1.Rosa palustris, a Model Species for Fragrance Investigations James McDaniel and Nancy Cowden, Ph.D., Biology Department and Priscilla Gannicott, Ph.D., Chemistry Department and DeAnne MooreLynchburg College, 1501 Lakeside Drive, Lynchburg, VA 24501

2. ABSTRACT The flowering plants Rosa palustris and Rosa palustris var. scandens havedistinguishing characteristics such as their habitat and floral anatomy thatset them apart. With a five-petaled bloom, R. palustris thrives in swampsor bogs while R. palustris var. scandens is a double-petaled hybridcultivated for gardening purposes. From June 2010 to June 2011, weinvestigated the fragrance composition of these flowering plants. A totalof 16 samples were collected from R. palustris and R. palustris var.scandens flowers, using solid-phase microextraction (SPME). Total ionchromatograms were produced by a Thermo electron-focus GC/DSQ MSand XCalibur 1.4 software. Mass spectra were obtained from thesechromatograms using NIST MS Search 2.0, which allowed us to identify thevolatile organic compounds found in these roses. The most abundantcompounds found in R. palustris were 1-(R)-alpha-pinene, phenylethylalcohol, geraniol acetate, trans-geraniol, and cis-geraniol. Those found inR. palustris var. scandens were phenylethyl alcohol, nonanal, 1-(R)-alpha-pinene, and cis-geraniol. Ultimately, characterizing their fragrancesignatures is important because it has the potential to aid in selectingfragrant varieties for new hybridizing endeavors in the future. 3. INTRODUCTION The genus Rosa includes approximately 200 species as well as more than 18,000cultivated varieties (Guterman, 2002). Species roses, such as Rosa palustris, havebeen cultivated for centuries to produce hybrid varieties. In the garden setting,hybrid varieties known as cultivars are selectively propagated for desirablecharacteristics. Some of these desirable characteristics include the following: longvase life, flower shape, color, and scent (Shalit, 2003). In the field, it is visuallyevident that the garden cultivar Rosa palustris var. scandens is propagated for itsflower shape because it has more petals than R. palustris and no prickles whereasthe native rose, R. palustris, has only a single bloom and curved prickles (Figs. 1and 2). It is not quite clear, however, whether or not R. palustris var. scandens ispropagated for its scent because its fragrance composition has not beencharacterized previously. Scent is important because it is an ancient connectionamong flowering plants, their pollinators, and enemies (Raguso, 2008). We not only investigated the fragrance composition of the native wetland species,R. palustris, but also that of the garden cultivar R. palustris var. scandens as ameans of comparison. This summer, we plan to further examine the relationshipbetween the fragrance of R. palustris and R. palustris var. scandens as well as theirpollinators. By examining this relationship, pollination efficiency can be examinedover time, which can be dependent upon visitor abundance, visitor activity, andpollen loads deposited on receptive stigmas and seed set (Yeboah Gyan, 1987).Determination of pollination efficiency coupled with the characterization of thefragrance of each rose could aid in selecting fragrant varieties for new hybridizingendeavors in the future. 4. INTRODUCTION Figure 1. Rosa palustris in a wetland, Figure 2. Rosa palustris var. Bedford Co., VA. scandens in the Antique RoseEmporium, Brenham, TX. 5. MATERIALS AND METHODSIn 2010 and 2011, the month of June provided us with the opportunity to collect atotal of 15 floral fragrance samples from Rosa palustris at the Claytor Nature StudyCenter, Bedford Co., Virginia. Additionally, one floral fragrance sample of Rosapalustris var. scandens was obtained in June of 2011 at the Old City Cemetery,Lynchburg, Virginia. All of the samples were collected using static-headspace,solid-phase microextraction (SPME) via 50/30 mm DVB/carboxen fibers (SUPELCOAnalytical, Bellefonte, Pa.). To collect the samples, theSPME fibers were placedabove the floral headspace of each individually bagged bloom (Figure 3). Over thecourse of an hour, the SPME fibers absorbed the volatile organic fragrancecompounds emitted from each rose bloom. After an hour had passed, these fiberswere retracted back into their protective casings and transported to LynchburgCollege for further analysis on the same day that the samples were obtained. At Lynchburg College, desorption of the volatile organic compounds was achievedin the laboratory by inserting the individual samples into the injection port of aThermo-Scientific Focus GC/DSQ MS set at 250C. Total ion chromatograms wereanalyzed to confirm the identity of each volatile organic compound by comparingmass spectra to a NIST MS Search 2.0 database. 6. MATERIALS AND METHODS Figure 3. Static-headspace SPME field sampling technique. 7. RESULTS When comparing the Rosa palustris samples, it became evident thatsome of them not only differed in the number of volatile organiccompounds that were present, but also by the relative percentages(percent area and percent height) of each compound. Although other compounds were identified using the NIST MSsearch 2.0 database, we consistently identified 1-(R)-alpha-pinene,phenylethyl alcohol, cis-geraniol, trans-geraniol, and geraniolacetate as the volatile, organic fragrance compounds thatconstituted the greatest relative percentages out of all of thefragrance compounds found in R. palustris (Figs. 4 and 5). Inaddition, the NIST MS search 2.0 database determined that 1-(R)-alpha-pinene, nonanal, phenylethyl alcohol and cis-geraniolconstituted the greatest relative percentages out of all of thefragrance compounds found in R. palustris var. scandens (Figs. 6 and7). 8. RESULTSRT: 8.00 - 18.00 12.69NL: 1005.20E8 9514.70TIC F: MS 90 rcarolina62810mix1 85Phenylethyl alcohol 80 75 Trans-geraniol 70 65 60Cis-geraniol 16.42 55 50 14.33 45 40 35 30Geraniol acetate 25 20 1514.95 10 13.77 15.1810.40 11.4017.185 8.62 9.4313.20 15.27 9.5908910 11 12 131415161718 Time (min) Figure 4. A representative total ion chromatogram Figure 5. Relative percentages of all fragrance of a Rosa palustris SPME sample (6/28/2010).compounds found in a fresh Rosa palustris bloom (6/28/2010). 9. RESULTSRT: 8.00 - 18.0013.63 NL: 1001.16E8 Phenylethyl alcohol 95 TIC F: MS 90 PalustrisScandens06032 85 011mixedpg13.32 1 80 75 70 65 60 55 50Nonanal 45 40 Cis-geraniol 35 3016.26(R)-alpha-pinene 25 14.78 2013.93 15.74 17.57 15 10.4412.40 17.19 109.5111.28 12.7215.295 8.539.71 16.400891011 12 13 1415 16 1718Time (min) Figure 6. A representative total ion chromatogram of aFigure 7. Relative percentages of all Rosa palustris var. scandens SPME sample (6/3/2011).fragrance compounds found in a fresh Rosa palustris var. scandens bloom (6/3/2011). 10. DISCUSSION More than 400 volatile, organic compounds are known to be related to rose scent(Shalit, 2003). Among these are alcohols described as having a rose like odorthat are frequently found in rose species (Chemicalland21.com, 2010). Phenylethylalcohol, cis-geraniol, and trans-geraniol are all alcohols which appeared in massspectra in 13, 12, and 11 samples, respectively, out of 15 total Rosa palustrissamples. The ester, geraniol acetate, which is described as having a floral orfruity rose aroma (Chemicalland21.com, 2010), appeared in mass spectra in tenout of 15 R. palustris samples. (R)-alpha pinene, which is a bridged hydrocarbon,appeared in all 15 R. palustris samples and is also described as a volatile rosecompound. For Rosa palustris var. scandens, the alcohols phenylethyl alcohol andcis-geraniol appeared in the mass spectrum obtained. Also present were thealdehyde nonanal, which is described as having a strong floral or fruity aroma,and (R)-alpha-pinene. Combined, our work represents the first known analysis ofthe volatile organic fragrance compounds found in R. palustris and R. palustris var.scandens. Future research will allow us to correlate each rose with its pollinators and toexamine how fragrance signatures affect insect visitors responses to the scentsproduced. 11. SELECTED LITERATUREAroKor Holdings, Inc. 2000 2010. Chemicalland21.com. Retrieved April 7, 2012, from: http://www.chemicalland21.comThe Taunton Press, Inc. 2012. Fine Gardening. Retrieved April 7, 2012, from: http://www.finegardening.com/plantguide/rosa-palustris-var-scandens-swamp-rose.aspxGuterman, I., M. Shalit, N. Menda, D. Piestun, M. Dafny-Yelin, G. Shalev, E. Bar, O. Davydov, M.Ovadis, M. Emanuel, et al. 2002. 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