Molecular genetic studies on Brassica napus L.

The feasibility of using two different methods of assaying for DNA polymer phisms has been assessed. They were Restriction fragment length polymorphisms (RFLPs) as revealed by a range of characterised Brassica cDNA sequences and Random amplified polymorphic DNA (RAPD). These techniques have been shown to reveal DNA polymorphisms between varieties of Brassica napus L.. Further more, the sequence and organisation of a Hind III family of highly repetitive DNA sequences were also studied on Brassica napus L. RFLPs associated with rape extensin, ext A, and Brassica oleraceae self- incompatibility genes were observed when DNA samples from 19 commercial varieties of B. napus were analysed using the cDNA probes pRR566 (coding for root-specific extensin) and pBOS2 (coding for S(_5) self-incompatibility allele in B. oleraceae). Both cDNA clones were able to reveal RFLP patterns with varying degrees of polymorphism depending on the restriction enzymes used in the digestion of genomic DNAs. Although both probes could generate complex RFLP band patterns, those revealed by pB0S2 were generally easier to analyse and more suitable for DNA fingerprinting while those revealed by pRR566 were less distinct as a result of extensive background hybridisations. The probe pRR566, with certain restriction enzymes generated simpler RFLP band patterns that were more suitable for segregation analyses. Segregation analysis of F(_1) individuals revealed additive RFLP band patterns of both parental varieties, while that of F(_2) individuals revealed RFLP band patterns of each parental varieties as well as the additive pattern. When analysed for possible association with varietal glucosinolate content, none of the RFLP band patterns showed such linkage. A cDNA library was constructed from pod material of a high glucosinolate variety in an attempt to obtain clones which could reveal RFLP patterns associated with glucosinolate content. Differential screening using total cDNAs from pod materials of high and low glucosinolate varieties failed to isolate any cDNA clones useful as RFLP markers. Another DNA polymorphism assay studied, RAPD, was able to detect inter- and intraspecific variation in Brassica sp.. Analysis of six phylogenetically-related but distinct Brassica sp. revealed extensive variation in the RAPD band patternsof amplification products; with some amphidiploid species sharing conserved band patterns with their ancestral species. RAPD analysis on 17 varieties of rape revealed polymorphic as well as highly conserved RAPD band patterns depending on the primer used. One of the primers was able to amplify a polymorphic band which could be associated with low glucosinolate varieties i.e. present almost exclusively in low glucosinolate varieties. Species-specific as well as variety-specific band patterns were also observed during the RAPD analysis. Finally, sequence and organisation of a Hind III family of repetitive sequences was studied. The monomeric and polymeric forms (trimer and tetramer) of the repetitive sequences were successfully cloned into pUCl8. Sequence analysis of the two clones containing the polymeric forms revealed that the monomers were arranged in tandem array and that all internal Hind III recognition sites were lost due to point mutation(s) which occurred within the six basepair recognition site. A consensus monomeric sequence was deduced from sequence comparison of the 8 copies of the monomeric sequences present in the 3 clones and the deviation from the consensus sequence of each of the eight monomers was less than 3%. No two monomeric sequences were identical. It was estimated that the number of copies of the monomeric sequences in a haploid genome was approximately 0.3 million copies. Estimates of the proportional representation of each of the polymeric sequences based on the number of copies of the monomers in each polymer were also calculated

The best method for isolated total RNA from durian tissues

Plant tissues, especially durian tissues contain high content of polysaccharides, polyphenols and other secondary metabolites which can co-precipitate with RNA causing problem in further transcriptomic study. In this experiment, three basic chaotic agents, CTAB, SDS and guanidine are used in three basic protocols for RNA isolation. The effectiveness of each method was determined by spectrophotometer, denaturing agarose gels analysis and northern blot hybridization. CTAB combining with additional sodium acetate precipitation step showed highest yield and best quality of isolated RNA which was free from contaminations of polysaccharides, polyphenols and other secondary metabolites. Furthermore, the total RNA from 4-month old durian flesh of clone D24 was successfully used to construct a cDNA library. In conclusion, CTAB method is effective to isolate total RNA on various types of durian tissues for further gene expression analysis

Studies on the genetic variation of the green unicellular alga Haematococcus pluvialis (Chlorophyceae) obtained from different geographical locations using ISSR and RAPD molecular marker.

Haematococcus pluvialis (Flotow) is a unicellular green alga, which is considered to be the best astaxanthin-producing organism. Molecular markers are suitable tools for the purpose of finding out genetic variations in organisms; however there have been no studies conducted on ISSR or RAPD molecular markers for this organism. The DNA of 10 different strains of H. pluvialis (four strains from Iran, two strains from Finland, one strain from Switzerland and three strains from the USA) was extracted. A genetic similarity study was carried out using 14 ISSR and 12 RAPD primers. Moreover, the molecular weights of the bands produced ranged from 0.14 to 3.4 Kb. The PCA and dendrogram clustered the H. pluvialis strains into various groups according to their geographical origin. The lowest genetic similarity was between the Iran2 and USA2 strains (0.08) and the highest genetic similarity was between Finland1 and Finland2 (0.64). The maximum numbers of bands produced by the ISSR and RAPD primers were 35 and 6 bands, respectively. The results showed that ISSR and RAPD markers are useful for genetic diversity studies of Haematococcus as they showed geographical discrimination

Performance of air-cathode microbial fuel cell with wood charcoal as electrodes

Local wood charcoal was used as the main component of the electrodes of an air-cathode microbial fuel cell (air-cathode MFC) in current study. The air cathode was build with fnely milled charcoal powder and cement plaster as binder; while anode was made up of a packed bed of charcoal granules. Mangrove estuary brackish water was inoculated in the anodic chamber as the fuel and a source of exoelectrogens. The constructed fuel cell was monitored by measuring the potential over time. The MFC generated a stable power density at 33mW/m2(0.5V) under a load of 200Ω after 72 hours of operation. An open circuit voltage (OCV) of 0.7mV was obtained after 15 hours operating under open circuit. The result of power generation by the constructed fuel cell indicating that wood charcoal could be used as electrode in an MFC and that brackish water contained potential exoelectrogens. However, further investigation and modifcation is required to increase the performance of the fuel cell

Effect of Exserohilum monoceras (Drechslera) leonard & suggs on the competitiveness of Echinocloa cruss-galli (L.) P. Beauv

The use of bioherbicide to reduce interference by barnyard grass in rice cropping system has been suggested but has not been reported. Against this conceptual background, a mini-plot study was conducted to simulate the efficacy of Exserohilum monoceras to reduce competitiveness of barnyard grass in rice using replacement series experiment. The effect of E. monoceras on rice was negligible, as it did not cause any infection. Severe infection was observed on barnyard grass inoculated with this fungus at all plant densities as indicated by high AUDPC values (ranges from 610.35-468.28 unit2) and fast disease progress rates (r L= 0.48 logit/day). Rice biomass in mixture with diseased weed was higher than in the presence of healthy weed, and is not significantly different from rice biomass in the non-weedy control. In the inoculated experiment, at lower weed density, competition between barnyard grass and rice was not apparent despite the fact that the weed growth was reduced. As the weed density increased, rice continued to grow, but barnyard grass was suppressed; the growth difference was bigger and more measureable. In the non-inoculated control, the interaction between barnyard grass and rice was observed at 2:2 ratio, but at 3:1 in the inoculated experiment, indicating that rice was more competitive over barnyard grass. It took three barnyard grass to equal the shoot dry weight of one rice plant. This study provides strong evidence of the ability of E. monoceras in reducing the competitive ability of barnyard grass and thus provides new opportunities for the future of biological weed control in Malaysia

Detection of aerolysin and hemolysin genes in Aeromonas spp. isolated from environmental and shellfish sources by polymerase chain reaction

Polymerase chain reaction (PCR) technique was used to assay for the detection of specific genes in the genomes of the Aeromonas spp. isolated from environmental and shellfish sources, particularly aero and hlyA genes, responsible for aerolysin and hemolysin toxins production in this genus. The results showed that: (i) the 1500 bp amplicon of the hlyA gene was detected in 20/38 of the Aeromonas hydrophila, 13/38 of the A. caviae and 6/9 of the A. veronii biovar sobria isolates; (ii) the 690 bp amplicon of the aero gene was detected in 20/38 of A. hydrophila, 17/38 of A. caviae and 6/9 of A. veronii biovar sobria isolates; (iii) the nucleotide blast results of aerolysin gene sequences of the representative strains of A. hydrophila, A. caviae and A. veronii biovar sobria revealed a high homology of 94%, 95% and 95% with published sequences, respectively and; (iv) the protein blast showed 97%, 94% and 96% homology when compared to the published sequences, respectively. The finding of A. hydrophila virulence genes in other members of the genus Aeromonas, may give a new perspective to the significance of these results. The method described here may be a useful detection tool to assist in further investigation of aero and hlyA genes in the genus Aeromonas, especially for food microbiologist

Relationship between Induction of Novel Somaclonal Variants and Types of Organogenesis in Muskmelon (Cucumis melo L.)

A comparative study on induction of somaclonal variation in muskmelon (Cucumis melo L.) cv. Birdie regenerants obtained through direct and indirect organogenesis was carried out. Two types of non-meristematic explants (e.g. cotyledon and petiole) were used for this study. A significantly lower (p<0.05) frequency of variation was observed in muskmelon somaclones regenerated via direct organogenesis (MS medium with BAP) compared to indirect (MS medium with BAP and 2,4-D). Morphological study revealed that the somaclones regenerated from proximal cotyledon, petiole and distal cotyledon explants through direct organogenesis did not show any variation in elongation medium at the concentrations of BAP 0.1, 0.3 and 0.5 mg-l, respectively. In contrast, higher number of morphologically somaclonal variants was obtained from these explants at the same concentration of BAP obtained through indirect organogenesis. Other concentrations of BAP, on the other hand, added to the elongation medium showed higher percentage of somaclones with different types of novel variations, e.g. early flowering including higher number of flowers, slow growth of shoots with variant shape of leaves having long and thick petioles, and stubby shoot apices including flattened stem. These variations could be the prime genetic materials to develop new varieties of muskmelon, e.g. high yielding variety, early, late variety, dwarf variety, and variety with desirable body configurations. The results suggest that specific concentrations of BAP or combinations of BAP and 2,4-D have a significant (p<0.05) influence on the induction of novel somaclonal variations in muskmelon regenerants. Future cytogenetic and molecular studies reveal that the novel genetic variations at the chromosome level in somaclonal variants can exist