An efficient system for high-quality large-scale micropropagation of Miscanthus × giganteus plants

Miscanthus?×?giganteus is a perennial grass considered to be one of the most promising biofuel and bioenergy crops because of its high biomass and quality, and low requirement for fertilizers and pesticides. Market demand for Miscanthus is rapidly increasing. However, M.?×?giganteus is a triploid that cannot produce viable seeds, and it has traditionally been propagated through rhizome division, which is low throughput and labor-intensive. Plant tissue culture provides the potential to propagate M.?×?giganteus in vitro while maintaining the original plant characteristics. Although protocols exist for M.?×?giganteus micropropagation, the multiplication rate and plant quality need to be improved to meet commercial demands. For this research, we have assessed callus induction, callus multiplication, plantlet regeneration, shoot multiplication, shoot quality improvement, rooting, plant acclimatization, and survival in the greenhouse and in the field. Through these studies, we have developed an efficient system for high-quality, large-scale micropropagation of M.?×?giganteus. The plants produced from our protocol exhibited more than 99% survival in soil due to the production of vigorous shoots and roots.     

Improving micropropagation of Mentha × piperita L. using a liquid culture system

In the current study, in vitro shoot proliferation and plant regeneration of Mentha × piperita L. (peppermint) cultivar ‘Black Mitcham’ was compared in semi-solid and liquid culture systems. Shoot tips from field-grown plants were used as explants to study shoot proliferation response on either Murashige and Skoog (MS) or Chee and Pool (C2D) medium containing varying levels of 6-benzylaminopurine (BAP), kinetin, and 6-γ,γ-dimethylallyl aminopurine (2iP). Differences in leaf ultrastructure and antioxidant capacity of greenhouse-grown and micropropagation-derived plants were studied to identify potential changes occurring during in vitro culture. Among the various media treatments tested, the maximum number of shoots was produced on the C2D medium with 4.0 μM BAP (40.7) followed by the MS medium with 4.0 μM BAP (32.2). Among the rooting treatments, shoots on the MS medium with 1.0 μM indole-3-butyric acid (IBA) produced the maximum number of roots (14.4). The number of shoots produced in Liquid Lab Rocker® (LLR) vessels containing liquid C2D medium with BAP (103.4) was significantly higher than that produced on semi-solid medium (40.7). No differences were observed in the leaf ultrastructure and antioxidant capacity of leaf extracts obtained from greenhouse-grown and micropropagation-derived plants. The study indicates that the liquid culture system under the described conditions can enhance peppermint micropropagation, with plant material being potentially valuable for use in herbal supplements and essential oil production.

     

Is a Persistent Global Bias Necessary for the Establishment of Planar Cell Polarity?

Planar cell polarity (PCP)–the coordinated polarisation of a whole field of cells within the plane of a tissue–relies on the interaction of three modules: a global module that couples individual cellular polarity to the tissue axis, a local module that aligns the axis of polarisation of neighbouring cells, and a readout module that directs the correct outgrowth of PCP-regulated structures such as hairs and bristles. While much is known about the molecular components that are required for PCP, the functional details of–and interactions between–the modules remain unclear. In this work, we perform a mathematical and computational analysis of two previously proposed computational models of the local module (Amonlirdviman et al., Science, 307, 2005; Le Garrec et al., Dev. Dyn., 235, 2006). Both models can reproduce wild-type and mutant phenotypes of PCP observed in the Drosophila wing under the assumption that a tissue-wide polarity cue from the global module persists throughout the development of PCP. We demonstrate that both models can also generate tissue-level PCP when provided with only a transient initial polarity cue. However, in these models such transient cues are not sufficient to ensure robustness of the resulting cellular polarisation.     

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2) of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes. Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gyno- genetic progeny of allotetraploid hybrids of red crucian carp × common carp had important signifi- cances in both biological evolution and production application.     

Correction to: Improving micropropagation of Mentha × piperita L. using a liquid culture system

In Vitro Cellular & Developmental Biology - Plant - This correction reflects Sadanand A. Dhekney’s updated e-mail address and affiliation.     

DNA microarray technology: a new tool for the epidemiological typing of bacterial pathogens?

Genomic hybridization on whole genome arrays detects the presence or absence of similar DNA regions in sufficiently related microorganisms, allowing genome-wide comparison of their genetic contents. A whole genome array is based on a sequenced bacterial isolate, and is a collection of DNA probes fixed on a solid support. In a single hybridization experiment, the absence/presence status of all genes of the sequenced microbe in the queried isolate can be examined. The objective of this minireview is to summarize the past usage of DNA microarray technology for microbial strain characterizations, and to estimate its future utilization in epidemiological studies and molecular typing of bacterial pathogens. The studies reviewed here confirm the usefulness of microarray technology for the detection of genetic polymorphisms. However, the construction or purchase of DNA microarrays and the performance of strain to strain hybridization experiments are still prohibitively expensive for routine application. Future use of arrays in epidemiology is likely to depend on the development of more cost-effective protocols, more robust and simplified formats, and the adequate evaluation of their performance (efficacy) and convenience (efficiency) compared with other genotyping methods. It seems more likely that a more focused assay, concentrating on genomic regions of variability previously detected by genome-wide microarrays, will find broad application in routine bacterial epidemiology.     

Establishment of a cell-based assay for examining the expression of tumor necrosis factor alpha (TNF-α) gene

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-alpha is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-alpha in macrophages, we attempt to establish a reporter system to assess TNF-alpha gene expression through measuring luciferase activity. In this study, mouse macrophage cell line RAW 264.7 was stably transfected with a luciferase reporter pGL3-TNFPro-UTR, which contains TNF-alpha promoter and 3'-untranslated region (3'-UTR). The TNF-alpha-luciferase reporter cell line is used for assessing the expression of TNF-alpha gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-alpha such as dexamethasone and emodin, and also for measuring change of expression of TNF-alpha gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-alpha. The luciferase activity correlated well with the ELISA results for TNF-alpha production, therefore, the TNF-alpha-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-alpha gene.     

Importance of the iron chelate formula for micropropagation of Rosa hybrida L. ‘Moneyway’

In vitro propagation of the rose rootstock Moneyway was investigated on the following media: Murashige and Skoog (MS), Quoirin and Lepoivre (QL) and Woody Plant (WP). Growth, which was measured as length of shoots after a 6-week period, was faster on MS and QL than on WP. In spite of the better growth, chlorosis of newly formed leaves occurred from the third week on and was correlated with a lower chlorophyll content of shoots. Replacement of FeEDTA by FeEDDHA in QL and MS resulted in the development of green shoots for more than 3 months. The occurrence of chlorosis was not pH directed since the pH of QL with FeEDTA or FeEDDHA had not changed after 6 weeks of growth. Addition of the light absorbing dye fast yellow 9 to QL with FeEDTA also resulted in green shoots with a higher chlorophyll content. It is suggested that FeEDDHA is a more photostable chelate than FeEDTA, resulting in a higher availability of iron for the rose shoots. The impact of the iron chelate formula on the micropropagation of plant species that are susceptible to iron deficiency is discussed.Abbreviations BA 6-benzyladenine, fast yellow 9-4-amino-1,1-azobenzene-3,4-disulfonic acid - FeEDTA ferric ethylenediamine tetraacetate - FeEDDHA ferric ethylenediamine di(o-hydroxyphenylacetate) - IAA indole-3-acetic acid - IBA indole-3-butyric acid - LSD least significant difference - NAA -naphthaleneacetic acid - P probability     

Thermal denaturation: is solid-state fermentation really a good technology for the production of enzymes?

The potential for thermal denaturation to cause enzyme losses during solid-state fermentation processes for the production of enzymes was examined, using the protease of Penicillium fellutanum as a model system. The frequency factor and activation energies for the first-order denaturation of this enzyme were determined as 3.447 x 10(59) h(-1) and 364,070 Jmol(-1), respectively. These values were incorporated into a mathematical model of enzyme deactivation, which was used to investigate the consequences of subjecting this protease to temporal temperature profiles reported in the literature for mid-height in a 34 cm high packed-bed bioreactor of 150 mm diameter. In this literature source, temperature profiles were measured for 5, 15 and 25 liters per minute of air and enzyme activities were measured as a function of time. The enzyme activity profiles predicted by the model were distributed similarly, one relative to the other, as had been found in the experimental study, with substantial amounts of denaturation being predicted when the substrate temperature exceeded 40 degrees C, which occurred at the lower two airflow rates. A mathematical model of a well-mixed bioreactor was used to explore the difficulties that would be faced at large scale. It suggests that even with airflows as high as one volume per volume per minute, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation. This work highlights the extra care that must be taken in scaling up solid-state fermentation processes for the production of thermolabile products.     

Modified single node culture method—a new micropropagation method for chickpea

Summary Chickpea was micropropagated by axillary shoot proliferation (ASP) and modified single node culture (MSNC) methods. Maximum propagule proliferation occurred on Murashige and Skoog (MS) medium enriched with 1–10 μM N⁶-benzyladenine and 0.01 μM α-naphthaleneacetic acid. The propagules were rooted on MS medium containing 1 μM 3-indolebutyric acid and B₅ vitamins. Regenerated plants were fertile and phenotypically similar to control plants grown from seed. The MSNC method was four times more efficient than the ASP method in terms of the number of plants produced per explant.     

Proposal for the Establishment of the Core Zones in the Biosphere Reserve of Tehuacán-Cuicatlán, Mexico

This work proposes the establishment of core zones in the Biosphere Reserve of Tehuacán-Cuicatlán (BRTC), based on plant species richness and endemism. A total of 561 species of the four most important plant families in the region (Asteraceae, Cactaceae, Leguminosae and Poaceae) as well as 174 endemic species of these and other families were used in the analyses. Distribution of these taxa was analyzed using two different iterative complementarity methods. Significant correlations were found between patterns of species richness and endemic plants distribution in the study area. These results were combined with other analysis where two different indices (species richness index and human population index) were used. The results suggest the delimitation of four core zones within the Biosphere Reserve, covering a total area of 105,300 ha. The core zones represent 21.8% of the area, and would protect 72.54% of the species from the selected plant families and 67.8% of endemic species.     

Design and analysis of a replicated server architecture for supporting IP–host mobility

Mobility support in IP networks requires servers to forward packets to mobile hosts and to maintain information pertaining to a mobile host's location in the network. In the mobile Internet Protocol (mobile-IP), location and packet forwarding functions are provided by servers referred to as home agents. These home agents may become the bottleneck when there are a large number of mobile hosts in the network. In this paper, we consider the design and analysis of a replicated server architecture in which multiple home agents are used to provide mobility support. In order to minimize the delay across the home agents, one of the key aspects is the design of load balancing schemes in which a home agent may transfer the control of a mobile host to another home agent in the same network. The methods for triggering the transfer and the policy for selecting the next home agent define various load balancing schemes which have different performance characteristics. In this paper, we design a protocol that forms the building block for implementing such load balancing schemes, and we then study the performance characteristics of three selection schemes, namely, random, round-robin, and join the shortest queue (JSQ), and three transfers policies, namely, timer-, counter- and threshold-based. The key results of this study are as follows: (1) The results show that both random and round-robin selection policies can yield modest load balancing gains, and that these gains increase when the traffic is more bursty (burstiness is defined as the ratio of the peak arrival rate to the mean arrival rate) as well as when there are more home agents. (2) The threshold-based transfer policy performs better than timer-based and counter-based policies, since in threshold-based policies transfers are made only when the queue is overloaded, unlike counter- and timer-based policies in which transfers can be made from an unloaded home agent to an overloaded home agent. This revised version was published online in August 2006 with corrections to the Cover Date.     

Establishment of a pancreatic β cell proliferation model in vitro and a platform for diabetes drug screening

Diabetes, a disease resulting from loss of functional β cells, is globally an increasingly important condition. Based on the islet-differentiation ability of ductal epithelial cells and stimulating β cell proliferation ability of the Reg Iα gene, we aimed to establish an in vitro pancreatic β cell proliferation model for screening therapeutic drugs of diabetes in the future. Pancreatic ductal epithelial cells were isolated from male Wistar rats, and induced to differentiate into pancreatic β cells. Immunofluorescence staining assay, western blot, RT-PCR analysis, and dithizone staining were used to characterize the cells. Rat Reg Iα protein was transiently expressed in vitro by transfection of HEK 293 cells with the PCMV6-entry-REG Ia plasmid, and expression was verified by RT-PCR analysis, proliferation assay, and apoptosis assay. The pancreatic β cell proliferation model was further validated by a proliferation assay using differentiated pancreatic β cells treated with transfection supernatant. Finally, we have successfully established an in vitro pancreatic β cells proliferation model using transiently expressed rat Reg Iα protein and differentiated pancreatic β cells from pancreatic ductal epithelial cells. This model could be used as a platform to screen new drugs for islet neogenesis to cure diabetes, especially Chinese herbal drugs in the future.     

In vitro micropropagation of a Cucurbita interspecific hybrid cultivar – a root stock plant

An efficient in vitro micropropagation protocol was developed for direct shoot growth of interspecific Cucurbita hybrid variety using shoot–tips of 5-day-old explants. The excised shoot–tips were cultured on Murashige and Skoog (MS) medium containing two plant growth regulators (6-benzyladenine and naphthaleneacetic acid) with various combinations and concentrations for the study of shoot induction. The best condition for shoot growth was with 3 mg l^–1 6-benzyladenine (BA) in MS medium. The shooting frequency was 84% and five shoots were obtained from each explant after 30 days of culture. Shoots (11.5 cm length) were rooted most effectively in 1 mg l^–1 indole-3 butyric acid (IBA)-supplemented MS medium. The highest root formation rate was 93% and all rooted shoots were transplanted into soil.     

Phenotypic variation during micropropagation of the chimeralRhododendron ‘President Roosevelt’

The putative periclinal chimeraRhododendron xlimbatum President Roosevelt was used to study the origin of shoots in vitro. Genotypic segregation readily occurred in vitro. Numerous phenotypes were observed, although most shoots were either entirely green or maintained the original variegation pattern. Derivatives of the third apical layer were rarely involved in shoot formation. A reversed chimeral form was isolated. Adventitious shoots were usually miniaturized and rapidly proliferating, but axillary shoots had thicker stems, larger leaves and proliferated more slowly. Corolla tissue produced stunted, leafy shoots; no variegated shoots were produced from floret explants. In shoot tip cultures the addition of 40M 2iP without IBA resulted in the greatest number of shoots. Explant choice was the most critical factor for maintenance of foliar variegation.     

Biotic wetland connectivity—supporting a new approach for wetland policy

Wetlands are key habitats connected physically and socially with processes occurring over a much wider territory. The biotic connection through dispersal mechanisms among wetlands is of primary importance to wetland management and policies. However, traditional wetland conservation approaches are based on the preservation of isolated sites considered to be of special importance (typically owing to their importance for concentrations of migratory waterbirds). Research linking local species richness and bird migration suggests that the effect of wetland loss on regional diversity might be much larger than what would be expected from direct habitat loss. Since the biotic connection among wetlands serviced by waterbirds appears to be more efficient within a limited range, the distribution of wetlands in space is a key aspect determining wetland connectedness even in the absence of direct hydrologic links. Protected areas should thus be defined with regard to waterfowl movements and waterbird migration as functional processes contributing to aquatic species migration and local species richness. This calls for a regional approach to wetland management within a continental context. This paper aims at defining an operational view of the dispersion function of wetlands and its implication for conservation policies. For this purpose, we examined the conservation policies of the Ramsar Convention (the international treaty that protects wetlands) and the European Union (as an example of relevant continental level policy-making) from the viewpoint of bird-mediated dispersal of aquatic organisms. We propose nine specific avenues for the inclusion of bird-mediated dispersal in the policy documents examined. Non-governmental organisations and other organisations working in waterbird conservation should also recognise the importance of their policies for aquatic biodiversity at broader levels and avoid compartmentalising their conservation activities.     

Reconsideration of the term ‘vitrification’ as used in micropropagation

The term vitrification is currently used to describe two types of processes related to tissue-cultured plant material. The first is used to describe organs and tissues having an abnormal morphological appearance and physiological function. The second is used to describe the transition from liquid to solid state, i.e. the formation of ice during low temperature storage of in vitro cultured cells, tissues and organs. Use of the same term to define two greatly different processes in the same research area can only lead to confusion, especially for key words. Thus it is appropriate to reconsider the usage of vitrification in the first sense mentioned above. It is recommended that the term vitrification should no longer be used to indicate plant material with an abnormal morphological appearance and physiological function, and should be substituted by the term hyperhydricity.This paper was written as a follow up of the discussions held during the workshop on Vitrification at the IAPTC-Congress in Amsterdam (1990). Different eminent research workers in the field of vitrification were invited to formulate their preference for a new terminology, and they came to a consensus.