1.1 Physical properties of Aquilaria malaccensis There are fifteen species in the genus Aquilaria and eight are known to produce agarwood (Ng et al., 1997). Recently it was mainly produced from A. malaccensis, A. agallocha and A. secundaria (Broad, 1995). A. crassna and A. sinensis are the other two members of the genus that are usually harvested. Aquilaria Malaccensis has been cultivated as part of the Agar plantation project in the eastern hill regions in the division of Sylhet, Chittagong, Chittagong Hilltracts and Cox?s Bazaar forests of Bangladesh (A. Faruque, Chief Conservator of Forests and Management Authority for CITES, Bangladesh, in litt. to TRAFFIC Southeast Asia, April 2003).
Aquilaria malaccensis is a large, evergreen tree, up to 20 - 40 m tall (Adelina, 2004) and 1.5-2.5 m wide, with a moderately straight and often fluted stem bearing thin, pointed leaves, 5-8 cm long and with several parallel veins (Chakrabarty et al., 1994). Flowers are hermaphroditic, up to 5 mm long, fragrant and yellowish green or white which blooms at June. Fruits which appear in August, are green, egg-shaped capsule, leathery covered with fine hairs, 4 cm long, and 2.5 cm wide. There are two seeds per fruit.
Aquilaria has a single layer epidermis which is composed of parenchymatous cells. This tree has an unusual anatomy and specialized cells within the xylem that produce the resin (Norsuzieana, 2009). Unlike most trees in the Angiospemae that produce phloem cells external to the xylem (growing out from the circumference of the cambium), Aquikiaria produce bundles of phloem cells throughout the xylem as well as in a layer external to the xylem. This means that the xylem (consisting of vessels, fiber and parenchyma cells) also contains groups of phloem cells called included phloem or interxlyary phloem of the diffuse (forminate) type. Centrally a pith region is located. When trees are wounded they respond by forming new wood cells by the cambium. These cells differentiate and close the wound with newly produced cells (Blanchette, 1992).
2 Agarwood inoculation methods Aquilaria is unique in produceing phloem bundles within the xylem. This network of phloem and parenchyma produce and distribute the resin around affected areas as a tree defense reaction (Blanchette and Van Beek, 2005). There are two kinds of inoculations. i) Natural Inoculation ii) Artificial Inoculation
2.1 Natural inoculation In natural forest, only about 7% of the trees are infected naturally (Ng et al., 1997). Formation of agar wood can be initiated by natural injuries (by ant, snails or fungus) and mostly obtained at the junctures of broken branches.
2.2 Artificial inoculation Artificial inoculation technique developed and standardized in lab-scale is found to be the most effective and reliable method for enhancement of agar wood formation. Some major methods are described here. (a) Nailing method: Hammering of nails into the trunks has been used widely in the past, but the yield from this treatment is generally of poor quality which cannot meet the desired market demand (Persoon, 2007). Iron nails are placed into the trunk spirally in this method. (b) Drilling method: A drill is usually employed to make holes in the trunks and main branches of mature trees (Akter et al., 2013). The drilled pores were placed in a spiral fashion on the tree from the ground line up into the crown. Wounds were placed 3 to 5 cm apart and inoculated with resinous agarwood powder or kept open to ease access of natural agents into the pores. Pores are checked and rewounded every 2-3 months (Blanchette, 2006)
(c) Aeration method, (d) Agar-wit method, (e) Burning-Chisel-Drilling method, (f) Fungi-Inoculation method, (g) Partly-Trunk-Pruning method, (h) bark peeling off.
(c) Aeration method: Aeration means a device inserted into the wound to prevent healing the pores and establish prolonged infection (Liu et al., 2013). This treatment causes tree to respond both physical and chemical defense mechanisms. The aeration device may contain aeration holes in it, and/or it may contain grooves on its exterior surface. It may be made of plastic, bamboo, wood, or other organic material, or metal, such as iron. It may be about 2 cm in diameter. When inserted, the aeration device may extend out about 2 to 15 cm from the exterior of the tree (http://forestpathology. coafes.umn.edu).
This method may also involve applying a resin-inducing agent to cells surrounding the wound. It may kill live parenchyma cells around the wounded region of the xylem. It can be, for example, sodium bisulfate, NaCl, ferric chloride, ferrous chloride, chitin, cellobiose, iron powder or yeast extract. In particular, it may be 1:1:3 sodium bisulfate, Difco yeast extract and iron powder (Blanchette, 2006). Alternatively, or additionally, the resin-inducing agent may be an organism, such as an insect or microb, such as a fungus such as Deuteromyota sp., Ascomycota sp., Basidiomycota sp. This artificial induction could yield agarwood ten times faster than natural formation (Blanchette, 2006).
(d) Agar-wit method: The agarwood induced via Agar-Wit showed its characteristics similar to those of high-grade wild agarwood in terms of texture, chemical constituents, essential oil content, and ethanol-soluble extract content within 20 months (Zhang et al., 2012). Small holes deep into the xylem were drilled above 50 cm from the ground of the main trunk and the inducer was conveniently injected into the Aquilaria trees through a transfusion set. Due to water transpiration pull, the inducer was transported to the whole tree (root to branch) in 2 to 3 h and consequently led to internal wounds. Resinous wood formed around the wounds over several months. Substance like formic acid that have a low pH and NaCl that has a high pH both can disrupt live cells and induce large amount of agarwood.
(e) Burning-Chisel-Drilling method (BCD): To prevent the invasion of environmental microbes, Aquilaria trees were induced to produce agarwood in a closed system by a typical physical wounding method, the burning-chisel-drilling (BCD) treatment (Zhang et al., 2014). The holes in the trunk from approximately 50 cm above the ground to the top of the trunk were achieved by a burning and red-hot iron drill bit (approximately 600°C and 1.2 cm wide). The drilled holes, approximately 20 cm apart, were immediately sealed with sterilized paraffin wax to prevent microorganism invasion.