Genetic Transformation of Orchid Dendrobium Sonia-17 Using the Biolistic Method

The ever-changing tastes and preferences of orchid consumers initiated the need to create new and better varieties. Progress in molecular biology has allowed genetically well defined characteristics to be added to the gene pools, thereby increasing the potential for genetic improvement. However, such effort at creating a custom-made flower has yet to be realised in orchids. The present study aims at developing a genetic transformation system for the introduction of specific foreign genes into orchid. Protocorm-like-bodies (PLBs) of orchid hybrid, Dendrobium Sonia-17, were established to be suitable target tissues for the introduction of foreign genes using the biolistic method. They were easily micropropagated in vitro that provided plenty of materials to work with and were a reliable source of potentially regenerabIe tissues. The effect of blasting on the growth of the PLBs was evaluated by subjecting the PLBs to bombardment with uncoated gold microparticles. One month following bombardment, fresh weights gained by the PLBs were recorded. The results showed that bombarded PLBs had higher weight increments compared to non-bombarded treatments, indicating that subsequent lethal responses by the PLBs on antibiotic selections were mainly due to the selection pressure and not as a result of injuries inflicted during the bombardment

Phenol toxicity and phytoremediation

Widespread applications of phenol in manufacturing industries and oil refineries had resulted in unprecedented leakage of phenol into the environment, which can cause serious health effects such as tissue necrosis and cardiac arrhythmia upon contact or ingestion. Plants exposed to phenol had reduced seed germination index, inhibited growth or even fatality. There are many technologies currently practised to remediate phenol pollution such as physiochemical methods (adsorption to activated carbon and chemical oxidation), biological methods (biodegradation by bacteria or fungus, and soil bioaugmentation), and phytoremediation method (using hairy roots of plants). As physiochemical and microbial phenol degradation are destructive and costly, phytoremediation is widely studied as an alternative phenol remediator which is environmental friendly and cost effective. Microorganisms can detoxify the aromatic xenobiotic through the aerobic or anaerobic pathway. Aerobic degradation of phenol is through either the meta- or ortho-pathway of catechol cleavage while anaerobic degradation occurs through the benzoate pathway. In plants, degradation of phenol is also through catechol cleavage as in microorganisms. However, different enzyme systems were utilised in the different pathways involved

Glasshouse acclimatization of tissue-cultured Melastoma malabathricum plantlets

Tissue-cultured Melastoma malabathricum plants were acclimatized to glasshouse conditions by leaving them for 7 or 14 days in open jars in distilled water or washed and kept in a covered aquarium. Plants in the aquarium had a higher survival rate than those open jars and grew to greater heights

Phenol Toxicity and Remediation

Widespread applications of phenol in manufacturing industries and oil refinerieshaveresulted in unprecedented leakageof phenol into the environment. Its toxicity, remediation methods and degradation pathways were outlined in this review. Upon contact or ingestion, phenolcausesserious health effects such as tissuenecrosis and cardiac arrhythmia. Plantsexposed to phenolhavereducedseed germination index, inhibitedgrowthoreven fatality. There are many technologies currently practised to remediate phenol pollutionsuch asphysicochemical methods(adsorption to activated carbon and chemical oxidation), biological methods (biodegradation by bacteriaor fungus,andsoil bioaugmentation),and phytoremediation method (using hairy rootsof plants). As physicochemicaland microbial phenol degradation are destructive and costly, phytoremediation is widely studied as an alternative phenol remediator which is environmental friendly and cost-effective.Microorganisms can detoxify the aromatic xenobiotic through the aerobic or anaerobic pathway. Aerobic degradation of phenol is througheitherthe meta-or ortho-pathway of catechol cleavage while anaerobic degradation occurs through the benzoate pathway. In plants, degradation of phenol is also through catechol cleavage as in microorganisms. However, different enzyme systems areutilisedin the differentpathways involved

Isolation, cloning and characterisation of new fragrance-related floral transcripts of Vanda Mimi Palmer

A subtracted cDNA library of open flower was constructed using the suppression subtractive hybridization (SSH) technique, to identify fragrance-related transcripts of Vanda Mimi Palmer. In total, 107 transcripts up-regulated during blooming were identified and sequenced. Only 33 clones (4 singletons and 29 contigs) showed similarities to known sequences in the public database. Of these, thirty-two clones were transcripts encoding fragrance-related enzymes including sesquiterpene synthase, (±)-germacrene D synthase, tyrosine decarboxylase and putative acyltransferase. Two fragrance-related transcripts, VMPAAT encoding a putative alcohol acyltransferase and VMPSTS encoding a sesquiterpene synthase, were subjected to full-length cDNA isolation and characterization. The full length cDNA of VMPAAT has a 1343bp open reading frame (ORF) of 448 amino acid residues whereas VMPSTS is predicted to encode a polypeptide of 561 amino acid residues with 1682bp ORF. VMPAAT and VMPSTS show high homologies with plant alcohol acyltransferase and terpene synthase, respectively. Real time RT-PCR indicated that both transcripts were expressed preferentially in floral tissues, with high levels in blooming and full bloom flowers. VMPAAT and VMPSTS transcripts were expressed in a rhythmic pattern. The results presented in this study will be potentially useful in providing additional insights into the fragrance-related pathways of Orchidaceae members, which until today is still limited

Anthocyanin stability studies in Tibouchina semidecandra L.

The effects of pH, storage period, temperature, light and dark conditions on the stability of anthocyanins extracted from Tibouchina semidecandra flowers of different developmental stages was evaluated. Fully formed but unopened flower bud had the highest amount of total anthocyanin extracted from fresh petals. The anthocyanin contents for all flower developmental stages were stable at pH 0.5–3.0 but the colour of the extracts faded at higher pH values. Degradation percentages of total anthocyanins in the extracts kept at 25 °C were 7–20% lower than that maintained at 31 °C. Extracts stored in darkness at 25 °C maintained their purple colour for 26 days while light exposure reduced it to an average of 10 days. The study shows that suitable storage condition for coloured anthocyanin pigments in extracted form is in acidic conditions in the dark. This implies the potential usage of coloured anthocyanins as natural food colourants and shelf life indicator for acidic foods

Optimization of Agrobacterium-Mediated Transformation Parameters for Melastomatacea Spp. Using Green Fluorescent Protein(GFP) as A Reporter

Agrobacterium-mediated transformation for both Melastoma malabathricum and Tibouchinu semidecanda were optimized using green fluorescent protein (GFP) as a reporter. The binary vector pCAMBIA1304 harboring the modified green fluorescent protein (mgfp) gene driven by the CuMV 35S promoter was used. Parameters optimized were bacterial strain, bacterial concentration, pre-culture period, co-cultivation period,immersion time, acetosyring concentration and wounding type. Results obtained obtained were based on the percentage of (GFP expression which was observed 3 days post-transformation. Agrobacterium tumefaciens starin LBA4404 and EHA105 at concentration 1 X 107 cfu ml -1 (OD 600mm 0.8) showed the highest virulence on M. malabathricum and T.semidecandra, respectively. Four days of pre-culture and 2 days of co-cultivation were optimum for M.malabathricum transformation, while 3 days of pre-culture and co-cultivation fot T, semidecandra, . result also showed that 60 min of immersion and addition of 200 p.M acetosyringone gave the highest percentage of positive transformants for both M. malabathricum and T.semidecandra. Mild wounding also significantly increased the efficiency 0f M.malabathricum transformation

Agrobacterium-mediated transformation of melastoma malabathricum and tibouchina semidecandra with sense and antisense dihydroflavonol-4-reductase(DFR)genes.

Genetic engineering of a wide variety of plant species has led to the improvement of plant traits. In this study, the genetic transformation of two potentially important flowering ornamentals, Melastoma malabathricum and Tibouchina semidecandra, with sense and antisense dihydroflavonol-4-reductase (DFR) genes using the Agrobacterium mediated method was carried out. Plasmids pBETD10 and pBETD11, each harbouring the DFR gene at different orientations (sense and antisense) and selectable marker npt II for kanamycin resistance, were used to transform M. malabathricum and T. semidecandra under the optimized transformation protocol. Putative transformants were selected in the presence of kanamycin with their respective optimized concentration. The results indicated that approximately 4.0% of shoots and 6.7% of nodes for M. malabathricum regenerated after transforming with pBETD10, whereas only 3.7% (shoots) and 5.3%(nodes) regenerated with pBETD11 transformation. For the selection of T. semidecandra, 5.3% of shoots and 9.3% of nodes regenerated with pBETD10 transformation, while only 4.7% (shoots) and 8.3% (nodes) regenerated after being transformed with pBETD11. The presence and integration of the sense and antisense DFR genes into the genome of M. malabathricum and T. semidecandra were verified by polymerase chain reaction (PCR) and nucleotide sequence alignment and confirmed by southern analysis. The regenerated putative transformants were acclimatized to glasshouse conditions. Approximately 31.0% pBETD10-ransformed and 23.1% pBETD11-transformed M. malabathricum survived in the glasshouse, whereas 69.4% pBETD10-transformed and 57.4% pBETD11-transformed T. semidecandra survived. The colour changes caused by transformation were observed at the budding stage of putative T. semidecandra transformants where greenish buds were produced by both T. semidecandra harbouring the sense and antisense DFR transgenes. Besides that, the production of four-petal flowers also indicated another morphological difference of putative T. semidecandra transformants from the wild type plants which produce five-petal flowers

Molecular characterization of a new 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) transcript from Vanda Mimi Palmer.

A 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) transcript was successfully isolated from the floral cDNA library of Vanda Mimi Palmer (VMPDXR). The full-length cDNA of clone VMPDXR was predicted to encode a polypeptide of 473 amino acid residues with 15 bp of 5′ UTR and 230 bp of 3′ UTR including a poly-A tail. VMPDXR was predicted to have a molecular mass of 51.4 kD and a pI value of 6.04. It has two conserved domains, an N-terminal NADPH binding site (GSTGSIG) and an N-terminal proline-rich region(PPPPAWPGR). It also contains two highly homologous regions, a 78–207 amino acids stretch at the N-terminal and a 221–304 amino acids stretch at the C-terminal domain. The putative plastid transit peptide is not found in VMPDXR and it is clustered into the plant DXRs in the phylogenetic tree. VMPDXR was differentially expressed in roots, leaves, sepals, petals and column. The VMPDXR transcript levels were preferentially high in blooming and fully bloomed flowers compared to the bud. The expression of VMPDXR at different times did not appear in a rhythmic manner and no drastic fluctuation was observed at night except at 2 pm during the day

An overview of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) in plants

Isoprenoids biosynthesis in plants involves two separate pathways, mevalonate (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. A large group of isoprenoids are found to play crucial roles in common plant biochemical functions and have been produced on a large scale for commercial applications. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the key enzyme that catalyses the first committing step in the MVA pathway. In mammals and yeast, HMGR is a well-studied enzyme as many studies have been done on this enzyme due to its important function in the biosynthesis of cholesterol. In plants, many researches on HMGR have been done on different plant species, for example, Arabidopsis thaliana, tobacco, gingko, Zea mays, potato, rose, rubber tree, muskmelon, ginseng and others, in the past decades since it was discovered. Previous researches that worked on plant HMGR focused on the cloning and characterisation of its physiological functions. Little is known about the aspect of regulation and structural characteristics of plants’ HMGR. This review is aimed at providing an overview of the characteristics and structure of HMGR, the transcriptional and post-translational events related to HMGR that have been reported in plants, and proposes areas on the regulation event of HMGR in plants that can be explored to further enhance understanding towards HMGR regulatory interactions