Enhancement of Food Functionality of a Local Pungent Radish “Izumo Orochi Daikon” ‘Susanoo’ by Introduction of a Colored Root Character

Abstract

Purple and red colored root characteristics were introduced to a local pungent radish “Izumo orochi daikon” ‘Susanoo’, and several characteristics including food functional components were evaluated. The root and leaf phenotype, the pigment composition, the isothiocyanate and soluble solids contents, and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity in the root were investigated. These characteristics were compared between the mass selection breeding lines of purple, red roots and their parent cultivars. Although one-third of individuals in the M₆ and M₇ were non colored, the colored root characteristics was introduced into ‘Susanoo’. The main anthocyanins in the deep reddish purple colored purple root lines were acylated cyanidin 3-sophoroside-5-glucosides, and those in the vivid purplish red colored red root lines were acylated pelargonidin 3-sophoroside-5-glucosides, which corresponded to the main anthocyanins in their respective colored root parental cultivars. The isothiocyanate contents in the purple and red root lines were almost the same as that in ‘Susanoo’. The DPPH radical scavenging activity of the purple and red root lines was almost two times higher than that of ‘Susanoo’. These results showed that the food functionality was enhanced by introduction of a colored root characteristic in ‘Susanoo’.

Introduction

Japanese wild radish (Raphanus sativus L. f. raphanistroides Makino; Japanese name, Hama-daikon) grows widely in Japan and East Asia. It is used as a pungent condiment known as “Nodaikon” (wild radish) in the San’in area of Japan. “Izumo orochi daikon” is a newly developed local savory variety of pungent radish that was domesticated from a Japanese wild radish growing beside Lake Shinji and in the areas surrounding the Shimane Peninsula in Japan. It has been improved by selective breeding, focusing on a high isothiocyanate content, favorable root shape, and late-flowering characteristics (Ban et al., 2009; Kobayashi et al., in press). This newly developed radish was registered as ‘Susanoo’ (July, 2011, Register No. 20879; Ministry of Agriculture, Forestry and Fisheries, Japan), and commercially produced seeds have been released from the experimental farm of Shimane University under the university’s brand name. Because of its high pungency and unique savory and umami flavors, it is a popular condiment that is served with various Japanese dishes including soba noodles, sashimi raw fish, and meat dishes. Beside the roots, stuffed leaves are also edible. This new variety is now being cultivated mainly in the Shimane area and also in the metropolitan area as a representative local vegetable of Shimane Prefecture.

Brassica vegetables like broccoli (Brassica oleracea var. italica), kale (B. oleracea L. var. acephala DC.), cabbage (B. oleracea L.), and radish (Raphanus sativus L.) are rich in phytochemicals such as phenolics, vitamins, glucosinolates, and anthocyanins, which positively affect human health (Wei et al., 2011). Glucosinolates are hydrolyzed by myrosinase to form isothiocyanates, which react with various cellular targets and have antimicrobial (Hashem and Saleh, 1999), antimutagenic (Hamilton and Teel, 1996), and anticarcinogenic (Hecht, 1999) activities.

Anthocyanins can prevent oxidative damage to DNA, proteins, lipids, and other macromolecules caused by reactive oxygen species. de Pascual-Teresa and Sanchez-Ballesta (2008) suggested that anthocyanin-rich products are beneficial for human health, as they may prevent cardiovascular diseases and some types of cancer. Radish anthocyanins have bright colors over a wide pH range and their multiple acylation with hydroxycinnamic acids contribute to their remarkable heat stability in acidic environments (Jing et al., 2012). Radish anthocyanins have been widely used as natural colorants because of their strong natural color and health benefits, including antioxidant activity (Matsufuji et al., 2007; Rahman et al., 2006). The main anthocyanins of purple radish are acylated cyanidin 3-sophoroside-5-glucosides, whereas those of red radish are acylated pelargonidin 3-sophoroside-5-glucosides (Kato et al., 2013).

To enhance antioxidant ability and extend its appeal to color dishes with natural pigments, the colored root characteristics were introduced to ‘Susanoo’. Purple and red root mass selection breeding lines were constructed after crossing with colored root parental cultivars. In this study, the root and leaf phenotype, anthocyanin composition, isothiocyanate and soluble solids contents, and antioxidant activity were investigated in purple and red root breeding lines, and they were compared with their parental cultivars.

Materials and Methods

Breeding of plant materials and their characterization

The genealogical chart of purple and red root breeding lines is shown in Figure 1. To introduce the colored root characteristic into ‘Susanoo’, the purple root radish ‘Karaine aka’ (Watanabe seed Co., Ltd., Miyagi, Japan) which accumulated anthocyanin mainly in the root skin was used as a genetic source of cyanidin, and the red root radish ‘Chouan aomaru koshin’ (Takii & Co., Ltd., Kyoto, Japan) which accumulated anthocyanin in mainly the root xylem parenchyma was used as a genetic source of pelargonidin. The F₁ hybrid with purple roots was obtained from crosses between ‘Susanoo’ and each of these colored root cultivars in May 2006. Approximately seven individuals with colored roots and leaves, and a root shape similar to ‘Susanoo’ were selected from the F₁ progenies. The first mass selection generation (M₁) was obtained by open pollination of F₁ in 2007 to introduce pigmentation into the root skin and xylem parenchyma of ‘Susanoo’. The M₂ were obtained by mass-selection of approximately seven individuals with colored roots and leaves, root shape, and high isothiocyanate contents from the M₁ progenies in 2008. Approximately seven individuals with the desired purple and red root phenotypes were selected from among the M₂ progenies to establish the purple and red root breeding lines, respectively. From M₄ to M₈ were obtained by mass-selection of approximately fifteen purple and red root progenies with the desired root shape and contents from 2010 to 2014, respectively. The first selfing generations (S₁), S₂, and S₃ were obtained by bud-pollination of purple and red root M₅ progenies from 2012 to 2014.

Fig. 1

Genealogical chart of purple and red root breeding lines. Gray, black, and white boxes indicate wild species, cultivars and breeding lines, respectively.

M₆ and M₇ were grown at the experimental farm of Shimane University (Izumo, Japan). S₁ and S₂ were grown in a field of Shimane University (Matsue, Japan). All lines were grown from September until February for characterization.

We evaluated the following phenotypes of the mass selection lines, M₆ and M₇ in purple and red root breeding lines: leaf shape, taproot shape, lateral root level, and anthocyanin accumulation in the leaf and root. The inbred lines S₁ and S₂ in purple and red root breeding lines were evaluated only for root color. The evaluation standards were as follows: Leaf shape: lobed leaf, mid, or entire leaf. Taproot shape: Kameido type, Taibyo type, Nezumi type, Koshin type, and Kikon type. Lateral root level was classified according to a number of lateral roots; none (level 0), a few (level 1), moderate (level 2), and many (level 3) (Fig. S1). Anthocyanin pigmentation in leaves: none, weak, medium, and strong. Root color: wholly purple, partially purple (colored skin, cortex, or xylem parenchyma), wholly red, partially red, and white.

Analysis of root pigments in purple and red root breeding lines

‘Susanoo’, ‘Karaine aka’, ‘Chouan aomaru koshin’, M₆ of purple, and red root breeding lines were used for root pigments analysis. The color of the root skin and xylem parenchyma in ‘Susanoo’, purple and red root breeding lines were recorded by photographs (Fig. 2) and by the RHSCC (Royal Horticultural Society Colour Chart), and measured for lightness (L*) and two chromatic components a* and b* using a Color Reader (CR-10; Konica Minolta Sensing Inc., Tokyo, Japan). The root color of ‘Karaine aka’ and ‘Chouan aomaru koshin’ were investigated using the RHSCC and a Color Reader in the mainly pigmented parts. Refer to Mizuta et al. (2009) for details. For both breeding lines, root skin and xylem parenchyma were collected and dried overnight at 40°C, and then kept at 4°C until analysis. Anthocyanins were extracted from each sample (about 10 mg) in 1 mL MAW (MeOH-HOAc-H₂O, 4:1:5, v/v/v) at 4°C overnight, and then an aliquot of the extract was analyzed by HPLC. The HPLC system comprised an LC 10A instrument (Shimadzu, Kyoto, Japan) equipped with a Waters C18 (4.6 × 250 mm) column. The temperature was 40°C, the flow rate was 1 mL·min⁻¹, and product elution was monitored at 515 nm. The eluant was applied as a linear gradient for 40 min from 20% to 85% solvent B (1.5% H₃PO₄, 20% HOAc, 25% MeCN in H₂O) in solvent A (1.5% H₃PO₄ in H₂O).

Fig. 2

Photograph of ‘Susanoo’ (left) and purple and red root breeding lines (center and right) in 2012 (A) and their cross sections (B–D). Bar indicates 10 cm.

Evaluation of other root components and antioxidant activity

‘Taibyo sobutori’ (Takii & Co., Ltd.), ‘Karamaru’ (Sakata seed Co. Ltd. Yokohama, Japan), ‘Susanoo’, M₆ of purple and red root breeding lines, were cultivated in the experimental field of Shimane University (Matsue, Japan) from September 2012 until February 2013. ‘Taibyo sobutori’, ‘Karamaru’, ‘Susanoo’, ‘Karaine aka’, ‘Chouan aomaru koshin’, and M₈ of both breeding lines were cultivated in the above experimental field from September until December 2014. Only colored root individuals of purple and red lines were used for this analysis.

The isothiocyanate contents were analyzed using the methods of Esaki and Onozaki (1980) and Ishii and Saijo (1987). The fresh radish roots were washed with water, and then the central parts were shredded using a ceramic grater. The shredded samples were squeezed through double gauze into 50 mL tubes. Then, the squeezed extracts were incubated at 30°C for 30 min to synthesize isothiocyanates. To convert the produced isothiocyanates into thiourea derivatives, 5 mL isothiocyanate solution was added to 20 mL EtOH-ammonia solution (39:1, v/v) in a new 50 mL tube, and the mixture was incubated at 30°C for 60 min. Then, the 25 mL allylthiourea solution was neutralized with 1 mL 50% acetic acid and filtered through filter paper. A 1 mL aliquot of the filtered solution was added to 4 mL 25× diluted Grote reagent (Esaki and Onozaki, 1980) and then incubated at 37°C for 45 min before measuring the absorbance at 600 nm using a spectrophotometer (UV-1800; Shimadzu). The isothiocyanate contents were calculated from an allylthiourea calibration curve, using the following equation: isothiocyanate content (mg/100 g juice) = allylthiourea content (mg·mL⁻¹) × 26/5 × 99/116 × 100 (Ishii and Saijo, 1987).

To measure the soluble solids content (SSC), the central parts of fresh radish roots were shredded with a ceramic grater and then SSC was measured using a digital refractometer (PR-101α; ATAGO, Tokyo, Japan).

For anthocyanin extraction, the root skin and xylem parenchyma were homogenized in liquid nitrogen. Anthocyanins were extracted from 0.5 g of finely ground plant materials as described by Pattanaik et al. (2010) with minor modifications. Samples were extracted with 1.5 mL methanol of 1% HCl (v/v) overnight in a dark refrigerator, and then 1 mL of water and an equal volume of chloroform were added to remove chlorophyll. After centrifugation, the anthocyanins were collected by separation into the aqueous methanol phase. The anthocyanins were then measured at 520 and 657 nm in a spectrophotometer (UV-1800; Shimadzu). Quantification of anthocyanins was performed using the following equation: Q_Anthocyanins = (A₅₂₀ − 0.25 × A₆₅₇) × M⁻¹, where Q_Anthocyanins is the amount of anthocyanins, A₅₂₀ and A₆₅₇ is the absorption at the indicated wavelengths and M is the weight of the plant material used for extraction (g).

To evaluate the antioxidant activity, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity was estimated by the method using of Suda (2000). The fresh radish roots were washed with water, and then the central parts were shredded using a ceramic grater. Approximately 5 g shredded samples were added to 18 mL ethanol and ground using a mortar and pestle. After the ground samples were diluted to 50 mL with 80% ethanol, the sample solution was filtered through filter paper. The reaction mixture consisted of 2.4 mL DPPH mixture solution (400 µM DPPH in ethanol: a 2-morpholinoethanesulphonic acid (MES) buffer (pH 6.0): 20% ethanol, 1: 1: 1, v/v/v), x mL of sample solution and (0.8 − x) mL of 80% ethanol. The reaction was initiated by the addition of the sample solution. After the test tube stood for 20 min, the absorbance of the DPPH radical at 520 nm was measured using a spectrophotometer (UV-1800; Shimadzu). DPPH radical-scavenging activity was evaluated by the decrease in the absorbance at 520 nm and expressed as a Trolox equivalent per 100 g (Fresh weight) by using the calibration curve of Trolox.

Results

Characterization of purple and red root breeding lines

The leaf and root shape and color phenotypes are summarized in Tables 1 and 2. All of the purple and red root lines in the M₆ and M₇ had lobed leaves. The most common taproot shape in M₆ and M₇ was the Kameido type (Fig. S1) (74.2%–76.1% in the purple root line, and 56.3%–64.3% in the red root line) which was same as that of ‘Susanoo’. Most of the plants in M₆ and M₇ had level 2 or 3 lateral roots (65.3%–83.5% in the purple root line, and 73.4%–90.9% in the red root line; Fig. S1) which was similar to the level of ‘Susanoo’ (Table 1). In all breeding lines, at least 70% of individuals accumulated anthocyanins in their leaves (Table 2).

Table 1

Ratios of leaf and root shape characteristics in M₆ and M₇ of purple and red root breeding lines.

Table 2

Ratios of leaf and root color characteristics in M₆, M₇, S₁, and S₂ of purple and red root breeding lines.

In the purple root mass selection breeding lines, the root color of individuals in M₆ and M₇ could be divided into five classes; wholly purple, partially purple, wholly red, partially red, and white; the ratios were 55.8%–56.9%, 1.4%–2.0%, 2.1%, 0.5%–0.8%, and 38.8%–39.6%, respectively. In the purple root selfed lines, the root color of individuals in S₁ and S₂ could be divided into three classes; wholly purple, partially purple, and white (ratios of 73.0%–81.3%, 0%–10.2%, and 8.5%–27.0%, respectively) (Table 2). In the red root mass selection breeding lines, the root color of individuals in M₆ and M₇ could be divided into three classes; wholly red, partially red, and white; the red colored root (wholly red and partially red) ratio was 66.6%–74.2%. The red root selfed lines S₁ and S₂ could be also divided into three classes; wholly red, partially red, and white; the red colored root ratio was 82.2%–83.3% (Table 2).

Root color and pigments in purple and red root breeding lines

Both root skin (L*: 82.2, b*/a*: 11.29 ± 2.23) and xylem parenchyma (L*: 79.1, b*/a*: 8.34 ± 0.95) in ‘Susanoo’ were yellowish white (RHSCC No. NN155A; white group) whereas root skin (L*: 48.3, b*/a*: −0.47 ± 0.02) in ‘Karaine aka’ was soft reddish purple (N81D; Purple violet group), and that of root xylem parenchyma (L*: 36.3, b*/a*: −0.08 ± 0.01) in ‘Chouan aomaru koshin’ was vivid purplish red (64A; Red-purple group) (Table 3). The roots colors of purple and red root breeding lines were deep reddish purple and vivid purplish red, and similar to that of ‘Karaine aka’ and ‘Chouan aomaru koshin’, respectively (Table 3; Fig. 2).

Table 3

Evaluation of root color in cultivars and breeding lines.

The main anthocyanins of the root skin and xylem parenchyma in purple root breeding lines were cyanidin derivatives: cyanidin 3-[2-(glucosyl)-6-(trans-p-coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside] (P1), cyanidin 3-[2-(glucosyl)-6-(trans-feruloyl)-glucoside]-5-[6-(malonyl)-glucoside] (P2), and malonyl cyanidin 3-[2-(glucosyl)-6-(trans-caffeoyl)-glucoside]-5-glucoside (P3) (Fig. 3). The main anthocyanins of the root skin and xylem parenchyma in red root breeding lines were pelargonidin derivatives: pelargonidin 3-[2-(glucosyl)-6-(trans-feruloyl)-glucoside]-5-(6-malonyl-glucoside) (R1), pelargonidin 3-[2-(glucosyl)-6-(trans-p-coumaroyl)-glucoside]-5-(6-malonyl-glucoside) (R2), and pelargonidin 3-[2-(glucosyl)-6-(trans-caffeoyl)-glucoside]-5-(6-malonyl-glucoside) (R3) (Fig. 3).

Fig. 3

HPLC chromatograms of anthocyanin pigments in the root skin and xylem parenchyma of purple and red root breeding lines. Peaks are as follows; P1: Cyanidin 3-[2-(glucosyl)-6-(trans-p-coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside], P2: Cyanidin 3-[2-(glucosyl)-6-(trans-feruloyl)-glucoside]-5-[6-(malonyl)-glucoside], P3: Malonyl cyanidin 3-[2-(glucosyl)-6-(trans-caffeoyl)-glucoside]-5-glucoside, R1: Pelargonidin 3-[2-(glucosyl)-6-(trans-feruloyl)-glucoside]-5-(6-malonyl-glucoside), R2: Pelargonidin 3-[2-(glucosyl)-6-(trans-p-coumaroyl)-glucoside]-5-(6-malonyl-glucoside), R3: Pelargonidin 3-[2-(glucosyl)-6-(trans-caffeoyl)-glucoside]-5-(6-malonyl-glucoside).

Evaluation of root isothiocyanate, soluble solids contents, and DPPH radical-scavenging activity

The isothiocyanate content in the purple root line was approximately the same as that in ‘Susanoo’ and the pungent radish ‘Karamaru’, and tended to be higher than that in ‘Taibyo sobutori’. The isothiocyanate content of the red root line was significantly lower than that of ‘Susanoo’ in 2013, and was same as that in 2014 (Fig. 4A).

Fig. 4

Isothiocyanate content (A) and soluble solids content (SSC; B) in ‘Taibyo sobutori’ (TS), ‘Karamaru’ (KM), ‘Susanoo’ (SU), ‘Karaine aka’ (KA), ‘Chouan aomaru koshin’ (CAK), and purple and red root breeding lines (PBL and RBL). Bars are standard errors (n = 3). Different letters indicate significant differences at P < 0.05 (Tukey’s multiple comparison tests).

The SSC of the purple and red root lines was significantly higher than that of ‘Taibyo sobutori’ in 2013 and 2014, and similar to that of ‘Karamaru’ and parent cultivars in 2014 (Fig. 4B).

The DPPH radical-scavenging activity of ‘Susanoo’ was similar to that of ‘Karamaru’, ‘Karaine aka’, and ‘Chouan aomaru koshin’, and that of the purple and red root lines was almost two times higher than them. Comparing with the common Japanese radish ‘Taibyo sobutori’, the DPPH radical-scavenging activities of purple and red root lines in 2013 and 2014 were seven to ten times higher (Fig. 5).

Fig. 5

DPPH radical scavenging activity of ‘Taibyo sobutori’ (TS), ‘Karamaru’ (KM), ‘Susanoo’ (SU), ‘Karaine aka’ (KA), ‘Chouan aomaru koshin’ (CAK), and purple and red root breeding lines (PBL and RBL) in 2013 and 2014. Bars are standard errors (n = 3). Different letters indicate significant differences at P < 0.05 (Tukey’s multiple comparison tests).

Discussion

To introduce the colored root characteristic into ‘Susanoo’ and enhance food functionality, the M₆, M₇, and M₈ progeny of purple and red root breeding lines were obtained by mass selection breeding.

‘Karaine aka’ has purple skin and light-purple xylem parenchyma. Conversely, ‘Chouan aomaru koshin’ has green skin and red xylem parenchyma. To obtain ‘Susanoo’ with colored root skin and xylem parenchyma, purple and red root breeding lines were mass-selected from an open-pollinated population derived from crosses between ‘Susanoo’ and those cultivars (Fig. 1). Based on the root color phenotype of the M₃ open-pollinated population, excellent purple and red colored root individuals were selected and mass-selection was repeated from the M₃ to M₈ generation. As a result, a purple root breeding line with acylated cyanidin 3-sophoroside-5-glucosides and a red root breeding line with acylated pelargonidin 3-sophoroside-5-glucosides were constructed. Their anthocyanin profiles were the same as purple and red root parent cultivars, respectively (Kato et al., 2013).

Approximately one-third of individuals in the M₆ and M₇ were non-colored (Table 2). In the cross test between ‘Comet’ (red root) and ‘Shogoin’ (white root), all of the F₁ plants had purple roots, and the F₂ progenies were divided into three root color phenotypes: purple, red, and white (9:3:4) (Hoshi et al., 1963). Our results suggested that non colored root individuals appeared in the subsequent generation because most of the selected colored root individuals may be heterozygous for anthocyanin production genes in the purple and red root lines. Moreover, segregation of root color did not obey Mendelian inheritance in progenies between watermelon radish and white radish (So et al., 1919). The root colors of F₂ progenies between watermelon radish ‘Koshin ao’ and white radishes were colored and non colored roots, at a ratio of 2:1, respectively. Tatebe (1940) suggested the effect of a mutable gene which changed colored into non colored roots in these cross combinations. In this study, ‘Chouan aomaru koshin’ which was a genetically similar cultivar to ‘Koshin ao’ was also used as a genetic source of anthocyanin pigmentation, and therefore, the appearance of non colored root individuals may be maintained at a higher frequency in mass selection breeding lines. On the other hand, the colored root characteristic was fixed by self-pollination in the Japanese radish “Inuidani” which accumulates pelargonidin as the major anthocyanidin (Asako et al., 2011). In this study, self-pollination was effective to accumulate the homozygote of the colored root characteristic because the colored root ratios in S₁ and S₂ were higher than those in M₆ and M₇. In this breeding program, the fixation of the colored root characteristic will be obtained through the investigation of genes controlling root color and the repetition of self-pollination.

The morphological characteristics of ‘Susanoo’ are lobed leaves and needle shape Kameido-type roots with fibrous lateral roots. In the early generations of both breeding lines, lobed leaf and the entire leaf shape of ‘Chouan aomaru koshin’ were segregated (data not shown). As mass-selective generation advanced, the morphological characteristics of purple and red root lines stabilized the same as that of ‘Susanoo’. The morphological characteristics of both lines will be improved by repeated mass-selection.

Compared with the roots of ‘Susanoo’, those of the purple and red root lines had almost the same isothiocyanate content and SSC (Fig. 4). The antioxidant activities in the DPPH assay correlated well with the total phenolic contents and total anthocyanin contents in highly pigmented vegetables (Li et al., 2012). In normal ‘Mizu-nasu’ fruit, there was a positive correlation between anthocyanin concentration and the radical scavenging activity of the peel of ‘Mizu-nasu’ fruit (Kitsuda et al., 2005). DPPH radical scavenging activity of acylated anthocyanins was higher than those of pelargonidin and perlargonidin-3-glucoside (Matsufuji et al., 2007). Increased anthocyanin accumulation was associated with increased antioxidant activity in the red cabbage (Yuan et al., 2009). In this study, the total anthocyanin content of root skin and xylem parenchyma in purple and red root lines tended to be higher than that in ‘Karaine aka’ and ‘Chouan aomaru koshin’ (Fig. S2). The food functionality of ‘Susanoo’ was enhanced by introduction of the same anthocyanin composition as the parental cultivar because the DPPH radical scavenging activity of purple and red root lines was two times higher than that of ‘Susanoo’ (Fig. 5).

In conclusion, purple and red pigmentation was successfully introduced from parental cultivars into ‘Susanoo’ (Fig. 2A). In addition, the food functionality of ‘Susanoo’ was enhanced by anthocyanin accumulation of the same composition as the parental cultivar. It is expected that purple and red root breeding lines will be used for the coloring of dishes and as natural pigments. However, further breeding is required to fix the purple and red root phenotypes because some non colored individuals were still present in these generations, and the red root characteristic appeared in the M₆ and M₇ of purple root breeding lines. We reported that the lack of Flavonoid 3'-hydroxylase (F3'H) function which was caused by insertion of a retrotransposon in the first exon of the F3'H homolog contributed to pelargonidin-based anthocyanin accumulation in red radishes (Masukawa et al., 2018). The purple root characteristic can be fixed by selection of normal homozygous F3'H. From now on, we intend to investigate the cause of the non colored individuals in the mass selection breeding lines, and to develop a DNA marker for fixation of purple and red colored root characteristics.

Acknowledgements

The authors thank the faculty of Life and Environmental Science in Shimane University for help and financial support for publishing this report.

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