An efficient callus induction and in vitro regeneration were developed using plant growth regulators, carbon sources, and basal media for three selected Malaysian wetland rice varieties (MR220, MR220-CL2, and MR232) and one upland variety (Bario). Effect of plant growth regulator (PGR) was carried out using four different concentrations (1-4 mg/L) of 2,4-D (2, 4-dichlorophenoxyacetic acid), and NAA (1-naphthalene acetic acid) (2.5, 5.0, 7.5, and 10 mg/L) with optimized 2,4-D. Effects of carbon sources (maltose and sorbitol), and basal media (MS, N6, and LS) were also studied with optimized PGR to maximize the induction of regenerable calli. This study found that all four varieties exhibited high frequency of callus induction on MS (Murashige and Skoog) medium that was supplemented with 3 mg/L 2,4-D and 30 g/L maltose. Callus induction frequencies in the cases of MR220, MR220-CL2, MR232, and Bario were found to be 76%, 94%, 85%, and 42% respectively. Morphological analysis through scanning electron microscopy (SEM) and histological analysis revealed the embryogenicity of the induced callus. In the regeneration study, it was observed that combination of 2 mg/L BAP (6-benzylaminopurine), 2 mg/L Kin (Kinetin) and 0.5 mg/L NAA supplemented MS medium has the potential to promote regeneration of selected indica rice varieties with higher regeneration percentage, i.e., 82% (MR220-CL2), 68% (both in MR220 and MR232), and 40% (Bario). The optimized conditions for callus formation and regeneration can be useful for biotechnological practices for the genetic improvement of Malaysian indica rice.
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.
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.
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.
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