Clematis hybridas (Clematis florida Thunb.), also known as Tiexianmudan, Fanlian and Jinbaoyin in Chinese, belongs to Clematis genus in the family of Ranunculaceae, most of which are deciduous or evergreen herbaceous lianas. Its flowers are exquisite in shape, bright in color and large in quantity and it has climbing ability, which has high ornamental value. Once it blooms, it will be very spectacular. Therefore, it is known as the “Queen of Lianas”, and is called the “Three Treasures of the Garden” together with rose and hydrangea.

With the progress of various cultivation technologies and biological genetic engineering technologies, clematis hybridas has also developed from the original single petal type to the current double and multilayer petals. Clematis hybridas with double and multilayer petals are the star series of clematis hybridas (Wang et al., 2020). With high beauty and popularity, they have become important planting materials for excellent vertical greening, pot planting and compound cultivation with ground cover plants in modern gardens. Clematis hybridas has a broad prospect in landscape application, and its flower color has unique research value. However, at present, the research on clematis hybridas with double petals mainly focuses on systematic classification and evolution (Wang, 2005), resource investigation and evaluation (Lei et al., 2022), tissue rapid propagation (Gao et al., 2021) and so on. There are few reports on the research of color mechanism.

Flower color, as one of the decisive traits of ornamental landscape plants, is the most adaptive phenotypic trait in the process of plant natural evolution (Dai and Hong, 2016). Anthocyanins are one of the important pigments that determine the color of flowers. They are widely distributed in more than 90% of angiosperms. Different anthocyanin components and their relative contents will cause changes in flower colors. It is speculated that the rich changes in the color of clematis hybridas with double petals may be related to the anthocyanins in the calyx petals. Anthocyanins are flavonoid substances, and their stability is easily affected by vacuolar pH value, epidermal cell shape, external temperature, light, etc. (Gan et al., 2020). There are at least 650 kinds of anthocyanins identified in nature, including 6 common ones, including petunidin, cyanidin, delphinidin, malvin, pelargonidin and peonidin. The synthetic pathway of anthocyanins has been basically clear, and more detailed studies have also been carried out in some plants, such as eustoma (Eustoma grandiflorum) (Gao and Li, 2020), common freesia (Freesia refracta) (Yu et al., 2020), and freesia (Freesia hybrida) (Xu et al., 2016). However, the relationship between the flower color of clematis hybridas and the composition and content of anthocyanins is still unclear, and still needs to be tested and analyzed.

In this study, the calyx petals of 7 clematis hybridas varieties with double petals from 4 color systems were selected as the research object, and the quantitative analysis of anthocyanin monomers was carried out by HPLC-MS to find out the rule of the effect of anthocyanin monomer content on the flower color phenotype, thus, to provide a theoretical basis for the flower color breeding, new variety cultivation and variety identification, protection and classification of excellent varieties of clematis hybridas with double petals.

1 Results and Analysis

1.1 Preparation of standard curve of 6 anthocyanin monomers

A certain amount of standard samples of petunidin, cyanidin, delphinidin, malvin, pelargonidin and peonidin were prepared with ultrapure water into standard mixed solutions of different concentrations, respectively injected into high-performance liquid chromatography for analysis, and the standard curve (Figure 1) was drawn with peak area (Y) and corresponding concentration (X), and the regression equation of the standard curve (Table 1) was obtained. The results showed that the linear correlation coefficient R² of petunidin, cyanidin, delphinidin and malvin was larger than 0.995, the linear correlation coefficient R² of pelargonidin and peonidin was also larger than 0.990, and the linearity of the 6 anthocyanin monomers was good, which could meet the detection requirements within the determination range.

1.2 Quantitative determination of anthocyanin monomers in clematis hybridas with double petals

7 samples of clematis hybridas with double petals from 4 color systems, namely red, purple, blue and white (Figure 2) were collected, and high-performance liquid chromatography (HPLC) tandem mass spectrometer with 150×4.6 mm×3.5 μm C18 chromatographic column was used to separate and determine the sample preparation solution. Through constant gradient elution by changing the concentration ratio of mobile phase (Table 2), with mass spectrometry parameter (Table 3), the 6 anthocyanin maps of 7 samples of clematis hybridas with double petals were obtained (Figure 3). The peak value of the chromatogram was substituted into the regression equation of the standard curve, the liquid chromatography-mass spectrometer was used to automatically obtain the sample concentration, and then according to concentration×dilution volume/mass to obtain the content of the sample (Table 4). The results showed that there were anthocyanins in the calyx petals of 7 clematis hybridas varieties with double petals from 4 color systems, namely red, purple, blue and white, but the content of 6 anthocyanin monomers was significantly different, and the dominant anthocyanin monomer in the calyx petals of clematis hybridas in different color systems were different.

1.3 Relationship between flower color and anthocyanin content of clematis hybridas

1.3.1 Relationship between flower color and anthocyanin content of red clematis hybridas

According to the test, the dominant monomers in the anthocyanins of the pale purple-red “LadyKyoko” and the dark pink “Josephine” was peonidin, with the contents of 606.29 ng/g and 669.77 ng/g respectively, followed by pelargonidin, and the content of petunidin and malvidin was low (Table 5). According to the total amount of 6 anthocyanins measured, the total amount of 6 anthocyanins in pale purple-red “LadyKyoko” was 1 191.97 ng/g, and the total amount of 6 anthocyanins in dark pink “Josephine” was 1 013.38 ng/g (Table 5). The darker the color was, the higher the content was. Compared with the other color systems, the total amount of anthocyanins in the two samples of red color system was the highest; The content of peonidin was significantly higher than that of purple and blue systems, up to 30.7 times; The content of cyanidin was also significantly higher than that of purple and blue systems, up to 13.0 times (Table 4), which is speculated that peonidin and cyanidin may be related to the red coloration of the calyx petals of clematis hybridas.

1.3.2 Relationship between flower color and anthocyanin content of purple clematis hybridas

The test results (Table 6) showed that the dominant monomer in the blue purple “Taiga” and the purple blue “Sodertalje” was pelargonidin, with the content of 421.46 ng/g and 365.42 ng/g respectively, followed by peonidin, and the content of malvidin was relatively low. According to the total amount of 6 anthocyanins determined, the total amount of 6 anthocyanins in the blue purple “Taiga” was 651.38 ng/g, and the total amount of 6 anthocyanins in the purple blue “Sodertalje” was 687.90 ng/g (Table 6), which is relatively close. Compared with the other color systems, the total amount of anthocyanins in the two samples of the purple system was second only to the red system, and the content of delphinidin was higher than the red system, but lower than the blue system (Table 4).

1.3.3 Relationship between flower color and anthocyanin content of blue clematis hybridas  

According to the test, the dominant monomer in the pale blue-purple “MultiBlue” and light blue “BlueLight” was delphinidin, with the content of 63.25 ng/g and 59.80 ng/g respectively, followed by peonidin (Table 7). From the total amount of 6 anthocyanins determined, the total amount of 6 anthocyanins in “MultiBlue” was 113.58 ng/g, and the total amount of 6 anthocyanins in the light blue “BlueLight” was 122.60 ng/g (Table 5), which is relatively close. Compared with the other color systems, the content of delphinidin in the two samples of blue color system was the highest (Table 4), which was speculated that the content of delphinidin may affect the change of flower color from purple to blue.

1.3.3 Relationship between flower color and anthocyanin content of white clematis hybridas

The test results (Table 8) showed that only a small amount of cyanidin was detected in the white “Yukiokoshi”, and no other 5 anthocyanins were detected. It was speculated that the color of clematis hybridas was related to anthocyanins, and the pure white clematis hybridas may not contain anthocyanins, and a small amount of cyanidin detected may be related to the slight red at the end of the calyx petals of “Yukiokoshi”.

2 Discussion

Anthocyanin content is the main factor affecting flower color. In this study, the HPLC method was established for the determination of 6 anthocyanins in 7 clematis hybridas varieties with double petals from 4 color systems. The results showed that the content of peonidin and cyanidin in red samples was much higher than that in purple and blue samples, indicating that the content of peonidin and cyanidin was related to the red coloration of calyx petals, which was consistent with the coloration mechanism of flowers such as Jinxiudujuan (Rhododendron × pulchrum) (Xia et al., 2022); The content of delphinidin in purple samples was higher than that in red samples, and the highest in blue samples, indicating that the content of delphinidin may affect the change of flower color from purple to blue, which was consistent with the research results of Li et al. (2020); Except for a small amount of cyanidin, there were no other 5 anthocyanins detected in the white samples, which indicates that the pure white clematis hybridas may not contain anthocyanins, and the detected small amount of cyanidin may be related to the slight red at the end of the calyx petals, which was consistent with the results detected in flowers such as Mudan (Paeonia × suffruticosa) (Gan et al., 2020). In addition, the total amount of 6 anthocyanins in red clematis hybridas was much higher than that in the other color systems, and the total amount of anthocyanins in the same color systems was relatively close, indicating that the different color of calyx petals has a great relationship with the total amount of anthocyanins.

At present, the red, purple, blue and white clematis hybridas varieties are common, but the orange and other color are rare. How to innovate the flower color is one of the key objectives of cultivating new clematis hybridas varieties. As a large resource country of clematis hybridas, China should actively invest in the development and cultivation of new varieties, so as to provide more choices for the garden application of clematis hybridas. This study preliminarily revealed the relationship between the flower color and anthocyanin content of clematis hybridas, which can provide a theoretical basis for the flower color breeding and new variety breeding of clematis hybridas. There are many purple to blue varieties of clematis hybridas, which may also be related to the combination of flavonoids, polyphenols and anthocyanins to produce color enhancement and redshift, the specific mechanism and effect results of which still need further study.

Although the main anthocyanin content in the calyx petals of 7 clematis hybridas varieties with double petals from 4 color systems, namely red, purple, blue and white, has been determined by HPLC-MS in this study, due to the complexity of color rendering, it is necessary to further clarify its color rendering mechanism through biotechnology means, such as molecular markers, RNA interference, real-time fluorescence quantitative PCR, mRNA differential display, gene cloning and screening.

3 Materials and Methods

3.1 Test materials

7 clematis hybridas varieties with double petals (LadyKyoko, Josephine, Taiga, Sodertalje, MultiBlue, BlueLight and Yukiokoshi) from 4 color systems of red, purple, blue and white were used as test materials. The test materials were taken from Clematis Hybridas Breeding Base of Jiangsu Vocational College of Agriculture and Forestry (119°16′32″N, 31°55′53″E) in the first ten days of May 2022, when the clematis hybridas varieties to be tested entered the full flowering period with more than 75% of the plants were flowering (Liu et al., 2021). During sampling, 10 to 20 plump buds were selected from each plant, and the middle part of fresh calyx petals were removed with tweezers, and then placed respectively in centrifuge tubes for ultra-low temperature storage.

3.2 Main reagents

1) Formic acid: chromatographically pure, methanol: chromatographically pure; 2) Standard samples of petunidin, cyanidin, delphinidin, malvin, pelargonidin and peonidin, with purity of 96%; 3) Standard mixed solution: accurately weigh each standard samples and prepare standard mixed solution of different concentrations with ultrapure water. 4) Sample solution: properly weigh calyx petal samples of various colors, add purified water to a 50 mL volume, shake well and then stand still, and use 0.22 μm membrane filtration for standby.

3.3 Test instruments

Shimadzu LC-30AD high-performance liquid chromatography, ABSCIEX QTRAP6500+ mass spectrometer, 1/10000 analytical balance, water bath, centrifuge, oscillator.

3.4 Determination methods

Preparation of standard curve: Inject 6 standard mixed solutions into the high-performance liquid chromatography for analysis, and take the concentration of standard mixed solution of anthocyanins as the abscissa (X) and the peak area measured by the instrument as the ordinate (Y) to obtain the standard curve.

Determination of anthocyanin monomer: by referring to the previous determination method (Lin et al., 2013), with 150×4.6 mm×3.5 μm C18 chromatographic column, column temperature (35℃), flow rate (1 mL/min), injection volume (2 µL), mobile phase A being water (containing 0.1% formic acid) and mobile phase B being acetonitrile (0.1% acetonitrile), the sample solution was injected into the high-performance liquid chromatography for separation and determination. The test concentration of the sample was automatically calculated by high-performance liquid chromatography by substituting the peak value into the regression equation of standard curve, and the content of the sample was obtained according to concentration×dilution volume/mass.

3.5 Data analysis

Excel software was used for data analysis.

Authors’ contributions

YMX was the experimental designer and implementor of this study, responsible for the writing and revision of the paper; SW was the designer and leader of the project. ZJ and CTS participated in some data statistics and experiments; LHH guided some experimental operations. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by the Open Competition Mechanism to Select the Best Candidates Project for Seed Industry Revitalization in Jiangsu Province (JBGS(2021)092) and Science and Technology Plan Project of Jiangsu Vocational College of Agriculture and Forestry (2022kj36).

References

Dai S.L., and Hong Y., 2016, Molecular breeding for flower colors modification on ornamental plants based on the mechanism of anthocyanins biosynthesis and coloration, Zhongguo Nongye Kexue (Scientia Agricultura Sinica), 49(3): 529-542.

Gan L.X., Li H.H, Yang C., Ming L., Han M.L., and Xin Z.X., 2020, Analysis of the content and composition of peony pigments in different colors, Northern Horticulture, 44(5): 67-73.

Gao S., and Li T.L., 2020, Relationship between the composition of anthocyanins and flower color variation in Lisianthus (Eustoma grandiflorum), Shenyang Nongye Daxue Xuebao (Journal of Shenyang Agricultural University), 51(4): 410-416.

Gao Y., Mo J.B., Fu Y.R., and Feng S.C., 2021, Tissue culture and plant regeneration of clematis ‘Julka’, Nanjing Linye Daxue Xuebao (Journal of Nanjing Forestry University (Natural Sciences Edition), 45(3): 109-116.

Lei L., Zhou Y.X., Jing R., and Chen L.Q., 2022, Investigation on wild germplasm resources of clematis plants in Shanxi Province, Zhongzi (Seed), 41(6): 50-54, 63.

Li X., Wu Y.Y., and Feng Y., 2022, Analysis of anthocyanin content and related gene expression in different varieties of Hyacinthus orientalis, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 18(14): 4562-4571.

Lin Y.S., Liu X.M., Yang R.L., Chen Z.Y., Yang C.Y., Zhao X.L., and Wang S.Y., Determination of polyphenols and anthocyanins contents in mulberry tablet, 2013, Xiandai Shipin Keji (Modern Food Science and Technology), 29(4): 890-893.

Liu H., Ren Y., Lin B.G., Zhu J.F., Cheng H., Zhang D.Q., Wang J.W., and Hua S.J., 2021, Dissection of petal pigment components in colored rapeseed (Brasscia napu L.) genotypes, Henongxue Bao (Journal of Nuclear Agricultural Sciences), 35(4): 837-845.

Wang J.Y., Qiao Q., Zhang J., and Hu F.R., 2020, Research progress and landscape application of Clematis L., Shandong Nongye Daxue Xuebao (Journal of Shandong Agricultural University (Natural Science Edition), 51(2): 217-221.

Wang W.C., and Li L.Q., 2015, A new system of classification of the genus Clematis (Ranunculaceae), Zhiwu Fenlei Xuebao (Acta Phytotaxonomica Sinica), 43(5): 431-488.

Xia X., Gong R., and Zhang C.Y., 2022, Anthocyanin composition and coloration mechanism in petals of Rhododen-dron pulchrum with different colors, Jiangsu Nongye Xuebao (Jiangsu Journal of Agricultural Sciences), 38(1): 207-213.

Xu Y. Q., Yuan Y., Tao X.H., Yang J., Shi Y.M., and Tang D.Q., 2016, Main Anthocyanin Profiles in Petals of Freesia hybrida, Zhiwu Yanjiu (Bulletin of Botanical Research), 36(2): 184-189.

Yu J.J., Tang D.Q., and Li X., 2020, Anthocyanin compositions in petals of Freesia hybrida, Guangxi Zhiwu (Guihaia), 40(5): 687-695.