Bioefficacy of crude extract of Cyperus aromaticus (Family: Cyperaceae) cultured cells, against Aedes aegypti and Aedes albopictus mosquitoes.

OBJECTIVE: To evaluate the growth inhibition activity of the crude extract of Cyperus aromaticus (C. aromaticus) cultured cells against the 3rd instar larvae of Aedes aegypti (Linn.) and Aedes albopictus Skuse (Ae. albopictus) under laboratory conditions, and determine the sublethal effects (EI50) of the crude extract of C. aromaticus cultured cells on some biological and morphological parameters of both Aedes mosquito species during two generations as well. METHODS: The cell suspension cultures of C. aromaticus were activated from five callus lines (P4, Pa, Z1, Z6 and Ml) derived from the root explants of in vitro plantlets. The cultured cells were extracted in chloroform and used as plant material for the present study. For detection of juvenile hormone III, the crude extracts were analyzed by HPLC. Then the crude extracts of the three C. aromaticus cultured cell lines which contained varied amounts of juvenile hormone III [high level (P4 cell line), medium level (Z1 cell line) and low level (Ml cell line)] were tested against Aedes mosquito species. Laboratory evaluation was performed against late third instar larvae of the Vector Control Research Unit strains of Ae. aegypti and Ae. albopictus using the standard WHO method. The effects of EI50 of the C. aromaticus cultured P4 cells on fecundity, fertility, growth period, sex ratio, adult size and longevity of Aedes mosquitoes were assessed. RESULTS: Bioassay tests presented the remarkable growth inhibition activity of the crude extracts of C. aromaticus cultured cells against the two Aedes mosquitoes. Between the two mosquito species, Ae. albopictus was more susceptible to the crude extracts with lower EI50 values. EI50 of the crude extract of C. aromaticus cultured cells (P4) increased the sterility indices in the parental generation females in both Aedes mosquito species. A significant delay in the pupal formation and adult emergence were observed in the parental generation of the both mosquito species. The sex ratio of the adult population either parental or F1 generation of the Aedes mosquito species was not significantly affected by the EI50 dosage of the crude extract of C. aromaticus cultured P4 cells. A significant decrease in the wing length of the treated adult (female and male) of Aedes aegypti as well as the treated female of Ae. albopictus were observed. Longevity of the adult female of the parental generation of both Aedes mosquitoes as well as females of F1 generation of Ae. albopictus were significantly decreased. CONCLUSIONS: The present study revealed the potential of the crude extract of C. aromaticus cultured cells in controlling vector mosquito populations in the effort to reduce the transmission of vector borne diseases.

Effects of ascorbic acid on PVS2 cryopreservation of Dendrobium Bobby Messina's PLBs supported with SEM analysis.

Regrowth of the cryopreserved protocorm-like bodies (PLBs) of Dendrobium Bobby Messina was assessed based on the plant vitrification solution 2 (PVS2) optimisation conditions. The optimized protocol obtained based on TTC spectrophotometrical analysis and growth recovery were 3-4 mm of PLBs size precultured in 0.2 M sucrose for 1 day, treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min and subsequently dehydrated with PVS2 at 0 °C for 20 min prior to storage in liquid nitrogen. Following rapid warming in a water bath at 40 °C for 90 s, PLBs were treated with unloading solution containing half-strength liquid MS media supplemented with 1.2 M sucrose. Subsequently, the PLBs were cultured on half-strength semi-solid MS media supplemented with 2 % (w/v) sucrose without any growth regulators and resulted in 40 % growth recovery. In addition, ascorbic acid treatment was used to evaluate the regeneration process of cryopreserved PLBs. However, growth recovery rates of non-cryopreserved and cryopreserved PLBs were 30 and 10 % when 0.6 mM ascorbic acid was added. Scanning electron microscopy analysis indicates that there are not much damages observed on both cryopreserved and non-cryopreserved PLBs in comparison to PLBs stock culture.

Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae).

Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of various ailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production. The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time on cell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of different concentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50 mg/L) had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30 g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45 g/L sucrose without MeJA showed the highest pigment content (0.69 +/- 0.22 CV/g-FCM). The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5 mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40 CV/g-FCM). This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures.

Effects of Environmental Factors on Growth and Artemisinin Content of Artemisia annua L.

Seeds of two selected clones of Artemisia annua L., TC1 and TC2, were germinated in a greenhouse. Four-week-old seedlings from both clones were grown in the Thù Dúc province of Ho Chi Minh City on 2(nd) January 2009 and Dà Lat on 20(th) January 2009. During this study period in Thù Dúc province, which is situated 4-5 m above sea level, was experiencing a tropical, dry season with temperatures ranging from 26.2°C-32.8°C. Dà Lat, situated at 1500-2000 m above sea level, was having temperate, dry season with lower temperatures, ranging from 10.5°C-18.0°C. The high temperatures and low elevation in Thù Dúc Province led to slow vegetative growth for all of the plants from the two different clones and the artemisinin contents were significantly reduced. The temperate environment of Dà Lat supported robustly growing plants, with plant heights and branch lengths 4-5 times taller and longer that those planted at Thù Dúc Province. The artemisinin contents of A. annua planted at Dà Lat were 3-4 times greater than those cultivated at Thù Dúc Province. Hence, this study indicated that the variations observed in plant growth and artemisinin contents were due to temperature effects because the two selected clones were genetically homogenous. The cold weather of Dà Lat was suitable for planting of A. annua as opposed to the tropical weather of Thù Dúc Province.

Microbial Inoculation Improves Growth of Oil Palm Plants (Elaeis guineensis Jacq.).

Introduction of diazotrophic rhizobacteria to oil palm tissues during the in vitro micropropagation process establishes an early associative interaction between the plant cells and bacteria. In the association, the diazotrophs provide the host plants with phytohormones and fixed nitrogen. This study was conducted to observe growth of bacterised tissue cultured oil palm plants under ex vitro conditions after 280 days of growth. Root dry weight, shoot dry weight, root volume, bacterial colonisation, leaf protein and chlorophyll content of the host plants were observed. The results revealed that the inocula successfully colonised roots of the host plants. Plants inoculated with Acetobacter diazotrophicus (R12) had more root dry weight and volume than plants inoculated with Azospirillum brasilense (Sp7). Leaf protein and chlorophyll content were higher in the bacterised plants compared to Control 2 plants (inoculated with killed Sp7). These results suggest that the diazotrophs successfully improved the growth of the host plant (oil palm) and minimised the amount of N fertiliser necessary for growth.