In vitro cultures of Aquilaria malaccensis for agarwood production

This thesis describes the results of a series of plant tissue culture, chemical and molecular based experiments aimed at developing an in vitro system to study the fundamental changes in chemical composition or activation of specific chemical pathways which take place during the onset and production of agarwood in Aquilaria malaccensis. Cell suspension cultures were established using callus initiated from shoot and leaf segments excised from in vitro grown plantlet of Aquilaria malaccensis. Callus was successfully established and maintained by culturing leaf segments on MS medium supplemented with 3% sucrose, 0.3% phytagel, 2. 2µM of 2,4-D and 2.3 µM of BAP, and cultured under ambient culture condition i.e. 28 ± 2 oC under continuous dark conditions. Cell suspensions were initiated from the callus lines using the same medium composition, but without the gelling agent and placed on a rotary shaker at 75 rpm. Leaf-derived callus (CS 11) was identified as the preferred source of callus due to the formation of a more homogenous cell suspension cultures which maintained continuous growth after many rounds of sub-culturing. Cell line CS 11 was used for further studies, i.e. determining the effect of elicitation on cell growth, biochemical change and gene expression. In order to effectively study the biochemical changes (sesquiterpenes and chromones production) within the cells in cultures, it would first be necessary to devise suitable analytical methods which would enable the analysis of the effect of elicitation, and to study the chemical profile of each cell culture lines. Various analytical techniques were evaluated using agarwood oil extracts (as standards) and cell cultures as target material. Solid phase micro extraction (SPME) was found to be the most effective technique in detecting the presence of sesquiterpenes and chromones within the cells in cultures. Four sesquiterpenes (alpha humulene, delta guaiene, beta caryophyllene, alpha guaiene) and four chromones (6-methoxy-2-(2-phenylethyl)- chromone, 5,8-dihydroxy-2-(2-(4-methoxyphenylethyl)-chromone, 7-hydroxy-2-(2-phenyl ethyl] chromone and 6-methoxy-2-[2-(3-methoxyphenyl)ethyl] chromones) were found to be produced in unstressed cell suspensions. However it was important to note that although chromones were detected there was no consistent production of any chromones in cell cultures. Overall, the production of sesquiterpenes in cell suspension cultures was found to be higher following elicitation using methyl jasmonate, salicylic acid and ethanol. While salicylic acid was found to enhance cell growth, methyl jasmonate was found to suppress the growth of cells. Unexpectedly the addition of alcohol (0.17µM), the solvent used to dissolve methyl jasmonate was found to have an effect on the production of sesquiterpenes specifically when applied separately where, it was found to induce higher concentration of alpha guaiene and alpha humulene as compared to methyl jasmonate or salicylic acid treatments. The correlation of increase in the production of sesquiterpenes in relation to sesquiterpene synthase expression was also explored in a preliminary study done using the ACL 154 primers whereby the increase in alpha humulene production was found to correlate with an increase in delta guaiene synthase activity suggesting that delta guaiene synthase may be responsible for alpha humulene production in Aquilaria malaccensis. In summary, the combined results of the above studies led to the development of a series of analytical methods and the establishment of an in vitro model system for Aquilaria malaccensis using cell cultures. This represents the first study successfully examining the simulated effect of artificially induced wound (elicitation), in terms of its direct influence on sesquiterpenes profile expressed, and an insight to gene expression patterns which take place within cell cultures of Aquilaria malaccensis.