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Volatiles in Different Floral Organs, and Effect of Floral Characteristics on Yield of Extract fromBoronia megastigma(Nees)
Annals of Botany 80 : 305–311, 1997
Volatiles in Different Floral Organs, and Effect of Floral Characteristics on Yield of Extract from Boronia megastigma (Nees) H A Z E L S. M A C T A V I S H* and R O B E RT C. M E N A R Y Department of Agricultural Science, Uniersity of Tasmania, G.P.O. Box 252-54 Hobart, Tasmania, Australia 7001 Received : 6 February 1997
Accepted : 4 April 1997
The relative amounts of volatile compounds in the extract and headspace from each floral organ were assessed in order to identify the main organs for accumulation and emission. The mass of flowers}organs, the number}density of oil glands and yield of volatiles were examined for their relationship with extract yield, in clonal and non-clonal plants. Boronia flowers were divided into component organs and the solvent extractable product and headspace above each organ type was quantified. The petals comprised 50 % of the weight of the flowers, and the stigma 20 % ; however, the stigma contributed 70 % of the total volatile compounds to extract from the whole flower. Proportionately more β-ionone and dodecyl acetate were emitted from the stigma and anthers than were contained in the extract, compared with other volatiles. The sexual organs are morphologically equipped for emission of volatiles to attract pollinators. Between non-clonal plants, there was a lower coefficient of variation for extract yield than for values relating to extract composition, indicating that the former is more heritable than the latter. Variation between clonal plants was reduced compared with variation between non-clonal plants. The environment modifies yield and quality of extract in clonal plants, indicating that both have relatively low heritability. No significant relationships between any floral characteristics and extract yield were found. Biosynthetic potential to accumulate extract is therefore of prime importance, and the effect of environment on this potential should be the subject of future work. # 1997 Annals of Botany Company Key words : Boronia megastigma, brown boronia, Rutaceae, essential oils, flower, stigma, oil gland, β-ionone.
INTRODUCTION Boronia megastigma Nees. (brown boronia, family Rutaceae) is an endemic Australian shrub grown commercially in Tasmania for production of a highly valued floral extract. The strongly perfumed red-brown and golden boronia flowers are harvested in September and extracted by solvent to yield between 0±3 and 0±7 % (by fresh flower weight) of a yellow-brown waxy concrete. Boronia extract (concrete) is an extremely complex mixture of compounds including monoterpenes, cinnamates, norisoprenoids including ionones, related epoxides and dihydro compounds, acetates of decyl, dodecyl and tetradecyl alcohols and methyl jasmonate isomers and triterpenes (Guenther 1974 ; Davies and Menary 1983 ; Weyerstahl et al., 1994). β-ionone is the major volatile (12–30 % of total volatiles). The abaxial surface of the four petals of boronia flowers is usually red-brown or purple and dotted with translucent glands, the adaxial surface is bright yellow. The flowers are dominated by a large, brown, four-lobed stigma. Pollen is produced by four small, yellow, petaline anthers (functional), however no pollen is formed by the four large, redbrown, sepaline anthers (non-functional). Flowers also comprise four sepals and a calyx, considered together in this study. The small fruits do not set in Tasmania, presumably because the pollinating vectors are absent (MacTavish, 1995). Plantations are established from vegetatively propagated clonal plants. * For correspondence.
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Usually all floral organs contain volatiles typical of that species (Mihailova, Atanasova and Balinova-Tsvetkova, 1977). However, quantitative differences between attractive, non-food structures (perianth) and attractive food sources such as the androecium and gynoecium are common (Francis and Allcock, 1969 ; Dobson, Bergstro$ m and Groth, 1990). Generally, petals and female organs have similar oil compositions but a lower yield than stamens which have markedly different compositions (Attaway, Pieringer and Barabas, 1966 ; Mihailova et al., 1977 ; Pichersky et al., 1994). Quantitative differences in the volatiles emitted from flower parts may be important in effecting pollinator attraction (Dobson et al., 1990 ; Hansted, Jakobsen and Olsen, 1994). Typical floral volatiles are found in all floral organs of boronia flowers (Wilson, 1982), however studies on the contribution of each organ to the extract obtained from the whole flower have so far been preliminary and not quantitative (Bussell, Considine and Spadek, 1995). Analysis of volatiles emitted into the atmosphere (so-called ‘ headspace ’) above boronia flowers or extract has not previously been reported. Vegetative essential oils are usually contained within glands or trichomes ; in many cases there is a positive relationship between the number of glands and essential oil yield (Henderson et al., 1970 ; Tanaka et al., 1989). In rose flowers there are no specialized glands for accumulation of extract, the simple glandular epidermal tissue is the site of terpene biosynthesis (Loomis and Croteau, 1973 ; Stubbs and Francis, 1971). In such cases, one would expect to find a correlation between yield of
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MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma
extract per flower and factors such as flower and}or petal size and}or weight, however Patra et al. (1987) found no such correlations in rose. Sharma and Farooqi (1990) found that in damask rose, the concentration of extract in flowers of differing mass was constant and hence, flowers with greater mass contained more extract per flower. In boronia, flower yield per hectare is directly correlated with flower size, the number of flowers per node, the number of flowers per plant and the number of plants per hectare (Roberts, 1989). To date, most of the published work on boronia has concentrated on flower production. Oil glands have been noted on the epidermis of boronia flower petals (Haberlandt, 1928) and small numbers of glands have been observed on other organs including the receptacle, ovary and nectary (Wilson, 1982 ; Bussell et al., 1995). The structure and ultrastructure of the oil glands on the petals has been described (MacTavish, 1995) ; glands appear to develop lysigenously. It has been assumed that most of the floral extract is contained within these glands ; Wilson (1982) raised the question of whether the volume or number of oil glands on the petals or the size of the stigma may be related to extract yield from boronia flowers. The effect of various morphological characteristics on extract yield have not been examined. A more detailed knowledge of the factors that affect extract yield and composition is required. A quantitative study of the contribution of each organ to the extract has been undertaken. Floral characteristics, which may be indicative of extract yield, were studied. These included factors such as flower yield per plant, the average flower weight, the average weight of the stigma and petals and the number of oil glands per petal. The identification of floral characteristics which are positively linked with extract yield may greatly benefit selection for new plants to clone for commercial production.
MATERIALS AND METHODS Plant material Unless otherwise stated, plant material used in this study comprised clonal plants of brown boronia (Boronia megastigma Nees), developed by the University of Tasmania and grown on commercial plantations in Tasmania over several growing seasons.
Solents Two solvents were used : re-distilled technical grade petroleum ether (b.p. 40–60 °C) (pet. ether) and analytical grade hexane (Mallinckrodt).
Floral extracts Small scale extracts. Extracts of all floral organs were made using pooled, dissected organs from 40 flowers (2±0 g). Flower material was extracted in hexane with an octadecane internal standard for 12 h at 4 °C. After removal of the
plant material, the hexane was concentrated under nitrogen gas and analysed by GC. Laboratory scale extracts. Twenty grams of frozen flowers were extracted in triplicate using pet. ether. The flower} solvent mixture was homogenized for 10–15 s using an Ultra-Turrax (Janke and Kunkel). The flowers were extracted at room temperature during two washes of 2 h each, followed by a final rinse of 5 min. Solvent from the three washes was pooled and evaporated to dryness at reduced pressure on a rotary vacuum evaporator at 60 °C. The yield of extract was calculated as a percentage of the fresh flower weight. Gas chromatography A Hewlett Packard 5890 Series II gas chromatograph equipped with a flame ionisation detector (FID), a splitinjection system and an HP-1 cross linked methyl silicon gum column 30 m¬0±2 mm i.d., film thickness 0±33 microns, was used. (Carrier gas : N ! 2 ml min−", head pressure # 12 psi and split ratio of 1 : 50. Oven temperature programme : 50 °C for 1 min, then 10 °C min−" to 250 °C. Injector temperature : 250 °C, detector temperature : 280 °C. Injection volume : 1µl.) Peaks were initially identified by GC}MS (Davies and Menary, 1983). Quantitative peak estimation was achieved by addition of octadecane as an internal standard, an FID response factor of one was assumed. Total volatiles were calculated as the fraction of the GC-analysable material eluting before n-heneicosane (Davies and Menary, 1983). Headspace analysis Twelve flowers for headspace analysis were selected from one lateral of one plant, dissected into their component organs (four petals, four sepals one calyx, four functional (petaline) anthers, four non-functional (sepaline) anthers and one stigma), and sealed into 10 ml glass headspace sample vials. Vials were left at room temperature to equilibrate for 10 min. Subsequently, 20 ml of the headspace (two injections of 10 ml each with a 1 min equilibration period between samples) was injected onto a cryotrap and subsequently onto the GCMS. A Hewlett Packard 5890 Series II GC equipped with MS (HP 5970), a split-less injection system and a BP-1 cross linked methyl silicon gum column 50 m¬0±2 mm i.d., film thickness 0±33 microns, was used. (Carrier gas : Helium ! 3 ml min−", head pressure 10 psi. Oven temperature programme : 30 °C for 2 min, then 6 °C min−" to 150 °C, then 10 °C min−" to 250 °C. Injector temperature : 250 °C, detector temperature : 260 °C, ion source : 70eV, selected ion monitoring was used to detect peaks.) No internal standards were used because of the lack of appropriate standards for the solid-sample type used. The peak areas of identified volatiles in blank samples were subtracted from all other samples. Enumeration of oil glands Non-clonal plants from one site in one season were used. The weight of petals as a proportion of the total fresh flower
MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma weight was assessed using 50 flowers per plant. Twenty randomly selected petals per plant were mounted in glycerine onto glass slides, abaxial surface uppermost, and examined under a binocular dissecting microscope. Photographs were taken from which oil glands were counted. The oil glands appeared as non-pigmented areas in the dark red-brown abaxial epidermis. Natural pigmentation obscured counting on some petals, therefore these petals were extracted with acidified ethanol, and then re-stained with crystal violet and photographed as described previously.
Surface area of petals From enlarged photocopies of the photographs of the petals, the area of the petal was cut out and weighed. By comparison between the weight of a known area of tonerblackened photocopy paper, the area of the petal was calculated, from which the oil gland density was subsequently calculated.
307
A. Weight of component organs
C. Total volatiles in headspace from component organs
B. Total volatiles in extract from component organs
Floral characteristics and extract yield studies
RESULTS Weight of indiidual floral organs The petals of open flowers comprised approx. 50 % of the total flower by weight ; the stigma and calyx (sepals and pedicel) were equivalent in weight, comprising about 22 % of the total flower weight each (Fig. 1 A). The mass of nonfunctional anthers was five times greater than that of functional anthers.
Extract and headspace emission from component organs Sixty five percent of the volatiles in the extract from the whole flower were contributed by the stigma, only 20 % were present in the petals (Fig. 1 B). The calyx, however, contributed 51 % of total volatiles emitted into the headspace from the whole flower (Fig. 1 C). Other organs contributed in the order stigma (16 %) " functional anther (15 %) " non-functional anther (14 %) " petal (3 %). Volatiles were chosen with different volatilities namely αpinene, β-ionone, dodecyl acetate, methyl jasmonate and methyl epijasmonate (together) and (Z)-heptadec-8-ene. These compounds were expressed as a percentage of the total volatiles in the extract and headspace of each organ
F. 1. Contribution of each floral organ to A, mass of the whole flower (g) ; B, extract per flower (mg) ; C, volatiles emitted into the headspace from the whole flower. Petals, 4 (+) ; stigma, 1 (8) ; functional anthers, 4 (7) ; non-functional anthers, 4 ( ) ; calyx including sepals, 1 (*).
100 % of each volatile in extract from each organ
Studies on the relationship between flower yield per plant and extract yield used 15 clonal plants grown at one site in one season. Studies on the relationship between extract yield and average flower weight, oil gland distribution and weight of component organs (petals, stigmas) used non-clonal plants from two sites over two seasons. The average weight of 50 individual flowers was assessed. The stigmas from 25 flowers were removed at a natural point of abscission, and the separated stigmas and flowers-without-stigmas were weighed.
80 60 40 20 0
Petals
Stigma Functional NonCalyx + anthers functional sepals anthers
F. 2. Relative amounts of selected volatiles in extracts from each floral organ, expressed as a % of the total volatiles in the extract from each organ type. α-pinene (9) ; β-ionone (:) ; dodecyl acetate (+) ; (Z)-heptadec-8-ene ( ) ; methyl jasmonate and methyl epijasmonate (8) ; total other volatiles (*).
type (Figs 2 and 3, respectively), the jasmonates were not calculated in the headspace due to low detection in some organs. Extract from all organs contained all volatiles examined (Fig. 2). Highly volatile compounds such as αpinene predominated in the headspace from the petals and calyx, although other, less volatile compounds were present in extract from petals at relatively higher levels. (Z)heptadec-8-ene, which is not particularly volatile, nevertheless comprised significant proportions of the emission
MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma
% of each volatile in headspace from each organ
308
T 2. Summary of flower and extract yield per plant, and extract concentration and composition from 15 clonal plants at one site in one season
100 80 60
Value
40 20 0
Petals
Stigma Functional NonCalyx + anther functional sepals anther
F. 3. Relative amounts of selected volatiles in the headspace from each floral organ, expressed as a % of the total headspace from each organ type. α-pinene (9) ; β-ionone (:) ; dodecyl acetate ( ) ; (Z)heptadec-8-ene (7) ; total other volatiles (*).
T 1. Summary of yield of extract and extract composition from 61 genetically different plants Value
Total volatiles (% by f.wt)
Yield of extract (% by f. wt) Total volatiles (% by f. wt¬10−$) β-ionone (% by f. wt¬10−$
Mean
s.d.
Coefficient of variation
0±4584
0±0665
14±515
0±291
Range
59±831
22±995
38±433
140±235
14±486
8±030
55±434
56±304
0.175 0.150 0.125 0.100 0.075 0.050 0.025 0 0.30
0.35 0.40 0.45 0.50 0.55 0.60 Yield of extract (% by fresh weight)
0.65
F. 4. Relationship between yield of extract (% by fresh weight) and total volatiles (mg}100 g flower material) in the extract from 61 genetically different plants.
Mean
Flower yield per plant (g) 146±44 Extract yield (% by 0±35 f. wt) Total extract per plant 0±52 (g) Total volatiles (% by f. wt¬10−$) 40±98 β-ionone (% by f. wt¬10−$) 14±91
s.d.
Coefficient of variation Range
51±13 0±02
34±92 4±73
0±19 0±06
0±19
36±88
0±69
6±45
15±74
23±00
2±30
15±45
8±91
ionone, than for yield of extract (% by f. wt). There was an increase in the total amount of volatiles (% by f. wt) in flowers with yields of extract between 0±32 and 0±48 % (Fig. 4). Subsequent increases in extract yield did not produce increased volatiles in flower material, however, plants with particularly high yields of extract and volatiles were observed (Fig. 4). Flower yield per plant The variation in the flower and extract yield between clonal plants at one site in 1 year was analysed (Table 2). The yield of extract per plant was equal to flower yield per plant multiplied by the concentration of extract in the flower material. There was, therefore, a significant positive relationship between flower yield per plant and extract yield (mg) per plant (R# ¯ 0±986). The yield of flower material per plant (and hence extract yield per plant) varied widely between plants, however, the concentration of extract in flower material varied to a lesser extent. Variation between plants in the concentration of volatiles including β-ionone was greater than the variation in extract concentration. There were no significant relationships between the yield of flower material per plant and either the concentration of extract (% by f. wt) or the yield of extract per flower (Table 3). Aerage flower weight
from floral organs, particularly the sexual organs. Relatively high levels of β-ionone and dodecyl acetate were emitted from the sexual organs compared with proportional levels in the extract from these organs.
There was a trend for heavier flowers to contain more extract per flower (R# ¯ 0±470) (Table 3). There were, however, no significant relationships between the average weight of a fresh, open flower and either extract yield per flower or extract concentration (% by f. wt) in non-clonal plants (Table 3).
Variation in non-clonal plants
Weight of petals and number of oil glands
Sixty one non-clonal plants were examined for yield of extract and composition (Table 1). The coefficients of variation and standard deviations were greater for values relating to extract composition i.e. total volatiles and β-
There were no significant relationships between the weight of the petals as a proportion of the flower and extract yield per flower or extract concentration (Table 3). The surface area of petals and the variation in the density of oil glands
MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma
309
T 3. Correlation coefficients between arious floral characteristics and yield of extract per flower and extract concentration (% by f. wt) Extract per flower (mg)
Extract concentration (% by f. wt)
0±203 0±470
0±198 0±028 (negative slope)
0±210 0±051 (negative slope)
0±411 0±298 (negative slope)
not calculated
0±427 (negative slope)
Ratio of stigma weight : flower weight
0±00
Average stigma weight (mg)
0±348
0±475 (negative slope) 0±638 (negative slope)
Value Flower yield per plant (g) Average flower weight (g) Ration of petal weight : flower weight No. of oil glands per petal Density of oil glands (glands mm−#)
T 4. Surface area of petals, oil gland distribution and extract yield in genetically different plants Value Surface area of petals (mm#) Number of oil glands}petal Density of oil glands (no mm−#) Yield of extract (mg}flower)
Mean
s.d.
Coefficient of variation
181±64
20±59
11±33
204±29
39±16
19±17
1±12
0±16
14±05
194±71
8±70
14±13
and extract yield per flower in non-clonal plants is presented (Table 4). There was no relationship between the number of oil glands per petal and the concentration of extract (% by f. wt) (Table 3). There was a trend for petals with an increased density of oil glands to have a reduced concentration of extract. There were no significant relationships between the number of oil glands per petal and the composition of extract. Weight of the stigma There were no significant relationships between the weight of the stigma (or the weight of the stigma as a proportion of the whole flower) and either extract per flower, or extract concentration (% by f. wt) (Table 3). DISCUSSION The petals of boronia flowers are not the major source of extract or volatile compounds. The important role of the stigma in the composition of the extract is evident : it comprises only 22 % of the weight of the flower but contains 70 % of the volatiles ; there are few, if any, distinct glands present on this organ (Leggett 1979 ; MacTavish, 1995). The
anthers and stigma also emitted relatively high amounts of ‘ floral ’ volatiles such as β-ionone and dodecyl acetate. A comparison of the levels of such compounds in the extract and headspace from all organs, shows that the sexual organs emit relatively higher levels of these compounds than other, sometimes more volatile compounds which also occur in the extracts from these organs. The morphology of the sexual organs (MacTavish, 1995) suggests that they have a glandular epidermis (Francis and Allcock, 1969). It appears that glandular epidermis is capable of accumulating relatively higher concentrations of volatiles than discrete glands, such as those found on the epidermis of the petals. This supports other work on this species (Bussell et al., 1995). The headspace emitted from different floral organs demonstrates the dominance of more volatile compounds, such as α-pinene, over less volatile compounds such as βionone. The low threshold of β-ionone and related compounds, particularly jasmonates, has a dominant effect on fragrance perception, even though emission levels may be relatively minuscule. Boronia megastigma is believed to be pollinated by a moth, probably in the family Heliozelidae (MacTavish, 1995). Therefore, relatively high levels of emission of compounds such as dodecyl acetate, an active component of moth pheromones, in preference to other, more volatile compounds from the anthers may play an important ecological role. Between non-clonal plants, the yield of extract had a lower coefficient of variation than the level of volatiles. In plants where higher yields of extract occurred, higher levels of volatiles also occurred, indicating that particular plants contained more, or more active biosynthetic enzymes for all components of the extract. Relatively low variability in extract yield compared with extract composition may be the result of low variability in compounds which comprise the bulk of the extract such as waxes, pigments and other nonvolatile compounds, some of which may be products of primary metabolism. Secondary compounds present in aromatic extracts may arise by one-step processes from primary compounds, or from small modifications to
310
MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma
ubiquitous secondary compounds such as geranial. As such, secondary compounds may vary more between plants as a result of minor genetic and environmental differences. It is likely that β-ionone, the dominant volatile in boronia flowers, is produced by a one or two step process from carotenoids such as β-carotene (Enzell, 1985 ; Kanasawud and Crouzet, 1990 ; Lutz and Winterhalter, 1992), and therefore variation in the levels of this compound may be affected by differences in the amount or availability of substrate, or the activity of cleavage enzyme(s). The analytical methods used for determination of extract concentration generally produce larger variation than the methods used for quantitation of volatiles, therefore experimental technique is unlikely to produce the results observed. Extract yield and composition varied less between clonal plants than between non-clonal plants. There are large numbers of four distinct clonal types grown in Tasmania ; their yield and extract varies between sites (MacTavish, 1995). This indicates that phenotypic expression and environmental conditions predominate over genotype to a certain extent. Environmental factors were not considered in this study. The concentration of extract (% by fresh flower weight) appears to be negatively correlated with the density of oil glands on the petals, suggesting that glands may be smaller or less full when large numbers occur, although the trend is not significant (R# ¯ 0±427). The effect of the size and volume of oil glands was not studied. Since there are no floral characteristics which are positively correlated with extract yield, clonal selection must be based on the performance of plants with regard to extract yield and composition, as well as other agronomic factors, at several sites characteristic of potential commercial sites. Once suitable plants are selected, increased yields of extract per hectare can be obtained by increasing the number of flowers initiated each year, possibly by the use of appropriate fertilizer application (Roberts and Menary, 1994). Accumulation of extract in non-gland bearing tissues in boronia flowers indicates that activity of biosynthetic enzymes is a more significant factor contributing to increased yields of extract than the number of glands present on the petals. Further, boronia flowers with larger organs with an active glandular epidermis, such as the stigma, do not necessarily produce high yields of floral extract, indicating that there are other limits to accumulation of extract, one of which may be toxicity of products when not localized in glandular structures (Brown, Hegarty and Charlwood, 1987). There are large amounts of glycosidically bound volatiles in boronia flowers, including glycosides of βionone (MacTavish, 1995), which may act as non-toxic storage of products in a water soluble form. The localization of glycosides within organs, and the relationship between free and bound volatiles and extract yield have yet to be considered, but will be the subject of future publications. The work presented here furthers our understanding of the limitations to extract production in boronia flowers and provides a basis for more directed research, namely the identification of enzymes active in the synthesis of important components of boronia extract.
A C K N O W L E D G E M E N TS The authors acknowledge support from an Australian Postgraduate Research Award (Industry), with industry collaboration from Essential Oils of Tasmania Pty. Ltd. and Natural Plant Extracts Co-operative Pty. Ltd. We thank Noel Davies of the Central Science Laboratory at the University of Tasmania for help with headspace analysis. LITERATURE CITED Attaway JA, Pieringer AP, Barabas LJ. 1966. The origin of citrus flavor components–II. Identification of volatile components from citrus blossoms. Phytochemistry 5 : 1273–1279. Brown JT, Hegarty PK, Charlwood BV. 1987. The toxicity of monoterpenes to plant cell cultures. Plant Science 48 : 195–201. Bussell BM, Considine JA, Spadek ZE. 1995. Flower and volatile oil ontogeny in Boronia megastgima. Annals of Botany 76 : 457–463. Davies NW, Menary RC. 1983. Volatile constituents of Boronia megastigma flowers. Perfumer and Flaorist 8 : 3–8. Dobson HEM, Bergstro$ m G, Groth I. 1990. Differences in fragrance chemistry between flower parts of Rosa rugosa Thunb. (Rosaceae). Israel Journal of Botany 39 : 143–156. Enzell C. 1985. Biodegradation of carotenoids – an important route to aroma compounds. Pure and Applied Chemistry 57 : 693–700. Francis MJO, Allcock C. 1969. Geraniol β--glucoside ; occurrence and synthesis in rose flowers. Phytochemistry 8 : 1339–1347. Guenther E. 1974. Oil of Boronia megastigma (Boronia flower oil). In : Guenther E. The essential oils. Vol. 3 – Indiidual essential oils of the plant families Rutaceae and Labiatea. R. E. Krieger Publishing Company, 364–367. Haberlandt G. 1928. Physiological plant anatomy. London : Macmillan and Co. Ltd. 518–521. Hansted L, Jakobsen HB, Olsen CE. 1994. Influence of temperature on the rhythmic emission of volatiles from Ribes nigrum flowers in situ. Plant, Cell and Enironment 17 : 1069–1072. Henderson W, Hart JW, How P, Judge J. 1970. Chemical and morphological studies on sites of sesquiterpene accumulation in Pogostemon cablin (Patchouli). Phytochemistry 9 : 1219–1228. Kanasawud P, Crouzet JC. 1990. Mechanism of formation of volatile compounds by thermal degradation of carotenoids in aqueous medium. 1. β-Carotene degradation. Journal of Agricultural and Food Chemistry 38 : 237–243. Leggett GWW. 1979. Preliminary inestigations of the production of an oil extract from Boronia megastigma Nees. B. Agr. Sci. Hons. Thesis, University of Tasmania. Loomis WD, Croteau R. 1973. Biochemistry and physiology of lower terpenoids. In : Runeckles VC, Mabry TJ, eds. Recent adances in phytochemistry 6 Terpenoids ; Structure, biogenesis and distribution. 147–185. Lutz A, Winterhalter P. 1992. Isolation of additional carotenoid metabolites from quince fruit (Cydonia oblonga Mill.). Journal of Agricultural and Food Chemistry 40 : 1116–1120. MacTavish HS. 1995. Factors affecting yield and composition of floral extract from Boronia megastigma Nees. PhD Thesis, University of Tasmania. Mihailova J, Atanasova R, Balinova-Tsvetkova A. 1977. Direct gas chromatography of essential oil in the separate parts of the flower of the Kazanlik rose (Rosa damascena Mill. f. trigintipetala Dieck.). VII International Congress of Essential Oils, Kyoto, Japan, 219–220. Patra NK, Srivastava HK, Srivastava RK, Naqvi AA. 1987. Association of oil content with floral characteristics of Rosa damascena. Indian Journal of Agricultural Sciences 57 : 938–940. Pichersky E, Raguso RA, Lewinshohn E, Croteau R. 1994. Floral scent production in Clarkia (Onagraceae). Plant Physiology 106 : 1533–1540. Roberts NJ. 1989. Morphological, physiological and biochemical aspects of flower initiation and deelopment in Boronia megastigma Nees. PhD Thesis, University of Tasmania. Roberts NJ, Menary RC. 1994. Effect of nitrogen on growth, flower
MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma yield and oil composition and yield in Boronia megastigma Nees. Journal of Plant Nutrition 17 : 2035–2052. Sharma S, Farooqi AHA. 1990. Effect of 2-chloroethylphosphonic acid on economic yield of damask rose (Rosa damascena). Indian Journal of Agricultural Science 60 : 691–692. Stubbs JM, Francis MJO. 1971. Electron microscopical studies of rose petals cells during flower maturation. Planta Medica 20 : 211–218. Tanaka S, Yamaura T, Shigemoto R, Tabata M. 1989. Phytochrome-
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Volatiles in Different Floral Organs, and Effect of Floral Characteristics on Yield of Extract from Boronia megastigma (Nees) H A Z E L S. M A C T A V I S H* and R O B E RT C. M E N A R Y Department of Agricultural Science, Uniersity of Tasmania, G.P.O. Box 252-54 Hobart, Tasmania, Australia 7001 Received : 6 February 1997
Accepted : 4 April 1997
The relative amounts of volatile compounds in the extract and headspace from each floral organ were assessed in order to identify the main organs for accumulation and emission. The mass of flowers}organs, the number}density of oil glands and yield of volatiles were examined for their relationship with extract yield, in clonal and non-clonal plants. Boronia flowers were divided into component organs and the solvent extractable product and headspace above each organ type was quantified. The petals comprised 50 % of the weight of the flowers, and the stigma 20 % ; however, the stigma contributed 70 % of the total volatile compounds to extract from the whole flower. Proportionately more β-ionone and dodecyl acetate were emitted from the stigma and anthers than were contained in the extract, compared with other volatiles. The sexual organs are morphologically equipped for emission of volatiles to attract pollinators. Between non-clonal plants, there was a lower coefficient of variation for extract yield than for values relating to extract composition, indicating that the former is more heritable than the latter. Variation between clonal plants was reduced compared with variation between non-clonal plants. The environment modifies yield and quality of extract in clonal plants, indicating that both have relatively low heritability. No significant relationships between any floral characteristics and extract yield were found. Biosynthetic potential to accumulate extract is therefore of prime importance, and the effect of environment on this potential should be the subject of future work. # 1997 Annals of Botany Company Key words : Boronia megastigma, brown boronia, Rutaceae, essential oils, flower, stigma, oil gland, β-ionone.
INTRODUCTION Boronia megastigma Nees. (brown boronia, family Rutaceae) is an endemic Australian shrub grown commercially in Tasmania for production of a highly valued floral extract. The strongly perfumed red-brown and golden boronia flowers are harvested in September and extracted by solvent to yield between 0±3 and 0±7 % (by fresh flower weight) of a yellow-brown waxy concrete. Boronia extract (concrete) is an extremely complex mixture of compounds including monoterpenes, cinnamates, norisoprenoids including ionones, related epoxides and dihydro compounds, acetates of decyl, dodecyl and tetradecyl alcohols and methyl jasmonate isomers and triterpenes (Guenther 1974 ; Davies and Menary 1983 ; Weyerstahl et al., 1994). β-ionone is the major volatile (12–30 % of total volatiles). The abaxial surface of the four petals of boronia flowers is usually red-brown or purple and dotted with translucent glands, the adaxial surface is bright yellow. The flowers are dominated by a large, brown, four-lobed stigma. Pollen is produced by four small, yellow, petaline anthers (functional), however no pollen is formed by the four large, redbrown, sepaline anthers (non-functional). Flowers also comprise four sepals and a calyx, considered together in this study. The small fruits do not set in Tasmania, presumably because the pollinating vectors are absent (MacTavish, 1995). Plantations are established from vegetatively propagated clonal plants. * For correspondence.
0305-7364}97}09030607 $25.00}0
Usually all floral organs contain volatiles typical of that species (Mihailova, Atanasova and Balinova-Tsvetkova, 1977). However, quantitative differences between attractive, non-food structures (perianth) and attractive food sources such as the androecium and gynoecium are common (Francis and Allcock, 1969 ; Dobson, Bergstro$ m and Groth, 1990). Generally, petals and female organs have similar oil compositions but a lower yield than stamens which have markedly different compositions (Attaway, Pieringer and Barabas, 1966 ; Mihailova et al., 1977 ; Pichersky et al., 1994). Quantitative differences in the volatiles emitted from flower parts may be important in effecting pollinator attraction (Dobson et al., 1990 ; Hansted, Jakobsen and Olsen, 1994). Typical floral volatiles are found in all floral organs of boronia flowers (Wilson, 1982), however studies on the contribution of each organ to the extract obtained from the whole flower have so far been preliminary and not quantitative (Bussell, Considine and Spadek, 1995). Analysis of volatiles emitted into the atmosphere (so-called ‘ headspace ’) above boronia flowers or extract has not previously been reported. Vegetative essential oils are usually contained within glands or trichomes ; in many cases there is a positive relationship between the number of glands and essential oil yield (Henderson et al., 1970 ; Tanaka et al., 1989). In rose flowers there are no specialized glands for accumulation of extract, the simple glandular epidermal tissue is the site of terpene biosynthesis (Loomis and Croteau, 1973 ; Stubbs and Francis, 1971). In such cases, one would expect to find a correlation between yield of
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# 1997 Annals of Botany Company
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MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma
extract per flower and factors such as flower and}or petal size and}or weight, however Patra et al. (1987) found no such correlations in rose. Sharma and Farooqi (1990) found that in damask rose, the concentration of extract in flowers of differing mass was constant and hence, flowers with greater mass contained more extract per flower. In boronia, flower yield per hectare is directly correlated with flower size, the number of flowers per node, the number of flowers per plant and the number of plants per hectare (Roberts, 1989). To date, most of the published work on boronia has concentrated on flower production. Oil glands have been noted on the epidermis of boronia flower petals (Haberlandt, 1928) and small numbers of glands have been observed on other organs including the receptacle, ovary and nectary (Wilson, 1982 ; Bussell et al., 1995). The structure and ultrastructure of the oil glands on the petals has been described (MacTavish, 1995) ; glands appear to develop lysigenously. It has been assumed that most of the floral extract is contained within these glands ; Wilson (1982) raised the question of whether the volume or number of oil glands on the petals or the size of the stigma may be related to extract yield from boronia flowers. The effect of various morphological characteristics on extract yield have not been examined. A more detailed knowledge of the factors that affect extract yield and composition is required. A quantitative study of the contribution of each organ to the extract has been undertaken. Floral characteristics, which may be indicative of extract yield, were studied. These included factors such as flower yield per plant, the average flower weight, the average weight of the stigma and petals and the number of oil glands per petal. The identification of floral characteristics which are positively linked with extract yield may greatly benefit selection for new plants to clone for commercial production.
MATERIALS AND METHODS Plant material Unless otherwise stated, plant material used in this study comprised clonal plants of brown boronia (Boronia megastigma Nees), developed by the University of Tasmania and grown on commercial plantations in Tasmania over several growing seasons.
Solents Two solvents were used : re-distilled technical grade petroleum ether (b.p. 40–60 °C) (pet. ether) and analytical grade hexane (Mallinckrodt).
Floral extracts Small scale extracts. Extracts of all floral organs were made using pooled, dissected organs from 40 flowers (2±0 g). Flower material was extracted in hexane with an octadecane internal standard for 12 h at 4 °C. After removal of the
plant material, the hexane was concentrated under nitrogen gas and analysed by GC. Laboratory scale extracts. Twenty grams of frozen flowers were extracted in triplicate using pet. ether. The flower} solvent mixture was homogenized for 10–15 s using an Ultra-Turrax (Janke and Kunkel). The flowers were extracted at room temperature during two washes of 2 h each, followed by a final rinse of 5 min. Solvent from the three washes was pooled and evaporated to dryness at reduced pressure on a rotary vacuum evaporator at 60 °C. The yield of extract was calculated as a percentage of the fresh flower weight. Gas chromatography A Hewlett Packard 5890 Series II gas chromatograph equipped with a flame ionisation detector (FID), a splitinjection system and an HP-1 cross linked methyl silicon gum column 30 m¬0±2 mm i.d., film thickness 0±33 microns, was used. (Carrier gas : N ! 2 ml min−", head pressure # 12 psi and split ratio of 1 : 50. Oven temperature programme : 50 °C for 1 min, then 10 °C min−" to 250 °C. Injector temperature : 250 °C, detector temperature : 280 °C. Injection volume : 1µl.) Peaks were initially identified by GC}MS (Davies and Menary, 1983). Quantitative peak estimation was achieved by addition of octadecane as an internal standard, an FID response factor of one was assumed. Total volatiles were calculated as the fraction of the GC-analysable material eluting before n-heneicosane (Davies and Menary, 1983). Headspace analysis Twelve flowers for headspace analysis were selected from one lateral of one plant, dissected into their component organs (four petals, four sepals one calyx, four functional (petaline) anthers, four non-functional (sepaline) anthers and one stigma), and sealed into 10 ml glass headspace sample vials. Vials were left at room temperature to equilibrate for 10 min. Subsequently, 20 ml of the headspace (two injections of 10 ml each with a 1 min equilibration period between samples) was injected onto a cryotrap and subsequently onto the GCMS. A Hewlett Packard 5890 Series II GC equipped with MS (HP 5970), a split-less injection system and a BP-1 cross linked methyl silicon gum column 50 m¬0±2 mm i.d., film thickness 0±33 microns, was used. (Carrier gas : Helium ! 3 ml min−", head pressure 10 psi. Oven temperature programme : 30 °C for 2 min, then 6 °C min−" to 150 °C, then 10 °C min−" to 250 °C. Injector temperature : 250 °C, detector temperature : 260 °C, ion source : 70eV, selected ion monitoring was used to detect peaks.) No internal standards were used because of the lack of appropriate standards for the solid-sample type used. The peak areas of identified volatiles in blank samples were subtracted from all other samples. Enumeration of oil glands Non-clonal plants from one site in one season were used. The weight of petals as a proportion of the total fresh flower
MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma weight was assessed using 50 flowers per plant. Twenty randomly selected petals per plant were mounted in glycerine onto glass slides, abaxial surface uppermost, and examined under a binocular dissecting microscope. Photographs were taken from which oil glands were counted. The oil glands appeared as non-pigmented areas in the dark red-brown abaxial epidermis. Natural pigmentation obscured counting on some petals, therefore these petals were extracted with acidified ethanol, and then re-stained with crystal violet and photographed as described previously.
Surface area of petals From enlarged photocopies of the photographs of the petals, the area of the petal was cut out and weighed. By comparison between the weight of a known area of tonerblackened photocopy paper, the area of the petal was calculated, from which the oil gland density was subsequently calculated.
307
A. Weight of component organs
C. Total volatiles in headspace from component organs
B. Total volatiles in extract from component organs
Floral characteristics and extract yield studies
RESULTS Weight of indiidual floral organs The petals of open flowers comprised approx. 50 % of the total flower by weight ; the stigma and calyx (sepals and pedicel) were equivalent in weight, comprising about 22 % of the total flower weight each (Fig. 1 A). The mass of nonfunctional anthers was five times greater than that of functional anthers.
Extract and headspace emission from component organs Sixty five percent of the volatiles in the extract from the whole flower were contributed by the stigma, only 20 % were present in the petals (Fig. 1 B). The calyx, however, contributed 51 % of total volatiles emitted into the headspace from the whole flower (Fig. 1 C). Other organs contributed in the order stigma (16 %) " functional anther (15 %) " non-functional anther (14 %) " petal (3 %). Volatiles were chosen with different volatilities namely αpinene, β-ionone, dodecyl acetate, methyl jasmonate and methyl epijasmonate (together) and (Z)-heptadec-8-ene. These compounds were expressed as a percentage of the total volatiles in the extract and headspace of each organ
F. 1. Contribution of each floral organ to A, mass of the whole flower (g) ; B, extract per flower (mg) ; C, volatiles emitted into the headspace from the whole flower. Petals, 4 (+) ; stigma, 1 (8) ; functional anthers, 4 (7) ; non-functional anthers, 4 ( ) ; calyx including sepals, 1 (*).
100 % of each volatile in extract from each organ
Studies on the relationship between flower yield per plant and extract yield used 15 clonal plants grown at one site in one season. Studies on the relationship between extract yield and average flower weight, oil gland distribution and weight of component organs (petals, stigmas) used non-clonal plants from two sites over two seasons. The average weight of 50 individual flowers was assessed. The stigmas from 25 flowers were removed at a natural point of abscission, and the separated stigmas and flowers-without-stigmas were weighed.
80 60 40 20 0
Petals
Stigma Functional NonCalyx + anthers functional sepals anthers
F. 2. Relative amounts of selected volatiles in extracts from each floral organ, expressed as a % of the total volatiles in the extract from each organ type. α-pinene (9) ; β-ionone (:) ; dodecyl acetate (+) ; (Z)-heptadec-8-ene ( ) ; methyl jasmonate and methyl epijasmonate (8) ; total other volatiles (*).
type (Figs 2 and 3, respectively), the jasmonates were not calculated in the headspace due to low detection in some organs. Extract from all organs contained all volatiles examined (Fig. 2). Highly volatile compounds such as αpinene predominated in the headspace from the petals and calyx, although other, less volatile compounds were present in extract from petals at relatively higher levels. (Z)heptadec-8-ene, which is not particularly volatile, nevertheless comprised significant proportions of the emission
MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma
% of each volatile in headspace from each organ
308
T 2. Summary of flower and extract yield per plant, and extract concentration and composition from 15 clonal plants at one site in one season
100 80 60
Value
40 20 0
Petals
Stigma Functional NonCalyx + anther functional sepals anther
F. 3. Relative amounts of selected volatiles in the headspace from each floral organ, expressed as a % of the total headspace from each organ type. α-pinene (9) ; β-ionone (:) ; dodecyl acetate ( ) ; (Z)heptadec-8-ene (7) ; total other volatiles (*).
T 1. Summary of yield of extract and extract composition from 61 genetically different plants Value
Total volatiles (% by f.wt)
Yield of extract (% by f. wt) Total volatiles (% by f. wt¬10−$) β-ionone (% by f. wt¬10−$
Mean
s.d.
Coefficient of variation
0±4584
0±0665
14±515
0±291
Range
59±831
22±995
38±433
140±235
14±486
8±030
55±434
56±304
0.175 0.150 0.125 0.100 0.075 0.050 0.025 0 0.30
0.35 0.40 0.45 0.50 0.55 0.60 Yield of extract (% by fresh weight)
0.65
F. 4. Relationship between yield of extract (% by fresh weight) and total volatiles (mg}100 g flower material) in the extract from 61 genetically different plants.
Mean
Flower yield per plant (g) 146±44 Extract yield (% by 0±35 f. wt) Total extract per plant 0±52 (g) Total volatiles (% by f. wt¬10−$) 40±98 β-ionone (% by f. wt¬10−$) 14±91
s.d.
Coefficient of variation Range
51±13 0±02
34±92 4±73
0±19 0±06
0±19
36±88
0±69
6±45
15±74
23±00
2±30
15±45
8±91
ionone, than for yield of extract (% by f. wt). There was an increase in the total amount of volatiles (% by f. wt) in flowers with yields of extract between 0±32 and 0±48 % (Fig. 4). Subsequent increases in extract yield did not produce increased volatiles in flower material, however, plants with particularly high yields of extract and volatiles were observed (Fig. 4). Flower yield per plant The variation in the flower and extract yield between clonal plants at one site in 1 year was analysed (Table 2). The yield of extract per plant was equal to flower yield per plant multiplied by the concentration of extract in the flower material. There was, therefore, a significant positive relationship between flower yield per plant and extract yield (mg) per plant (R# ¯ 0±986). The yield of flower material per plant (and hence extract yield per plant) varied widely between plants, however, the concentration of extract in flower material varied to a lesser extent. Variation between plants in the concentration of volatiles including β-ionone was greater than the variation in extract concentration. There were no significant relationships between the yield of flower material per plant and either the concentration of extract (% by f. wt) or the yield of extract per flower (Table 3). Aerage flower weight
from floral organs, particularly the sexual organs. Relatively high levels of β-ionone and dodecyl acetate were emitted from the sexual organs compared with proportional levels in the extract from these organs.
There was a trend for heavier flowers to contain more extract per flower (R# ¯ 0±470) (Table 3). There were, however, no significant relationships between the average weight of a fresh, open flower and either extract yield per flower or extract concentration (% by f. wt) in non-clonal plants (Table 3).
Variation in non-clonal plants
Weight of petals and number of oil glands
Sixty one non-clonal plants were examined for yield of extract and composition (Table 1). The coefficients of variation and standard deviations were greater for values relating to extract composition i.e. total volatiles and β-
There were no significant relationships between the weight of the petals as a proportion of the flower and extract yield per flower or extract concentration (Table 3). The surface area of petals and the variation in the density of oil glands
MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma
309
T 3. Correlation coefficients between arious floral characteristics and yield of extract per flower and extract concentration (% by f. wt) Extract per flower (mg)
Extract concentration (% by f. wt)
0±203 0±470
0±198 0±028 (negative slope)
0±210 0±051 (negative slope)
0±411 0±298 (negative slope)
not calculated
0±427 (negative slope)
Ratio of stigma weight : flower weight
0±00
Average stigma weight (mg)
0±348
0±475 (negative slope) 0±638 (negative slope)
Value Flower yield per plant (g) Average flower weight (g) Ration of petal weight : flower weight No. of oil glands per petal Density of oil glands (glands mm−#)
T 4. Surface area of petals, oil gland distribution and extract yield in genetically different plants Value Surface area of petals (mm#) Number of oil glands}petal Density of oil glands (no mm−#) Yield of extract (mg}flower)
Mean
s.d.
Coefficient of variation
181±64
20±59
11±33
204±29
39±16
19±17
1±12
0±16
14±05
194±71
8±70
14±13
and extract yield per flower in non-clonal plants is presented (Table 4). There was no relationship between the number of oil glands per petal and the concentration of extract (% by f. wt) (Table 3). There was a trend for petals with an increased density of oil glands to have a reduced concentration of extract. There were no significant relationships between the number of oil glands per petal and the composition of extract. Weight of the stigma There were no significant relationships between the weight of the stigma (or the weight of the stigma as a proportion of the whole flower) and either extract per flower, or extract concentration (% by f. wt) (Table 3). DISCUSSION The petals of boronia flowers are not the major source of extract or volatile compounds. The important role of the stigma in the composition of the extract is evident : it comprises only 22 % of the weight of the flower but contains 70 % of the volatiles ; there are few, if any, distinct glands present on this organ (Leggett 1979 ; MacTavish, 1995). The
anthers and stigma also emitted relatively high amounts of ‘ floral ’ volatiles such as β-ionone and dodecyl acetate. A comparison of the levels of such compounds in the extract and headspace from all organs, shows that the sexual organs emit relatively higher levels of these compounds than other, sometimes more volatile compounds which also occur in the extracts from these organs. The morphology of the sexual organs (MacTavish, 1995) suggests that they have a glandular epidermis (Francis and Allcock, 1969). It appears that glandular epidermis is capable of accumulating relatively higher concentrations of volatiles than discrete glands, such as those found on the epidermis of the petals. This supports other work on this species (Bussell et al., 1995). The headspace emitted from different floral organs demonstrates the dominance of more volatile compounds, such as α-pinene, over less volatile compounds such as βionone. The low threshold of β-ionone and related compounds, particularly jasmonates, has a dominant effect on fragrance perception, even though emission levels may be relatively minuscule. Boronia megastigma is believed to be pollinated by a moth, probably in the family Heliozelidae (MacTavish, 1995). Therefore, relatively high levels of emission of compounds such as dodecyl acetate, an active component of moth pheromones, in preference to other, more volatile compounds from the anthers may play an important ecological role. Between non-clonal plants, the yield of extract had a lower coefficient of variation than the level of volatiles. In plants where higher yields of extract occurred, higher levels of volatiles also occurred, indicating that particular plants contained more, or more active biosynthetic enzymes for all components of the extract. Relatively low variability in extract yield compared with extract composition may be the result of low variability in compounds which comprise the bulk of the extract such as waxes, pigments and other nonvolatile compounds, some of which may be products of primary metabolism. Secondary compounds present in aromatic extracts may arise by one-step processes from primary compounds, or from small modifications to
310
MacTaish and Menary—Volatiles in Different Floral Organs of Boronia megastigma
ubiquitous secondary compounds such as geranial. As such, secondary compounds may vary more between plants as a result of minor genetic and environmental differences. It is likely that β-ionone, the dominant volatile in boronia flowers, is produced by a one or two step process from carotenoids such as β-carotene (Enzell, 1985 ; Kanasawud and Crouzet, 1990 ; Lutz and Winterhalter, 1992), and therefore variation in the levels of this compound may be affected by differences in the amount or availability of substrate, or the activity of cleavage enzyme(s). The analytical methods used for determination of extract concentration generally produce larger variation than the methods used for quantitation of volatiles, therefore experimental technique is unlikely to produce the results observed. Extract yield and composition varied less between clonal plants than between non-clonal plants. There are large numbers of four distinct clonal types grown in Tasmania ; their yield and extract varies between sites (MacTavish, 1995). This indicates that phenotypic expression and environmental conditions predominate over genotype to a certain extent. Environmental factors were not considered in this study. The concentration of extract (% by fresh flower weight) appears to be negatively correlated with the density of oil glands on the petals, suggesting that glands may be smaller or less full when large numbers occur, although the trend is not significant (R# ¯ 0±427). The effect of the size and volume of oil glands was not studied. Since there are no floral characteristics which are positively correlated with extract yield, clonal selection must be based on the performance of plants with regard to extract yield and composition, as well as other agronomic factors, at several sites characteristic of potential commercial sites. Once suitable plants are selected, increased yields of extract per hectare can be obtained by increasing the number of flowers initiated each year, possibly by the use of appropriate fertilizer application (Roberts and Menary, 1994). Accumulation of extract in non-gland bearing tissues in boronia flowers indicates that activity of biosynthetic enzymes is a more significant factor contributing to increased yields of extract than the number of glands present on the petals. Further, boronia flowers with larger organs with an active glandular epidermis, such as the stigma, do not necessarily produce high yields of floral extract, indicating that there are other limits to accumulation of extract, one of which may be toxicity of products when not localized in glandular structures (Brown, Hegarty and Charlwood, 1987). There are large amounts of glycosidically bound volatiles in boronia flowers, including glycosides of βionone (MacTavish, 1995), which may act as non-toxic storage of products in a water soluble form. The localization of glycosides within organs, and the relationship between free and bound volatiles and extract yield have yet to be considered, but will be the subject of future publications. The work presented here furthers our understanding of the limitations to extract production in boronia flowers and provides a basis for more directed research, namely the identification of enzymes active in the synthesis of important components of boronia extract.
A C K N O W L E D G E M E N TS The authors acknowledge support from an Australian Postgraduate Research Award (Industry), with industry collaboration from Essential Oils of Tasmania Pty. Ltd. and Natural Plant Extracts Co-operative Pty. Ltd. We thank Noel Davies of the Central Science Laboratory at the University of Tasmania for help with headspace analysis. LITERATURE CITED Attaway JA, Pieringer AP, Barabas LJ. 1966. The origin of citrus flavor components–II. Identification of volatile components from citrus blossoms. Phytochemistry 5 : 1273–1279. Brown JT, Hegarty PK, Charlwood BV. 1987. The toxicity of monoterpenes to plant cell cultures. Plant Science 48 : 195–201. Bussell BM, Considine JA, Spadek ZE. 1995. Flower and volatile oil ontogeny in Boronia megastgima. Annals of Botany 76 : 457–463. Davies NW, Menary RC. 1983. Volatile constituents of Boronia megastigma flowers. Perfumer and Flaorist 8 : 3–8. Dobson HEM, Bergstro$ m G, Groth I. 1990. Differences in fragrance chemistry between flower parts of Rosa rugosa Thunb. (Rosaceae). Israel Journal of Botany 39 : 143–156. Enzell C. 1985. Biodegradation of carotenoids – an important route to aroma compounds. Pure and Applied Chemistry 57 : 693–700. Francis MJO, Allcock C. 1969. Geraniol β--glucoside ; occurrence and synthesis in rose flowers. Phytochemistry 8 : 1339–1347. Guenther E. 1974. Oil of Boronia megastigma (Boronia flower oil). In : Guenther E. The essential oils. Vol. 3 – Indiidual essential oils of the plant families Rutaceae and Labiatea. R. E. Krieger Publishing Company, 364–367. Haberlandt G. 1928. Physiological plant anatomy. London : Macmillan and Co. Ltd. 518–521. Hansted L, Jakobsen HB, Olsen CE. 1994. Influence of temperature on the rhythmic emission of volatiles from Ribes nigrum flowers in situ. Plant, Cell and Enironment 17 : 1069–1072. Henderson W, Hart JW, How P, Judge J. 1970. Chemical and morphological studies on sites of sesquiterpene accumulation in Pogostemon cablin (Patchouli). Phytochemistry 9 : 1219–1228. Kanasawud P, Crouzet JC. 1990. Mechanism of formation of volatile compounds by thermal degradation of carotenoids in aqueous medium. 1. β-Carotene degradation. Journal of Agricultural and Food Chemistry 38 : 237–243. Leggett GWW. 1979. Preliminary inestigations of the production of an oil extract from Boronia megastigma Nees. B. Agr. Sci. Hons. Thesis, University of Tasmania. Loomis WD, Croteau R. 1973. Biochemistry and physiology of lower terpenoids. In : Runeckles VC, Mabry TJ, eds. Recent adances in phytochemistry 6 Terpenoids ; Structure, biogenesis and distribution. 147–185. Lutz A, Winterhalter P. 1992. Isolation of additional carotenoid metabolites from quince fruit (Cydonia oblonga Mill.). Journal of Agricultural and Food Chemistry 40 : 1116–1120. MacTavish HS. 1995. Factors affecting yield and composition of floral extract from Boronia megastigma Nees. PhD Thesis, University of Tasmania. Mihailova J, Atanasova R, Balinova-Tsvetkova A. 1977. Direct gas chromatography of essential oil in the separate parts of the flower of the Kazanlik rose (Rosa damascena Mill. f. trigintipetala Dieck.). VII International Congress of Essential Oils, Kyoto, Japan, 219–220. Patra NK, Srivastava HK, Srivastava RK, Naqvi AA. 1987. Association of oil content with floral characteristics of Rosa damascena. Indian Journal of Agricultural Sciences 57 : 938–940. Pichersky E, Raguso RA, Lewinshohn E, Croteau R. 1994. Floral scent production in Clarkia (Onagraceae). Plant Physiology 106 : 1533–1540. Roberts NJ. 1989. Morphological, physiological and biochemical aspects of flower initiation and deelopment in Boronia megastigma Nees. PhD Thesis, University of Tasmania. Roberts NJ, Menary RC. 1994. Effect of nitrogen on growth, flower
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