Conferred resistance to Botrytis cinerea in Lilium by overexpression of the RCH10 chitinase gene.

KEY MESSAGE: Transgenic Lilium lines have been generated by Agrobacterium -mediated transformation that have enhanced resistance to Botrytis cinerea as a consequence of ectopic expression of a rice chitinase gene. The production of ornamentals is an important global industry, with Lilium being one of the six major bulb crops in the world. The international trade in ornamentals is in the order of £60-75 billion and is expected to increase worldwide by 2-4% per annum. The continued success of the floriculture industry depends on the introduction of new species/cultivars with major alterations in key agronomic characteristics, such as resistance to pathogens. Fungal diseases are the cause of reduced yields and marketable quality of cultivated plants, including ornamental species. The fungal pathogen Botrytis causes extreme economic losses to a wide range of crop species, including ornamentals such as Lilium. Agrobacterium-mediated transformation was used to develop Lilium oriental cv. 'Star Gazer' plants that ectopically overexpress the Rice Chitinase 10 gene (RCH10), under control of the CaMV35S promoter. Levels of conferred resistance linked to chitinase expression were evaluated by infection with Botrytis cinerea; sporulation was reduced in an in vitro assay and the relative expression of the RCH10 gene was determined by quantitative reverse transcriptase-PCR. The extent of resistance to Botrytis, compared to that of the wild type plants, showed a direct correlation with the level of chitinase gene expression. Transgenic plants grown to flowering showed no detrimental phenotypic effects associated with transgene expression. This is the first report of Lilium plants with resistance to Botrytis cinerea generated by a transgenic approach.

Meristematic cell proliferation and ribosome biogenesis are decoupled in diamagnetically levitated Arabidopsis seedlings.

BACKGROUND: Cell growth and cell proliferation are intimately linked in the presence of Earth's gravity, but are decoupled under the microgravity conditions present in orbiting spacecraft. New technologies to simulate microgravity conditions for long-duration experiments, with stable environmental conditions, in Earth-based laboratories are required to further our understanding of the effect of extraterrestrial conditions on the growth, development and health of living matter. RESULTS: We studied the response of transgenic seedlings of Arabidopsis thaliana, containing either the CycB1-GUS proliferation marker or the DR5-GUS auxin-mediated growth marker, to diamagnetic levitation in the bore of a superconducting solenoid magnet. As a control, a second set of seedlings were exposed to a strong magnetic field, but not to levitation forces. A third set was exposed to a strong field and simulated hypergravity (2 g). Cell proliferation and cell growth cytological parameters were measured for each set of seedlings. Nucleolin immunodetection was used as a marker of cell growth. Collectively, the data indicate that these two fundamental cellular processes are decoupled in root meristems, as in microgravity: cell proliferation was enhanced whereas cell growth markers were depleted. These results also demonstrated delocalisation of auxin signalling in the root tip despite the fact that levitation of the seedling as a whole does not prevent the sedimentation of statoliths in the root cells. CONCLUSIONS: In our model system, we found that diamagnetic levitation led to changes that are very similar to those caused by real- [e.g. on board the International Space Station (ISS)] or mechanically-simulated microgravity [e.g. using a Random Positioning Machine (RPM)]. These changes decoupled meristematic cell proliferation from ribosome biogenesis, and altered auxin polar transport.

Characterization of the fungal gibberellin desaturase as a 2-oxoglutarate-dependent dioxygenase and its utilization for enhancing plant growth.

The biosynthesis of gibberellic acid (GA(3)) by the fungus Fusarium fujikuroi is catalyzed by seven enzymes encoded in a gene cluster. While four of these enzymes are characterized as cytochrome P450 monooxygenases, the nature of a fifth oxidase, GA(4) desaturase (DES), is unknown. DES converts GA(4) to GA(7) by the formation of a carbon-1,2 double bond in the penultimate step of the pathway. Here, we show by expression of the des complementary DNA in Escherichia coli that DES has the characteristics of a 2-oxoglutarate-dependent dioxygenase. Although it has low amino acid sequence homology with known 2-oxoglutarate-dependent dioxygenases, putative iron- and 2-oxoglutarate-binding residues, typical of such enzymes, are apparent in its primary sequence. A survey of sequence databases revealed that homologs of DES are widespread in the ascomycetes, although in most cases the homologs must participate in non-gibberellin (GA) pathways. Expression of des from the cauliflower mosaic virus 35S promoter in the plant species Solanum nigrum, Solanum dulcamara, and Nicotiana sylvestris resulted in substantial growth stimulation, with a 3-fold increase in height in S. dulcamara compared with controls. In S. nigrum, the height increase was accompanied by a 20-fold higher concentration of GA(3) in the growing shoots than in controls, although GA(1) content was reduced. Expression of des was also shown to partially restore growth in plants dwarfed by ectopic expression of a GA 2-oxidase (GA-deactivating) gene, consistent with GA(3) being protected from 2-oxidation. Thus, des has the potential to enable substantial growth increases, with practical implications, for example, in biomass production.

Microgravity simulation by diamagnetic levitation: effects of a strong gradient magnetic field on the transcriptional profile of Drosophila melanogaster.

BACKGROUND: Many biological systems respond to the presence or absence of gravity. Since experiments performed in space are expensive and can only be undertaken infrequently, Earth-based simulation techniques are used to investigate the biological response to weightlessness. A high gradient magnetic field can be used to levitate a biological organism so that its net weight is zero. RESULTS: We have used a superconducting magnet to assess the effect of diamagnetic levitation on the fruit fly D. melanogaster in levitation experiments that proceeded for up to 22 consecutive days. We have compared the results with those of similar experiments performed in another paradigm for microgravity simulation, the Random Positioning Machine (RPM). We observed a delay in the development of the fruit flies from embryo to adult. Microarray analysis indicated changes in overall gene expression of imagoes that developed from larvae under diamagnetic levitation, and also under simulated hypergravity conditions. Significant changes were observed in the expression of immune-, stress-, and temperature-response genes. For example, several heat shock proteins were affected. We also found that a strong magnetic field, of 16.5 Tesla, had a significant effect on the expression of these genes, independent of the effects associated with magnetically-induced levitation and hypergravity. CONCLUSIONS: Diamagnetic levitation can be used to simulate an altered effective gravity environment in which gene expression is tuned differentially in diverse Drosophila melanogaster populations including those of different age and gender. Exposure to the magnetic field per se induced similar, but weaker, changes in gene expression.

Somatic hybrid plants of Nicotiana x sanderae (+) N. debneyi with fungal resistance to Peronospora tabacina.

BACKGROUND AND AIMS: The genus Nicotiana includes diploid and tetraploid species, with complementary ecological, agronomic and commercial characteristics. The species are of economic value for tobacco, as ornamentals, and for secondary plant-product biosynthesis. They show substantial differences in disease resistance because of their range of secondary products. In the last decade, sexual hybridization and transgenic technologies have tended to eclipse protoplast fusion for gene transfer. Somatic hybridization was exploited in the present investigation to generate a new hybrid combination involving two sexually incompatible tetraploid species. The somatic hybrid plants were characterized using molecular, molecular cytogenetic and phenotypic approaches. METHODS: Mesophyll protoplasts of the wild fungus-resistant species N. debneyi (2n = 4x = 48) were electrofused with those of the ornamental interspecific sexual hybrid N. × sanderae (2n = 2x = 18). From 1570 protoplast-derived cell colonies selected manually in five experiments, 580 tissues were sub-cultured to shoot regeneration medium. Regenerated plants were transferred to the glasshouse and screened for their morphology, chromosomal composition and disease resistance. KEY RESULTS: Eighty-nine regenerated plants flowered; five were confirmed as somatic hybrids by their intermediate morphology compared with parental plants, cytological constitution and DNA-marker analysis. Somatic hybrid plants had chromosome complements of 60 or 62. Chromosomes were identified to parental genomes by genomic in situ hybridization and included all 18 chromosomes from N. × sanderae, and 42 or 44 chromosomes from N. debneyi. Four or six chromosomes of one ancestral genome of N. debneyi were eliminated during culture of electrofusion-treated protoplasts and plant regeneration. Both chloroplasts and mitochondria of the somatic hybrid plants were probably derived from N. debneyi. All somatic hybrid plants were fertile. In contrast to parental plants of N. × sanderae, the seed progeny of somatic hybrid plants were resistant to infection by Peronospora tabacina, a trait introgressed from the wild parent, N. debneyi. CONCLUSIONS: Sexual incompatibility between N. × sanderae and N. debneyi was circumvented by somatic hybridization involving protoplast fusion. Asymmetrical nuclear hybridity was seen in the hybrids with loss of chromosomes, although importantly, somatic hybrids were fertile and stable. Expression of fungal resistance makes these somatic hybrids extremely valuable germplasm in future breeding programmes in ornamental tobacco.

Cryopreservation of scalps of Malaysian bananas using a pre-growth method.

The effect of preculture with different sugars and mannitol on cryopreservation of scalps of the banana (Musa) cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak was investigated. Scalps (0.3 square cm) were precultured on semi-solid MS-based medium, containing 0.4 or 0.5 M sucrose, glucose, fructose, trehalose or mannitol, for 14 days under a 16 h light and 8 h dark photoperiod prior to rapid cooling and storage in liquid nitrogen. Explants were rewarmed rapidly in a water bath at 40 degree C for 1 min, followed by recovery on two layers of sterile filter paper overlaying 25 ml aliquots of semi-solid MS-based medium with 5 mg per liter benzylaminopurine, 0.2 mg per liter indole acetic acid and 10 mg per liter ascorbic acid (PM8 medium) for 2 days in the dark. Subsequently, scalps were transferred onto 25 ml aliquots of semi-solid PM8 medium and incubated in the dark for 1 week prior to incubation in the light. Shoot regeneration from 5 - 48 percent of cryopreserved scalps of all the banana cvs., was observed only following preculture with 0.4 or 0.5 M glucose or fructose, and with 0.4 M trehalose for the cvs. Pisang Berangan and Pisang Awak. Preculture with 0.4 M glucose resulted in maximum shoot regeneration of cryopreserved scalps of 10 percent, 13 percent, 42 percent and 48 percent for the cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak, respectively. Concentrations of 0.5 M trehalose, or 0.4 and 0.5 M sucrose or mannitol were extremely toxic to scalps of all the cvs. investigated.

Isolation, culture, and plant regeneration from leaf protoplasts of Passiflora.

The family Passifloraceae contains many species exploited in the food, pharmaceutical, and ornamental plant industries. The routine culture of isolated protoplasts (naked cells) followed by reproducible plant regeneration, is crucial to the genetic improvement of Passiflora spp. by somatic cell technologies. Such procedures include somatic hybridization by protoplast fusion to generate novel hybrid plants, and gene introduction by transformation. Seedling leaves are a convenient source of totipotent protoplasts. The protoplast-to-plant system developed for Passiflora edulis fv. flavicarpa is summarized in this chapter. The procedure involves enzymatic degradation of leaf tissue using commercially-available Macerozyme R10, Cellulase R10, and Driselase. Isolated protoplasts are cultured in Kao and Michayluk medium, semi-solidified with agarose. The medium containing the suspended protoplasts is dispensed as droplets or thin layers and bathed in liquid medium of the same composition. Shoot regeneration involves transfer of protoplast-derived tissues to Murashige and Skoog-based medium. The protocols developed for P. edulis are applicable to other Passiflora spp. and will underpin the future biotechnological exploitation of a range of species in this important plant family.

Metabolite fingerprinting in transgenic lettuce.

Metabolite fingerprinting has been achieved using direct atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) and linked gas chromatography (GC-APCI/EI-MS) for transgenic lettuce (Lactuca sativa L. cv. Evola) plants expressing an IPT gene under the control of the senescence-specific SAG12 promoter from Arabidopsis thaliana (P(SAG12)-IPT). Mature heads of transgenic lettuce and their azygous controls were maintained under defined conditions to assess their shelf life. Transgenic lettuce plants exhibited delayed senescence and significant increases (up to a maximum of threefold) in the concentrations of three volatile organic compounds (VOCs), corresponding to molecular masses of 45, 47 and 63, when compared with heads from azygous plants. These VOCs were identified as acetaldehyde (45), ethanol (47) and dimethyl sulphide (63). The increase in dimethyl sulphide was paralleled by an accumulation of reactive oxygen species (ROS) in the heads of transgenic plants. These results demonstrate the applicability of metabolic fingerprinting techniques to elucidate the underlying pleiotropic responses of plants to transgene expression.

Plant protoplasts: status and biotechnological perspectives.

Plant protoplasts ("naked" cells) provide a unique single cell system to underpin several aspects of modern biotechnology. Major advances in genomics, proteomics, and metabolomics have stimulated renewed interest in these osmotically fragile wall-less cells. Reliable procedures are available to isolate and culture protoplasts from a range of plants, including both monocotyledonous and dicotyledonous crops. Several parameters, particularly the source tissue, culture medium, and environmental factors, influence the ability of protoplasts and protoplast-derived cells to express their totipotency and to develop into fertile plants. Importantly, novel approaches to maximise the efficiency of protoplast-to-plant systems include techniques already well established for animal and microbial cells, such as electrostimulation and exposure of protoplasts to surfactants and respiratory gas carriers, especially perfluorochemicals and hemoglobin. However, despite at least four decades of concerted effort and technology transfer between laboratories worldwide, many species still remain recalcitrant in culture. Nevertheless, isolated protoplasts are unique to a range of experimental procedures. In the context of plant genetic manipulation, somatic hybridisation by protoplast fusion enables nuclear and cytoplasmic genomes to be combined, fully or partially, at the interspecific and intergeneric levels to circumvent naturally occurring sexual incompatibility barriers. Uptake of isolated DNA into protoplasts provides the basis for transient and stable nuclear transformation, and also organelle transformation to generate transplastomic plants. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, synthesis of pharmaceutical products, and toxicological assessments. This review focuses upon the most recent developments in protoplast-based technologies.

Selectable markers: antibiotic and herbicide resistance.

The low efficiencies of most plant transformation methods necessitate the use of selectable marker genes to identify those cells that successfully integrate and express transferred DNA. Genes conferring resistance to various antibiotics or herbicides are commonly used in laboratory transformation research. They encode proteins that detoxify corresponding selection agents and allow the preferential growth of transformed cells. This chapter describes the application of two selection systems on the transformation of wheat. One is based on the nptII gene and corresponding aminoglycoside antibiotics, the other is based on the bar gene and corresponding glufosinate ammonium herbicides.

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers.

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems, and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intracellularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers, we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs.

Salinity tolerance and antioxidant status in cotton cultures.

This investigation focuses upon cell growth and antioxidant status in cultured cells of cotton (Gossypium herbaceum) cvs. Dhumad (salt-tolerant, TOL), H-14 (medium salt-tolerant, MED), and RAhs-2 (salt-sensitive, SEN) exposed to saline stress (50-200 mM NaCl). Mean (+/- SEM) callus fresh weight (f.wt.) and dry weight (d.wt.) gains were significantly (p <.05) greater on Murashige and Skoog (MS) [1]-based medium with 50 mM NaCl for the TOL cv. (62% and 16%, respectively) over NaCl-free controls (2020 +/- 45 and 166 +/- 4 mg, respectively); comparable differences were not observed for the MED cv. A significant (p <.05) decrease in mean f.wt. occurred with the SEN cv. exposed to 50 mM NaCl. For all cvs., there were (p <.05) reductions in mean f.wts. in medium with >or=100 mM NaCl. At 200 mM NaCl, mean f.wt. decreases were 52% (TOL), 89% (MED), and 91% (SEN), respectively. A strong correlation existed between antioxidant status and growth of cells with NaCl. Superoxide dismutase and glutathione reductase activities increased with increasing salinity in the TOL cv. to maximum values of 26.3 +/- 1.1 U mg(-1) protein and 1.05 +/- 0.01 AB(340 nm) min(-1) mg(-1) protein, respectively, at 150 mM NaCl; for the MED and SEN cvs., there were no changes in activities of these enzymes between control and salt treatments. Catalase activity decreased progressively with increasing salt concentration in all cvs. except for SEN with 100 mM NaCl, where mean catalase activity (1.75 +/- 0.04 AB(240 nm) min(-1) mg(-1) protein) was greater (p <.05) than control (1.13 +/- 0.08). Overall, cultured cotton cells provide an experimental system for investigating the role of antioxidants in salt tolerance at the cellular level.

Effects of N(6)-benzylaminopurine and Indole Acetic Acid on In Vitro Shoot Multiplication, Nodule-like Meristem Proliferation and Plant Regeneration of Malaysian Bananas (Musa spp.).

Different concentrations of N(6)-benzylaminopurine (BAP) and indole acetic acid (IAA) in Murashige and Skoog based medium were assessed for their effects on shoot multiplication, nodule-like meristem proliferation and plant regeneration of the Malaysian banana cultivars Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak. BAP at 1-14 mg L(-1) with or without 0.2 mg L(-1) IAA, or BAP at 7-14 mg L(-1) with the same concentration of IAA, was evaluated for shoot multiplication from shoot tips and the proliferation of nodule-like meristems from scalps, respectively. Plant regeneration from scalps was assessed using 1 mg L(-1) BAP and 0.2 mg L(-1) IAA separately, or a combination of these two growth regulators. Data on shoot multiplication, the proliferation of nodule-like meristems with associated plant regeneration were recorded after 30 days of culture. A maximum of 5 shoots per original shoot tip was achieved on medium supplemented with BAP at 5 mg L(-1) (Pisang Nangka), 6 mg L(-1) (Pisang Mas and Pisang Berangan), or 7 mg L(-1) (Pisang Awak), with 0.2 mg L(-1) IAA. BAP at 11 mg L(-1) with 0.2 mg L(-1) IAA induced the most highly proliferating nodule-like meristems in the four banana cultivars. Plant regeneration from scalps was optimum in all cases on medium containing 1 mg L(-1) BAP and 0.2 mg L(-1) IAA. This is the first report on the successful induction of highly proliferating nodule-like meristems and plant regeneration from scalps of the Malaysian banana cultivars Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak.

Effect of magnetically simulated zero-gravity and enhanced gravity on the walk of the common fruitfly.

Understanding the effects of gravity on biological organisms is vital to the success of future space missions. Previous studies in Earth orbit have shown that the common fruitfly (Drosophila melanogaster) walks more quickly and more frequently in microgravity, compared with its motion on Earth. However, flight preparation procedures and forces endured on launch made it difficult to implement on the Earth's surface a control that exposed flies to the same sequence of major physical and environmental changes. To address the uncertainties concerning these behavioural anomalies, we have studied the walking paths of D. melanogaster in a pseudo-weightless environment (0g*) in our Earth-based laboratory. We used a strong magnetic field, produced by a superconducting solenoid, to induce a diamagnetic force on the flies that balanced the force of gravity. Simultaneously, two other groups of flies were exposed to a pseudo-hypergravity environment (2g*) and a normal gravity environment (1g*) within the spatially varying field. The flies had a larger mean speed in 0g* than in 1g*, and smaller in 2g*. The mean square distance travelled by the flies grew more rapidly with time in 0g* than in 1g*, and slower in 2g*. We observed no other clear effects of the magnetic field, up to 16.5 T, on the walks of the flies. We compare the effect of diamagnetically simulated weightlessness with that of weightlessness in an orbiting spacecraft, and identify the cause of the anomalous behaviour as the altered effective gravity.

Diamagnetic levitation enhances growth of liquid bacterial cultures by increasing oxygen availability.

Diamagnetic levitation is a technique that uses a strong, spatially varying magnetic field to reproduce aspects of weightlessness, on the Earth. We used a superconducting magnet to levitate growing bacterial cultures for up to 18 h, to determine the effect of diamagnetic levitation on all phases of the bacterial growth cycle. We find that diamagnetic levitation increases the rate of population growth in a liquid culture and reduces the sedimentation rate of the cells. Further experiments and microarray gene analysis show that the increase in growth rate is owing to enhanced oxygen availability. We also demonstrate that the magnetic field that levitates the cells also induces convective stirring in the liquid. We present a simple theoretical model, showing how the paramagnetic force on dissolved oxygen can cause convection during the aerobic phases of bacterial growth. We propose that this convection enhances oxygen availability by transporting oxygen around the liquid culture. Since this process results from the strong magnetic field, it is not present in other weightless environments, e.g. in Earth orbit. Hence, these results are of significance and timely to researchers considering the use of diamagnetic levitation to explore effects of weightlessness on living organisms and on physical phenomena.

Micropropagation of poinsettia by organogenesis.

Poinsettia (Euphorbia pulcherrima) is one of the most popular ornamental pot plants. Conventional propagation is by cuttings, generally focused on a period prior to the most intensive time of sales. Rapid multiplication of elite clones, the production of pathogen-free plants and more rapid introduction of novel cultivars (cvs.) with desirable traits, represent important driving forces in the poinsettia industry. In recent years, different strategies have been adopted to micropropagate poinsettia, which could assist breeders to meet consumer demands. The development of reliable in vitro regeneration procedures is likely to play a crucial role in future production systems. Stem nodal explants cultured on semi-solid MS-based medium supplemented with benzylaminopurine (BAP) and naphthalene acetic acid (NAA) develop shoots from adventitious/axillary buds after 7 weeks of culture. Rooting of in vitro regenerated shoots is achieved in semi-solid MS-based medium containing the auxin indole-3-acetic acid (IAA). Four to six weeks after transfer to root-inducing medium, regenerated plants can be transferred to compost and acclimatized in the glasshouse. Direct shoot regeneration from cultured explants is important to minimize somaclonal variation in regenerated plants.