Effects of plant growth regulators, carbon sources and pH values on callus induction in Aquilaria malaccensis leaf explants and characteristics of the resultant calli

The endangered tropical tree, Aquilaria malaccensis, produces agarwood for use in fragrance and medicines. Efforts are currently underway to produce valuable agarwood compoundsn tissue culture. The purpose of this study was to develop an optimal growth medium, specifically, the best hormone combination for callus suspension culture. Using nursery-grown A. malaccensis, sterilized leaf explants were first incubated on basic Murashige and Skoog (MS) gel medium containing 15g/L sucrose and at pH 5.7. Different auxin types including 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-butyric acid (IBA), were tested at various concentrations (0.55, 1.1 and 1.65 μM) using the basic medium. Leaf explants were incubated for 30 days in the dark. Callus induced by 1.1 μM NAA had the highest biomass dry weight (DW) of 17.3 mg; however the callus was of a compact type. This auxin concentration was then combined with either 6-benzylaminopurine (BAP) or kinetin at 0.55, 1.1, 2.2 or 3.3 μM to induce growth of friable callus. The 1.1μM NAA + 2.2μM BAP combination produced friable callus with the highest biomass (93.3mg DW). When testing the different carbon sources and pHs, sucrose at 15g/L and pH at 5.7 yielded highest biomasses at 87.7mg and 83mg DW, respectively. Microscopic observations revealed the arrangement of the friable cells as loosely packed with relatively large cells, while for the compact callus, the cells were small and densely packed. We concluded that MS medium containing 15 g/L sucrose, 1.1 μM NAA + 2.2 μM BAP hormone combination, and a pH of 5.7 was highly effective for inducing friable callus from leaf explants of A. malaccensis for the purpose of establishing cell suspension culture

An improved surface sterilization technique for introducing leaf, nodal and seed explants of Aquilaria malaccensis from field sources into tissue culture

A critical stage in the introduction of plants into tissue culture is to obtain cultures free from microbial contamination. This study investigated different sterilization regimes for leaf and nodal explants from Aquilaria malaccensis grown in the shade house under natural environmental conditions, and for seeds from wild mature trees. We found that pre-sterilization using 0.2% Benomyl for 15 minutes improved the number of ‘clean and alive’ individuals of all types of explants, especially when followed by surface sterilization using mercury chloride (HgCl2). Treatment with 0.1 % HgCl2 for 15 and 30 seconds yielded the best results for leaf and nodal explants, respectively. Maximum percentage of ‘clean and alive’ seeds was observed when using 0.2 % HgCl2 for 12 minutes. Treatment with Clorox® bleach (5.25% sodium hypochlorite as the active ingredient) even at high concentration (50% Clorox®) alone was not sufficient to control fungal and bacterial contamination in the explants. We conclude that HgCl2 coupled with Benomyl pre-treatment produced a highly efficient sterilization method producing 83 – 90% ‘clean’ leaf, nodal and seed explants of A. malaccensis from natural sources after fourteen days in culture

Development of an enzyme-based fiber optic biosensor for detection of haloalkanes

Purpose – The main purpose of this study is to demonstrate the development of an enzyme-based sensor for haloalkane detection. Haloalkane is a toxic compound that is found as genotoxic impurities in pharmaceutical products and contaminants in waste. The need to investigate the genotoxic level in pharmaceutical manufacturing is very crucial because of its toxicity effects on human health. The potential of mini protein as an alternative bioreceptor was explored with the aim to be more effective and stable under extreme conditions. Design/methodology/approach – Mini proteins of haloalkane dehalogenase (HLD) were computationally designed and experimentally validated. Tapered multimode fiber (TMMF) was bio-functionalized with a bioreceptor either native (positive control) or mini protein. The absorbance-based sensor resulting from the binding interaction of mini protein with haloalkane was monitored through a wavelength range of 200-1,300 nm. Findings – An increment of the UV absorption is observed at 325 nm when haloalkane interacted with the immobilized bioreceptors, native or mini protein. Both biosensors displayed a continuous response over the range of 5-250 mM haloalkane. They also had the capability to detect haloalkanes below 1 min and with an operational stability of up to seven days without significant loss of sensitivity. Practical implications – The results indicate the potential viability of the enzyme HLD-based sensor to monitor the existence of haloalkane in both pharmaceutical and environmental products. Originality/value – The paper describes an outcome of experimental work on TMMF-based biosensor coated with HLD for label-free haloalkane detection. Mini protein can be used as an effective bioreceptor with some structural modification to improve functionality and stability

Optimization of culture parameters for flavonoid production from callus and suspension cultures of the karas tree (Aquilaria malaccensis Lam.)

Plant tissue culture technique has been shown to be efficient for producing flavonoids at higher amount and lesser time period compared to intact plant. This study aimed at producing flavonoids in elevated amounts using callus and cell suspension cultures of A. malaccensis. Aquilaria trees not only produce secondary metabolites known as agarwood (gaharu) but also rich in flavonoids. To introduce plant materials into tissue culture, different sterilization regimes for leaf, nodes and seeds of A. malaccensis, were investigated. Pre-sterilization using 0.2% Benomyl for 15 minutes improved the number of „sterile and survived‟ individuals for all types of explants (83-90%), after surface sterilization using mercury chloride (0.1 to 0.2 % HgCl2). The most favorable explants for producing highest flavonoid cell lines were determined between leaf and nodal explants by using spectrophotometer and HPLC analysis. Result showed that flavonoid content in the leaf explants (8.85 ± 0.76 mg/g dry weight (DW)) was 7-fold higher than that of nodal explants (1.25 ± 0.53 mg/g DW). Leaf contained high amount of quercetin (16.19 ± 1.02 μg/g DW) then rutin (0.21 ± 0.31 μg/g DW). Nodes explants contained quercetin and rutin at 0.94 ± 0.07 μg/g DW and 0.06 ± 0.01 μg/g DW, respectively. Kaempherol was not detected in any of the explants. Leaf was used as explant for callus induction due to high flavonoid content and placed on different types of plant hormones to indicate the type of hormone use to induce callus. Callus grew best on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg/L 6-benzylaminopurine (BAP). Total flavonoid content in MS medium supplemented with 2.0 mg/L BAP and 0.5 mg/L was 6- fold higher than in phytohormone- free MS medium. Using this optimal hormone combination, callus growth was observed on different strengths of MS medium, carbon sources and pH. Full-strength MS medium supplemented with 2.0 mg/L 2,4-D and 0.5 mg/L BAP, 20 g/L sucrose, in pH 5.7, significantly yielded the highest biomass (231 ± 0.13 mg DW /culture) of callus. These calli were used to initiate the cell suspension cultures. To enhance the flavonoid production in callus and cell suspension cultures, examine phenylalanine (Phe) was used as a precursor in ranged of 20 to 100 mg/L. Cell suspension culture added 60 mg/L Phe yielded the highest amount of total flavonoid (37.92 ± 0.77 mg/g DW), 4.8-fold higher than Phe-free cell culture, and 2-fold higher than that in calli (19.11 ± 0.36 mg/g DW). The suspension culture with 60 mg/L Phe had the highest amount of rutin (17.69 ± 1.37 μg/g DW), quercetin (3.46 ± 0.97 μg/g DW) and kaempherol (2.28 ± 1.67 μg/g DW). When compared the cell suspension culture with 60 mg/L Phe to Phe-free cell culture, only rutin and quercetin were detected and at lower levels, 1/10-fold and 1/4-fold, respectively. This study demonstrated that callus and cell suspension cultures of A. malaccensis produced flavonoids, and feeding the cultures with Phe elevated flavonoids levels and induced production of additional compound which is kaempherol

Development of a novel mini peptide-based bioreceptor for haloalkane detection

Haloalkanes are the reactants in pharmaceutical manufacturing, which can be found in the final active pharmaceutical ingredients (APIs) and in the waste as impurities. Due to their toxicity effect to organisms’ health, the concern towards haloalkanes is increased. The conventional detection method is time-consuming, costly, lab-based, difficult to operate and not practical for continuous monitoring. Thus, this increases the demand for a simple and rapid device for direct detection of the compounds. Haloalkane dehalogenase (HLD) can be used as the specific bioreceptor to detect the presence of haloalkanes. But, the uses of native HLD are less efficient at extreme condition. Therefore, this study aims to develop a mini protein of HLD as an alternative bioreceptor focusing on sensitivity and stability. A novel mini peptidebased bioreceptor based on HLD from Xanthobacter autotrophicus (PDB ID: 2DHC) as template was developed for haloalkane biosensor. Yet Another Scientific Artificial Reality Application (YASARA) software was utilized to create the mini proteins by downsizing approach. Residues were removed gradually to obtain the mini protein while retaining the three active site residues; Asp-124 (nucleophile), His-289 (base), Asp-260 (acid) and two halide stabilizing residues; Trp-125, Trp-175. Five mini proteins comprising 283 amino acids or less, with the highest binding energy (enzymesubstrate complex) and distance of less than 4 Å between Asp-124 and three haloalkanes were chosen as the best validated designs. The recombinant mini proteins were constructed using pET vector and Escherichia coli BL21 (DE3) as the expression vector and host, respectively. The smallest mini protein, with 86 amino acids (model 5) was chosen for His-tag affinity purification and subsequent analysis as it could be expressed in soluble form. No catalytic activity was detected with haloalkane substrate. Isothermal titration calorimetry (ITC) showed there was binding interaction between the mini protein and haloalkane. Thermal stability study with circular dichroism (CD) had proven the mini protein possessed higher Tm value at 83.73 °C than the native HLD at 43.97 °C. Optical sensor with tapered multimode glass fiber (TMMF) was fabricated. Protein was immobilized on TMMF with the action of aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA). The interaction of haloalkane and the immobilized mini protein showed an increment of the UV absorption at 325 nm. Optical sensor proved that the mini protein could act as a potential bioreceptor. However, it demonstrated low sensitivity for haloalkane at 0.0002 μM-1 (R2: 0.9832) with limit of detection (LOD) at 80 μM and low stability. Thus, screen-printed carbon electrodes (SPEs) was used to look for the interaction via electrochemical sensor, to enhance the sensitivity and stability. To improve the stability, mini protein structure was mutated with cysteine at residues 49 and 78 to form a disulfide bridge. Bare SPE was modified with gold nanowires coated on the working electrode surface, followed by self-assembly of L-cysteine (Cys) and GA for protein immobilization. The interaction of the mutated mini protein immobilized on SPEs was studied and compared to native HLD immobilized SPEs as positive control. Electrocatalytic oxidation of haloalkane was examined with cyclic voltammetry (CV) and differential pulse voltammetry (DPV) at working potential 0.03 V and -0.1 V, respectively. Electrochemical impedance spectroscopy (EIS) was also performed to detect the binding interaction of haloalkane with the fabricated mutated mini protein. An electrochemical sensor with DPV analysis presented a more sensitive (0.2118 μM- 1, R2: 09741) detection with low LOD at 6 μM. The sensor also demonstrated good repeatability (RSD 4.3%) and reproducibility (RSD 5%) for haloalkane detection. The mutated mini protein based sensor with modified SPEs provided higher sensitivity and better detection of haloalkane than the native HLD. It can be a potential tool in haloalkane detection for immediate application