5 research outputs found

    Crude extract of Trichoderma elicits agarwood substances in cell suspensionculture of the tropical tree, Aquilaria malaccensis Lam.

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    Agarwood is the precious fragrant wood produced by the tropical tree Aquilaria, often after elicitation by wounding or fungal attack. In this study we established a cell suspension culture of A. malaccensis from leaf-derived callus and induced agarwood production in the culture using fungal elicitors. Elicitors were made from crude mycelial extracts of two fungal species from the genera Trichoderma and Lasidiplodia. The elicitors were added to the cell suspension culture, initiated with 2 g of fresh calli, at concentrations ranging from 2 to 10 mg L-1. A light agarwood scent was detected from the suspension culture elicited with 8 mg L-1 Trichoderma extract. To increase scent intensity, cell suspension cultures were initiated from 2 to 8 g of calli and treated with 8 mg L-1 Trichoderma extract. The combination of 8 g of calli inoculum and 8 mg L-1 Trichoderma extract produced the most intense fragrance, one comparable to agarwood scent. The cell culture was harvested, extracted in methanol, and analyzed using GC-MS. Several important agarwood compounds were detected including 8-epi-.gama.-eudesmol, á-guaiene, and alloaromadendrene oxide-1. Trichoderma appeared to be a suitable inducer for agarwood production when used at an optimal concentration and in combination with a cell suspension culture of Aquilaria

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

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    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

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    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

    Agarwood induction in cell suspension culture of Aquilaria malaccensis Lam.

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    Most species of Aquilaria in the Thymelaeaceae family produce agarwood or ‘gaharu’. In Malaysia, the primary producer of agarwood is Aquilaria malaccensis, a tree species locally known as ‘karas’. Agarwood is one of the precious woods on earth and prized for its rich and wonderful fragrance. This study aimed at inducing agarwood production in cell suspension culture of Aquilaria malaccensis, by investigating the optimal growth medium, optimal inocula size and finding suitable elicitors. To establish cell suspension culture, callus was first induced from nursery-derived leaf explants incubated on basic Murashige and Skoog (MS) solid medium containing 1.5% sucrose, 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). Compact type callus was induced by 1.1 μM NAA with highest biomass dry weight (DW) of 17.3 mg. This auxin concentration was then combined with either 6-benzylaminopurine (BAP) or kinetin, at 0.55, 1.1, 2.2 and 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). Microscopic observations revealed the arrangement of the friable callus as loosely packed with relatively large cells. However, for the compact callus, the cells were small and densely packed. Further investigation on effects of elicitor in agarwood production were studied using cell suspension culture with initial an inocula of 4% (fresh weight, FW) of callus. The culture was challenged with fungal elicitor prepared from two species of fungi, Trichoderma and Lasiodiplodia. Fungal elicitor in the form of mycelial crude extract was added to the medium to a final concentration of 2, 4, 6, 8, and 10 mg/L, respectively. A light scent of agarwood was detected from the culture after it had been challenged for 20 days with 8mg/L Trichoderma extract. To increase scent intensity, cell suspension cultures were initiated from 4%, 8%, 16% and 32% (FW) of calli and elicited with 8 and 16 mg/L Trichoderma extract, respectively. The combination of 16% of inocula and 8 mg/L Trichoderma extract produced the most intense fragrance comparable to agarwood scent. Several important agarwood compounds were detected using GC-MS including 8-epi-.gama.-eudesmol, á-guaiene, alloaromadendrene oxide-1 and chromone,5-hydroxy-6,7,8-trimethoxy-2,3-dimethyl. Trichoderma appeared to be a suitable elicitor for agarwood production. In conclusion, the presence of these compounds was evidence that agarwood had been induced in the fungal inoculated cell suspension culture
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