobin (Erythrogen) enhanced microcallus formation from protoplasts of Indica rice (Oryza sativa L.).

The beneficial effects have been studied of supplementing culture medium with 1:100-1:500 (v:v) of a commercial haemoglobin solution (Erythrogen) on the mitotic division of cell suspension-derived protoplasts of Indica rices (Oryza sativa L.). Protoplasts were cultured in liquid medium, at densities of 1.5 x 10(6) or 2.5 x 10(6) ml(-1), on nitrocellulose membranes overlaying a semi-solidified medium layer that was supplemented with both Erythrogen and nurse cells of Lolium multiflorum. The mean final plating efficiencies (FPEs) of rice cv. BR26 protoplasts cultured with 1:200 (v:v) Erythrogen, at 1.5 x 10(6) ml(-1) (0.018+/-0.003%; n = 8) and 2.5 x 10(6) ml(-1) (0.016+/-0.002%; n = 8), were both significantly (P < 0.05) greater than controls lacking Erythrogen (0.0058+/-0.002%; n = 8 and 0.0041+/-0.001%; n = 8, respectively). Similarly, the mean FPEs of cv. Bini protoplasts cultured with 1:200 (v:v) Erythrogen at 1.5 x 10(6) ml(-1) (0.012+/-0.003%; n = 6) and 2.5 x 10(6) ml(-1) (0.017+/-0.001%; n = 6) were also significantly (P < 0.05) greater than their respective controls (0.003+/-0.001%, n = 6 and 0.002+/-0.001%, n = 6). In contrast, supplementation with 1:100 or 1:500 (v:v) Erythrogen did not lead to sustained mitotic division and microcallus formation in both rice cultivars.

Effects of P(SAG12)-IPT gene expression on development and senescence in transgenic lettuce.

An ipt gene under control of the senescence-specific SAG12 promoter from Arabidopsis (P(SAG12)-IPT) significantly delayed developmental and postharvest leaf senescence in mature heads of transgenic lettuce (Lactuca sativa L. cv Evola) homozygous for the transgene. Apart from retardation of leaf senescence, mature, 60-d-old plants exhibited normal morphology with no significant differences in head diameter or fresh weight of leaves and roots. Induction of senescence by nitrogen starvation rapidly reduced total nitrogen, nitrate, and growth of transgenic and azygous (control) plants, but chlorophyll was retained in the lower (outer) leaves of transgenic plants. Harvested P(SAG12)-IPT heads also retained chlorophyll in their lower leaves. During later development (bolting and preflowering) of transgenic plants, the decrease in chlorophyll, total protein, and Rubisco content in leaves was abolished, resulting in a uniform distribution of these components throughout the plants. Homozygous P(SAG12)-IPT lettuce plants showed a slight delay in bolting (4-6 d), a severe delay in flowering (4-8 weeks), and premature senescence of their upper leaves. These changes correlated with significantly elevated concentrations of cytokinin and hexoses in the upper leaves of transgenic plants during later stages of development, implicating a relationship between cytokinin and hexose concentrations in senescence.

Beneficial effects of Pluronic F-68 and artificial oxygen carriers on the post-thaw recovery of cryopreserved plant cells.

The storage of prokaryotic and eukaryotic cells at ultra-low temperature in liquid nitrogen (-196 degrees C) is a procedure that has assumed an increasingly important role in underpinning many aspects of biotechnology. For eukaryotic cells, the transition from a cryopreserved state to physiologically normal temperatures and oxygen tensions, induces respiratory imbalances that may stimulate the production of toxic oxygen radicals causing impaired cellular functions. Novel treatments, that focus specifically on enhancing oxygen delivery to cells, are important in maximising post-thaw recovery. Recently, several approaches have been evaluated with suspension cultured plant cells as a model, yet biotechnologically-important, totipotent eukaryotic cell system. Such treatments include non-ionic surfactants, primarily Pluronic F-68, and artificial oxygen carriers, the latter based on inert perfluorochemical liquids or chemically-modifed haemoglobin, as supplements to culture medium used during the post-thaw recovery phase of cell growth. When used either alone or in combination, such novel treatments stimulate significantly the post-thaw viability and biomass production of cultured plant cells. Many of these technologies will be exploitable in cryopreservation protocols for eukaryotic cells in general.

Pluronic F-68 enhanced shoot regeneration in a potentially novel citrus rootstock.

The effects have been studied in vitro of the non-ionic, co-polymer surfactant, Pluronic F-68, on shoot regeneration and bud induction in epicotyl and cotyledon explants of Citrus depressa, a potential alternative rootstock to C. jambhiri for commercial Citrus. Supplementation of Murashige and Skoog (1962)-based, agar-solidified shoot regeneration/bud induction (SRBI) medium with 1.0 mg l(-1) 6-benzylaminopurine and 0.5% (w/v) Pluronic F-68 significantly (P < 0.05) increased mean fresh weight by a maximum of 60%, the proportion of explants exhibiting shoot/bud regeneration by 25% and the mean number of shoots per epicotyl explant by 184%, compared to untreated controls. Similarly, 0.5% (w/v) Pluronic F-68 significantly (P < 0.05) enhanced the mean percentage bud induction (91%) and the number of buds regenerated (>4-fold) per cotyledon explant. Interestingly, the mean fresh weight gain for both explants was unaffected across the range of concentrations (0.001-0.1% w/v) of Pluronic F-68 evaluated. Regenerated plants from epicotyl explants were transferred and acclimatized to glasshouse conditions.

Effect of Erythrogen on post-thaw recovery of cryopreserved cell suspensions of indica rice (Oryza sativa L.).

This study shows that adding haemoglobin solution (Erythrogen) to post-thaw medium of Indica rice (Oryza sativa L.) cells enhances survival following cryopreservation. Haemoglobin (1:50 - 1:200 v:v) had a beneficial effect on post-thaw viability and subsequent cell growth. A key finding was that the successful recovery from cryopreservation of cell suspensions of the Indica rice cvs. BR26 and Pajam, and their re-establishment in AA2 medium, reflected a requirement for such supplementation of the post-thaw recovery medium with Erythrogen.

Combined PI-DAPI staining (CPD) reveals NOR asymmetry and facilitates karyotyping of plant chromosomes.

This paper presents a preparative and staining procedure for plant mitotic chromosomes that uses a combination of PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindol) and which reveals a pattern of high-affinity regions for these fluorochromes. Nucleolar organiser regions (NORs), telomeres and centromeric regions exhibit high PI affinity (red), whereas other chromosomal regions exhibit high affinity for either PI (red) or DAPI (blue). NOR-bearing and other chromosomes are readily distinguished, facilitating karyotyping. The dual staining pattern was observed in all the plants tested. Aspects of NOR size, number and occurrence are discussed. A karyotype of rice metaphase chromosomes is presented, based on their fluorescent banding patterns.

Induction of dwarfism in transgenic Solanum dulcamara by over-expression of a gibberellin 20-oxidase cDNA from pumpkin.

The gibberellin (GA) 20-oxidase (CmGA20ox1) from immature pumpkin seed produces predominantly inactive tricarboxylic acid GAs. We expressed CmGA20ox1 under the control of the CaMV 35S promoter in Solanum dulcamara to assess the usefulness of this gene for reducing GA content in transgenic plants. All transgenic plants obtained were semi-dwarfs with smaller, deep-green leaves and highly pigmented stems compared to the wild-type. Such transformants flowered earlier than the wild-type plants and produced more fruit and more seeds per fruit. The transgene was efficiently expressed, producing high levels of CmGA20ox1 transcript and protein. Furthermore, the concentration of GA(1) was reduced in leaves of the transformants to approximately 20% or less of that in the wild-type and to about 40% or less in stems. The concentrations of other 13-hydroxylated GAs were also reduced, except for the tricarboxylic acid, GA(17), which accumulated in the transformants due to 13-hydroxylation of GA(25). By contrast, the concentrations of non-13-hydroxylated GAs, GA(4) and GA(34), were not consistently reduced, indicating that the effect of expressing the pumpkin gene may not be predictable. Transcript abundance for a native GA 20-oxidase gene was higher in the leaves and stems of S. dulcamara transformed with the pumpkin gene than in wild-type, reflecting the feedback control of 20-oxidase gene expression that serves as a homeostatic mechanism for GAs.

Viability of plant cell suspensions exposed to homogeneous ultrasonic fields of different energy density and wave type.

Exposure of Petunia hybrida cell suspensions to ultrasound at a frequency of 2.43 MHz in a standing wave field at an energy density of 70 Jm-3 (pressure amplitude of 0.78 MPa) decreased their mean viability to 35% after 20 min of sonication. A comparison of propagating wave and standing wave treatments at equal frequency (2.15 MHz) and energy density (8.5 Jm-3) showed, in the first case, a rapid decline in mean viability of cells (to 30% after 10 min of sonication) and, in the second case, a retaining of the initial viability (95%), respectively. Cells sonicated 4 days after subculture were more sensitive than cells sonicated 2 or 6 days after transfer to new culture medium. It was concluded that cellular viability depends primarily on the acoustic energy density, the exposure time, and the mechanical properties of the cells determined by age. As a consequence of the trapping of cells in the anti-node planes of the standing wave, propagating wave fields reduced cellular viability compared with standing wave fields at equal energy density.

Culture of cells at perfluorocarbon-aqueous interfaces.

Protoplasts (wall-less cells) isolated enzymatically from leaf tissues of Manihot esculenta, Passiflora edulis and Petunia parodii, and from cell suspensions of Oryza sativa, Passiflora giberti, Petunia hybrida and Salpiglossis sinuata, were cultured for up to 35 d at an interface between the inert, oxygen-gassed perfluorocarbon (PFC) liquid, perfluorodecalin, overlaid with liquid or semi-solidified aqueous media. The maximum increase in mitotic division, as assessed by initial plating efficiency (IPE) occurred with protoplasts of O. sativa, which showed a 4-fold increase above the control over 35 d. Similar, but less pronounced increases in IPE of 90-103% occurred with S. sinuata, P. giberti and P. parodii following culture with oxygenated PFC. The least responsive species was M. esculenta, where the mean IPE after 25 d was increased by 33% over control. For those totipotent protoplast systems (e.g. P. edulis, P. giberti, O. sativa and P. parodii) phenotypically normal plants were regenerated following initial culture with oxygenated PFC. The advantages of such an interface system include (1) ease of sterilisation of the PFC by autoclaving, (2) the recycleability and, hence, recovery of the PFC, thereby offsetting the high initial costs, and (3) the ability to aspirate cells at the interface.

Haemoglobin (Erythrogen)-enhanced post-thaw growth of cryopreserved cells.

Supplementation of semi-solid R2 culture medium with a commercial bovine haemoglobin (Hb) solution (Erythrogen) at 1:50-1:500 (v:v), had beneficial effects on the growth, following cryopreservation, of cells of the Indica rice, Oryza sativa cv. Pusa Basmati 1. The mean absorbance, as assessed by triphenyl tetrazolium chloride reduction, of rice cells at 8 d post-thawing, was increased by up to 60% (P < 0.05), compared to cells recovered in the absence of Hb. Erythrogen (1:50-1:500 v:v) promoted an increase in biomass, of up to 25% over control (P < 0.05), at 24 d post-thawing. Cell suspensions, re-established by transfer to liquid medium of cells initially thawed and cultured with Erythrogen for 24 d, exhibited increased (up to 2-fold) growth rates over a subsequent 20-d period, compared to cells recovered without Hb.