Somatic embryogenesis, organogenesis and plant regeneration in taro (Colocasia esculenta var. esculenta)

Callus was initiated in three different ‘‘esculenta’’ taro cultivars by culturing corm slices in the dark on half-strength MS medium supplemented with 2.0 mg/l 2,4- dichlorophenoxyacetic acid (2,4-D) for 20 days followed by subculture of all corm slices to half-strength MS medium containing 1.0 mg/l thidiazuron (TDZ). Depending on the cultivar, 20–30% of corm slices produced compact, yellow, nodular callus on media containing TDZ. Histological studies revealed the presence of typical embryogenic cells which were small, isodiametric with dense cytoplasms. Somatic embryos formed when callus was transferred to hormone-free medium and *72% of the embryos germinated into plantlets on this medium. Simultaneous formation of roots and shoots during germination, and the presence of shoot and root poles revealed by histology, confirmed that these structures were true somatic embryos. Plants derived from somatic embryos appeared phenotypically normal following 2 months growth in a glasshouse. This method is a significant advance on those previously reported for the esculenta cultivars of taro due to its efficiency and reproducibility.

Initiation of embryogenic cell suspensions of taro (Colocasia esculenta var. esculenta) and plant regeneration

Embryogenic callus was initiated by culturing in vitro taro corm slices on agar-solidified half-strength MS medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by transfer to 1.0 mg/L thidiazuron (TDZ). Callus was subsequently proliferated on solid medium containing 1.0 mg/L TDZ, 0.5 mg/L 2,4- D and 800 mg/L glutamine before transfer to liquid medium containing the same components but with reduced glutamine (100 mg/L). After 3 months in liquid culture on an orbital shaker, cytoplasmically dense cell aggregates began to form. Somatic embryogenesis was induced by plating suspension cells onto solid media containing reduced levels of hormones (0.1 mg/L TDZ, 0.05 mg/L 2,4-D), high concentrations of sucrose (40–50 g/L) and biotin (1.0 mg/L). Embryo maturation and germination was then induced on media containing 0.05 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-acetic acid (IAA). Histological studies of the developing embryos revealed the presence of typical shoot and root poles suggesting that these structures were true somatic embryos. The rate of somatic embryos formation was 500–3,000 per mL settledcell volume while approximately 60% of the embryos regenerated into plants.

A cost-based optimization of a fiberboard pressing plant using Monte-Carlo simulation (a reliability program)

In this research the reliability and availability of fiberboard pressing plant is assessed and a cost-based optimization of the system using the Monte- Carlo simulation method is performed. The woodchip and pulp or engineered wood industry in Australia and around the world is a lucrative industry. One such industry is hardboard. The pressing system is the main system, as it converts the wet pulp to fiberboard. The assessment identified the pressing system has the highest downtime throughout the plant plus it represents the bottleneck in the process. A survey in the late nineties revealed there are over one thousand plants around the world, with the pressing system being a common system among these plants. No work has been done to assess or estimate the reliability of such a pressing system; therefore this assessment can be used for assessing any plant of this type.

A myosin from a higher plant has structural similarities to class V myosins

In plant cells, myosin is believed to be the molecular motor responsible for actin-based motility processes such as cytoplasmic streaming and directed vesicle transport. In an effort to characterize plant myosin, a cDNA encoding a myosin heavy chain was isolated from Arabidopsis thaliana. The predicted product of the MYA1 gene is 173 kDa and is structurally similar to the class V myosins. It is composed of the highly-conserved NH2-terminal "head" domain, a putative calmodulin-binding "neck" domain an alpha-helical coiled-coil domain, and a COOH-terminal domain. Northern blot analysis shows that the Arabidopsis MYA1 gene is expressed in all the major plant tissues (flower, leaf, root, and stem). We suggest that the MYA1 myosin may be involved in a general intracellular transport process in plant cells.

Modelling cell separation during plant organ abscission

A simple mathematical model is presented to describe the cell separation process that plants undertake in order to deliberately shed organs. The focus here is on modelling the production of the enzyme polygalacturonase, which breaks down pectin that provides natural cell-to-cell adhesion in the localised abscission zone. A coupled system of three ordinary differential equations is given for a single cell, and then extended to hold for a layer of cells in the abscission zone. Simple observations are made based on the results of this preliminary model and, furthermore, a number of opportunities for applied mathematicians to make contributions in this subject area are discussed.

Cystine-mediated oxidative defence in Lactobacillus reuteri BR11

Lactobacillus reuteri BR11 possesses an abundant cystine uptake (Cyu) ABC-transporter that was previously found to be involved in a novel mechanism of oxidative defence mediated by cystine. The current study aimed to elucidate this mechanism with a focus on the role of the co-transcribed cystathionine ã-lyase (Cgl). Growth studies of wild-type L. reuteri BR11 and mutants inactivated in cgl and the cystine-binding protein encoding gene cyuC showed that in contrast to the Cyu transporter, whose inactivation led to growth arrest in aerated cultures, Cgl is not crucial for oxidative defence. However, the role of Cgl in oxidative defence became apparent in the presence of severe oxidative damage and cysteine deprivation. Cysteine was found to be protective against oxidative stress, and the action of Cgl in both cysteine biosynthesis and degradation poses a seemingly futile pathway that deprives the intracellular cysteine pool. To further characterise the relationship between Cgl activity and cysteine and their roles in oxidative defence, enzymatic assays were performed on purified Cgl, and intracellular concentrations of cysteine, cystathionine and methionine were determined. Cgl was highly active towards cystine and cystathionine and less active towards cysteine in vitro, suggesting the main function of Cgl to be cysteine biosynthesis. Cysteine was found at high concentrations in the cell, but the levels were not significantly affected by inactivation of cgl or growth under aerobic conditions. It was concluded that both anabolic and catabolic activities of Cgl towards cysteine contribute to oxidative defence, the former by maintaining an intracellular reservoir of thiol analogous to glutathione, and the latter by producing H2S which is readily secreted, thus creating a reducing extracellular environment. The significance of the Cyu transporter to the physiology of L. reuteri BR11 prompted a phylogenetic study to determine its presence in bacteria. Orthologs of the Cyu transporter that are closest matches to the Cyu transporter are only limited to several species of Lactobacillus and Leuconostoc. Outside the Lactobacillales order, the closest matching orthologs belong to Proteobacteria, and there are more orthologs in Proteobacteria than non-Lactobacillales Firmicutes, suggesting that the Cyu transporter locus was present in the ancestor of the Proteobacteria and Firmicutes, and over evolutionary time has been lost or diverged in many Firmicutes. The clustering of the Cyu transporter locus with a gene encoding a Cgl family protein is even rarer. It was only found in L. reuteri, Lactobacillus vaginalis, Weissella paramesenteroides, the Lactobacillus casei group, and several Campylobacter sp. An accompanying phylogenetic study of L. reuteri BR11 using multi-locus sequence analysis showed that L. reuteri BR11 had diverged from more than 100 strains of L. reuteri isolated from various hosts and geographical locations. However, comparison with other Lactobacillus species supported the current classification of BR11 as L. reuteri. The most closely related species to L. reuteri is L. vaginalis or Lactobacillus antri, depending on the housekeeping gene used for analysis. The close evolutionary relationship of L. vaginalis to L. reuteri and the high degree of sequence identity between the cgl-cyuABC loci in both species suggest that the Cyu system is highly likely to perform similar functions in L. vaginalis. In search of other genes that function in oxidative defence, a number of mutants which were inactivated in genes that confer increased resistance to oxidative stress in other bacteria were constructed. The genes targeted were ahpC (peroxidase component of the alkyl hydroperoxide reductase system), tpx (thiol peroxidase), osmC (osmotically induced protein C), mntH (Mn2+/Fe2+ transporter), gshA (ã-glutamylcysteine synthetase) and msrA (methionine sulfoxide reductase). The ahpC and mntH mutants had slightly lower minimum inhibitory concentrations of organic peroxides, suggesting these genes might be involved in resistance to organic peroxides in L. reuteri. However, none of the mutants exhibited growth defects in aerated cultures, in stark contrast to the cyuC mutant. This may be due to compensatory functions of other genes, a hypothesis which cannot be tested until a robust protocol for constructing markerless multiple gene deletion mutants in L. reuteri is developed. These results highlight the importance of the Cyu transporter in oxidative defence and provide a foundation for extending the research of this system in other bacteria.

Sensitivity of Dot-ELISA on Nitrocellulose Membranes in Comparison with ELISA on Polystyrene Plates for the Detection of Four Plant Viruses

ABSTR.4CT Senitivity of dot-immunobindinding ELf SA on nitrocellulose membrane (DotELISA)was compared with double-antibody sandwich ELISA (DAS-ELlSA) on polystyrene plates for the detection of bean yellow mosaic virus (BYMV), broad bean stain virus (WMV-2). Dot-ELISA was 2 and 1O times more sensitive than DAS-ELISA for the detection of BBSV and WMV-2, respectively, whereas DAS-ELISA was more sensitive than Dot-ELiSA for {he detection of BYMV. Both techniques were equally sensitive for the detection of BYDV. Using one day instead uf the two-day procedure, the four viruses were still detectable and the ralative sensitivity of both techniques remained the same.