In Vitro Propagation of Aquilaria Species and Induction of Gaharu in Calli and Cell Suspension Cultures

Zul Helmey, Mohamad Sabdin (2016) In Vitro Propagation of Aquilaria Species and Induction of Gaharu in Calli and Cell Suspension Cultures. Masters thesis, Universiti Malaysia Sarawak(UNIMAS).

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Abstract

Aquilaria species such as A. malaccensis and A. microcarpa are considered as a popular local tree species in Malaysia for production of gaharu. In addition to conserving the trees in their natural habitat and ensuring sustainable supply of the agarwood, in vitro techniques such as production of plantlets and induction for resin production in callus need to be developed. The present study started with an attempt to establish contamination-free culture for explants of the two species of Aquilaria using different concentration of Clorox and exposure time. Results showed that samples dipped in 10% Clorox for 15 minutes produced higher axenic leaf and shoot tips explants of the two species of Aquilaria, while treatment with 10% of Clorox and dipped for 20 minutes was the best to produce axenic explants for rachis explants. Lateral buds explants of the two species of Aquilaria produced higher number of axenic tissue after sterilization in 15% Clorox for 10 minutes. For results callus induction from leaf and rachis explants of the Aquilaria species, 100% leaf explants of the two species of Aquilaria produce callus after six weeks of culturing on MS medium supplemented with 2.0 and 3.0 mg/L 2,4-D alone or their combination with 0.5 mg/L BAP. MS medium supplemented with 1.0 and 2.0 mg/L 2,4-D generate rachis to produce callus explants while the MS medium supplemented with 2.0 mg/L 2,4-D alone or in combination with 0.5 mg/L BAP also succeed to produce callus for explants rachis for two species of Aquilaria after six weeks of culturing. The best protocol to establish fast growing cell suspension cultures of the Aquilaria species were cultured in flask containing liquid MS medium supplemented with 2.0 mg/L 2,4-D plus 0.5 mg/L BAP and 40 g/L of sucrose. For result shoot induction, BAP. The lower concentration of BAP (0.25 mg/L) was the best for bud multiplication of explants for both species of the Aquilaria. For lateral buds explants of A. malaccensis and A. microcarpa, the higher number of shoot multiplication was on MS media supplemented with 0.25 mg/L BAP and 0.5 mg/L BAP. In indirect organogenesis using clumps of callus, the higher number of shoot multiplication for both species of the Aquilaria was on MS supplemented with BAP at 1.5 mg/L and Napthaleneacetic α- acid (NAA) at 0.25 mg/L. For results rooting of the regenerated shoots for both of the Aquilaria species, half strength MS medium supplemented with 0.5 mg/L of indole-3-butyric acid (IBA) was the most effective for rooting for both of the plant species. The regenerated plantlets both of the Aquilaria species may successfully adapt to media, such as Jiffy-7, and grew well after planting in mixed media in polybag containing peat, soil and sand in a ratio of 1: 1: 1. For resin production, elicitors P2 and P3 are expected enhancing the production of fragrant in the callus cultures and cell suspension cultures for both of the Aquilaria species. Brown colour or exudate from callus on solid media can be seen and sweet smell also produce in callus on solid and in liquid media for both species of Aquilaria treated with three elicitors after 28 days of incubation. Several important gaharu compounds that were detected in this study including alpha-guaiene, alpha-humulene, jinkoh-eremol, agarospirol and 6,7-dimethoxy-2-(2-pheneylethyl) chromone.

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