Materials and Methods

Plant materials and growing conditions.

Twenty-five clones and cultivars were used (Table 1). Of these, 18 clones and one cultivar belonged to R. damascena Mill. f. trigintipetala Dieck. developed using various methods, including selection of natural spontaneous mutations from the Kazanlak oil-bearing rose population (Table 1). The selected clones were chosen using molecular markers and identified as belonging to R. damascena Mill. (Rusanov et al., 2005b; Todorova et al., 2004). We also included three rose species used for commercial oil production in other countries (Table 1). Cultivar Kooperatorka was developed by Maichenko (Nedkov and Attanassova, 2004). Cultivar Iskra from R. damascena was used as the standard.

Table 1.

List of the rose species, hybrids, and accessions used in this study with indication of the methods they were developed.

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The field experiments were conducted in the experimental fields of the Research Institute for Roses and Medicinal Plants in Kazanlak, Bulgaria, during the 2000 to 2004 cropping seasons using a randomized complete block design with three blocks. Individual plots were 16 m² with 20 rose bushes in every plot planted at 0.8 m within row and 2.8 m between rows to reflect common agricultural practices of rose production in the region. To eliminate border effects, of the 20 plants in each individual plot, only the 10 inside plants were harvested. Plants from all clones and cultivars were propagated through rooting of green cuttings in greenhouses in the spring of 2000 following the established protocol for the production of standard rose cuttings (Zlatev et al., 2001) and transplanted in the field in Nov. 2000. However, because rose bushes do not flower during the first growing season (2001), data were not available for 2001. During the four cropping seasons (2001 to 2004), a standardized agricultural protocol developed by the researchers at the Research Institute for Roses and Medicinal Plants for industrial rose plantations was used (Kovatcheva et al., 2004; Nedkov and Attanassova, 2004). Soil was deluvial-meadow sandy (European Digital Archive of Soil Maps of the World, 2009) with relatively low concentrations of available nutrients and pH of 4.9 in the soil profile 0 to 20 cm and pH of 5.09 in the deep soil profile 20 to 40 cm.

Biometrical measurements, oil content, and oil composition analysis.

Plant height, plant diameter, number of flowering buttons per flowering branch, weight, diameter, number of developed and undeveloped petals per individual flower, and essential oil content were taken every year at flowering by picking the rose flowers in early morning (0500 to 0700 hr) when the content and composition of the essential oil are optimal (Topalov, 1962, 1978).

The essential oil content from each plot was measured using samples of 600 g fresh flowers through water distillation in Clevenger-type glass distillation equipment (Furnis et al., 1989; Topalov, 1962). The essential oil obtained was measured in cubic millimeters, multiplied by 0.85, which is the relative weight of the rose oil (Topalov, 1962), and was calculated as percentage of oil content in 100 g fresh flowers. Rose oil from the field experiments and the chemical standards were analyzed by gas chromatograph (GC) PYE Unicam. The GC was equipped with a EKONO-CAP™EC™-1 fused silica capillary column (30 m × 0.32 mm) operated under the following conditions: injector temperature 300 °C, column temperature, 70 to 230 °C at 8 °C·min⁻¹, then held at 230 °C for 5 min; carrier gas, hydrogen at 1.3 mL·min⁻¹ flow rate; injection volume of 0.1 μL (splitless). Internal chemical standards were used to identify and quantify the individual constituents. The GC analyses identified the major and some minor oil constituents as required by the international standard ISO 9842-2004.

Antimicrobial, antimalarial, and antileishmania activity and cytotoxicity.

Assays for antimicrobial, antimalarial, and antileishmania activity and cytotoxicity were performed as described previously (Mikus and Steverding, 2000; Zheljazkov et al., 2008). As a result of the high costs of conducting these assays, at least one representative essential oil sample from each rose species [R. damascena × R. gallica, R. gallica subsp. Eriostyla var. austriaca, R. damascena (R. kazanlika), R. centifolia, R. alba, and R. gallica] was tested for antifungal, antimicrobial, antimalarial, and antileishmania activity against a panel of human pathogens in an in-house testing facility at the National Center for Natural Products Research at the University of Mississippi. In the primary in-house screens for antimicrobial, antifungal, antileishmania, and antimalarial activity, none of the samples showed greater than 50% growth inhibition of the tested organisms at 50 μg/mL and were therefore deemed to be inactive.

Statistical analyses.

Essential oil content response was analyzed as a randomized complete block design with 25 treatments (19 clones, three hybrids, and three species) and nine blocks. The blocks were combinations of the three blocks in the field and the 3 years. The analysis of variance was completed using the GLM procedure of SAS 9.2 (SAS Institute Inc., 2008). Because the effect of treatment was highly significant with a P value < 0.001, multiple means comparison was done using the least significant difference method to generate letter groupings at the 5% level of significance. The validity of normal distribution and constant variance assumptions on the error terms was also verified.

Results and Discussion

Rainfall and temperature data for the period 2002 to 2004 suggest the most favorable conditions occurred in the 2004 cropping season (Table 2) with sufficient rainfall and mild temperature that increased consistently in May and June.

Table 2.

Average monthly temperatures and rainfall for the four growing seasons in Kazanlak, Bulgaria.

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The means of rose bushes height for all species ranged from 74 to 128 cm. Within the R. damascena clones, the tested clones and cultivars could be divided into several groups: 1) short, with bush height from 90 to 100 cm (#831, #6/79, #7/79, #K-IV, Iskra, #809, Svejen 188, Svejen 74, and Svejen 72); 2) medium height roses, 100 to 110 cm (#1151, #101, #148/75, #70/74, #601, #9/77 and the standard improved population #5); and 3) high-bush roses with height greater than 110 cm (Aleksandrovo 112, #1071, and #51) (data not shown). The most homogenous for height was Svejen 188. Among the species, the tallest bushes were found in R. centifolia.

The size of the rose bushes is associated with their productivity, size increase in subsequent years (sometimes up to the 10th year) corresponds to increase in flower production. However, overall, the steepest increases in the size and productivity of rose bushes occur in the first 3 years (Topalov, 1962, 1978). Plant height and the diameter of the rose bushes are important for the industry because the rose flowers are picked manually. Despite the rigorous research on mechanizing flower picking, a feasible mechanical flower picking technology is yet to be developed (Nedkov and Attanassova, 2004).

Mean bush diameter for all tested roses ranged from 32 to 105 cm (data not shown). R. damascena clones and cultivars could be grouped in relation to their diameter and form of the bushes: 1) upright bushes, 80 cm or less in diameter (#1151, #831, #101, #7/79, #K-IV, Svejen 188, #809, #9/77, Iskra, Svejen 74, improved population #5); 2) medium spread type, 80 to 100 cm diameter (#601, #148/75, #70/74, Svejen 72, Aleksandrovo 112, and #1071); and 3) widely spread type, greater than 100 cm in diameter (#51 and #6/79). Generally, rose bushes with erect growth habits are preferred because the flowers are easy to pick (Topalov, 1962). Rose flowers are picked manually between 0400 and 0700 hr, when the rose flowers open and the essential oil is present in the highest amount with the best quality (Topalov, 1962, 1978). Previous research on other cultivars of R. damascena found a positive correlation between the height of the rose bushes and their productivity (Singh and Kayiyar, 2001). In our study, the clones and cultivars with the highest bushes belong to the first two groups or relatively upright-type roses; however, for reasons indicated previously, producers prefer upright and relatively short bushes such as #831, #7/79, #K-IV, Svejen 188, #809, and Iskra. With the exception of R. alba, the tested hybrids and species had measured values below those measured in R. damascena clones (data not shown).

Rose flower morphology is one of the most conservative phenotypic traits, generally with significant differences between rose species, although the environment could have a modifying effect on the expression of this trait (Tabaei-Aghdaei et al., 2007; Topalov, 1978). The weight of an individual flower in oil-bearing roses is critically important because it is directly related to the efficiency of flower picking and the overall economy of the rose production (Topalov, 1978). Our results demonstrated that the weight of individual flowers of R. damascena was between 2.35 and 2.78 g with the highest values being the flowers of the control cultivar Iskra (data not shown). Of the hybrids, the heaviest flowers were found in #836/61, whereas of the species, the heaviest flowers were found in R. centifolia. The results from this study were similar to those of Tabaei-Aghdaei et al. (2007), who found rose flower weight variation between 1.39 and 2.78 g.

One of the main objectives of the selection and breeding work in oil-bearing roses is the development of cultivars with high essential oil content, possibly greater than 0.05% in flowers. For the three cropping seasons, the average essential oil content in fresh flowers of R. damascena varied between 0.032% and 0.049%, that for hybrid rose flowers between 0.03% and 0.039%, whereas the oil content of the other rose species was 0.012% to 0.021% (Fig. 1). Our results demonstrated that Svejen 74 had higher essential oil concentration relative to the control cultivar Iskra. Furthermore, the essential oil concentration of rose accessions #831, #101, #6/79, and #70/74 was not different from that of Svejen 74 or from that of the control cultivar Iskra (Fig. 1). The essential oil concentration of the rest of the rose accessions from R. damascena was not different from that of the control cultivar Iskra. The essential oil concentration of hybrid 90/67 and the other species (R. centifolia, R. gallica, and R. alba) was significantly lower than that of the control cultivar Iskra (Fig. 1).

Mean essential oil content (%) from 19 R. damascena clones, three hybrids, and three species. Means sharing the same letter are not significantly different at the 5% level of significance.

Citation: HortScience horts 46, 5; 10.21273/HORTSCI.46.5.710

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Mean essential oil content (%) from 19 R. damascena clones, three hybrids, and three species. Means sharing the same letter are not significantly different at the 5% level of significance.

Citation: HortScience horts 46, 5; 10.21273/HORTSCI.46.5.710

• Download Figure

Mean essential oil content (%) from 19 R. damascena clones, three hybrids, and three species. Means sharing the same letter are not significantly different at the 5% level of significance.

Citation: HortScience horts 46, 5; 10.21273/HORTSCI.46.5.710

• Download Figure

Furthermore, the oil content of Bulgarian clones of R. damascena was higher than that of native R. damascena clones collected in Iran, which was reported to vary between 0.017% and 0.035% (Tabaei-Aghdaei et al., 2007). Of the hybrid roses, high essential oil content was found in #836/61 and Kooperatorka.

The rose essential oil composition must meet the Bulgarian State Standard that was recently recognized as the international one (ISO 9842). We found differences in the number of the major essential oil constituents of the tested species and cultivars (Table 3).

Table 3.

Oil constituents of the studied roses: means for 2002, 2003, and 2004 growing seasons.

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L-citronellol, geraniol, and nerol are considered the main constituents of the rose oil (Topalov, 1978). These three components constitute the so-called “eleopten” part of the rose oil and provide the high fixing ability, which is directly related to the longevity of the perfume aroma and fragrances of the rose oil. The ISO 9842 rose oil standard requires the L-citronellol content to be within concentration ranges of 20% to 34% of the oil, nerol 5% to 12%, and geraniol 15% to 22%. The ratio among the three rose constituents, especially the sum of citronellol plus nerol, is important for rose oil quality and its marketing price. Our results indicated that within R. damascena, with the exception of #7/79 and Aleksandrovo, all cultivars and clones meet this quality requirement (Table 3). The concentration of geraniol indicates the differences existing among various cultivars and clones within one genotype. Regarding the concentration of paraffins (heptadecane, nonadecane, heneicosane), variation was also found, in some instances above the upper limit of ISO 9842 (Table 3).

The essential oils obtained from each of the rose species in this study did not show greater than 50% growth inhibition of the tested organisms at 50 μg/mL and were therefore deemed to be inactive with respect to antimicrobial, antibacterial, antifungal, antileishmania, and antimalarial activities (Table 4). Our results contradict earlier reports on antibacterial effect of R. damascena essential oil (Basim and Basim, 2003; Ozkan et al., 2004), most probably as a result of relatively lower concentrations tested in our study.

Table 4.

Antiprotozoal screening assays for antileishamia, antimalaria, antifungal, and antibacterial activities of rose essential oils from this study.

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Summary and Conclusions

Although with a similar genotype, the various R. damascena clones expressed different phenotypes. Our results suggest that clones #831, #7/79, K-IV, Svejen 188, #809, and Iskra are the most suitable for the establishment of commercial rose plantations in the region. Some of the clones of R. damascena have the potential to provide up to 0.05% average essential oil content. Hybrid #836/61 had not only high essential oil content, but also a composition that is not typical for R. damascena. Hence, this clone may have the potential to be developed as a cultivar representing another type of rose essential oil. Generally, the essential oil content of R. damascena and the two hybrid roses, #836/61 and cv. Kooperatorka, were similar and varied between 0.03% and 0.05%, whereas the essential oil content of hybrid #90/67 was lower than that of most clones. Overall, the highest essential oil content was found in R. damascena Svejen 74 (higher than in the control cultivar Iskra) and the lowest in R. alba. Essential oil constituents of all clones were determined. The essential oils from the rose species did not show greater than 50% activity and were deemed to be inactive with respect to antibacterial, antifungal, antimalarial, and antileishmania activities. Our research demonstrated that R. alba has a similar oil composition to that of R. damascena, making it a suitable genetic material for the development of new varieties.