B. megastigma plants bear a profusion of strongly scented bell-like flowers (Fig. 1). Petals are yellow on the inside, while outer petal color varies from yellow (cv Lutea) through red to dark brown. Outer petal color may be solid or in vertical red (cv Harlequin) or brown stripes (Plummer 1995). Essential oils are extracted from the flowers with hexane to yield about 0.4%-0.8% concrete. Concrete is washed with alcohol or distilled to yield 60% boronia absolute (Guenther 1949; Penfold and Willis 1954). Boronia absolute is valued at between US$4,000 to $10,000/kg, depending on quality and purity. It can be used in perfumes, cosmetics, and as a food flavoring where it enhances most fruit essences (Davies and Menary 1984; Weyerstahl et al. 1994). There are over 150 compounds in boronia concrete. ß-Ionone is by far the most important, but other compounds such as dodecyl acetate enhance the perfume. The monoterpene hydrocarbons, a-pinene, ß-pinene, and limonene detract from the aroma. Three cultivated genotypes have been shown to have a high degree of variability in their essential oil components (Davies and Menary 1984) but the diversity within the species has not been examined.
Cut flowering stems of B. heterophylla have been exported from Australia to Japan for 7 years and markets are growing in southeast Asia, Europe, and Canada. B. heterophylla is an upright shrub which produces a spectacular display of vibrant pink bell-shaped flowers each about 1 cm long in early spring. Cultivated plants can yield 8-12 stems 12-15 months from planting, 20-30 stems in the third year, and 30-60 stems in the fourth year with a commercial life of 5-6 years. Stems are usually 60 cm long and are picked with >50% of the flowers open (Plummer 1995). Although demand currently exceeds supply, the limited flowering season (1-2 weeks) and lack of flower color options are obstacles to future expansion.
Demand for both boronia absolute from B. megastigma and cut flowering stems of B. heterophylla is increasing. The export market potential is substantial and is likely to continue its rapid growth. Exports from Western Australia alone are expected to increase by 20% per annum. Boronia exports from Western Australia were valued at US$46,000 FOB (freight on board) in 1992/93 while the total value of boronia exports from Australia was US$180,000 FOB in the same year (Anon. 1994). Current wholesale prices for B. heterophylla range from US$2.00 to $3.50 per bunch, depending on supply. Wholesale prices for B. megastigma blossom for essential oil production range from US$10 to $15/kg.
Commercial production of both species is dependent on a few cultivars and selection of superior genotypes would be beneficial. The aim of this study was to investigate variation in the content of essential oil components of B. megastigma within and between populations, and over different seasons, and to extend the color range and production period of B. heterophylla.
Details of flower color, plant age, and plant vigor were collected for all plants. Plant age was determined using the number of years since the last fire or major soil disturbance as an approximate age. Seedlings were determined as less than 1 year old, young plants 1-2 years, mature plants 2 to 6 years and old plants greater than 6 years. Plant vigor was determined by the density of leaves and flowers on each plant. Weak plants had few flowers and little foliage, average plants had moderate amounts of foliage and flowers, and vigorous plants had abundant flowers and lush green foliage. Oil component contents within each category were compared using analysis of variance and means were separated using Fisher's PLSD. The amount of shading by surrounding vegetation was determined for 273 of the plants sampled in 1993 using a Model-A forest densiometer (Lemmon 1957).
Seven to 10 plants from 12 natural populations initially sampled in 1993 were re-sampled in 1994 in order to investigate the quantity of oil components produced by individual plants over successive years. Comparisons were made using paired 2-tailed t-test.
For oil analysis 12 flowers collected from each plant were extracted with absolute ethanol (10 ml). Collection vials were weighed before and after flower collection and the difference used to determine flower fresh weight. Flowers were extracted for at least 24 h before oil analysis. An aliquot (4 ml) of the extract was analysed without further purification or concentration using a gas liquid chromatograph (Hewlett Packard 5890A) fitted with dual columns (50 m length, 0.25 mm internal diameter BPX70 from SGE International and 50 m length, 0.2 mm internal diameter ULTRA1 from Hewlett Packard) connected to a single injection port and twin ionization detectors. The injector and detector temperatures were set at 275°C. An initial oven temperature of 60°C was held for 5 minutes and then increased at a rate of 8°C min-1 to a final temperature of 260°C which was held for 10 min. Ethyl un-decanoate (1 µg ml-1) was used as an internal standard. The quantity (µg g-1 fresh weight of flowers) of a-pinene, ß-pinene, limonene, a-ionone, ß-ionone, and dodecyl acetate was determined using the areas produced by the ULTRA 1 column. The BPX70 column was used to confirm the presence or absence of a-ionone, ß-ionone and dodecyl acetate. a-Pinene, ß-pinene, and limonene co-eluted with the solvent peak on this column.
Principal components analyses were performed on the oil data. The first analysis used the oil data from all individuals sampled to plot the orthogonal factor scores and determine the relationship between the different oil components. The second analysis used the mean data for each oil component from each population to determine the relationship between different populations in terms of the 5 oil components.
The mean quantity of oil components varied considerably between populations. a-pinene, ß-pinene and limonene were commonly absent from individual plants, but each of these compounds was present in at least one plant in each population. Location had some influence on the content of the monoterpene hydrocarbons. Southeastern populations had quite high concentrations of a-pinene, ß-pinene, and limonene, while northern populations all had low concentrations. Location was not always a good indicator of the content of oil components. In the Jarrahwood area, Jarrahwood-3 produced more than twice the amount of a-pinene and ß-pinene as Jarrahwood-2 and Jarrahwood-4, even though the three populations are only a short distance apart. Location did not influence ß-ionone and dodecyl acetate. The highest mean ß-ionone content for a population was 736 µg g-1 at Albany-1 and the lowest was 227 µg g-1 at Porongurup-1.
The quantity of oil components from plants within populations varied considerably. The Walpole-1 population sampled in 1993 was typical of the variation in ß-ionone content within populations. ß-Ionone concentration ranged from very low (175 µg g-1) to extremely high (1342 µg g-1).
The relationship between oil components based on their quantitative presence in plants was investigated using principal components analysis. Results indicated that ß-ionone and dodecyl acetate were closely related and quite distinct from the monoterpene hydrocarbons. The was also a close relationship among the monoterpene hydrocarbons. a-Pinene and ß-pinene, and to a lesser extent, limonene were closely related.
Principal components analysis was also used to investigate the relationship between B. megastigma populations based on the mean content of oil components at each site. Results failed to show any distinct relationships between populations. However, among the populations sampled in each year there were certain populations which were closely related in terms of the mean content of the 5 oil components. Some populations in close proximity to each other were similar in oil composition, but others were distinct. Boyup Brook, Palgarup-1 and Palgarup-2 were within 33 km of each other and were closely related (1993 data). Jarrahwood-2 and Jarrahwood-4 were closely related, but Jarrahwood-3 was quite distinct (1994 data).
Canopy cover above B. megastigma plants affected concentrations of the monoterpene hydrocarbons but not ß-ionone (y = 0.6x + 365; r2 = 0.005) and dodecyl acetate (y = 0.8x + 273; r2 = 0.009). Plants shaded by higher densities of canopy cover produced lower concentrations of -pinene (y = -3x + 267; r2 = 0.09), ß-pinene (y = -2.2x + 156; r2 = 0.09), and limonene (y = -2.1x + 160; r2 = 0.09).
Plant age, plant vigor, and flower color each affected the concentration of at least one of the five essential oil components investigated. Similar trends occurred in both years but only 1993 data is presented. Age affected four essential oil components (Table 2). Young plants contained less alpha-pinene and limonene than mature plants and less ß-pinene than seedlings, mature and old plants. Old plants had the most dodecyl acetate. Seedlings had more ß-pinene and limonene, and less ß-ionone and dodecyl acetate than older plants but, due to the low sample size of seedlings, the differences were not significant. Vigorous plants tended to produce more ß-pinene and limonene than weak plants (Table 3). Red flowers contained the lowest concentrations of ß-ionone and dodecyl acetate (Table 4). There was a tendency for red/brown flowers to have less ß-ionone and dodecyl acetate than brown flowers. Yellow flowers contained no ß-pinene.
In plants sampled in both years ß-ionone and dodecyl acetate content differed between the 1993 and 1994 sampling. The mean quantity of ß-ionone and dodecyl acetate was higher in 1994 than 1993, while a-pinene, ß-pinene and limonene content did not differ (Table 5). The mean quantity of the majority of oil components for each population sampled in 1994 was the same as for 1993. Where differences did occur, they could be attributed to either large differences in an individual plant, or numerous small differences in several plants within the population.
Twenty six plants were found to have no a-pinene in at least 1 of the years sampled. Of the plants found to have no a-pinene present in either the 1993 or 1994 analysis, 69% were found to have no a-pinene in both years. No ß-pinene was found in 53 plants in at least 1 of the years sampled. Of these plants, 85% had no ß-pinene in both years. Of the plants lacking limonene, only 21% had no limonene in both years. Many plants consistently had two or more of the hydrocarbon monoterpenes absent, and several were lacking all three.
The relationships between ß-ionone and dodecyl acetate and a-pinene, ß-pinene, and limonene is supported by the work of Bussell et al. (1995) on volatile oil ontogeny in B. megastigma. The stigma, stamen, and staminode tissue contained dodecanol, ß-ionone, dodecyl acetate, and heptadecene among the principal components. ß-Ionone and dodecyl acetate concentrations were highest in the stigma extracts. Extracts of the combined ovary, nectary, and receptacle tissue contained primarily a-pinene, ß-pinene, and limonene. The petal tissue contained relatively low concentrations of all of the above-mentioned volatile compounds.
The recorded characteristics of B. megastigma and B. heterophylla varied considerably between populations. Although current populations were quite isolated from one another, this is likely to be a recent event due to a number of factors, such as over-picking, frequent fires, and land clearing for agriculture. Boronia flowers are pollinated by an unidentified small moth (Bussell et al. 1995). It is possible that as populations have become more isolated, the movement of pollinators could be restricted to individual populations or a few close populations. This would result in distant populations becoming more genetically isolated over time. For example B. megastigma populations in the southeastern area of their range had quite high concentrations of a-pinene, ß-pinene, and limonene while northern populations all had low concentrations.
Populations in close proximity may remain genetically similar not only due to pollen transfer but also water flow which could wash seeds down river from one population to another. Populations within the same water catchment area, e.g. (B. megastigma) Jarrahwood-2 and Jarrahwood-4, appeared closely related, while nearby populations located within different catchment areas e.g. Jarrahwood-3 were occasionally quite distinct. However, location did not always explain the differences between populations. There are many environmental factors which may affect flower color, oil content, and plant vigor.
Canopy cover was used as a crude measure of shading levels. In open conditions with little canopy cover and shade, B. megastigma produced high amounts of a-pinene, ß-pinene, and limonene. In dense forest where canopy cover and shade levels were high, monoterpene hydrocarbons were produced in lower quantities. A number of essential oil species have decreased yields when they are grown under shade. In peppermint (Mentha piperita) high oil yield, including the production of limonene, results from high light intensity (Clark and Menary 1980). Reduced oil production occurs under low radiation in Japanese mint (Mentha arvensis) (Duriyaprapan and Britten 1982). In contrast to our results, experiments by Bussell (1996) showed that 70% shade cloth (30% of ambient light) had no effect on a-pinene and limonene concentrations (ß-pinene was not tested) in three clones of B. megastigma and ß-ionone and dodecyl acetate concentrations increased. Although the genetic material used by Bussel (1996) was different, the shade levels much higher and the plants grown under cultivation, these differences highlight the need to further investigate the influence of shading. Light may also affect flower color in B. heterophylla. Anthocyanins contribute to red flower color and anthocyanin production in many species is modified by light quality and intensity. Flowers of B. heterophylla are known to fade during senescence (Plummer 1995) but the influence of light on pigment intensity remains unknown.
Plant age, vigor, and flower color affected the concentration of oil components in B. megastigma. Unfortunately concentrations of oil components produced by seedlings was not a good indication of production in the adult plant. This reduces the possibility of screening seedling material for high quality oil in a breeding program. Red-flowering plants are unlikely to be used in commercial production of oil as they produced low amounts of the favorable compounds, ß-ionone and dodecyl acetate. Plants with yellow flowers produced very little limonene and no ß-pinene but relatively high levels of ß-ionone and dodecyl acetate. Bussell (1996) also found ß-pinene to be absent from yellow flowering B. megastigma. Apart from these trends flower color does not appear to be a useful tool in selecting plants with high oil quality.
Some differences in oil content were detected between 1993 and 1994. The concentrations of the undesirable oil components, a-pinene, ß-pinene, and limonene were fairly consistent which permits genetic selection for low or no production. The change in ß-ionone and dodecyl acetate content was only marginal but offers the possibility of environmental manipulation, such as site selection, to increase yield.
Boronia heterophylla plants with a range of solid and striped flower colors, and plants which flower outside the usual two-week flowering period were identified. Most of these plants could be propagated successfully. Plants will need to be examined in cultivation to determine if these characteristics are genetically controlled. Plants will also need to be assessed for their ability to produce numerous high quality stems and withstand the substantial pruning at harvest. Selected plants with these attributes will increase the colour range and harvest period.
Although many of the original populations of B. megastigma and B. heterophylla no longer exist much variation exists in the remaining plants. Natural pollination of boronias requires the presence of a pollinator moth and her movements tend to ensure out-crossing. Thus the remaining plants probably contain most of the original genetic diversity. Boronias can be manually self and cross pollinated (Plummer and Considine 1995) and a breeding program has commenced. B. megastigma plants with high yields of ß-ionone and dodecyl acetate and low or no monoterpene production were identified. Collection from natural stands is inadequate to meet demand and expansion of cultivation is underway. Currently boronia absolute is only used in the food flavoring sector and development of new uses will require expanded production. B. heterophylla plants with a range of pink flower tones and different flowering periods were identified. Although these characteristics will need to be confirmed under conditions of commercial cultivation they show promise for the improvement of product quality and the expansion of production of these new crops.
| Oil content (µg g-1 FW) | ||||||
| Rank | % of total plants | a-Pinene | ß-Pinene | Limonene | ß-Ionone | Dodecyl acetate |
| Extremely high | 5 | 420-1001 | 298-940 | 264-893 | 803-1787 | 577-1291 |
| Very high | 5 | 303-419 | 207-297 | 184-263 | 646-802 | 518-576 |
| High | 10 | 193-302 | 140-206 | 118-183 | 548-645 | 427-517 |
| Medium | 60 | 0-192 | 0-139 | 33-117 | 260-547 | 178-426 |
| Low | 10 | 0 | 0 | 25-32 | 199-259 | 115-177 |
| Very low | 5 | 0 | 0 | 15-24 | 162-198 | 84-114 |
| Extremely low | 5 | 0 | 0 | 0-14 | 70-161 | 0-83 |
| Component (µg g-1 FW) | |||||
| Plant age | a-Pinene | ß-Pinene | Limonene | ß-Ionone | Dodecyl acetate |
| Seedling | 156ab | 231b | 125ab | 234a | 169ab |
| Young | 90a | 29a | 44a | 395a | 274a |
| Mature | 159b | 93b | 99b | 382a | 297a |
| Old | 144ab | 87b | 60ab | 391a | 364b |
| Component (µg g-1 FW) | |||||
| Plant vigor | a-Pinene | ß-Pinene | Limonene | ß-Ionone | Dodecyl acetate |
| Weak | 120a | 76ab | 48a | 411a | 347a |
| Average | 145a | 80a | 84a | 383a | 303a |
| Vigorous | 207a | 161b | 157b | 379a | 265a |
| Component (µg g-1 FW) | |||||
| Flower color | a-Pinene | ß-Pinene | Limonene | ß-Ionone | Dodecyl acetate |
| Yellow | 287a | 0 | 27a | 380ab | 273abc |
| Red | 121a | 100a | 86a | 263a | 172a |
| Red/brown | 140a | 79a | 84a | 378b | 280b |
| Brown | 157a | 88a | 90a | 407b | 338c |
| Component (µg g-1 FW) | |||||
| Year | a-Pinene | ß-Pinene | Limonene | ß-Ionone | Dodecyl acetate |
| 1993 | 157±17 | 86±11 | 80±7 | 415±16 | 323±16 |
| 1994 | 142±15 | 81±10 | 84±7 | 452±16 | 398±17 |
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Fig. 1. Young (2-year-old) Boronia megastigma plant in cultivation in Nannup, Western Australia. |
Fig. 2. Boronia megastigma populations in the south west of Western Australia. l denotes populations sampled in 1993, and u denotes populations sampled in 1994.