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

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1 The Hortiulture Journal 87 (3): doi: /hortj.OKD-132 JSHS The Japanese Soiety for Hortiultural Siene Enhanement of Food Funtionality of a Loal Pungent Radish Izumo Orohi Daikon Susanoo y Introdution of a Colored Root Charater Takanori Masukawa 1, Masayuki Kadowaki 2, Toshikazu Matsumoto 2, Akira Nakatsuka 2, Kyeong-Seong Cheon 2 **, Kazuhisa Kato 3 ***, Fumi Tatsuzawa 3 and Nouo Koayashi 2 * 1 The United Graduate Shool of Agriultural Siene, Tottori University, Tottori , Japan 2 Faulty of Life and Environmental Siene, Shimane University, Matsue , Japan 3 Faulty of Agriulture, Iwate University, Morioka , Japan Purple and red olored root harateristis were introdued to a loal pungent radish Izumo orohi daikon Susanoo, and several harateristis inluding food funtional omponents were evaluated. The root and leaf phenotype, the pigment omposition, the isothioyanate and solule solids ontents, and the 1,1-diphenyl-2- pirylhydrazyl (DPPH) radial savenging ativity in the root were investigated. These harateristis were ompared etween the mass seletion reeding lines of purple, red roots and their parent ultivars. Although one-third of individuals in the M 6 and M 7 were non olored, the olored root harateristis was introdued into Susanoo. The main anthoyanins in the deep reddish purple olored purple root lines were aylated yanidin 3-sophoroside-5-gluosides, and those in the vivid purplish red olored red root lines were aylated pelargonidin 3-sophoroside-5-gluosides, whih orresponded to the main anthoyanins in their respetive olored root parental ultivars. The isothioyanate ontents in the purple and red root lines were almost the same as that in Susanoo. The DPPH radial savenging ativity of the purple and red root lines was almost two times higher than that of Susanoo. These results showed that the food funtionality was enhaned y introdution of a olored root harateristi in Susanoo. Key Words: anthoyanin, DPPH radial savenging ativity, isothioyanate, mass seletion, Raphanus sativus. Introdution 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 ondiment known as Nodaikon (wild radish) in the San in area of Japan. Izumo orohi daikon is a newly developed loal savory variety of pungent radish that was domestiated from a Japanese wild radish growing eside Lake Shinji and in the areas surround- Reeived; August 8, Aepted; Novemer 13, First Pulished Online in J-STAGE on January 6, This study was supported y a Grant-in-Aid for Sientifi Researh (KAKENHI No. 16K07597) from the Japan Soiety for the Promotion of Siene (JSPS). * Corresponding author ( nkoayashi@life.shimane-u. a.jp). ** Present address: Genomis Division, National Institute of Agriultural Sienes, RDA, Jeonju 54874, Repuli of Korea *** Present address: Graduate Shool of Agriultural Siene, Tohoku University, Sendai , Japan ing the Shimane Peninsula in Japan. It has een improved y seletive reeding, fousing on a high isothioyanate ontent, favorale root shape, and lateflowering harateristis (Ban et al., 2009; Koayashi et al., in press). This newly developed radish was registered as Susanoo (July, 2011, Register No ; Ministry of Agriulture, Forestry and Fisheries, Japan), and ommerially produed seeds have een released from the experimental farm of Shimane University under the university s rand name. Beause of its high pungeny and unique savory and umami flavors, it is a popular ondiment that is served with various Japanese dishes inluding soa noodles, sashimi raw fish, and meat dishes. Beside the roots, stuffed leaves are also edile. This new variety is now eing ultivated mainly in the Shimane area and also in the metropolitan area as a representative loal vegetale of Shimane Prefeture. Brassia vegetales like rooli (Brassia oleraea var. italia), kale (B. oleraea L. var. aephala DC.), aage (B. oleraea L.), and radish (Raphanus sativus L.) are rih in phytohemials suh as phenolis, vita The Japanese Soiety for Hortiultural Siene (JSHS), All rights reserved.

2 Hort. J. 87 (3): mins, gluosinolates, and anthoyanins, whih positively affet human health (Wei et al., 2011). Gluosinolates are hydrolyzed y myrosinase to form isothioyanates, whih reat with various ellular targets and have antimiroial (Hashem and Saleh, 1999), antimutageni (Hamilton and Teel, 1996), and antiarinogeni (Heht, 1999) ativities. Anthoyanins an prevent oxidative damage to DNA, proteins, lipids, and other maromoleules aused y reative oxygen speies. de Pasual-Teresa and Sanhez-Ballesta (2008) suggested that anthoyaninrih produts are enefiial for human health, as they may prevent ardiovasular diseases and some types of aner. Radish anthoyanins have right olors over a wide ph range and their multiple aylation with hydroxyinnami aids ontriute to their remarkale heat staility in aidi environments (Jing et al., 2012). Radish anthoyanins have een widely used as natural olorants eause of their strong natural olor and health enefits, inluding antioxidant ativity (Matsufuji et al., 2007; Rahman et al., 2006). The main anthoyanins of purple radish are aylated yanidin 3-sophoroside-5- gluosides, whereas those of red radish are aylated pelargonidin 3-sophoroside-5-gluosides (Kato et al., 2013). To enhane antioxidant aility and extend its appeal to olor dishes with natural pigments, the olored root harateristis were introdued to Susanoo. Purple and red root mass seletion reeding lines were onstruted after rossing with olored root parental ultivars. In this study, the root and leaf phenotype, anthoyanin omposition, isothioyanate and solule solids ontents, and antioxidant ativity were investigated in purple and red root reeding lines, and they were ompared with their parental ultivars. Materials and Methods Breeding of plant materials and their haraterization The genealogial hart of purple and red root reeding lines is shown in Figure 1. To introdue the olored root harateristi into Susanoo, the purple root radish Karaine aka (Watanae seed Co., Ltd., Miyagi, Japan) whih aumulated anthoyanin mainly in the root skin was used as a geneti soure of yanidin, and the red root radish Chouan aomaru koshin (Takii & Co., Ltd., Kyoto, Japan) whih aumulated anthoyanin in mainly the root xylem parenhyma was used as a geneti soure of pelargonidin. The F 1 hyrid with purple roots was otained from rosses etween Susanoo and eah of these olored root ultivars in May Approximately seven individuals with olored roots and leaves, and a root shape similar to Susanoo were seleted from the F 1 progenies. The first mass seletion generation (M 1 ) was otained y open pollination of F 1 in 2007 to introdue pigmentation into the root skin and xylem parenhyma of Susanoo. The M 2 were otained y mass-seletion of approximately seven individuals with olored roots and leaves, root shape, and high isothioyanate ontents from the M 1 progenies in Approximately seven individuals with the desired purple and red root phenotypes were seleted from among the M 2 progenies to estalish the purple and red root reeding lines, respetively. From M 4 to M 8 were otained y mass-seletion of approximately fifteen purple and red root progenies with the desired root shape and ontents from 2010 to 2014, respetively. The first selfing generations (S 1 ), S 2, and S 3 were otained y ud-pollination of purple and red root M 5 progenies from 2012 to M 6 and M 7 were grown at the experimental farm of Shimane University (Izumo, Japan). S 1 and S 2 were grown in a field of Shimane University (Matsue, Japan). All lines were grown from Septemer until Feruary for haraterization. We evaluated the following phenotypes of the mass seletion lines, M 6 and M 7 in purple and red root reeding lines: leaf shape, taproot shape, lateral root level, and anthoyanin aumulation in the leaf and root. The inred lines S 1 and S 2 in purple and red root reeding lines were evaluated only for root olor. The evaluation standards were as follows: Leaf shape: loed leaf, mid, or entire leaf. Taproot shape: Kameido type, Taiyo Japanese wild radish M 1 M 2 M 3 M (Year) Susanoo Karaine aka (F 1 hyrid) F 1 M 7 M 8 M 3 M 4 M 5 M 6 Purple root reeding line S 1 S 2 S 3 M 1 M 2 M F 1 Chouan aomaru koshin :Mass seletion generation :F 1 hyrid F 1 M 7 M 8 M 3 M 4 M 5 M 6 Red root reeding line S 1 S 2 S 3 S :Selfing generation Fig. 1. Genealogial hart of purple and red root reeding lines. Gray, lak, and white oxes indiate wild speies, ultivars and reeding lines, respetively.

3 358 T. Masukawa, M. Kadowaki, T. Matsumoto, A. Nakatsuka, K.-S. Cheon, K. Kato, F. Tatsuzawa and N. Koayashi type, Nezumi type, Koshin type, and Kikon type. Lateral root level was lassified aording to a numer of lateral roots; none (level 0), a few (level 1), moderate (level 2), and many (level 3) (Fig. S1). Anthoyanin pigmentation in leaves: none, weak, medium, and strong. Root olor: wholly purple, partially purple (olored skin, ortex, or xylem parenhyma), wholly red, partially red, and white. Analysis of root pigments in purple and red root reeding lines Susanoo, Karaine aka, Chouan aomaru koshin, M 6 of purple, and red root reeding lines were used for root pigments analysis. The olor of the root skin and xylem parenhyma in Susanoo, purple and red root reeding lines were reorded y photographs (Fig. 2) and y the RHSCC (Royal Hortiultural Soiety Colour Chart), and measured for lightness (L*) and two hromati omponents a* and * using a Color Reader (CR-10; Konia Minolta Sensing In., Tokyo, Japan). The root olor 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 oth reeding lines, root skin and xylem parenhyma were olleted and dried overnight at 40 C, and then kept at 4 C until analysis. Anthoyanins were extrated from eah sample (aout 10 mg) in 1 ml MAW (MeOH-HOA-H 2 O, 4:1:5, v/v/v) at 4 C overnight, and then an aliquot of the extrat was analyzed y HPLC. The HPLC system omprised an LC 10A instrument (Shimadzu, Kyoto, Japan) equipped with a Waters C18 ( mm) olumn. The temperature was 40 C, the flow rate was 1 ml min 1, and produt elution was monitored at 515 nm. The eluant was applied as a linear gradient for A B C D Fig. 2. Photograph of Susanoo (left) and purple and red root reeding lines (enter and right) in 2012 (A) and their ross setions (B D). Bar indiates 10 m. 40 min from 20% to 85% solvent B (1.5% H 3 PO 4, 20% HOA, 25% MeCN in H 2 O) in solvent A (1.5% H 3 PO 4 in H 2 O). Evaluation of other root omponents and antioxidant ativity Taiyo soutori (Takii & Co., Ltd.), Karamaru (Sakata seed Co. Ltd. Yokohama, Japan), Susanoo, M 6 of purple and red root reeding lines, were ultivated in the experimental field of Shimane University (Matsue, Japan) from Septemer 2012 until Feruary Taiyo soutori, Karamaru, Susanoo, Karaine aka, Chouan aomaru koshin, and M 8 of oth reeding lines were ultivated in the aove experimental field from Septemer until Deemer Only olored root individuals of purple and red lines were used for this analysis. The isothioyanate ontents 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 entral parts were shredded using a erami grater. The shredded samples were squeezed through doule gauze into 50 ml tues. Then, the squeezed extrats were inuated at 30 C for 30 min to synthesize isothioyanates. To onvert the produed isothioyanates into thiourea derivatives, 5 ml isothioyanate solution was added to 20 ml EtOH-ammonia solution (39:1, v/v) in a new 50 ml tue, and the mixture was inuated at 30 C for 60 min. Then, the 25 ml allylthiourea solution was neutralized with 1 ml 50% aeti aid 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 inuated at 37 C for 45 min efore measuring the asorane at 600 nm using a spetrophotometer (UV-1800; Shimadzu). The isothioyanate ontents were alulated from an allylthiourea aliration urve, using the following equation: isothioyanate ontent (mg/100 g juie) = allylthiourea ontent (mg ml 1 ) 26/5 99/ (Ishii and Saijo, 1987). To measure the solule solids ontent (SSC), the entral parts of fresh radish roots were shredded with a erami grater and then SSC was measured using a digital refratometer (PR-101α; ATAGO, Tokyo, Japan). For anthoyanin extration, the root skin and xylem parenhyma were homogenized in liquid nitrogen. Anthoyanins were extrated from 0.5 g of finely ground plant materials as desried y Pattanaik et al. (2010) with minor modifiations. Samples were extrated 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 hloroform were added to remove hlorophyll. After entrifugation, the anthoyanins were olleted y separation into the aqueous methanol phase. The anthoyanins were then measured at 520 and 657 nm in a spetrophotometer (UV-1800; Shimadzu). Quantifiation of anthoyanins was performed using the following

4 equation: Q Anthoyanins = (A A 657 ) M 1, where Q Anthoyanins is the amount of anthoyanins, A 520 and A 657 is the asorption at the indiated wavelengths and M is the weight of the plant material used for extration (g). To evaluate the antioxidant ativity, the 1,1- diphenyl-2-pirylhydrazyl (DPPH) radial-savenging ativity was estimated y the method using of Suda (2000). The fresh radish roots were washed with water, and then the entral parts were shredded using a erami 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 reation mixture onsisted of 2.4 ml DPPH mixture solution (400 µm DPPH in ethanol: a 2-morpholinoethanesulphoni aid (MES) uffer (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 reation was initiated y the addition of the sample solution. After the test tue stood for 20 min, the asorane of the DPPH radial at 520 nm was measured using a spetrophotometer (UV-1800; Shimadzu). DPPH radial-savenging ativity was evaluated y the derease in the asorane at 520 nm and expressed as a Trolox equivalent per 100 g (Fresh weight) y using the aliration urve of Trolox. Hort. J. 87 (3): Tale 1. Ratios of leaf and root shape harateristis in M 6 and M 7 of purple and red root reeding lines. Results Charaterization of purple and red root reeding lines The leaf and root shape and olor phenotypes are summarized in Tales 1 and 2. All of the purple and red root lines in the M 6 and M 7 had loed leaves. The most ommon taproot shape in M 6 and M 7 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) whih was same as that of Susanoo. Most of the plants in M 6 and M 7 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) whih was similar to the level of Susanoo (Tale 1). In all reeding lines, at least 70% of individuals aumulated anthoyanins in their leaves (Tale 2). In the purple root mass seletion reeding lines, the root olor of individuals in M 6 and M 7 ould e divided into five lasses; 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%, respetively. In the purple root selfed lines, the root olor of individuals in S 1 and S 2 ould e divided into three lasses; wholly purple, partially purple, and white (ratios of 73.0% 81.3%, 0% 10.2%, and 8.5% 27.0%, respetively) (Tale 2). In the red root mass seletion reeding lines, the root olor of individuals in M 6 and M 7 ould e divided into three lasses; wholly Breeding lines Purple root Numer of individuals Tale 1. Ratios of leaf and root shape harateristis in M 6 and M 7 of purple and red root reeding lines. Leaf shape type (%) Taproot shape type z (%) Lateral root level z (%) Loed leaf Middle leaf Entire leaf Kameido Taiyo Nezumi Koshin Kikon Level 0 Level 1 Level 2 Level 3 M M Red root M M Tale z Morphology 2. Ratios was of shown leaf and in root Figure olor S1. harateristis in M 6, M 7, S 1, and S 2 of purple and red root reeding lines. Tale 2. Ratios of leaf and root olor harateristis in M 6, M 7, S 1, and S 2 of purple and red root reeding lines. Breeding lines Purple root Numer of individuals Anthoyanin pigmentation of leaf (%) Root olor (%) None Weak Medium Strong Wholly purple Partially purple Wholly red Partially red M M S S Red root M M S S White

5 360 T. Masukawa, M. Kadowaki, T. Matsumoto, A. Nakatsuka, K.-S. Cheon, K. Kato, F. Tatsuzawa and N. Koayashi red, partially red, and white; the red olored root (wholly red and partially red) ratio was 66.6% 74.2%. The red root selfed lines S 1 and S 2 ould e also divided into three lasses; wholly red, partially red, and white; the red olored root ratio was 82.2% 83.3% (Tale 2). Root olor and pigments in purple and red root reeding lines Both root skin (L*: 82.2, */a*: ± 2.23) and xylem parenhyma (L*: 79.1, */a*: 8.34 ± 0.95) in Susanoo were yellowish white (RHSCC No. NN155A; white group) whereas root skin (L*: 48.3, */a*: 0.47 ± 0.02) in Karaine aka was soft reddish purple (N81D; Purple violet group), and that of root xylem parenhyma (L*: 36.3, */a*: 0.08 ± 0.01) in Chouan aomaru koshin was vivid purplish red (64A; Red-purple group) (Tale 3). The roots olors of purple and red root reeding lines were deep reddish purple and vivid purplish red, and similar to that of Karaine aka and Chouan aomaru koshin, respetively (Tale 3; Fig. 2). The main anthoyanins of the root skin and xylem parenhyma in purple root reeding lines were yanidin derivatives: yanidin 3-[2-(gluosyl)-6-(trans-poumaroyl)-gluoside]-5-[6-(malonyl)-gluoside] (P1), yanidin 3-[2-(gluosyl)-6-(trans-feruloyl)-gluoside]- 5-[6-(malonyl)-gluoside] (P2), and malonyl yanidin 3- [2-(gluosyl)-6-(trans-affeoyl)-gluoside]-5-gluoside (P3) (Fig. 3). The main anthoyanins of the root skin and xylem parenhyma in red root reeding lines were pelargonidin derivatives: pelargonidin 3-[2- (gluosyl)-6-(trans-feruloyl)-gluoside]-5-(6-malonylgluoside) (R1), pelargonidin 3-[2-(gluosyl)-6-(transp-oumaroyl)-gluoside]-5-(6-malonyl-gluoside) (R2), and pelargonidin 3-[2-(gluosyl)-6-(trans-affeoyl)- gluoside]-5-(6-malonyl-gluoside) (R3) (Fig. 3). Evaluation of root isothioyanate, solule solids ontents, and DPPH radial-savenging ativity The isothioyanate ontent in the purple root line was approximately the same as that in Susanoo and the Tale pungent 3. Evaluation radish of Karamaru, root olor in ultivars and tended and reeding to e lines. higher than that in Taiyo soutori. The isothioyanate ontent of the red root line was signifiantly lower than that of Susanoo in 2013, and was same as that in 2014 (Fig. 4A). The SSC of the purple and red root lines was signifiantly higher than that of Taiyo soutori in 2013 and 2014, and similar to that of Karamaru and parent ultivars in 2014 (Fig. 4B). The DPPH radial-savenging ativity 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 ommon Japanese radish Taiyo soutori, the DPPH radial-savenging ativities of purple and red root lines in 2013 and 2014 were seven to ten times higher (Fig. 5). Disussion To introdue the olored root harateristi into Susanoo and enhane food funtionality, the M 6, M 7, and M 8 progeny of purple and red root reeding lines were otained y mass seletion reeding. Karaine aka has purple skin and light-purple xylem parenhyma. Conversely, Chouan aomaru koshin has green skin and red xylem parenhyma. To otain Susanoo with olored root skin and xylem parenhyma, purple and red root reeding lines were massseleted from an open-pollinated population derived from rosses etween Susanoo and those ultivars (Fig. 1). Based on the root olor phenotype of the M 3 open-pollinated population, exellent purple and red olored root individuals were seleted and massseletion was repeated from the M 3 to M 8 generation. As a result, a purple root reeding line with aylated yanidin 3-sophoroside-5-gluosides and a red root reeding line with aylated pelargonidin 3- sophoroside-5-gluosides were onstruted. Their anthoyanin profiles were the same as purple and red root parent ultivars, respetively (Kato et al., 2013). Approximately one-third of individuals in the M 6 and M 7 were non-olored (Tale 2). In the ross test etween Comet (red root) and Shogoin (white root), all Tale 3. Evaluation of root olor in ultivars and reeding lines. Cultivars and reeding lines Skin Xylem parenhyma RHSCC (group name) w L* */a* v RHSCC (group name) L* */a* v Susanoo z NN155A (White group) ± 2.23 NN155A (White group) ± 0.95 Karaine aka yx N81D (Purple violet group) ± 0.02 Chouan aomaru koshin yx 64A (Red-purple group) ± 0.01 Purple root reeding lines z 77A (Purple group) ± 0.03 N78B (Purple group) ± 0.04 Red root reeding lines z 67B (Red-purple group) ± A (Red-purple group) ± 0.01 z Cultivated from Septemer to Deemer in y Cultivated from Septemer to Deemer in x Measured in the mainly pigmented part of the ultivar. w Royal Hortiultural Soiety Colour Chart. v Mean ± SE.

6 Hort. J. 87 (3): A Isothioyanate ontent (mg / 100g juie) a a a a a a Fe De a a a B a SSC ( Brix) 6 4 a a 2 0 TS KM SU KA CAK PBL RBL Fig. 4. Isothioyanate ontent (A) and solule solids ontent (SSC; B) in Taiyo soutori (TS), Karamaru (KM), Susanoo (SU), Karaine aka (KA), Chouan aomaru koshin (CAK), and purple and red root reeding lines (PBL and RBL). Bars are standard errors (n = 3). Different letters indiate signifiant differenes at P < 0.05 (Tukey s multiple omparison tests). Fig. 3. HPLC hromatograms of anthoyanin pigments in the root skin and xylem parenhyma of purple and red root reeding lines. Peaks are as follows; P1: Cyanidin 3-[2-(gluosyl)-6- (trans-p-oumaroyl)-gluoside]-5-[6-(malonyl)-gluoside], P2: Cyanidin 3-[2-(gluosyl)-6-(trans-feruloyl)-gluoside]-5-[6- (malonyl)-gluoside], P3: Malonyl yanidin 3-[2-(gluosyl)-6- (trans-affeoyl)-gluoside]-5-gluoside, R1: Pelargonidin 3-[2- (gluosyl)-6-(trans-feruloyl)-gluoside]-5-(6-malonyl-gluoside), R2: Pelargonidin 3-[2-(gluosyl)-6-(trans-p-oumaroyl)- gluoside]-5-(6-malonyl-gluoside), R3: Pelargonidin 3-[2- (gluosyl)-6-(trans-affeoyl)-gluoside]-5-(6-malonyl-gluoside). DPPH radial-savenging ativities (μmol Trolox eq/100g FW) a a Fe De a TS KM SU KA CAK PBL RBL Fig. 5. DPPH radial savenging ativity of Taiyo soutori (TS), Karamaru (KM), Susanoo (SU), Karaine aka (KA), Chouan aomaru koshin (CAK), and purple and red root reeding lines (PBL and RBL) in 2013 and Bars are standard errors (n = 3). Different letters indiate signifiant differenes at P < 0.05 (Tukey s multiple omparison tests). a of the F 1 plants had purple roots, and the F 2 progenies were divided into three root olor phenotypes: purple, red, and white (9:3:4) (Hoshi et al., 1963). Our results suggested that non olored root individuals appeared in the susequent generation eause most of the seleted olored root individuals may e heterozygous for anthoyanin prodution genes in the purple and red root lines. Moreover, segregation of root olor did not oey Mendelian inheritane in progenies etween watermelon radish and white radish (So et al., 1919). The root olors of F 2 progenies etween watermelon radish Koshin ao and white radishes were olored and non olored roots, at a ratio of 2:1, respetively. Tatee (1940) suggested the effet of a mutale gene whih hanged olored into non olored roots in these ross ominations. In this study, Chouan aomaru koshin whih was a genetially similar ultivar to Koshin ao was also used as a geneti soure of anthoyanin pigmentation, and therefore, the appearane of non olored root individuals may e maintained at a higher frequeny in mass seletion reeding lines. On the other hand, the olored root harateristi was fixed y selfpollination in the Japanese radish Inuidani whih

7 362 T. Masukawa, M. Kadowaki, T. Matsumoto, A. Nakatsuka, K.-S. Cheon, K. Kato, F. Tatsuzawa and N. Koayashi aumulates pelargonidin as the major anthoyanidin (Asako et al., 2011). In this study, self-pollination was effetive to aumulate the homozygote of the olored root harateristi eause the olored root ratios in S 1 and S 2 were higher than those in M 6 and M 7. In this reeding program, the fixation of the olored root harateristi will e otained through the investigation of genes ontrolling root olor and the repetition of selfpollination. The morphologial harateristis of Susanoo are loed leaves and needle shape Kameido-type roots with firous lateral roots. In the early generations of oth reeding lines, loed leaf and the entire leaf shape of Chouan aomaru koshin were segregated (data not shown). As mass-seletive generation advaned, the morphologial harateristis of purple and red root lines stailized the same as that of Susanoo. The morphologial harateristis of oth lines will e improved y repeated mass-seletion. Compared with the roots of Susanoo, those of the purple and red root lines had almost the same isothioyanate ontent and SSC (Fig. 4). The antioxidant ativities in the DPPH assay orrelated well with the total phenoli ontents and total anthoyanin ontents in highly pigmented vegetales (Li et al., 2012). In normal Mizu-nasu fruit, there was a positive orrelation etween anthoyanin onentration and the radial savenging ativity of the peel of Mizu-nasu fruit (Kitsuda et al., 2005). DPPH radial savenging ativity of aylated anthoyanins was higher than those of pelargonidin and perlargonidin-3-gluoside (Matsufuji et al., 2007). Inreased anthoyanin aumulation was assoiated with inreased antioxidant ativity in the red aage (Yuan et al., 2009). In this study, the total anthoyanin ontent of root skin and xylem parenhyma in purple and red root lines tended to e higher than that in Karaine aka and Chouan aomaru koshin (Fig. S2). The food funtionality of Susanoo was enhaned y introdution of the same anthoyanin omposition as the parental ultivar eause the DPPH radial savenging ativity of purple and red root lines was two times higher than that of Susanoo (Fig. 5). In onlusion, purple and red pigmentation was suessfully introdued from parental ultivars into Susanoo (Fig. 2A). In addition, the food funtionality of Susanoo was enhaned y anthoyanin aumulation of the same omposition as the parental ultivar. It is expeted that purple and red root reeding lines will e used for the oloring of dishes and as natural pigments. However, further reeding is required to fix the purple and red root phenotypes eause some non olored individuals were still present in these generations, and the red root harateristi appeared in the M 6 and M 7 of purple root reeding lines. We reported that the lak of Flavonoid 3'-hydroxylase (F3'H) funtion whih was aused y insertion of a retrotransposon in the first exon of the F3'H homolog ontriuted to pelargonidinased anthoyanin aumulation in red radishes (Masukawa et al., 2018). The purple root harateristi an e fixed y seletion of normal homozygous F3'H. From now on, we intend to investigate the ause of the non olored individuals in the mass seletion reeding lines, and to develop a DNA marker for fixation of purple and red olored root harateristis. Aknowledgements The authors thank the faulty of Life and Environmental Siene in Shimane University for help and finanial support for pulishing this report. Literature Cited Asako, Y., Y. Owaki, Y. Ozeki, N. Sasaki, Y. Ae, T. 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