Improvement of shoot regeneration of potentially medicinal plant melaleuca alternifolia via axillary shoot

The aim of this study is to establish an improved shoot regeneration of potential medicinal plants, Melaleuca alternifolia. The essential oil of these plants is useful in the cosmetic and pharmaceutical industries. Despite the importance of this species, in vitro regeneration was limited. Therefore, the present study use various concentrations of plant growth regulators to improve shoot regeneration of these plants. The present study shows that axillary shoot gave the best response to the treatment containing 0.5mg/L BAP and 0.1mg/L NAA. The average number of shoots is 23 with 77% of shooting. For rooting experiment, 35% of axillary shoot were rooted when cultured on MS medium supplemented with 1.5mg/L IBA. In vitro plantlet was later developed after 3 months

ITS2 secondary structure data improves authentication of Moringa oleifera tea products when using with DNA barcoding

Adulteration is a severe issue affecting the herbal industry. Therefore, a robust tool is needed to address this problem. In the current study, Moringabased products (tea) authentication was conducted using DNA barcoding. Two different barcode regions, rbcL and ITS2, were investigated in terms of their effectiveness in authenticating the herbal products. To proceed with the DNA barcoding, a good quality of gDNA is a prerequisite for PCR amplification. Hence, two lysis buffers, PL1 and PL2, were compared to obtain good quality gDNA. Results revealed that a higher gDNA yield was obtained from the fresh plants using PL2, but a lower gDNA yield was obtained for the tea products except for sample P3. The PCR reaction was successfully conducted by amplifying two different barcodes, rbcL and ITS2. The amplicon size for the fresh plant was 643 bp for rbcL and 404 bp for ITS2. In contrast, the generated rbcL amplicon sizes for herbal tea products were 672 bp for P1, 679 bp for P2, 679 bp for P3, and 674 bp for P4. For rbcL and ITS2 amplicon sizes were 395 bp for P1, 406 bp for P2, 398 bp for P3, and 413 bp for P4. The amplified PCR products were analyzed using bioinformatic tools. The neighbour-joining (NJ) analysis for the rbcL barcode indicated that the P2, P3, and P4 tea products were categorized in the same clade with the M. oleifera sequence obtained from GenBank. Simultaneously, P1 was clustered individually with other closely related species. The analysis for the ITS2 barcode showed that all samples were grouped in the same clade. Incorporating secondary structure prediction after NJ analysis improved the discrimination between species

Isolation of high quality RNA from plant rich in flavonoids, Melastoma decemfidum Roxb ex. Jack.

Chalcone synthase (CHS) is a plant-specific enzyme that synthesises naringenin chalcone, an essential precursor of the flavonoid biosynthetic pathway. Naringenin and kaempferol are two flavonoids that have been demonstrated to inhibit the proliferation of HeLa cells. To study chalcone synthase gene regulation in Melastoma decemfidum, we developed a high-yield total RNA isolation method to assemble a partial putative CHS cDNA sequence. Our results indicated that a modified CTAB method produced the highest total RNA yield (8.26±3.99 μg/gFW) compared to other methods. Thus, we used this method to isolate total RNA from different types of tissues from this plant. Our improved protocol produced high-quality total RNA from different tissues, including the mature leaf (7.02±2.60 μg/gFW), stem (4.27±1.72 μg/gFW), flower bud (37.54±10.61 μg/gFW), flower (21.31±5.20 μg/gFW), and root (3.38±1.89 μg/gFW). The total RNA was then converted into cDNA, and a putative CHS gene product (~1049 bp fragment) was amplified using degenerate primers. A partial CHS gene sequence shared 80% homology with an Anthurium andraeanum CHS gene sequence (AY232492) and 92% homology with the amino acid sequence of the Acer maximowiczianum CHS gene (AEK80412.1), as determined using BlastN and BlastX, respectively. This study shows that our modified CTAB method allows for the isolation of high-quality and high-yield total RNA from various tissues of M. decemfidum. A partial putative CHS gene was amplified,thus confirming that the modified CTAB method is suitable for RT-PCR and gene isolation

In vitro regeneration of Garden Balsam, impatiens balsamina using cotyledons derived from seedlings

Garden balsam, Impatiens balsamina, is one of an important ornamental plant in Malaysia. An effort to develop a reliable tissue culture system for this Impatiens species was carried out for future genetic manipulation of this plant for useful traits. Parts of cotyledons and hypocotyl derived from different age of seedlings, from surface sterilized seeds, were subjected to shoot induction on MS media containing BAP, TDZ or combination of BAP and NAA in different ratios. Roots were developed from the above shoots on full- or halfstrength MS media containing IAA, IBA or NAA of different concentrations. Finally fully-developed plantlets were produced on hormone free MS media or full- or half-strength MS media containing specific hormones. It was observed that the most efficient shoot induction (93%) was obtained from proximal part of cotyledon derived from 7 days old seedlings on MS media supplemented with 1mg/L BAP. Roots were most efficiently produced (92%), in term of percentage and morphology, on half-strength MS media supplemented with 0.1 mg/L IAA. Further regeneration of plantlets, approximately 8-10 cm in height, was achieved after 3 weeks on hormone-free MS media. Finally, full regeneration system of Impatiens balsamina was developed, using MS media supplemented with 1 mg/L BAP for shoot and 0.1 mg/L IAA for roots, within 8 weeks in culture

Targeted biolistics for improved transformation of impatiens balsamina

A transgenesis programme has been developed for Impatiens balsamina that will allow elucidation of the roles played by individual genes in the flower reversion phenomenon shown by this model species. The lack of explants exhibiting adventitious shooting in I. balsamina hinders Agrobacterium-based transformation, but the multiple shoots that arise from cotyledonary nodes present a suitable target for biolistics. These tissues can be disrupted by the helium blast effect associated with conventional biolistic devices, so we have utilised modifications to the PDS 1000/He equipment originally developed for transformation of fragile insect tissues. By loading microcarriers on to a rigid, rather than flexible, macrocarrier, the blast effect is largely eliminated, and the use of a focussing nozzle allows the bombardment to be concentrated on the target tissues. This approach reduces waste of plasmid DNA and gold microcarriers and achieves transfection at lower, less disruptive helium pressures than would otherwise be necessary to efficiently penetrate below the shoot epidermis and generate heritable transgenic lines

Effect of 2,4-d on embryogenic callus induction of Malaysian indica rice (oryza sativa l) cultivars mr123 and mr127

The aim of this study is to study the effect of various concentrations of 2,4-D on embryogenic callus induction of indica rice MR123 cultivar and MR127 cultivar using mature seeds. Optimal media for induction of callus of both cultivars was MS basal media supplemented with 30g/L sucrose, 500mg/L glutamine and 500mg/L proline supplemented with 2.5 mg/L 2,4-D. The highest percentage of callus induction was 70% and 76% for MR123 and MR127 respectively. Both cultivars produced an embryogenic callus from scutellum after a week in culture with white-yellowish in color. The viability tested using Evans blue show callus obtained was embryogenic. This simple protocol using staining method could be used for screening embryogenic callus before further experiments can be conducte

Chemical composition of eurycoma longifolia (Tongkat Ali) and the quality control of its herbal medicinal products

Eurycoma longifolia which is known as Tongkat Ali is commonly found in Asian countries such as Malaysia, Indonesia, Thailand, Myanmar and Cambodia. This plant is famously known for its various pharmacological activities. The plant is also reported to consist of various types of important bioactive compounds such as quassinoids, canthine-6-one alkaloids, triterpenes, squalene derivatives, $-carboline alkaloids etc which are mostly found in the root part. The presence of these important phytochemicals contributes to their different types of therapeutic effects more especially in terms of aphrodisiac properties which have resulted in a massive increase in demand and production of their Herbal Medicinal Products (HMP). These situations have resulted in the production of E. longifolia HMPs whose quality are questionable, which might be as a result of restricted of sources that might lead to some unethical activities carried out by suppliers and manufacturers in order to gain more profit. Therefore, this review focused on adulteration issues such as contamination and substitution of E. longifolia HMP. The review also includes the possible solutions on how to improve the quality of these HMP so as they can be safe for consumption. Embracing pharmacovigilance in the preparation of the HMP, proper implementation of agricultural practices such as Good Agricultural and Collection Practices (GACP) and Good Manufacturing Practices (GMP) together with the establishment of effective regulatory bodies would undoubtedly improve the quality of E. longifolia HMP sold in the market. The detailed knowledge about the main composition of the E. longifolia HMP will help to ascertain their quality, efficacy and safety as these are very important toward quality control

Comparative evaluation of different DNA extraction methods from E. Longifolia herbal medicinal product

The aphrodisiac property of Eurycoma longifolia has led to an increase in the demand for its Herbal Medicinal Products (HMPs). However, the efficiency of such HMPs depends on the usage of their genuine raw materials. The conventional methods cannot identify species in processed form. The authentication of HMPs can be achieved effectively using DNA barcoding as the method species-specific. However, the use of this method solely relied on the extraction of high-quality DNA from the HMPs. Therefore, it is necessary to establish a satisfactory method for extracting high-quality DNA from the HMPs. Here, four DNA extraction methods were compared to evaluate the best protocol in yield, purity, polymerase chain reaction (PCR) amplification, sequencing, and species identification. The spectrophotometer analysis showed that the Nucleospin Plant II extraction kit has the best purity as this can be severely affected by the presence of various contaminants in the HMPs. Our findings reveal that DNA purity was more important as a predictor for PCR amplification than yield. Therefore, the present study results demonstrate that the Nucleospin Plant II extraction kit is the best because it produces the purest, amplifiable, and sequenceable DNA for identification and authentication of E. Longifolia HMPs

Biosynthesis of very long polyunsaturated Omega-3 and Omega-6 fatty acids in transgenic Japonica rice (Oryza sativa L)

Abstract In this research, the fatty acid pathway was modified to produce the C20 polyunsaturated fatty acid arachidonic acid (ARA) and eicosapentaenoic acid (EPA) by co-expression the genes encoding omega-3 fatty acid desaturase (SK-FAD3) from Saccharomyces kluyveri

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