Factors affecting yield and composition of floral extract from Boronia megastigma Nees

The Tasmanian boronia industry requires detailed information about biological and production factors which affect the yield and quality of floral extract obtained from Boronia megastigma Nees. This study included the sites and routes of biosynthesis and accumulation of particular secondary compounds, phenology of flowering and extract accumulation, genetic and environmental yield-limiting characteristics, conjugates between volatile compounds and sugars, the effect of various post-harvest storage conditions and the process of extraction of product. Cellular differentiation is not a prerequisite for accumulation of volatile compounds; typical floral volatiles are emitted from all floral organs, especially the stigma and anthers. Plastidial droplets and vesicles containing variably osmiophilic material occur within cells surrounding the central cavity of epidermal glands in the petals. The number and size of droplets increase with increasing proximity to the central cavity, and cellular disintegration becomes more apparent. As the content of epidermal glands increases, the cap cell rises above the surface of the epidermis. The septa on the cap cell(s) do not rupture until the petals have lost volatiles, moisture and pigments as a result of senescence, and have abscised and fallen to the ground. This indicates that active release mechanisms are not a prerequisite for fragrance release. There is evidence for enzyme controlled conversion of carotenoids into 13- ionone, most likely by a lipoxygenase. There is a high level of control for 0-ionone production which is lost as a result of senescence, when more random degradation of many compounds by other enzymes and via autoxidation occurs. The activity of biosynthetic enzymes is high in unopened buds and flowers just after anthesis, however activity declines with senescence. The loss of volatiles, pigments and moisture from flowers after anthesis is rapid, and may be dramatic even before obvious visual signs of senescence are apparent. The flowering pattern is an uneven one, and there is good reason not to wait until 100% of flowers are open before harvesting. In late flowering, the loss of flower and extract yield per plant through various catabolic reactions and the action of evaporation on mature flowers is significant. The negative effect on the total yield of product per plant is greater than the effect of reduced extract yield in immature buds which are present in the harvest earlier in the flowering season. In genetically different plants, the natural variation in extract yield is less than the variation in extract composition. The genetic predisposition to accumulate extract is more important than effects due to the environment, plant age or floral characteristics. There is no correlation between the yield of extract per flower and the number of epidermal glands per petal, or the weight of petals or stigmas as a proportion of flower weight. Non glandular production of extract components is probably as, if not more significant than glandular production. This indicates that one of the main factors contributing to high yielding clones which produce a good quality product (a high proportion of volatiles, particularly 0-ionone) is the enhanced activity or concentration of enzymes responsible for secondary metabolism of interest in these plants compared with lower yielding plants. Storage of flowers in enclosed boxes after harvest resulted in substantially higher levels of volatiles including p-ionone in the final product. The rate of respiration was highest in boxes that were regularly opened and disturbed. Post-harvest changes of this sort are not caused by hydrolysis of glycosidically bound volatiles, however a large increase in the level of volatile glycosides during extended storage is indicative of the role of glycosides in the catabolism of volatiles or in the transport of volatiles from flowers prior to abscission. Flowers harvested early in the flowering season have a greater potential for post-harvest changes than those harvested later, indicating that controlled biosynthesis of compounds is most likely to be the cause of the increase in volatiles observed. Freezer storage, prior to storage at room temperature, inhibits the increase in volatile content of flowers during this latter storage. During freezer storage for longer than one month, volatiles and extract yield are depleted. The process of squeezing or partially chopping flowers prior to extraction significantly increases the yield of extract obtained, indicating that some floral tissues are not completely extracted in the absence of this process due to the density of their tissues. A reduction in the solvent volume used during the extraction causes selective extraction of floral compounds such as 13-ionone in preference to more volatile compounds, and yields a product of differing organoleptic properties. The duration of extraction and the washing regimen can also be manipulated also to maximise extract yield and vary the nature of the final product. These results increase the duration of the optimum harvest 'window' and allow for choice of the best harvest date, given that different dates may enable maximal extract yield and increase the potential for post-harvest enhancement of volatile content. The production of an increased range of extracts to suit varying market demands is possible by several means.