Effects of electromagnetic field of 33 and 275 kV influences on physiological, biochemical and antioxidant system changes of leaf mustard (Brassica chinensis)

The effects of electromagnetic fields (EMF) from 33 and 275 kV high voltage transmission line on biochemical and antioxidant system changes in mustard leaf (Brassica chinensis) were investigated under field condition. Mustard leaves were exposed to EMF from power lines at distances of 0, 3, 6, 9, 10, 12, 15, 18, 20, 21, 30, 40, 50 and 60 m away from the 33 kV power line and at 0, 10, 20, 30, 40, 50, 60 and 70 m away from the 275 kV transmission lines. The effects of EMF from 33 kV power lines on leaf mustard planted at different distances from the line showed that leaf mustard planted within 20 m from the line had significantly (p< 0.05) higher protein, soluble protein, soluble nitrogen and chlorophyll contents due to the higher EMF strength which decreased with increasing distance from the line. Higher EMF strength nearer to the 275 kV power line resulted in higher peroxidase enzymatic activity, and chlorophyll content. Protein electrophoretic profile obtained from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis revealed no drastic alterations in the leaf mustard protein profiles. This suggests that electromagnetic field could be used as a tool to promote mustard growth via photosynthesis once the right EMF strength and duration of exposure has been established through future studies.Keywords: Mustard, electromagnetic field, biochemical marker

Factors affecting the accumulation of 9-methoxycanthin-6-one in callus cultures of Eurycoma longifolia.

A study was conducted to improve 9-methoxycanthin-6-one productivity (potential anti-tumour compound) from callus cultures of Eurycoma longifolia (Tongkat Ali). Several factors affecting 9-methoxycanthin-6-one production in callus cultures such as different medium compositions and physical factors were investigated and analyzed. Results show that a higher production of 9-methoxycanthin-6-one (3.84 mg'g-1 DW (Dry Weight)) is obtained from callus cultured in ¼ MS basal media. At fructose of 2% (w/v), the production of 9-methoxycanthin-6-one (4.59 mg'g-1 DW) is promoted to gain the highest yield, compared to other carbon sources tested. The addition of 2.0-mg'L-1 dicamba also increases 9-methoxycanthin-6-one production (12.3 mg'g-1 DW). Higher production of 9-methoxycanthin-6-one was obtained at pH 5.5 (1.53 mg'g-1 DW). Production of 9-methoxycanthin-6-one (2.34 mg'g-1 DW) in callus cultures is also increased when the medium is added with 1×10-1 μM phenylalanine. This study suggests that the successful production of 9-methoxycanthin-6-one in vitro cultures has a potential in large-scale production using bioreactor technology

Regeneration Rates of Dendrobium Bobby Messina Plbs with Ascorbic Acid Using PVS2 Vitrification

Cryopreservation techniques using PVS2 vitrification was applied on PLBs of Dendrobium Bobby Messina, with survival monitored through observations of growth rate and the 2,3,5-triphenyltetrazolium chloride (TTC) analyses. The parameters optimized were PLBs size, preculture concentration, preculture duration, PVS2 incubation temperature and duration. The optimized parameters obtained were 3-4mm of PLBs precultured in 0.2M sucrose for 1 day, treated with a mixture of 2M glycerol and 0.4M sucrose supplemented with half strength liquid MS media at 25°C for 20 minutes and subsequently dehydrated with plant vitrification solution 2 (PVS2) at 0°C for 20 minutes prior storage in liquid nitrogen. Following rapid warming in a water bath at 40°C for 90 seconds, PLBs were washed with a half strength liquid MS media supplemented with 1.2M sucrose. Subsequently, PLBs were cultured on half strength semi-solid MS media supplemented with 2% (w/v) sucrose in the absence of growth regulator. The optimized vitrification method was successful in preserving this orchid as it produced growth recovery in cryopreserved PLBs up to 40%. Ascorbic acid was added in the media to evaluate the regeneration process of cryopreserved PLBs. However, growth recovery rate was only 10% at 0.6mM ascorbic acid. RAPD analysis using 6 primers indicated that cryopreserved and non-cryopreserved PLBs from vitrification method were genetically faithful to the mother plant. However, 3 primers showed polymorphism and 1 primers indicated partial polymorphism between the cryopreserved and non-cryopreserved PLBs in comparative to the mother plant

Detection of somaclonal variation by random amplified polymorphic DNA analysis during micropropagation of Phalaenopsis bellina (Rchb.f.) Christenson

Phalaenopsis bellina (Rchb.f.) Christenson orchid species are known for their beautiful flower shape, graceful inflorescence and fragrance. Protocorm-like bodies (PLBs) of P. bellina were induced from leaf segments. The PLBs were then subjected to proliferation using ½ strength Murashige and Skoog (MS) media with two subcultures at three months intervals. Twelve decamer random amplified polymorphic DNA (RAPD) primers were used to study somaclonal variation among the mother plant, the initially induced PLBs and proliferated PLBs after 3 and 6 months in culture. Eight out of twelve primers produced 172 bands with 18 polymorphic bands in all the treatments. The amplified products varied between 125 to 8000 bp. Among the primers used, P 16 produced the highest number of bands (29), while primer OPU 10 produced the lowest number (15). The range of similarity coefficient was from 0.83 to 1.0 among the different sub-cultures and mother plant (MP). It was found that minimal or no changes occurred between the MP and the PLBs produced after 3 months of induction. The induced PLBs were then subcultured for six months for proliferation and this resulted in about 17% dissimilarity with MP. It is reported that micropropagation of P. bellina can be carried out successfully using ½ strength MS media for 6 months but further proliferation may result in somaclonal variation which might change the prolific characteristic of this orchids.Key word: Moth orchid, somaclonal variation, random amplified polymorphic DNA, protocorm-like bodies

The Effect of Strain Virulence on Agrobacterium Rhizogenes Transformation Efficiency in Eurycoma Longifolia

Eurycoma longifolia, known locally as ‘Tongkat Ali' in Malaysia, is popularly sought out as herbal remedy in many parts of South East Asia. Consequently, this has resulted in the indiscriminate collection of the taproot from the wild, leading to fears of extinction among conservationists. Agrobacterium rhizogenes is a naturally occurring soil bacterium that infects injured plants and causes a massive proliferation of roots, known as hairy roots. The objective of this study is to determine the transformation efficiency of 2 different strains of Agrobacterium rhizogenes on somatic embryos of Eurycoma longifolia using transient GUS expression as an indicator of successful transformation. Somatic embryos cultured in vitro for 4 to 5 weeks were used as explants and were pre-cultured for 2 days in full strength MS medium containing 0.5 mg/L IBA added with 1% PVP and 2mg/L DTT. The explants were transformed using Agrobacterium rhizogenes strains AR12 and AR14. GUS assay was carried out 1 week after transformation and observed. Observations indicate that AR12 is more effective in transforming somatic embryos of Eurycoma longifolia compared to AR14. Therefore, Agrobacterium rhizogenes strain AR12 is a potential candidate for the successful transformation of Eurycoma longifolia somatic embryos, leading to the induction of sustainable hairy root culture

In vitro induction and proliferation of protocorm-like bodies (PLBs) from leaf segments of Phalaenopsis bellina (Rchb.f.) Christenson

An in vitro culture procedure was established to induce protocorm-like bodies (PLBs) from leaf segments of the Phalaenopsis bellina (Rchb.f.) Christenson directly from epidermal cells without intervening callus on ½ strength modified Murashige and Skoog (MS) (in Physiol Plant 15:473–497, 1962) medium supplemented with 1-Naphthaleneacetic acid (NAA; 0, 0.1, 1 mg/l) and Thidiazuron (TDZ; 0, 0.1, 1, 3 mg/l). The best response was established at 3 mg/l TDZ which induced 78% of leaf segments to form a mean number of 14 PLBs per explant after 16 weeks of culture. No PLBs were found when leaf segments were cultured on ½ strength modified MS media supplemented with 0.1 and 1 mg/l NAA. The best induction percentage for auxin: cytokinin combination was at the combination of NAA and TDZ at 1.0 and 3.0 mg/l which gave 72% induction with 9 PLBs per explant. Semi-solid ½ strength MS and liquid Vacin and Went (VW) (in Bot Gaz 110:605–613, 1949) medium were used in order to find the highest survival and number of PLBs proliferation after 3 months in culture. Half strength MS showed an average of 9 PLBs in comparison with VW with an average of 5.3 PLBs per explants. Histological observations revealed that the regenerated PLBs were generally formed from the epidermal layers of the posterior regions of the leaf segments. Scanning electron micrograph of PLBs showed the origin of newly formed PLB from the peripheral region of leaf segments

A protocol for Agrobacterium-mediated transformation of banana with a rice chitinase gene.

A rice chitinase gene (RCC2) multiplied in Agrobacterium strain (EHA 101), was simultaneously introduced into single buds of in vitro grown banana cultivar, Rastali (AAB). Plasmid pBI333-EN4-RCC2 contained a hygromycin phosphotransferase gene (hptII) as the selectable marker and gusA gene as a reporter marker to identify the transformants.. Single buds derived from multiple bud clumps (Mbcs), were the target explants for transformation. Transformation frequency based on hygromycin selection (25 mg L-1) was higher, although no positive transformant was confirmed based on PCR and Southern blot analyses.. Stable gusA gene expression was detectable in transformed single buds, multiple bud clumps, shoots, leaves and roots derived from hygromycin selection at 50 mg L-1 ). An assay was performed to identify the minimum concentration of two antibiotics most effective against Agrobacterium EHA 101. Protein assay showed an increase in chitinase enzyme activity of transformed plantlets. The Agrobacterium-mediated transformation protocol reported here is suitable for future selection of banana meristem tissues resistant to fungal disease

Influence of plant growth regulators (PGRs) and various additives on in vitro plant propagation of Bambusa arundinacea (Retz.) Wild: A recalcitrant bamboo species

An efficient micropropagation protocol for high frequency plant regeneration was developed using nodal explants derived in vitro seedlings of Bambusa arundinacea which is an important multipurpose and edible bamboo species and recalcitrant to tissue culture. The nodal explants excised from 20-day-old seedlings were cultured on Murashige and Skoog (MS) medium fortified with various concentrations of 6-benzyl amino purine (BAP) and kinetin (KIN) (0.5–5.0 mg/l) alone and/or in combination with 0.5 mg/l of different auxins [indole-3-butyric acid (IBA) α-naphthalene acetic acid (NAA) and indole-3-acetic acid (IAA)] for shoot bud induction. The combination of BAP (3.0 mg/l) and IBA (0.5 mg/l) was found to be the best for the highest percent of shoot bud initiation (87.2%), with 24.2 shoots/explant. The highest frequency (95.2%) of shoot bud multiplication with maximum number of shoots (90.5 shoots/culture) was noticed on medium containing 4% coconut water with 4% sucrose. The regenerated shoot buds were cultured on MS medium supplemented with various concentrations of auxins alone and/or in combination with AgNO3 (0.5–4.0 mg/l) for in vitro rooting. Maximum percent of rooting (85%) was noticed on MS medium augmented with 3.0 mg/l IBA and 2.0 mg/l AgNO3 after 14 days of culture. Well rooted plantlets obtained were established in the field with 92% survival rate. The present plant regeneration protocol could be used for large scale propagation and ex-situ conservation of this important bamboo species in the near future

Variations in Hormones and Antioxidant Status in Relation to Flowering in Early, Mid, and Late Varieties of Date Palm (Phoenix dactylifera) of United Arab Emirates

The present study was carried out to assess the status of various hormones responsible for the flower induction of Nagal, Lulu, and Khalas date palm varieties in UAE. The nonenzymatic antioxidant compounds and the antioxidant enzymatic activities at preflowering, flowering, and postflowering stages of the date palm varieties were quantified. The ABA and zeatin concentrations were found to be significantly higher during the preflowering stage but gradually decreased during the flowering period and then increased after the flowering stage. Gibberellic acid (GA) concentrations were significantly higher in the early flowering varieties and higher levels of ABA may contribute to the delayed flowering in mid and late varieties. The results on hormone profiling displayed a significant variation between seasons (preflowering, flowering, and postflowering) and also between the three date palms (early, mid, and late flowering varieties). Ascorbic acid (AA) concentration was low at the preflowering stage in the early flowering Nagal (0.694 mg/g dw), which is similar with the late flowering Lulu variety (0.862 mg/g dw). However, Khalas variety showed significantly higher amount of AA content (7.494 mg/g dw) at the preflowering stage when compared to other varieties. In flowering stage, Nagal (0.814 mg/g dw) and Lulu (0.963 mg/g dw) were similar with respect to the production of AA, while the mid flowering variety showed significantly higher amount of AA (9.358 mg/g dw). The Khalas variety produced the highest tocopherol at 4.78 mg/g dw compared to Nagal and Lulu, at 1.997 and 1.908 mg/g dw, respectively, during the preflowering stage. In Nagal variety, the content of reduced glutathione (GSH) at the preflowering stage was 0.507 mg/g dw, which was not significantly different from the flowering and postflowering stages at 0.4 and 0.45 mg/g dw, respectively. The GSH was significantly higher in Khalas compared to Nagal and Lulu varieties, at 1.321 mg/g dw in the preflowering phase followed by 3.347 mg/g dw and 2.349 mg/g dw at the flowering and postflowering phases, respectively. Catalase activity increased with different stages of growth. The lowest catalase activity was observed at the preflowering stage in Khalas (0.116), with similar observations noted during flowering (0.110) and postflowering stage. This study provides an insight into the possible roles of endogenous hormones and antioxidants and in the activities of antioxidant enzymes in the regulation of flower development in date palm varieties