Comparative Analysis of Headspace Volatiles of Chinese Rosa rugosa

The floral headspace compounds of Chinese Rosa rugosa germplasms that were isolated by an automated headspace sampler with built-in trap, and followed by gas chromatography-mass spectrometry for identification and quantification. Up to 33 volatile compounds were identified from the 23 rose germplasms, including nine alcohols, five esters, three alkanes, 10 terpenes, three aldehydes, two ketones, and one ether. The main floral components identified were 2-phenylethanol, β-citronellol, ethanol, and n-hexane. ‘xizi’, ‘miaofengshan’, ‘xiangciguo’, and ‘tangbai’ contained the highest amounts of 2-phenylethanol at 84.66 μg·g-1, β-citronellol at 70.98 μg·g-1, ethanol at 83.87 μg·g-1, and n-hexane at 18.23 μg·g-1, respectively. ‘Rongchengyesheng’, ‘tanghong’, ‘xizi’, ‘miaofengshan’, and ‘baizizhi’ could be considered good materials for extracting rose oil and breeding new cultivars.


Introduction
Rosa rugosa, a deciduous shrub of the genus Rosa, has prickly stems, pinnately compound leaves, and variously colored, often fragrant flowers. The native range of R. rugosa includes Northeastern China [1], Northern Japan [2], the Korean Peninsula [2], and the Russian Far East [3]. In China, wild R. rugosa are naturally distributed on the coast and islands of Southern Liaoning Province, Eastern Shandong Province, and Tumen River estuary in Jilin Province, and is classified as an endangered species. In many European countries, it is also used to produce herbal medicines or foodstuffs rich in ascorbic acid, and is extensively used in the breeding of cultivated roses. Due to its hardy and diseaseresistant foliage, it has been widely used as a rootstock for grafted roses [4].
R. rugosa had a long history (over 1,300 years) of cultivation for perfume production in China and numerous varieties and cultivars have been developed mainly through hybridization, sport mutation, and seedling selection. Analytical research on the headspace compounds in rose essential oil has also been carried out for many years using different methods [26], but the content and distribution of the volatile components in the different Chinese R. rugosa germplasm resources are still unclear. Thus, identifying specific germplasms that have a high content of monoterpenes and acetate esters is necessary to take full advantage of the rich rose resources in China. In addition, the headspace constituents of rose flowers and essential oil vary and few studies on the headspace compounds in the fresh flowers of R. rugosa have been reported [26]. Therefore, this study aimed to evaluate and compare the floral headspace compounds and their content in different germplasms of Chinese R. rugosa, as well as to provide useful information regarding rose breeding. Table 1 lists the code numbers and names of the tested R. rugosa germplasms. Tables 2 and 3 list the volatile components identified from the flowers of R. rugosa, as well as their retention indices (RI), and their content in the different samples. In this study, 33 volatile compounds were identified in 23 rose germplasms, namely 9 alcohols, 5 esters, 3 aldehydes, 2 ketones, 1 ether, 10 terpenes, and 3 alkanes. Alcohols, alkanes, and esters were the major headspace compounds because they play dominant roles in the formation of the rose fragrance, whereas the other minor compounds, such as terpenes, aldehydes, ketones and ethers balance and form the specific fragrances of different R. rugosa germplasms. The main floral headspace components identified in all R. rugosa germplasms were as follows: ethanol, n-hexane, 2-phenylethanol and β-citronellol, the latter two compounds are the main constituents of 23 R. rugosa germplasms because they had the greatest aromatic intensity.

Analysis of floral headspace volatiles in different R. rugosa germplasms
The total alcohol varied content among the 23 germplasms, with 'xizi' having the highest content at 252.01 μg·g -1 , followed by 'tanghong' at 204.96 μg·g -1 , whereas 'zhongkeerhao' had the lowest content at 5.38 μg·g -1 . The most abundant alcohol compounds were 2-phenylethanol, β-citronellol, ethanol, and cis-geraniol, which were already reported as the major components in the fresh flowers and essential oil of Chinese R. rugosa [20,27].
Three alkanes were identified in the flowers of 23 R. rugosa germplasms, with n-hexane being the most abundant at an average content of 8.90 μg·g -1 whereas 'tangbai' and 'mupingyesheng' had the maximum and lowest content at 18.23 and 1.46 μg·g -1 , respectively. In addition, trans-rose oxide, an important constituent of the rose-specific aroma, was detected in 8 R. rugosa germplasms at an average of 1.60 μg·g -1 .

Analysis of floral headspace volatiles in some specific R. rugosa germplasms
'Mupingyesheng' and 'rongchengyesheng', two wild germplasms introduced from the coast and islands of Eastern Shandong Province of China, are classified as an endangered species [1]. Consequently, protecting and rationally using these precious wild germplasm resources is imperative. As shown in Tables 2 and 3, 13 volatile compounds, mainly alcohols, esters, aldehydes, ketones, terpenes and alkanes, were identified in 'mupingyesheng', whereas ethers were not detected. The main headspace compounds were β-citronellol, 2-phenylethanol, cis-geraniol, and ethanol, with β-citronellol as the most abundant at 11.14 μg·g -1 , compared with the other components.
Twelve compounds, including alcohols, aldehydes, ketones, terpenes, and alkanes, but not esters and ethers, were identified in 'rongchengyesheng', among which 2-phenylethanol, β-citronellol, cisgeraniol and ethanol were the major headspace compounds. The 2-phenylethanol content was the highest at 73.11 μg·g -1 . The total amount of headspace substances in 'rongchengyesheng' reached 124.88 μg·g -1 , 3.76 times that seen in 'mupingyesheng', and the alcohols were at levels up to 118.4 μg·g -1 or four times the level in 'mupingyesheng'. Consequently, 'rongchengyesheng' can be considered a potential high value germplasm.
Until now, the differences in headspace compounds between R. rugosa cultivar and its mutants have not been reported. 'Zizhi' had an important industrial and ornamental value with excellent features such as amaranthine flowers, amaranthine branches, and multi-seasonal flowering. 'Dahongzizhi', 'fenzizhi', and 'baizizhi' are sport mutations of 'zizhi', and their flowers are red, pink, and white, respectively. According to our results, 11, 10, seven, and 14 volatile compounds were identified in the flowers of 'zizhi', 'dahongzizhi', 'fenzizhi', and 'baizizhi', respectively. Only alcohols, esters, and alkanes were detected in 'zizhi', with alcohols being the major components. In contrast, only alcohols, esters, terpenes, and alkanes were detected in 'dahongzizhi', 'fenzizhi', and 'baizizhi', with alcohols and esters as their main components.

Plant material
A total of 23 R. rugosa germplasms were used in this study, including two wild germplasms and 21 cultivars. Table 1 shows the code numbers and names of the 23 germplasms that were collected from the R. rugosa germplasm repository of Shandong Agricultural University, China.

The headspace volatiles extraction
Three to five robust rose branches from each germplasm that had the most flowers were cut under water and immediately placed inside a vase with clean water, and were then quickly transported to the laboratory for testing. Five full freshly opened flowers were selected. Their petals were gently plucked and mixed. Up to 2 g of petals were quickly placed in 15 mL sample vials, internal standard (3-nonanone, 10 μL, 0.8 μg·μL -1 ) were added, and the vials were then sealed rapidly with septa (PTFE-butyl synthetic rubber). Extraction and concentration of the floral headspace volatiles from the fresh flowers were performed using a TurboMatrix 40 Trap (Automated headspace sampler with built-in trap, PerkinElmer, USA). The extraction conditions was as follows: The heating temperature was at 35 °C for 30 min, the temperature of the sampling needle and transmission line was 80 °C, the sample vial head pressure was 15 Pa, and trap was maintained for 5 min.

Gas chromatography with mass spectrometry (GC-MS) analysis
GC-MS was carried out using a GCMS-QP2010 Plus (Shimadzu Corporation, Japan). The mass spectral ionization temperature was set to 200 °C. The electron energy was 70 eV. Mass spectra were obtained by automatic scanning at m/z 45-450 amu.
The flow rate of the helium carrier gas on Restek Rtx-1 (30 m × 0.25 mm I.D., 0.25 μm film, Restek Corporation, USA) was 1.03 mL/min. Then, 1.0 μL of the sample was injected in the splitless mode at an injector temperature of 200 °C. The column temperature was programmed as follows: The initial temperature was maintained at 40 °C for 2 min, and then increased from 40 °C to 130 °C at 4 °C/min, maintained at 130 °C for 1 min, and finally increased to 230 °C at a rate of 7 °C/min, which was maintained for 4 min.

Qualitative and quantitative analysis
Statistical analysis was done using the Xcalibur software. Qualitative analysis was performed as follows: spectrometric data were compared with those obtained from the NIST HP59943C original library mass spectra (Hewlett-Packard) and the Wiley library, combined with the manual resolution of mass spectra and confirmed by comparing the Kovat's retention indices and relative reports from the literature. Only results identified with positive and negative matching values of more than 800 (maximum is 1,000) were shown in this article.
Quantitative analysis was done with 3-nonanone (10 μL at 0.8 μg·μL -1 ; Sigma Ltd. Co., St. Louis, MO, USA) as the internal standard. The selected ion monitoring technique was used for quantitative analysis of the headspace compounds, whereas the response factor method combined with the internal standard method was used to quantify these compounds. The mass fraction was calculated using the formula given below: Content of each component (μg·g -1 ) = [Peak area of each component/peak area of internal standard × Concentration of internal standard (μg·μL -1 ) × Volume of internal standard]/ Sample weight (g)

Conclusions
This study revealed a high level of floral headspace compound polymorphism in Chinese R. rugosa plants. Alcohols, alkanes, and esters were the major headspace compounds, whereas the main floral components that were identified in the 23 germplasms were ethanol, n-hexane, 2-phenylethanol, and βcitronellol. 'Tanghong', 'xizi', and 'miaofengshan' had high amounts of 2-phenylethanol, β-citronellol, cis-geraniol, citronellol acetate, and nerol acetate. 'Rongchengyesheng', a precious wild germplasm, is a potentially high-value germplasm because of its high aromatic intensity. After comparing the headspace compounds in a specific cultivar and its sport mutations, 'baizizhi' was verified as a significant source of rose oil due to its high concentrations of citronellol acetate and β-phenethyl acetate. In conclusion, 'rongchengyesheng', 'tanghong', 'xizi', 'miaofengshan', and 'baizizhi' are good materials for extracting rose oil and breeding new cultivars.