Sreeramanan Subramaniam

Preliminary investigation on the antibacterial activity of mango (Mangifera indica L: Anacardiaceae) seed kernel.

To evaluate the antibacterial activity of the methanolic extract of mango (Mangfera indica L.) seed kernel. Methods: Chokanan mango seed kernel and seed kernels from assorted mango varieties were collected, cleaned, dried and powered. Crude methanolic extracts of mango seed kernel were analyzed for the phytochemical constituents. The free radical scavenging activity was determined by 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assay. Antibacterial activity was evaluated by disc diffusion assay with three medically important bacterial pathogens such as methicillin resistant Staphylococcus aureus (S. aureus) (MRSA), Escherichia coli (F. coli) and Vibrio vulnificus (V. vulnificus). Results: Qualitative phytochemical analysis indicated the presence of important phytochemical compounds such as glycosides, saponins, flavanoids, tannins and alkaloids. There was no significant difference in the phytochemical content between the single and assorted mango seed kernels. However, the free radical scavenging study indicated that the assorted mango kernels showed slightly higher activity than the single species (P＜0.05). The crude methanolic extract of mango seed kernel at a concentration of 100mg/mL is found to have potential antimicrobial activity against MRSA and E. coli compared to V. vulnificus. Study on the antibacterial activity also indicated that there was no significant difference in the antibacterial activity of the single and assorted mango seed kernel extracts. Conclusions: The present study conclusively demonstrates the free radical scavenging activity and antibacterial activities of mango seed kernel. In addition, the results also indicated that there is no significant difference in the phytochemical content and biological activity of mango kernels from single and assorted mango varieties.

Toxicity of buprofezin on the survival of embryo and larvae of African catfish, Clarias gariepinus (Bloch).

Buprofezin is an insect growth regulator and widely used insecticide in Malaysia. The present study evaluated the toxic effects of buprofezin on the embryo and larvae of African catfish (Clarias gariepinus) as a model organism. The embryos and larvae were exposed to 7 different concentrations (0, 0.05, 0.5, 5, 25, 50 and 100 mg/L) of buprofezin. Each concentration was assessed in five replicates. Eggs were artificially fertilized and 200 eggs and larvae were subjected to a static bath treatment for all the concentrations. The mortality of embryos was significantly increased with increasing buprofezin concentrations from 5 to 100 mg/L (p< 0.05). However, the mortality was not significantly different (p<0.05) among the following concentrations: 0 (control), 0.05, 0.5 and 5 mg/L. Data obtained from the buprofezin acute toxicity tests were evaluated using probit analysis. The 24 h LC50 value (with 95% confidence limits) of buprofezin for embryos was estimated to be 6.725 (3.167-15.017) mg/L. The hatching of fish embryos was recorded as 68.8, 68.9, 66.9, 66.4, 26.9, 25.1 and 0.12% in response to 7 different concentrations of buprofezin, respectively. The mortality rate of larvae significantly (p<0.05) increased with increasing buprofezin concentrations exposed to 24-48 h. The 24 and 48 h LC50 values (with 95% confidence limits) of buprofezin for the larvae was estimated to be 5.702 (3.198-8.898) and 4.642 (3.264-6.287) mg/L respectively. There were no significant differences (p>0.05) in the LC50 values obtained at 24 and 48 h exposure times. Malformations were observed when the embryos and larvae exposed to more than 5 mg/L. The results emerged from the study suggest that even the low concentration (5 mg/L) of buprofezin in the aquatic environment may have adverse effect on the early embryonic and larval development of African catfish.

Preliminary study on cryopreservation of Dendrobium Bobby Messina protocorm- like bodies by vitrification

Although the delayed-type hypersensitivity (DTH) skin reaction to tuberculin is used worldwide for tuberculosis (TB) detection, it has poor diagnostic specificity due to the presence of common antigens in tuberculin shared by many mycobacterial species. The problem is noticed, especially in countries where the Bacillus Calmette-Gue´rin (BCG) vaccination is widely practiced. Thus, a new skin test antigen specific for the diagnosis of Mycobacterium tuberculosis (MTB) infection is urgently needed. CFP-10, a mycobacterial secretary protein that is absent in Mycobacterium bovis BCG and most other mycobacterial species including Mycobacterium avium, Mycobacterium intracellulare, has been shown to elicit cellular immune responses in MTB infected individuals and can be a good candidate for MTB specific diagnosis. We prepared recombinant MTB CFP-10, rCFP-10, and its utility as specific antigen for TB diagnosis was evaluated by skin testing in guinea pigs sensitized with M . tuberculosis, M. bovis, and M. bovis BCG. Our results show that the purified MTB rCFP-10 antigen elicits a positive skin response only in the guinea pigs sensitized with M. tuberculosis and M. bovis , and not in the animals sensitized with M. bovis BCG vaccine. The data presented in this study supports further testing of the use rCFP-10 as the specific antigen in the skin test for the diagnosis of MTB infection in humans. Key words : Recombinant CFP-10 protein, skin test, delayed-type hypersensitivity, tuberculosis infection, Mycobacterium tuberculosis, Mycobacterium bovis, Bacillus Calmette-Gue´rin.

The effect of cytokinins on in vitro shoot length and multiplication of Hymenocallis littoralis.

This study was performed to determine the effects of different cytokinins at various concentrations on the in vitro shoot formation of Hymenocallis littoralis. The bulb scales of H. littoralis were used as explants to establish the cultures. The explants were grown on semi-solid Murashige and Skoog (MS) medium supplemented with 2iP (2-isopentenyladenine), TDZ (thidiazuron) and Zeatin respectively at six different concentrations (2.25, 4.50, 9.00, 13.50, 18.00, and 22.50 μM). Two subcultures were performed at 30 days interval after the initial in vitro culture establishments. It was found that 2iP and concentration 13.50 μM respectively was the best for shoot elongation which was measured in length (cm). However, the three cytokinins and the six tested concentrations were recorded to have no significant difference in terms of shoot multiplication. Highest total chlorophyll content was observed in shoots grown on semi-solid MS medium supplemented with 2.25 μM of Zeatin.

Isolation and identification of salmonella from curry samples and its sensitivity to commercial antibiotics and aqueous extracts of Camelia sinensis (L.) and Trachyspermum ammi (L.).

OBJECTIVE To isolate Salmonella from curry samples and to evaluate the drug sensitivity of the food-borne Salmonella and its susceptibility to specific plant extracts. METHODS Salmonella was isolated from the curry samples by standard microbiological methods and was confirmed by biochemical tests. The antibiotic susceptibility test was conducted by disc diffusion method using commercially available antibiotics such as ampicillin, tetracycline, chloramphenicol, kanamycin, and penicillin. In addition, the susceptibility of the food-borne Salmonella was also evaluated against the aqueous extracts of Camelia sinensis (L.) Theaceae (tea leaves) and the Trachyspermum ammi (L.) Apiaceae ( ajwain or omum seeds). RESULTS Out of fifty curry samples, only seven samples were identified to have Salmonella contamination. The Salmonella isolates showed a significant drug resistance pattern except for kanamycin. The plant extracts showed a considerable antibacterial activity against the isolates, indicating the presence of antimicrobial principle which can be exploited after complete pharmacological investigations. CONCLUSIONS The present study demonstrates the occurrence of Salmonella in the curry samples, and shows significant drug resistance against most of the commercially available antibiotics, except kanamycin. Antimicrobial effect of the plant extracts against the food-bone Salmonella suggests that dietary including medicinal herbs would be one strategy to manage food borne pathogens.

Agrobacterium-Mediated Transformation of the Recalcitrant Vanda Kasem's Delight Orchid with Higher Efficiency

The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasems Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKDs PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A 600nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 𝜇M acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.

Storage of encapsulated oil palm polyembryoids: influence of temperature and duration

Encapsulation technology is an excellent substitute for traditional seed and micropropagation systems of clonally propagated plants because of the space requirements and high maintenance costs of germplasm repositories. The use of synthetic seeds appears to offer a channel for new plant lines, produced through biotechnological advances, to be delivered directly to the greenhouse or field. The high volume propagation potential of somatic embryos and the low delivery cost of synthetic seeds have opened new opportunities for clonal propagation in several commercially important plant species (Gray et al. 1995; Onay et al. 1996; Ara et al. 1999; Antonietta et al. 2007). Moreover, encapsulation technology using alginate coating is considered to be an efficient choice for both propagation and shortto mid-term storage. Interestingly, alginate coating on encapsulated explants acts as a shield for plant tissues, providing protection from physical and environmental injury during storage by rendering mechanical pressure to hold the explants inside the gel matrix (Ara et al. 2000). Since artificial seeds that are produced aseptically are free of pathogens, this facilitates transporting pathogen-free propagules across international borders because it overcomes the risk of spreading disease, which is a factor in bulk transportation of plants. When using tissue culture for plant propagation, loss of plant materials during the tissue culture process, due to the inability to arrest the growth and development of the explants, is arguably the most critical hurdle to overcome. There is no such obstacle involved in the production of synthetic seeds. Consequently, the production and storage of synthetic seeds, which not only arrests the precise developmental stage but also enables prompt regeneration after transportation, appears to be a more feasible method for exchange and preservation purposes. Therefore, storage conditions and storage periods that optimize viability need to be identified. Storage at low temperature has been used in the seed industry, wherein seeds are first desiccated to low water content followed by storage at low temperature of −18°C (Ellis et al. 1991). However, this is not possible with vegetative propagules as they are sensitive to desiccation, their cells are often highly vacuolated, and they face irreversible damage owing to removal of water. Despite the drawback, storage at 4°C has been used on clonal propagules such as orchid protocorm-like bodies and shoot tips with no loss in viability even after storage for 90 d (Sarmah et al. 2010; Gantait et al. 2012; Gantait and Sinniah 2013). Growth reduction is generally achieved by modifying the environmental conditions and/ or the culture medium. The most widely used technique is temperature reduction, which can be combined with a decrease in light intensity or culture in the dark. Oil palm (Elaeis guineensis Jacq.) is a monocotyledonous plant belonging to the family Arecaceae. It is an economically important source of vegetable oil that is traded in the international market (Corley and Tinker 2008). Oil palm is normally propagated from seeds, and currently, the ‘Dura’ (D)בPisifera’ (P) hybrids, produced by authorized agencies, are being used as the sources of seeds. However, clonal S. R. Palanyandy : S. Gantait : P. Suranthran :U. R. Sinniah (*) Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia e-mail: umarani@upm.edu.my

Larvicidal Efficacy of Different Plant Parts of Railway Creeper, Ipomoea cairica Extract Against Dengue Vector Mosquitoes, Aedes albopictus (Diptera: Culicidae) and Aedes aegypti (Diptera: Culicidae)

ABSTRACT. Natural insecticides from plant origin against mosquito vectors have been the main concern for research due to their high level of eco-safety. Control of mosquitoes in their larval stages are an ideal method since Aedes larvae are aquatic, thus it is easier to deal with them in this habitat. The present study was specifically conducted to explore the larvicidal efficacy of different plant parts of Ipomoea cairica (L.) or railway creeper crude extract obtained using two different solvents; methanol and acetone against late third-stage larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.) (Diptera: Culicidae). Plant materials of I. cairica leaf, flower, and stem were segregated, airdried, powdered, and extracted using Soxhlet apparatus. Larvicidal bioassays were performed by using World Health Organization standard larval susceptibility test method for each species which were conducted separately for different concentration ranging from 10 to 450 ppm. Both acetone and methanol extracts showed 100% mortality at highest concentration tested (450 ppm) after 24 h of exposure. Results from factorial ANOVA indicated that there were significant differences in larvicidal effects between mosquito species, solvent used and plant parts (F = 5.71, df = 2, P < 0.05). The acetone extract of I. cairica leaf showed the most effective larvicidal action in Ae. aegypti with LC50 of 101.94 ppm followed by Ae. albopictus with LC50 of 105.59 ppm compared with other fractions of I. cairica extract obtained from flower, stem, and when methanol are used as solvent. The larvae of Ae. aegypti appeared to be more susceptible to I. cairica extract with lower LC50 value compared with Ae. albopictus (F = 8.83, df= l, P <0.05). Therefore, this study suggests that the acetone extract of I. cairica leaf can be considered as plant-derived insecticide for the control of Aedes mosquitoes. This study quantified the larvicidal property of I. cairica extract, providing information on lethal concentration that may have potential for a more eco-friendly Aedes mosquito control program.

Effect of Plasmolysis on Protocorm-Like Bodies of Dendrobium Bobby Messina Orchid Following Cryopreservation with Encapsulation–Dehydration Method

Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates the cellular process of cryopreserved protocorm-like bodies (PLBs) was different comparative to non-cryopreserved PLBs. The cellular process was not only modified by the freezing and thawing effect but also due to the dehydration process itself during the cryopreservation procedure. Histological observation in Dendrobium Bobby Messina in encapsulation–dehydration method indicated that the degree of plasmolysis causes more cellular changes to the cryopreserved PLBs comparative to non-cryopreserved and stock culture PLBs. These results revealed higher amount of homogenous cell population and denser cytoplasm in cryopreserved PLBs. Histological analysis also revealed more voluminous nucleus in cryopreserved PLBs comparative to non-cryopreserved PLBs and PLBs stock culture. In contrast, scanning electron microscope analysis showed severe damages in cryopreserved PLBs and non-cryopreserved PLBs comparative to the PLBs stock culture which in return could be the possible reason of no regrowth in encapsulation–dehydration method. Damages incurred were on top part, side part, and at the stomata of the PLBs. Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates that the degree of plasmolysis causes changes in the cellular process of PLBs from cryopreserved PLBs was different comparative to non-cryopreserved PLBs.

Catalase and Superoxide Dismutase Activities and the Total Protein Content of Protocorm-Like Bodies of Dendrobium Sonia-28 Subjected to Vitrification

Dendrobium sonia-28 is an important ornamental orchid in the Malaysian flower industry. However, the genus faces both low germination rates and the risk of producing heterozygous progenies. Cryopreservation is currently the favoured long-term storage method for orchids with propagation problems. Vitrification, a frequently used cryopreservation technique, involves the application of pretreatments and cryoprotectants to protect and recover explants during and after storage in liquid nitrogen. However, cryopreservation may cause osmotic injuries and toxicity to cryopreserved explants from the use of highly concentrated additives, and cellular injuries from thawing, devitrification and ice formation. Reactive oxygen species (ROS), occurring during dehydration and cryopreservation, may also cause membrane damage. Plants possess efficient antioxidant systems such as the superoxide dismutase (SOD) and catalase (CAT) enzymes to scavenge ROS during low temperature stress. In this study, protocorm-like bodies (PLBs) of Dendrobium sonia-28 were assayed for the total protein content, and both SOD and CAT activities, at each stage of a vitrification exercise to observe for deleterious stages in the protocol. The results indicated that cryopreserved PLBs of Dendrobium sonia-28 underwent excessive post-thawing oxidative stress due to decreased levels of the CAT enzyme at the post-thawing recovery stage, which contributed to the poor survival rates of the cryopreserved PLBs.