Variability in seed storage components (protein, oil and fatty acids) in a Camellia germplasm collection

De Haro, A.* ¹, Obregón, S.¹, del Río², M., Font, R.², Mansilla, P.³, Salinero, M.C.³

¹ Instituto de Agricultura Sostenible, CSIC. Finca Alameda del Obispo, 14080 Córdoba, Spain.        
  E-mail: adeharobailon@ias.csic.es
² IFAPA Centro La Mojonera, Avda. del Mediterráneo, La Mojonera, Almería, Spain
³Estación Fitopatolóxica do Areeiro, Subida a la Robleda s/n, 36153 Pontevedra, Spain.

Introduction

      The genus Camellia is native to East Asia and includes a very large number (>200) of species. Notable among them are C. japonica, C. sinensis, C. sasanqua, C. reticulata and C. oleifera. The Japanese camellia tree (C. japonica) is native to Southern Japan where it is called Rose of Winter. Camellia sasanqua (Christmas Camellia) is a Camellia species native to China and Japan. Although Camellia is known worldwide for the production of tea, there is a growing industry that uses the oil derived from camellia seeds. Camellia oil, extracted from a number of different species including C. japonica, C. reticulata, C. sinensis and C. oleifera, has long been processed as industrial oil, for the production of medicines, cosmetics, soaps, and recently it is generating interest as a biofuel source (Lin and Fan, 2011). Camellia tea seeds have been utilized in China for more than a thousand years as an oil source. Tea oil is the main cooking oil in China’s southern provinces and Southeast Asia. Camellia oil is considered a high quality cooking oil, with high amounts of unsaturated fatty acids, mainly oleic and linoleic acids. This oil, called the Eastern olive oil by Long and Wang (2008) because it contains abundant oleic acid like olive oil, can be stored at room temperature. In addition to this, camellia oil is reputed to aid cholesterol reduction and resistance to stress (Fu & Zhou, 2003) and to protect against lipid peroxidation by elevating the expression of antioxidant enzymes (Lee et al. 2007). Camellia japonica oil has a long history of use as a cosmetic product to keep skin and hair healthy, with antibacterial activity and with anti-inflammatory properties (Kim et al. 2001, Kim et al. 2012). Also the antioxidant and antimicrobial features of virgin C. oleifera, C. reticulata and C. sasanqua oils have recently been demonstrated (Feas et al. 2013).
      The high oil content, (>30%) of Camellia seeds can vary depending on genetic and environmental factors (species, cultivars, temperature, rainfall, etc). Furthermore, fruit traits such as seed size and dry weight affect oil production in Camellia species (Li et al., 1992; Yanru and Zhangju, 2010, Huang et al. 2013).
      Galicia (NW Spain) is one of the most important Camellia producing-regions in Europe. Although camellias in Galicia are produced mainly as houseplants and for gardening purposes, recently interest has arisen in relation to the production of oil as a new market opportunity.
      The aims of this work were: a) to study  the chemical composition of seeds from different Camellia species grown in a live Camellia germplasm collection maintained at the Estacion Fitopatoloxica do Areeiro, in NW Spain, and b) to characterize the fatty acid composition of cold-pressed oil samples from different Camellia species produced at the E.F. do Areeiro.

Material and methods

Plant material

      The camellias used in this study were part of a live Camellia germplasm collection maintained at the Estacion Fitopatoloxica do Areeiro (Pontevedra, NW Spain). In 2004, seed samples from different accessions belonging to this germplasm bank were sent to the Plant Breeding Department of the Institute for Sustainable Agriculture (IAS, CSIC, Córdoba, SW Spain) to be analyzed for seed quality components. These materials consisted of 22 accessions of Camellia japonica, 2 accessions of C. sasanqua, 1 accession of C. reticulata x C. japonica, and 1 accession of C. sinensis.
      In 2013, seed oil samples of Camellia (obtained by cold-pressed method) of different accessions from the germplasm collection above cited were sent to the Plant Breeding Department of the IAS-CSIC to be analyzed for fatty acid composition. This selection consisted of 23 seed oil samples of C. japonica, 5 seed oil samples of C. sasanqua, 1 seed oil sample of C. reticulata, and 1 seed oil sample of C. hibrida.

Methods

      Analysis of germplasm (seed samples): five to ten seeds were randomly selected from each of the accessions received and individually analysed for the following seed quality components: protein content (% of dry matter), oil content (% of dry matter) and fatty acid composition (% of oil), according with the following standard methods for oilseeds: Kjeldahl method for protein content (N x 6.25), Nuclear Magnetic Resonance and Soxhlet method for oil content, and gas-liquid chromatography for fatty acid composition. The analytical data obtained from all the single seeds of each accession were combined to obtain the average content of the seed quality components for each accession.
      Analysis of Camellia seed oil: two different samples of seed oil from each of the accessions received were analysed for fatty acid composition by gas-liquid chromatography (GLC) of fatty acid methyl esters.
      Analysis of fatty acid composition: fatty acid methyl esters from seeds samples and from oil samples were obtained as described by Garces and Mancha (1993) and analysed on a Perkin-Elmer Autosystem gas–liquid chromatograph (Perkin-Elmer Corporation, Norwalk, CT, USA) equipped with a flame ionization detector (FID) and split injector. The chromatograph was equipped with a capillary column (25m × 0.25mm, i.d. 0.25 µm film) with acidified polyethylene glycol as the stationary phase. Oven temperature was programmed from 195 to 225°C at a rate of 2°C min-1. The temperature of the detector and injector were 275 and 250°C, respectively. Nitrogen was used as carrier gas. Fatty acids were identified by comparing the retention times of the Camellia methyl esters with those of known mixtures of standard fatty acids (Sigma) run on the same column under the same conditions.

Results and discussion

      Quality components of Camellia seed samples

      Fatty acids identities based on gas-liquid chromatography of Camellia germplasm are in Table 1.

Table 1. Nomenclature of Common Fatty Acids identified in seeds and oil of Camellia species

 (*) International Union of Pure and Applied Chemistry

      Data on protein content, oil content and fatty acid composition for the different species of Camellia accessions are summarized in Table 2. 

Table 2.  Protein content (% dry weight), Oil content (% dry weight) and Fatty acid composition (% of oil) of the seed samples from different Camellia species.

(*)  "a"=number of seed analyzed belonging to “b” different accessions

      The seed protein content of Camellia japonica accessions ranged from 8.71 to 14.88%, with an average content of 12.01%. The range of variation for the seed protein content found in the other species of Camellia germplasm is more limited than that found in C. japonica, possibly due to the smaller number of accessions analyzed.
      The seed oil content of Camellia japonica accessions ranged from 52.58 to 71.69%, with an average content of 61.44%.
      The protein and oil content of C. saluenensis seems to be similar to that of C. japonica. On the contrary the accession of C. sinensis is characterized by having higher seed protein content and lower seed oil content than that of C. japonica.
      The lowest oil content was found in all the individual seeds of the C. sinensis accession (mean oil content=38.76%). Nevertheless this oil content is higher than that found in Turkish germplasm by Yazicioglu and Karaali (1983).
      The seed oil content and the seed protein content of all the accessions studied from the different Camellia species are shown in Fig. 1.

Fig. 1. Oil and protein content in seeds of accessions from different Camellia species

      Most of the accessions of C. japonica exhibited oil contents above 60%. The accession br5b (Brava 5 white flower) had the highest mean seed oil content (71.69 %). The individual seed with the highest oil content (75%) was also found in the same accession br5b.
      The oil contents of C. sasanqua accessions are lower than that of C. japonica but higher than that of C. sinensis. More accessions of C. sasanqua and C. sinensis should be analyzed to confirm these preliminary results.
      The fatty acid composition of the seed oil from the accessions belonging to C. japonica, C. saluenensis, C. sasanqua and (C. reticulata x C. japonica) exhibit a similar pattern (see Table 1): about 8% in palmitic acid, about 2% in stearic acid, about 84% in oleic acid and about 5% in linoleic acid. On the contrary, all the seeds from the C. sinensis accessions studied show a different fatty acid pattern characterized by higher palmitic acid (about 14%), lower oleic acid (about 64%) and higher linoleic acid (18%) in relation to that of C. japonica accessions. The C. sasanqua samples of our study are higher in oleic acid content and lower in palmitic acid content than those from Vietnam samples described by Ucciani (1995), and similar to some C. oleifera germplasm analyzed by Tang et al. (1993). These differences in fatty acid composition are evident in the ratios saturated/unsaturated fatty acids, which range from 10.32% in C. reticulata x C. japonica to 20.44% in C. sinensis.
      The fatty acid composition of all the accessions studied from the different Camellia species are shown in Fig. 2.

Fig. 2. Fatty acid composition of the accessions from different Camellia species

      Many accessions of C. japonica exhibited oleic acid content above 85%, with the accession br5b among them.  The individual seed with the highest oleic acid content (89.39%) was found in the accession 16. The individual seed with the lowest oleic acid content (56.81%) and the highest linoleic acid content (26.81%) was found in the C. sinensis accession 143.  
      There is no correlation between the oil and the protein content of the accessions studied (Table 3). On the contrary, we have found a positive correlation between oil content and oleic acid content (0.81), and a negative correlation between oil and linoleic acid content (-0.78). Oleic acid content is negatively correlated both with palmitic acid content (-0.96) and with linoleic acid content (-0.98). According to these data, the selection of accessions having seeds with high oil content would allow us to improve the oil quality by simultaneously increasing the oleic acid content and decreasing the saturated palmitic acid content.

Table 3. Correlation coefficients between seed quality components

      Fatty acid composition of Camellia oil samples

      The fatty acid composition of the oil for the different samples and species of Camellia accessions is summarized in Table 4.

Table 4.  Fatty acid composition (% of oil) of the cold-pressed oil samples from different Camellia species.

(*)  "a"=number of oil samples analysed belonging to “b” different accessions

      Oil samples of C. japonica accessions have higher mean contents of palmitic acid (10.16%) and linoleic acid (6.25%), and lower mean content of oleic acid (80.59%) than those of the seed samples of the same species (mean contents of 8.59% for palmitic acid, 4.82% for linoleic acid, and 84.05% for oleic acid).  The same occurs with the fatty acid content of C. sasanqua oil samples in relation to those of C. sasanqua seed samples. The fatty acid composition of the Camellia oil samples in our study reflects the results obtained by Haiyan et al. (2007) by analyzing commercial samples of both refined Camellia oil and cold-pressed Camellia oil. The fatty acid composition of the C. japonica oil samples in our study match with that obtained by Salinero et al. (2012) with samples from the same germplasm collection.
      Similar to the fatty acid composition of seed samples, oil samples of C. sasanqua accessions have a slightly lower content in oleic acid and higher content in linoleic acid than those of C. japonica accessions. The fatty acid pattern of C. reticulata accession is similar to that of C. japonica accessions, although more accessions should be studied to confirm this trend.
      The fatty acid composition of the oil for all the accessions studied from the different Camellia species is shown in Fig. 3

Fig. 3. Fatty acid composition of the cold-pressed oil samples from different Camellia species.

      Oil samples from C. japonica accession 095-13 (Mision Biológica de Galicia) stand out as that with the highest linoleic acid content in this species (mean content = 9.06%). The highest content in oleic acid (84.22%) was found in the oil samples of the accession 108-13 from E.F. Areeiro. 
      Oil samples from C. sasanqua accession 118-13 (Areeiro) exhibits the lowest oleic acid content (73.83%) and the highest linoleic acid content (11.43%) of all the species analyzed.
      The highest stearic acid content (3.74%) has been found in the oil of the unique C. hibrida accession (148-13 Rubians 71) analyzed.
      The correlation coefficients between fatty acids of the different oil samples studied follow the same pattern as those obtained for the fatty acids of seed samples. We have found a negative correlation between oleic acid content and palmitic acid content (-0.72) and also an important negative correlation between oleic acid content and linoleic acid content (-0.82).
      These facts confirm the importance of exerting selection pressure on increasing the oleic acid content of Camellia seeds as the most suitable way for obtaining Camellia  varieties able to produce high quality ‘olive’ oil.

 

Conclusions

      A wide range of variation for all the characters analyzed was found, making it possible to select accessions with both high oil and high oleic acid content, and a fatty acid composition similar to that of high quality olive oil, specially within C. japonica accessions.
      All Camellia seed oils analyzed, regardless of the species involved, had high amounts of total unsaturated fatty acids (mainly oleic acid) and therefore are suitable for edible cooking or salad oils and for the manufacture of margarine.
      Results of the present work indicate that it is possible to exploit some varieties of Camellia spp. as an oilseed crop in NW Spain by selecting genotypes with high oil and high oleic acid content in their seeds.

Acknowledgements

      We thank Gloria Fernández (IAS-CSIC) for her technical assistance.

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