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Fruit Quality and Chemical Contents of Hybrid Boysenberry (Rubus ursinus) Lines Developed by Hybridization and Gamma Irradiation
Plant Breeding and Biotechnology 2017;5:228-236
Published online September 1, 2017
© 2017 Korean Society of Breeding Science.

Jaihyunk Ryu1, Soon-Jae Kwon1, Yeong Deuk Jo1, Hong-Il Choi1, Kyung-Yun Kang2, Bo mi Nam1, Dong-Gun Kim1, Chang-Hyun Jin1, Jin-Baek Kim1, Ee-Yup Kim3, Seung Cheol Oh3, Bo-Keun Ha4, and Si-Yong Kang1,*

1Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongup 56212, Korea, 2Suncheon Research Center for Natural Medicines, Suncheon 57922, Korea, 3Bioplus Co., Wanju 55310, Korea, 4Division of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University, Gwangju 61186, Korea
Correspondence to: Si-Yong Kang,, Tel: +82-63-570-3310, Fax: +82-63-570-3319
Received June 19, 2017; Revised August 17, 2017; Accepted August 18, 2017.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The Rubus fruit is an economically important berry crop that contains various functional compounds. The objective of this study was to analyze fruit qualities (i.e., pH, soluble solids content, titratable acidity, and mineral content) as well as fatty acid and phenolic compounds (i.e., ellagic acid and anthocyanins) among hybrid boysenberry lines developed by hybridization and gamma irradiation. There were no significant differences in the hybrid boysenberry fruit pH and titratable acidity (%) among the tested genotypes. However, the soluble solids content was higher in the BSA119 and BSA144 mutants than in the original genotype (BS_Hybrid). Meanwhile, linoleic acid was the most abundant fatty acid in the analyzed hybrid boysenberry fruits. The fatty acid composition did not differ significantly among the genotypes. The ellagic acid content of all genotypes ranged from 8.72 mg/100 g to 46.10 mg/100 g, with the highest concentration observed for the BSB127 genotype. Additionally, cyanidin-3-O-sophoroside (M-H+, 611 m/z) and cyanidin-3-O-glucoside (M-H+, 449 m/z) were the two major anthocyanins detected in the boysenberry and mutant genotypes, while cyanidin-3-O-glucoside was the predominant blackberry anthocyanin. The total anthocyanin concentrations of four mutant genotypes (i.e., BSA036, BSA078, BSA101, and BSB127) were significantly higher than that of the original genotype (382.0 mg/100 g). The highest total anthocyanin concentration was observed for the BSA078 genotype (467.9 mg/100 g). These results may be useful for identifying the optimal genotypes for breeding new cultivars with enhanced qualities and potential health benefits.

Keywords : Edible oils, GC-MS, LC-MS, Mutation breeding, Phenolic compounds, Phytochemicals, Thornless mutant

Boysenberry (Rubus ursinus Chamisso & Schlenhtendal) is a hybrid Rubus berry derived from a cross between loganberry (Rubus loganobaccus Bailey) and trailing blackberry (Rubus fruticosus L.) (Zafra-Stone et al. 2007). Rubus fruits are recognized for the health benefits of their natural phytochemicals, such as phenolics, vitamin C, minerals, and fatty acids (Kaume et al. 2012; Lee et al. 2012; Lee et al. 2014). These fruits are rich in phenolics, and the contribution of these compounds to antioxidant activities will be of interest to breeders whose goals include developing new varieties that produce higher quality and healthier fruits than what is currently available (Wada and Ou 2002; Parry et al. 2005; Guedes et al. 2013). Anthocyanins and ellagic acid are abundant phenolic compounds in Rubus fruits (Wada and Ou 2002; Cooney et al. 2004; Ryu et al. 2016). The potential health benefits of anthocyanins have led to a renewed interest in the consumption of foods rich in anthocyanins (e.g., berry fruits) and the bio-absorption of these phenolic compounds. Previous studies revealed that the possible positive health effects resulting from the intake of Rubus fruit anthocyanins include decreased oxidative stress, lowered serum cholesterol levels, and protection from liver ischemia-reperfusion injury (Cooney et al. 2004; Ghosh et al. 2006; Cho et al. 2015). Among the Rubus species, boysenberry has the most ellagic acid, which is a potential anticarcinogenic compound (Wada and Ou 2002). Additionally, specialty high-value Rubus fruits are gaining attention owing to their health benefits, which are linked to their abundance of polyunsaturated fatty acids (Parry et al. 2005; Van Hoed et al. 2009; Ferreira de Araujo et al. 2011).

Mutation breeding involves the use of a mutagen to create plants exhibiting a few new mutant characteristics that do not disturb other varietal traits. The fruit industry relies on a limited number of clonally propagated cultivars established based on specific fruit quality parameters and consumer familiarity with the product, and is very resistant to changes (Predieri et al. 2001; Kim et al. 2015; Jo et al. 2016; FAO/IAEA Mutant Variety Database []). This limits the application of cross-breeding in fruit-bearing plant species, as fruit cultivars are generally highly heterozygous, and progenies resulting from cross-breeding express many traits that are different from those of the parents (Predieri et al. 2001; Jo et al. 2016; FAO/IAEA Mutant Variety Database). In Rubus species, mutagenesis has been used to manipulate various traits, including the presence of spines, fruit size and color, disease resistance, timing of maturity, yield, and cold-induced resistance to pathogens (Donini 1982; Ryu et al. 2016; FAO/IAEA Mutant Variety Database). Improved thornless cultivars have been produced by breeders to meet the demands of growers who wish to maintain an economic advantage in the marketplace. Additionally, evaluating boysenberry fruit resources regarding phytochemical contents is important and may be considerably beneficial for the food and pharmaceutical industries (Predieri et al. 2001; Clark et al. 2007; Ryu et al. 2016).

The objective of this study was to investigate the nutritional characteristics [i.e., mineral content, soluble solids content (SSC), and soluble sugar content] and functional compounds (i.e., anthocyanins, ellagic acid, and fatty acid) of Rubus genotypes.


Plant materials and harvest

Eleven genotypes were analyzed in this study (Table 1). Among these genotypes, the BS_PI cultivars was introduced from Japan and produced spines, while the others were stabilized lines from advanced generations and were thornless (Fig. 1). The BS_Hybrid line was obtained from crosses between the thornless blackberry (Rubus fruticosus L. cv. V3) and boysenberry (Shin et al. 2008). V3 line was developed from spiny blackberry explants exposed to somaclonal variation. Six mutant genotypes (BSA036 to BSA144) were developed from hybrid boysenberry explants exposed to 20 Gy gamma radiation. Another two mutant genotypes (BSB032 and BSB127) were produced from hybrid boysenberry explants treated with 40 Gy gamma radiation. The eight mutant lines exhibited improved agronomic characteristics, including higher fruit yields and sugar contents than the original parent. Fully ripened fruits were collected from a 3 × 3 m2 quadrant. Three replicates were used for each sample.

Estimation of pH, soluble solids content, and titratable acidity

The pH was measured using a Docu-pH meter (Sartorius Inc., Göttingen, Germany). The SSC was determined using the PR-101 hand-held refractometer (Atago USA Inc., Bellevue, WA, U.S.A.). The titratable acidity (%) was calculated according to the AOAC (1995) method.

Mineral content

We determined the mineral contents using the AOAC (1995) methods. Samples (1.0 g) were weighed and subjected to dry ashing in a clean porcelain crucible at 550°C in a muffle furnace. The resultant ash was dissolved in 5.0 mL nitric acid: hydrogen chloride: purified water (1:2:3) and gently heated on a heating mantle until the brown fumes disappeared. Then, 5.0 mL distilled water was added to each sample, which was then heated until a colorless solution was obtained. The mineral solution was filtered into a 100 mL volumetric flask. The elemental composition of the solution was analyzed in triplicate using a Model 403 atomic absorption spectrophotometer (Perkin Elmer, Waltham, MA, U.S.A.).

Fatty acid composition

Fatty acid composition was analyzed by gas chromatography–mass spectrometry (GC–MS). According to the Soxhlet extraction procedure, 5 g crushed dried fruit (80 mashed) was packed into a thimble and the oils were extracted with diethyl ether for 6 h. The fruit lipid extracts were mixed with a 20% boron trifluoride-methanol reagent, and the fatty acids were converted into methyl ester derivatives. The methyl esters of the fatty acids were dissolved in chloroform and analyzed by GC–MS (Plus-2010; Shimadzu, Kyoto, Japan). The fruit fatty acid composition was analyzed using a GC–MS instrument equipped with an HP-88 capillary column (60 m × 0.25 mm; J & W Scientific, CA, U.S.A.) under the following conditions: ionization voltage, 70 eV; mass scan range, 35–450 mass units; injector temperature, 230°C; detector temperature, 230°C; injection volume, 1 μL; split ratio, 1:20; carrier gas, helium; and flow rate, 1.7 mL/minute. The column temperature program specified an isothermal temperature of 40°C for 5 minutes followed by an increase to 180°C at a rate of 5°C/minute and a subsequent increase to 230°C at a rate of 1°C/minute. We identified the substances present in extracts by comparing their mass spectra against a database of GC–MS spectra (Nist. 62 Library) and based on their retention indices.

Ellagic acid analysis

The lyophilized, powdered fruits were diluted with purified water and methanol (37.5:63.5). We then added 10 mL 1.2 mol hydrogen chloride to the diluted mixture, which was refluxed for 16 hours at 80 ± 5°C. The extract was cooled and filtered through a 0.45 μm membrane. The following gradients were used: 0–3 minutes, 10–20% B; 3–6 minutes, 20–30% B; 6–9 minutes, 30–50% B; 9–15 minutes, 50–60% B; 15–18 minutes, 60–70% B; 18–21 minutes, 70–50% B; 21–24 minutes, 50–30% B; and 24–27 minutes, 30–0% B. Ellagic acid was detected at 254 nm and identified using commercial standards (Sigma, St. Louis, MO, USA).

Anthocyanin analysis

Anthocyanins were extracted from samples as described by Ryu et al. (2016). Briefly, 0.5 g freeze-dried fruits were treated with 5 mL methanol:water:hydrochloric acid (70:29:1, v/v) at 4°C in darkness for 24 hours, with periodic vortexing. The material was filtered through a 0.45 μm membrane filter. Three extract replicates were analyzed for each sample. The extracted anthocyanins were analyzed using a 1260 series high-performance liquid chromatography system, a 380 evaporative light scattering detector, and a 6130 quadrupole mass spectrometry system (Agilent Technologies, USA) equipped with a Poroshell 120 SB-C18 column (150 mm × 4.6 mm internal diameter, 2.7 μm particle size; Agilent Technologies, CA, U.S.A.) with a compatible C18 gua