Alina Wagiran

Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas).

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L−1) and NAA (2 mg L−1) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5–1.5 mg L−1), BAP, NAA and 0.5 mg L−1 of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L−1), Kin (1.5 mg L−1), NAA (0.5 mg L−1) and TDZ (0.5 mg L−1) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots.

Review: DNA Barcoding and Chromatography Fingerprints for the Authentication of Botanicals in Herbal Medicinal Products

In the last two decades, there has been a tremendous increase in the global use of herbal medicinal products (HMPs) due to their claimed health benefits. This has led to increase in their demand and consequently, also, resulted in massive adulteration. This is due to the fact that most of the traditional methods cannot identify closely related species in a process product form. Therefore the urgent need for simple and rapid identification methods resulted in the discovery of a novel technique. DNA barcoding is a process that uses short DNA sequence from the standard genome for species identification. This technique is reliable and is not affected by external factors such as climates, age, or plant part. The difficulties in isolation of DNA of high quality in addition to other factors are among the challenges encountered using the DNA barcoding in the authentication of HMP. These limitations indicated that using DNA barcoding alone may ineffectively authenticate the HMP. Therefore, the combination of DNA barcoding with chromatographic fingerprint, a popular and generally accepted technique for the assessment and quality control of HMP, will offer an efficient solution to effectively evaluate the authenticity and quality consistency of HMP. Detailed and quality information about the main composition of the HMPs will help to ascertain their efficacy and safety as these are very important for quality control.

Assessing product adulteration of Eurycoma longifolia (Tongkat Ali) herbal medicinal product using DNA barcoding and HPLC analysis

Abstract Context: Eurycoma longifolia Jack (Simaroubaceae) commonly known as Tongkat Ali is one of the most important plants in Malaysia. The plant extracts (particularly roots) are widely used for the treatment of cough and fever besides having antimalarial, antidiabetic, anticancer and aphrodisiac activities. Objectives: This study assesses the extent of adulteration of E. longifolia herbal medicinal products (HMPs) using DNA barcoding validated by HPLC analysis. Materials and methods: Chloroplastic rbcL and nuclear ITS2 barcode regions were used in the present study. The sequences generated from E. longifolia HMPs were compared to sequences in the GenBank using MEGABLAST to verify their taxonomic identity. These results were verified by neighbor-joining tree analysis in which branches of unknown specimen are compared to the reference sequences established from this study and other retrieved from the GenBank. The HMPs were also analysed using HPLC analysis for the presence of eurycomanone bioactive marker. Results: Identification using DNA barcoding revealed that 37% of the tested HMPs were authentic while 27% were adulterated with the ITS2 barcode region proven to be the ideal marker. The validation of the authenticity using HPLC analysis showed a situation in which a species which was identified as authentic was found not to contain the expected chemical compound. Discussion and conclusions: DNA barcoding should be used as the first screening step for testing of HMPs raw materials. However, integration of DNA barcoding with HPLC analysis will help to provide detailed knowledge about the safety and efficacy of the HMPs.

Authenticity Testing and Detection of Eurycoma longifolia in Commercial Herbal Products Using Bar-High Resolution Melting Analysis

The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) and the nuclear ribosomal region- internal transcribed spacer 2 (ITS2), PCR amplification and HRM analysis using saturated Eva green dye as the source of fluorescence signals, was accomplished by employing a real-time cycler. The results were further validated by sequencing to identify unknown sequence from Genbank database and to generate phylogenetic tree using neighbour joint (NJ) analysis. Both of the DNA markers exhibited a distinguishable melting temperature and shape of the normalised curve between the reference and the adulterants. In the case of species identification, ITS2 was more successful in differentiating between species. Additionally, detection of admixture sample containing small traces of targeted E. longifolia DNA (w/v) can be detected as low as 5% for rbcL and less than 1% for ITS2, proving the sensitivity and versatility of the HRM analysis. In conclusion, the Bar-HRM analysis is a fast and reliable technique that can effectively detect adulterants in herbal products. Therefore, this will be beneficial for regulatory agencies in order to regulate food safety issues.

Molecular cloning and characterization of a cDNA encoding a polyketide synthase from Melastoma decemfidum

A famous plant polyketide synthase (PKS) is a chalcone synthase (CHS), which catalyzes the formation of naringenin chalcone, a limiting factor for flavonoid biosynthesis. Flavonoids such as naringenin and kaempferol-3-O-(2″,6″-di-O-p-trans-coumaroyl glucoside) have been identified from the flower extracts of Melastoma decemfidum RoxB. Therefore, a study was conducted to clone and characterize the pks gene from M. decemfidum. A newly isolated full-length cDNA containing the pks sequence (designated MdCHS; GenBank accession No. KF234569) was found to harbour a 1,355-bp open reading frame encoding 392 amino acids. The deduced MdCHS protein, which had a predicted molecular weight of approximately 42.6 kDa, exhibited 89% sequence identity to CHS from Anthurium andraeanum (GenBank accession No. AY232492) and 91% to those from Camellia sinensis (D26594) and Camellia chekiangoleosa (ADW11243). Southern blot analysis indicated that at least five pks genes are present in M. decemfidum. Additionally, transcription analysis revealed that pks gene expression was higher in roots than in other tissues (shoots, leaves, stems, flower buds and flowers). In conclusion, the characterization and cloning of the MdCHS gene further elucidates its role in flavonoid biosynthesis.