Christopher G. Jones*, Julie A. Plummer and Elizabeth L. Barbour

Abstract: East Indian sandalwood, Santalum album, is renowned for its fragrant heartwood. Current methods of heartwood oil assessment of standing trees remain largely untested and unreliable. Core sampling of 22 S. album trees at fixed heights followed by solvent extraction of core samples and subsequent analysis was correlated to distilled yields of wood from the same trees. Oil contents of 10-year-old sandalwood trees generally decreased exponentially up the height of the tree, with considerable variation amongst the population. From these trends, core sampling was used to estimate oil yields but large discrepancies existed, and only relative differences could be described. Gas chromatography yielded the most informative results for each core, particularly that the oil composition varied little at 30 cm or at 100 cm, however, there was a slightly higher proportion of sesquiterpene hydrocarbons in samples taken from 100 cm than those from 30 cm. The proportion of sesquiterpene alcohols was generally higher in 30 cm wood cores than in 100 cm samples.

East Indian sandalwood, Santalum album, is a root hemiparasite native to India and the Indonesian archipelago. The heartwood contains the fragrant essential oil that is highly valued. Oil yields of S. album heartwood are typically 3–8% (1). Sandalwood oil consists of the sesquiterpene alcohols (Figure 1) α-santalol [5], β-santalol [8], trans-α-bergamotol [6] and epi-β-santalol [7], as well as the corresponding sesquiterpenes α-santalene [1], trans-α-bergamotene [2], epi-β-santalene [3] and β-santalene [4] . The sapwood of S. album contains little, if any detectable oils (2). The very high value of sandalwood has led to unsustainable harvest rates. In India, natural regeneration of the species is particularly low (3). Plantations of S. album have been established around Southeast Asia and Australia. While the development of suitable silviculture is essential, breeding for reliable seed and superior trees is also required. Selection based on oil yield is difficult because the heartwood does not produce oil until after 10 years of growth and sandalwood trees are deemed mature when they are between 40 and 65 years old (3,4). The heritability of oil production in the heartwood of Santalum sp. is not fully understood, and warrants further investigation. Oil yield is usually based on steam distillation and is expressed as a percentage of dry ground heartwood (5). This is the industry standard but accurate determination of oil yield using this process requires a large sample size - ca. 100 g (6). Such a sample size cannot be removed from the tree without killing it. Core sampling of trees provides a less destructive sample method, but the amount of wood removed is typically about 20 g and this may not be representative of the whole tree. This quantity is insufficient for accurate distillation, however, other extraction and analytical techniques can be performed. Color of heartwood is regarded as an indicator of oil yield and quality (4) but its reliability has not been fully determined. Supercritical carbon dioxide has been used to extract oil from small wood samples of the sandalwood S. spicatum (7) yet the equipment for this analysis is often not readily available. Solvent extraction is particularly favorable due to high reproducibility and low cost, and the extracts possess approximately the same amounts of each component as steam distilled oils (8,9). Solvent extraction followed by some form of analysis provides an alternative to distillation and it can be applied to small sample sizes. Core sample analysis may not be indicative of the actual or overall oil yield, but may provide comparative information. The non-homogenous location of oil-bearing wood within the tree means predictions are error-prone. Thus core-sampling technique must also be standardized. Ideally, cores should be drilled through the central axis of the stem where the heartwood proportion would be greatest, and therefore detectable. The height of core sampling is particularly important, since the heartwood and oil content decreases up the height of the tree (7,10). Gas chromatography (GC) has long been employed for essential oil analysis. Identification of the oil components is often done using mass spectrometry. The main components of Indian sandalwood oil, the santalols, have been identified (11) and more recently the different isomers of these and other minor components have been confirmed. Gas chromatography provides an informative summary of the components and their concentrations. If standards are available, quantities can be very accurately determined with GC. The aim here was to develop a standardized approach towards reliable extraction and analysis of live sandalwood populations for breeding and selection purposes. Core sampling was performed on 10-year-old sandalwood trees and the most suitable heights from which to core were investigated. Wood from core samples were extracted and analyzed using UV-spectroscopy (6) and GC-FID. Chroma value of the ground core sample was also determined to assess the suitability of colour as an indicator of oil yield and composition. All analyses were compared to actual distilled yield of sandalwood from the same trees at the same heights. Composition of the extract solutions was examined. The suitability of the analyses and their application to tree breeding is discussed.