FAGOPYRUM Volume 36(2), June 2019 ISSN 0352 – 3020 Scientific Journal on Buckwheat Research International Buckwheat Research Association Slovenian Academy of Sciences and Arts FAGOPYRUM volume 36(2), June 2019 An international journal on buckwheat research published by The Slovenian Academy of Sciences and Arts, Ljubljana, Slovenia, under the auspices of The International Buckwheat Research Association (IBRA). Confirmed by the Class of Natural Sciences of the The Slovenian Academy of Sciences and Arts on November 15, 2018 and the presidency of the Academy on February 5, 2019. Managing Editorial Board Ivan Kreft (Editor-in-Chief) (Slovenia) Christian Zewen (Associate Editor) (Luxembourg) Blanka Vombergar (Associate Editor) (Slovenia) Mateja Germ (Associate Editor) (Slovenia) Kiyokazu Ikeda (Associate Editor) (Japan) Clayton Campbell (Language Editor) (Canada) Advisory Board Y. Asami, Ryukoko University, Ohtsu, Japan T. Bjorkman, Cornell Univerity, Geneva, USA C. Campbell, Chairperson of the 7thISB, Canada N. K. Chrungoo, North Eastern University, Shillong, India N. N. Fesenko, All-Russia Research Institute of Legumes and Groat Crops, Orel, Russia M. Germ, University of Ljubljana, Ljubljana, Slovenia H. Hayashi, Tsukuba University, Tsukuba, Japan Y. Honda, National Agriculture and Food Research Organization, Tsukuba, Japan S. Ikeda, Kobe Gakuin University, Kobe, Japan N. Inoue, Shinshu University, Minami-Minowa, Japan D. Janovska, Crop Research Institute, Praha, Czech R. Lin, Shanxi Academy of Agricultural Science, Taiyuan, China R. L. Obendorf, Cornell University, Ithaca, USA O. Ohnishi, Kyoto University, Kyoto, Japan R. Ohsawa, Tsukuba University, Tsukuba, Japan C. H. Park, Kangwon National University, Chunchon, Korea J. C. Rana, ICAR-National Bureau of Plant Genetic Resources, New Delhi, India G. N. Suvorova, All-Russia Research Istitute on Legumes and Groat Crops, Orel, Russia G. Wieslander, Department of Medical Sciences, Uppsala University and University Hospital, Uppsala, Sweden S.-H. Woo, Chungbuk National University, Cheongju, Korea Y. Yasui, Kyoto University, Kyoto, Japan Editor Emeritus: Toshiko Matano, Ohmi Ohnishi, Kiyokazu Ikeda Subscription Information: One volume per year: subscription price for printed issues 2018 is US $80.00 for individuals and scientific institutions. Authors, reviewers and editors of the issue receive free printed copies. Electronic versions are untill further freely available for academic and non-commercial use at http://www.sazu.si/publikacije-sazu FAGOPYRUM is open to everyone who is interested in buckwheat and will cover all aspects of buckwheat research: genetics, cytology, breeding, agronomy, nutrition, utilization, biochemistry, ethnobotany and others. FAGOPYRUM will accept manuscripts in English only, which meet the scientific requirements set by the Editorial Board and which have not been published or submitted for publication elsewhere. Announcements concerning the promotion of research on buckwheat (workshops, symposium and so on), bibliographies and other information related to buckwheat will also be published. Deadline for receiving manuscripts for volume 37: November 30, 2019. Page setup: Medija grafično oblikovanje d.o.o., Prečna ulica 6, 1000 Ljubljana. Front page photo: Memil guksu, Korean buckwheat noodles, 13th Int. Buckwheat Symposium, Korea Sept. 2016 (Photo I. Kreft). For the 14th IBS in Sept. 2019 in Shillong, India see: www.14isb.in FAGOPYRUM volume 36(2) (2019) CONTENTS ORIGINAL PAPERS Differentiation and growth of a growing point until the stage of flower bud appearance in leading common buckwheat and Tartary buckwheat varieties in northern Japan Shinya KASAJIMA, Ikumi YOSHIMARU, and Hirotake ITOH ............................................................................. 37 Changes of physicochemical properties and correlation analysis of common buckwheat starch during germination Licheng GAO, Meijuan XIA, Zhonghao LI, Pengke WANG, Meng WANG, Jinfeng GAO ....................................... 43 Variation of rutin and quercetin contents in Tartary buckwheat germplasm Je Hyeok YU, Soo Jeong KWON, Ju Young CHOI, Yong Hwan JU, Swapan Kumar ROY, Dong-Gyu LEE, Cheol Ho PARK, and Sun-Hee WOO ............................................................................................. 51 INFORMATION Information for authors ...................................................................................................................................... 66 Fagopyrum 36(2):37-41 (2019) 37 Research paper Differentiation and growth of a growing point until the stage of flower bud appearance in leading common buckwheat and Tartary buckwheat varieties in northern Japan Shinya KASAJIMA*, Ikumi YOSHIMARU, and Hirotake ITOH Faculty of Bioindustry, Tokyo University of Agriculture, Yasaka 196, Abashiri, Hokkaido 099-2493, Japan * Corresponding author, e-mail: s3kasaji@nodai.ac.jp Tel: +81-152-48-3823 Other e-mail addresses: Ikumi Yoshimaru, yoshimaru193@yahoo.co.jp, Hirotake Itoh, h-ito@nodai.ac.jp DOI https://doi.org/10.3986/fag0009 Received: April 11, 2019; accepted: June 8, 2019 Keywords: common buckwheat, developmental stage, growing point, Hokkaido, Tartary buckwheat ABSTRACT Studies regarding the developmental stage of common buckwheat (Fagopyrum esculentum Moench) have not been adequately performed despite its importance in studying the yield-determining process. In addition, the difference between common buckwheat and Tartary buckwheat (F. tataricum (L.) Gaertn.) is still unclear. In the present study, the differentiation and growth of the growing point until the stage of flower bud appearance were evaluated in the common buckwheat variety ‘Kitawasesoba’ and the Tartary buckwheat variety ‘Manten-Kirari’, which are the leading buckwheat varieties in Hokkaido, Japan. With some exceptions, the developmental stages of ‘Kitawasesoba’ and ‘Manten-Kirari’ can be distinguished. Thus, leaf primordia, axillary flower bud, and terminal flower bud differentiations and growths were observed. Both the common and Tartary buckwheat varieties did not exhibit large differences in the morphology of the growing point. However, the two varieties showed differences in the rates of differentiation and growth. Kasajima et al. (2019): Growing point of common and Tartary buckwheat 38 INTRODUCTION Buckwheat (family Polygonaceae) is a pseudocereal crop with beneficial effects on human health (Ikeda 2002; Ikeda et al. 2012). Common buckwheat (Fagopyrum escu- lentum Moench) and Tartary buckwheat (F. tataricum (L.) Gaertn.) are the well-known members of the Fagopyrum genus. Common buckwheat bears beautiful and abun- dant flowers. However, due to self-incompatibility, its seed set is generally low, implying poor seed yield (Woo et al. 2016). Currently, the seed yield of common buck- wheat in Japan is less than 1,000 kg ha-1. The yield of Tartary buckwheat is generally higher than that of com- mon buckwheat as Tartary buckwheat is an autogamous plant with high fertilization efficiency. However, the seed yield of Tartary buckwheat is still much lower than that of major grain crops such as wheat and rice.  In buckwheat, the seed yield per unit area is de- termined by the yield component, i.e., the number of bloomed florets per unit area, fertilization rate, the per- centage of ripened seeds, and seed weight (Ujihara and Matano 1975; Kasajima et al. 2011). The number of bloomed florets per unit area includes the number of flower clusters per unit area and the number of bloomed florets per flower cluster. In buckwheat, the number of flower clusters and the number of bloomed florets are considered to be important factors governing seed yield. In wheat, the grain number per spike is closely related to the formation and development of spikelets and florets (Miralles and Slafer 1999; Toyota et al. 2001). Similar- ly, in buckwheat, flower bud differentiation is thought to be related to the number of seeds based on the number of flower clusters and florets. Few studies have report- ed observations regarding flower bud differentiation in common buckwheat. Hagiwara et al. (1998) reported the criteria for determining the developmental stage of the growing point in common buckwheat. Although com- mon buckwheat is important for examining the yield-de- termining process, detailed studies assessing its develop- mental stage have not yet been conducted. In addition, the difference between common buckwheat and Tartary buckwheat is still unknown.  In Japan, the largest buckwheat-producing region is Hokkaido, which lies in the northern part of the coun- try. Common buckwheat production in Hokkaido ac- counts for approximately 40% of its total production in Japan (Morishita and Suzuki 2012). For approximate- ly past three decades, the common buckwheat variety ‘Kitawasesoba’ has been the main cultivated variety of buckwheat in Hokkaido. In addition, there has been an increased cultivation of Tartary buckwheat in Hokkaido. Suzuki et al. (2014) developed a new Tartary buckwheat variety, ‘Manten-Kirari’, the flour of which contains only trace amounts of rutinosidase and thus, lacks the bitter taste associated with buckwheat. The cultivation and use of ‘Manten-Kirari’ have rapidly grown in Hokkaido. Thus, ‘Kitawasesoba’ and ‘Manten-Kirari’ are the leading varieties of common and Tartary buckwheat, respective- ly, grown in Hokkaido. There is a lack of basic research from the viewpoint of developmental stages to realize high-yield capabilities of these varieties. The present study aimed to describe the differentia- tion and growth of a growing point until the stage of flow- er bud appearance in the leading common and Tartary buckwheat varieties ‘Kitawasesoba’ and ‘Manten-Kirari’, respectively, in Hokkaido, northern Japan. MATERIALS AND METHODS ‘Kitawasesoba’ and ‘Manten-Kirari’ varieties devel- oped by the NARO Hokkaido Agricultural Research Center were used in the present study (Inuyama et al. 1994; Suzuki et al. 2014). Under experimental condi- tions, the seeds of these varieties were sown in plastic planters measuring 56.5 × 17 × 16 cm (length × width × height) that were filled with commercial garden soil con- taining 0.374 g kg−1 N, 1.485 g kg−1 P2O5, and 0.242 g kg−1 K2O. The experiment was conducted in a greenhouse with all sides open at the Faculty of Bioindustry, the To- kyo University of Agriculture in Hokkaido, Japan, in the summer of 2018. Ten planters of each variety were placed in the greenhouse, and approximately 120 plants of each variety were grown in a completely randomized design with two replications. A depression measuring approxi- mately 2-cm in depth was made in the middle of the soil in the planters, and 24 seeds, equivalent to 250 seeds/ square meter, were sown per planter on June 10, 2018. Plants were irrigated daily with tap water, and no fertiliz- er was applied. Plants were sampled twice a week from the beginning of July to middle of August to observe the development of the growing point on the shoot. The growing points of three plant samples per plot were immediately dissected under a stereoscopic microscope to estimate the develop- mental stage of the growing points. The side views of the growing points were observed using a tablet in conjunc- tion with the microscope. The vertical length of the grow- Fagopyrum 36(2):37-41 (2019) 39 ing point was recorded using the software in the tablet. The definitions of the developmental stages of both the varieties generally followed those reported by Hagiwara et al. (1998). RESULTS AND DISCUSSION In the present study, the developmental stages of the common buckwheat variety ‘Kitawasesoba’ and the Tartary buckwheat variety ‘Manten-Kirari’ were distin- guished, with some exceptions, according to the criteria reported by in a study by Hagiwara et al. (1998) that used common buckwheat. Three primary developmental stag- es have been reported: (A) leaf primordia differentiation and growth period, (B) axillary flower bud differentiation and growth period, and (C) terminal flower bud differen- tiation and growth period. In the present study, the devel- opmental stages of both common buckwheat and Tartary buckwheat were almost consistent with the aforemen- tioned stages. The typical and easily-discernible pictures of each developmental stage are shown in Figs. 1–3. Fig. 1 depicts leaf primordia differentiation and growth period in ‘Manten-Kirari’. We did not observe the growing point with leaf primordia in ‘Kitawasesoba’. The growing point in Fig. 1 A0 showed the formation of only leaf primordia. An axillary bud was observed on the growing point along with the growth of leaf primordial (Fig. 2 A1). In higher plants, vegetative growth can be divided into juvenile and adult phases (Bäurle and Dean 2006; Yoshikawa et al. 2013). In the present study, the difference between the juvenile and adult phases was dif- ficult to discern, although the morphological character- istics of growing points were observed in the vegetative growth period. Fig. 2 depicts axillary differentiation and the growth period in ‘Kitawasesoba’ and ‘Manten-Kirari’. In Fig. 2 B0, the apical growing point and axillary bud primordia grew into a dome-shaped structure. However, the differentiat- ed dome on the growing point could not be observed in ‘Kitawasesoba’. As seen in Fig. 2 B1, numerous small pro- jections were formed on the surface of the dome differ- entiated in Fig. 2 B0. A flower bud grew and was covered with a bract (Fig. 2 B2). The flower bud grew, and floret clusters were found to be covered with a bract (Fig. 2 B3). In the growing point in B3, leaf primordia and flower buds simultaneously differentiated (Hagiwara et al. 1998). In general, buckwheat exhibits an indeterminate growth pattern that is based on indeterminate inflorescence. Fig. 1. Leaf primordia differentiation and growth period in ‘Manten-Kirari’. (A0) Growing point initiates only leaf primordia. The arrow shows the leaf primordia. (A1) Leaf primordia and axillary buds differentiate on the growing point. The arrow shows the axillary buds. Fig. 2. Axillary bud differentiation and growth period in ‘Kitawasesoba’ (left) and ‘Manten-Kirari’ (right). (B0) Apical growing point and axillary bud primordia grew into a dome-shaped structure. The arrow shows the dome differentiated on the growing point. (B1) Numerous small projections formed on the surface of the dome differentiated at B0. The arrow shows the small projections. (B2) A flower bud grows and is covered with bract, which is indicated by the arrow. (B3) Flower bud grows and floret clusters covered with bract are visible. The arrow shows a floret cluster. 1.06 mm 0.71 mm 0.29 mm 0.86 mm 0.49 mm 0.97 mm 1.72 mm 2.21 mm 2.09 mm Kasajima et al. (2019): Growing point of common and Tartary buckwheat 40 Conversely, simultaneous ripening is a characteristic of determinate varieties of common buckwheat (Martinen- co and Fesenko 1989). Therefore, the differentiation and growth of the growing point may vary between determi- nate and indeterminate common buckwheat varieties. Further studies that compare the growing point in deter- minate and indeterminate common buckwheat varieties are required to elucidate the effect of the overlapping period of vegetative and reproductive primordia on the agronomic characteristics of buckwheat. Fig. 3 depicts terminal flower bud differentiation and growth period in ‘Kitawasesoba’ and ‘Manten-Kirari’. As seen in Fig. 3 C0, the differentiation of the axillary flower Fig. 4. Mean vertical length of growing point in ‘Kitawasesoba’ and ‘Manten-Kirari’. bud ceased, and the apical growing point changed again into a dome-shaped structure. A dome-shaped growing point was found to be covered with small projections (Fig. 3 C1). Terminal flower bud growth and floret clus- ters formed from the small projections were visible (Fig. 3 C2). The floret clusters grew and were covered with bract (Fig. 3 C3). The results depicted in Figs. 1–3 show no large differences in the developmental morphology of the growing points of common buckwheat and Tartary buckwheat. Fig. 4 depicts the mean vertical length of the growing points in ‘Kitawasesoba’ and ‘Manten-Kirari’. The expan- sion of the size of the growing point in both ‘Kitawas- Fig. 3. Terminal flower bud differentiation and growth period in ‘Kitawasesoba’ (left) and ‘Manten-Kirari’ (right). (C0) Differentiation of axillary flower bud ceases, and the apical growing point changes again into the dome-shaped structure. The arrow shows the dome differentiated on the apical growing point. (C1) The dome-shaped growing point is covered with small projections (indicated by the arrow). (C2) Terminal flower bud growth and floret clusters formed from the small projections can be seen. The arrow shows visible floret clusters. (C3) Floret clusters grow and are covered with bract (indicated by the arrow). esoba’ and ‘Manten-Kirari’ showed an S-shaped curve. However, the expansion was delayed in ‘Manten-Kirari’ compared with that in ‘Kitawasesoba’. In addition, the onset of the expansion of the growing point was more rapid in ‘Kitawasesoba’ compared with that in ‘Mant- en-Kirari’, which may lead to a difficulty in identifying the developmental stage of the vegetative growth period in ‘Kitawasesoba’. Thus, the sampling frequency should be increased to observe the growing point during the vege- tative growth period of common buckwheat. In conclusion, there were no large differences in the morphology of the growing points of common buck- wheat and Tartary buckwheat. However, differences were observed in the rates of differentiation and growth in the two varieties. In Japan, buckwheat is very sensitive to en- vironmental stresses such as flooding stress during floral 1.20 mm 1.30 mm 3.14 mm 7.60 mm 5.68 mm 9.99 mm 7.32 mm 8.73 mm 4.73 mm 10.80 mm 7.11 mm 9.23 mm 13.27 mm Fagopyrum 36(2):37-41 (2019) 41 transition in the developmental stage. In further studies, we will conduct a more accurate evaluation of the grow- ing point during floral transition and of its association with abiotic environments. ACKNOWLEDGEMENTS We thank Dr. N. Inoue and Dr. M. Hagiwara of the Shinshu University for their valuable advice and Jinmon Co. Ltd. for providing ‘Manten-Kirari’ seeds. REFERENCES Bäurle, I. and C. Dean, 2006. The timing of developmental transitions in plants. CELL 125: 655-664. DOI: https://doi. org/10.1016/j.cell.2006.05.005 Hagiwara, M., N. Inoue and T. Matano, 1998. Variability in the length of flower bud differentiation period of common buckwheat. FAGOPYRUM 15: 55-64. Ikeda K, 2002. Buckwheat composition, chemistry, and processing. ADV FOOD NUTR RES 44: 395-434. DOI: https:// doi.org/10.1016/S1043-4526(02)44008-9 Ikeda, K., S. Ikeda, I. Kreft and R. Lin, 2012. Utilization of Tartary buckwheat. FAGOPYRUM 29: 27-30. Inuyama, S., Y. Honda, S. Furuyama, M. Kimura and H. Kasano, 1994. The breeding and characteristics of a buckwheat cul- tivar, “Kitawasesoba.” RES BULL HOKKAIDO NATL AGRIC EXP STN 159: 1-10. (In Japanese with English Summary) Kasajima, S. and H. Itoh, 2011. Effect of shading during different growth phases on yield parameters of common buck- wheat cv. Kitawasesoba in the northern region of Japan. FAGOPYRUM 28: 43-46. Martinenko, G. E. and N. V. Fesenko, 1989. Selection of determinant buckwheat varieties. INPROC 4TH INT SYMP ON BUCKWHEAT OREL, USSR 342-243. Morishita, T. and T. Suzuki, 2012. Buckwheat varieties and trend of cultivation in Hokkaido, Japan. RESEARCH JOUR- NAL OF FOOD AND AGRICULTURE 35: 9-12 (In Japanese). Miralles, D. J. and G. A. Slafer, 1999. Wheat development. In Satorre E. H. and G. A. Slafer (Eds.), Wheat: ecology and physiology of yield determination. pp. 13-43, Food Products Press, New York. Suzuki, T., T. Morishita, Y. Mukasa, S. Takigawa, S. Yokota, K. Ishiguro and T. Noda, 2014. Breeding of ‘Manten-Kirari’, a non-bitter and trace-rutinosidase variety of Tartary buckwheat (Fagopyrum tataricum Gaertn.). BREED SCI 64: 344- 350. DOI: https://doi.org/10.1270/jsbbs.64.344 Toyota, M., I. Tsutsui, A. Kusutani and K. Asanuma, 2001. Initiation and development of spikelets and florets in wheat as influenced by shading and nitrogen supply at the spikelet phase. PLANT PROD SCI 4: 283-290. DOI: https://doi. org/10.1626/pps.4.283 Ujihara, A. and T. Matano, 1975. Characteristics of flowering, fertilization and fructification - approaches to yielding process. J AGRI SCI 30: 406-408. (In Japanese) Woo, S. H., S. K. Roy, S. J. Kwon, S. W. Cho, K. Sarker, M. S. Lee, K. Y. Chung and H. H. Kim, 2016. Concepts, prospects, and potentiality in buckwheat (Fagopyrum esculentum Moench): a research perspective. In: Meiliang, Z., I. Kreft, S. H. Woo, N. Chrungoo and G. Wieslander (Eds.), Molecular Breeding and Nutritional Aspects of Buckwheat, pp. 21-49, Academic Press, London. Yoshikawa, T., S. Ozawa, N. Sentoku, J. Itoh, Y. Nagato S. Yokoi, 2013. Change of shoot architecture during juve- nile-to-adult phase transition in soybean. PLANTA 238: 229-237. DOI: https://doi.org/10.1007/s00425-013-1895-z IZVLEČEK Faze razvoja rastlin navadne ajde (Fagopyrum esculentum Moench) še niso bile ustrezno raziskane; so pa pomembne za formiranje pridelka ajde. Poleg tega še niso jasne morebitne razlike pri razvoju navadne in tatarske ajde (F. tataricum (L.) Gaertn.). V tej študiji sta bili proučeni rast in razvoj točke rasti vse do faze nastanka cvetnih brstov, pri navadni ajdi, sorta ‘Kitawasesoba’ in tatarski ajdi, sorta ‘Manten-Kirari’. Ugotovljene so razlike v fazah razvoja med obema kultivarje- ma. Obe proučevani sorti sta vodilni pri pridelovanju ajde na otoku Hokkaido, Japonska. Raziskani so listni primordiji, stranski in glavni cvetni brsti in apikalna točka rasti. Glede morfologije točke rasti ni bistvenih razlik med kultivarjema, se pa rastline obeh raziskanih kultivarjev razlikujejo v stopnjah diferenciacije in rasti. Fagopyrum 36(2):43-50 (2019) 43 Research paper Changes of physicochemical properties and correlation analysis of common buckwheat starch during germination Licheng GAO1, Meijuan XIA1, Zhonghao LI1, Pengke WANG1, Meng WANG2, Jinfeng GAO*1 1 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy, Northwest A&F University, Yangling, Shaanxi Province 712100, China 2 Yulin Institute of Agricultural Sciences, Yulin, Shaanxi Province 719000, China * Corresponding author, e-mail address: gaojf7604@126.com E-mail addresses: 1846756406@qq.com (Licheng Gao), 2631630429@qq.com (Meijuan Xia), 2817530110@qq.com (Zhonghao Li), (Pengke Wang), wangmeng3214@126.com (Meng Wang) DOI https://doi.org/10.3986/fag0010 Received: April 14, 2019; accepted: June 8, 2019 Key words: common buckwheat, germination, physicochemical properties, starch ABSTRACT In order to clarify the physicochemical properties of starch during germination of common buckwheat, Xinong9976 was selected as the experimental material to study the main nutrients, particle structure, particle size distribution, transparency, aging value, pasting properties and the correlation between pasting properties and starch composition and main nutrients. The results showed that main nutrients were significantly different. The diameter of starch gran- ules ranged from 2.36 to 8.89μm, and the shapes of starch granules were irregular with obvious holes and cracks on the surface. There were significant differences in starch transparency, aging value and pasting properties at different germi- nation stages. Peak viscosity, through viscosity and final viscosity of germinated common buckwheat was significantly positive correlated with amylopectin content (P < 0.05) and breakdown, final viscosity and setback were significantly negatively correlated with amylose content (P < 0.05). The correlation analysis of starch pasting properties and main nutrients showed that breakdown, setback and crude fat content were significantly negatively correlated (P < 0.01), peak viscosity, through viscosity and final viscosity were significantly negatively correlated with crude fat content (P < 0.05), while the starch pasting properties had no significant correlation with other nutrients. Gao et al. (2019): Common buckwheat starch during germination 44 INTRODUCTION Common buckwheat (Fagopyrum esculentum) belongs to Polygonaceae, it is an important minor grain crop in China (Nam et al. 2018). Common buckwheat is rich in nutrients, containing 10.6% -15.5% of protein, 1.2% - 2.8% of fat, 63% -71.2% of starch, as well as flavonoids, mineral elements and cysteine (Gimenez-Bastida and Zielinski 2015). Recently, with the improvement of peo- ple’s living standard, it has become fashionable to pursue a balanced diet with scientific based nutrition. There- fore, common buckwheat products with the reputation of “the same origin of food and medicine” will be widely welcomed by people. Besides, the development and re- search of common buckwheat healthy food have broad market prospects, high economic value and social value (Yoshimoto et al. 2004). There have been many studies on the cultivation methods, yield and protein content of common buckwheat in China and abroad (Salehi et al. 2018, Fang et al. 2018, Khan et al. 2012) and studies on the starch properties have also been reported. However, changes of physicochemical properties of germinated common buckwheat starch is not reported. Starch is the main nutritional component of common buckwheat. Its physicochemical properties affect the nutritional proper- ties of related products and are also related to the devel- opment of new uses of common buckwheat starch (Stibilj et al. 2004). Germination is a dynamic process where plants come from resting state to the state with many physiological activities. Germination treatments can enhance the res- piration of plants and significantly increase the species and number of enzymes (Mamilla and Mishra 2017). After germination, the starch content of mung bean de- creased (Liu et al. 2014), while the amylase activity of kidney bean increased, leading to the changes in starch structure and composition (Yanli et al. 2018). Studies have showed that buckwheat has a high peak viscosity, hot paste stability and cold paste stability (Hara et al. 2007). Tartary buckwheat flavone content significantly increased after germination (Xiao-Peng et al. 2015). In addition, germination treatment can improve the edible and health value of buckwheat, enhance the resistance of starch paste and improve the stability of starch paste (Hara et al. 2007). Therefore, the study on the charac- teristics of germinated common buckwheat starch is of great significance to the development and utilization. In this experiment, the main nutrients, particle structure, physicochemical properties and the correlation between gelatinization characteristic value and main nutrients of germinated common buckwheat starch were studied by using cv. Xinong9976 as material to provide basis for the development and utilization of common buckwheat sprouts and bean flour and the deep processing of starch. MATERIALS AND METHODS 1. Experimental materials Cv. Xinong9976, a common buckwheat variety, provided by small grain laboratory of Northwest A&F University, was used for the experiment. Common buckwheat seeds with full grain and no disease were se- lected and were sterilized with 0.1% H2O2 for 10 min and soaked in the distilled water for 24 h. Then seeds were placed in a petri dish with two layers of filter paper, the cultivation of the sample under 25˚C in dark for germina- tion, respectively taking sample after 2, 4, 6 d, removed shell, dried at 40˚C. The dried sample was grinded and passed through a 0.100 mm mesh. Added 80% ethanol, 50˚C, with the ul- trasonic treatment (500 w) 30 min to remove flavonoids, then added the volume of distilled water, heated under the condition of 30˚C for 24 h. Centrifuged (4000 rpm, 10 min) three times, scraped off the grayish-brown soft layer. Finally, dried at 40˚C for 48 h and sieved with a 0.150 mm mesh. 2. Measurement of physicochemical properties The morphology of starch granules was observed by scanning electron microscope (JSM-6390, Jeol Ltd, To- kyo Japan) at 2000 x magnification, the particle size dis- tribution was determined by a laser diffraction particle size analyzer and the pasting properties were measured using a rapid visco analyzer (RVA) (Newport Scientific, Pty Ltd, Warriewood, Australia). Starch transparency was determined using a modified version of (Zhou et al. 2014). 0.5g of the starch sample was blended with 50 mL distilled water and heated in a boiling water bath for 30 min. After the starch was com- pletely gelatinized by stirring, the starch was removed and cooled to room temperature. Distilled water was used as a blank for zero adjustment and the transparency was measured at the wavelength of 620 nm with a visi- ble-light spectrophotometer. The starch aging value was determined as follows: 0.5g of the starch sample was blended with 50 mL distilled wa- Fagopyrum 36(2):43-50 (2019) 45 Germination time/d Water/% Ash/% Crude fat/% Crude protein/% Total starch/% Amylose/% Amylopectin/% 0 12.26 ± 0.09 a 1.12 ± 0.03 c 1.37 ± 0.01 a 9.22 ± 0.06 c 68.41 ± 0.11 a 24.11±0.28 a 44.30±0.39 a 2 11.21 ± 0.03 b 1.36 ± 0.02 b 1.04 ± 0.01 b 9.42 ± 0.10 c 58.77 ± 0.81 b 22.89±0.17 b 35.88±0.97 b 4 9.78 ± 0.01 c 1.44 ± 0.01 b 0.92 ± 0.01 c 10.83 ± 0.17 b 52.80 ± 0.09 c 21.85±0.25 c 30.95±0.32 c 6 7.73 ± 0.01 d 1.69 ± 0.03 a 0.84 ± 0.01 d 13.79 ± 0.20 a 47.66 ± 0.11 d 19.77±0.20 d 27.89±0.31 d ter and heated in a boiling water bath for 30 min and added distilled water to keep the total volume constant. Removed and cooled to room temperature, refrigerated for 24h, and defrosted at room temperature, and then centrifuged at 4000 rpm for 10 min, finally weighed the sediment quali- ty. The starch aging value index was determined as follows: starch aging value = (weight of starch paste before centrif- ugation – weight of sediment quality) X 100. 3. Data analysis Three parallel tests were conducted in the experi- ment. SPSS 17.0 was used for statistical analysis, Origin 9.0 was used for drawing, and LSD minimum significant difference test was used for the determination of signifi- cance of differences. RESULTS AND DISCUSSION Changes of the concentration of main nutrients Main nutrients of common buckwheat were shown in table 1. The results showed that after germination, the contents of water, crude fat, total starch, amylose and amylopectin decreased significantly (P < 0.05), while the contents of ash and crude protein increased significant- ly (P < 0.05). Among them, the crude fat mass fraction decreased the most, from 1.37% to 0.84%, a decrease of 38.69% while the ash quality score increased the most, from 1.12% to 1.69%, increasing by 51%. In addition, the relative standard deviations of main nutrients in water, ash, crude fat, crude protein, total starch, amylose and amylopectin at different stages were 19.14%, 16.76%, 22.38%, 19.50%, 15.66%, 8.30% and 20.61%, respec- tively, indicating that the nutritional composition of the main nutrients in different stages of the germinated common buckwheat was significantly different. After germination, the crude protein mass fraction increased exponentially, which may be caused by the de- creased protease activity in the seeds of common buck- wheat during the germination process, which effectively weakened the hydrolysis of related proteins and thus pro- moted the protein accumulation (Ikeda et al. 1984). The decrease of total starch mass fraction may be due to the activation of α-amylase and β-amylase in the sprouting of common buckwheat, which could promote the degra- dation of starch and provide part of sugars needed for the germination (Mohan et al. 2010). The decrease of fat mass fraction might be due to the action of lipase, which could decompose part of the fat into the energy required for the germination and growth of common buckwheat seeds. Starch grain structure As can be seen on Fig. 1a, the starch particles of ma- ture common buckwheat seeds were complete with clear gaps, mostly spherical and oval in shape, with smooth surface and no holes or cracks. After the germination of 2 d, most starch granules were irregular in shape, while a few were spherical in shape. In addition, some of the crystalline structures of starch were destroyed, and a few starch granules showed cracks on the surface (Fig. 1b). In 4 d, the starch granules were disordered. A small number of starch granules were spherical in shape, while some starch granules were deformed and condensed together with the surrounding granules (Fig. 1c). And in 6 d, the starch granules were polygonal in shape with few of them being spherical. The crystalline structures of most starch granules were destroyed, and obvious cracks and holes appeared on the surface of most granules (Fig. 1d). The table 2 showed that in the process of germina- tion, starch granule size distribution was more dispersed, which indicated that the size of starch granules had ob- Table 1 Changes of main nutrients of common buckwheat before and after germination Note: different letters in the same column mean significant difference of P<0.05, the same as below. Gao et al. (2019): Common buckwheat starch during germination 46 Transparency can reflect the mutual solubility of starch and water, and the transparency of buckwheat starch is positively proportional to the absorbance value, the high- er the absorbance value is, the higher the transparency of the starch is (Li et al. 1997). As can be seen from Fig. 2, the transparency of common buckwheat starch first in- creased and then decreased with the extension of time in the germination process. And the transparency in dif- ferent stages was significantly different (P < 0.05), the most transparent stage was 2 d and the absorbance value was 1.75, indicating that starch particles were completely expanded at this time, and there was no mutual associ- ation among the starch molecules after gelatinization. While the lowest transparency period was 6 d and the absorbance value was 1.00, which decreased by 36.30% compared with that of mature seeds. The average absorb- ance of starch in different germination stages was 1.41. After germination of 2d, starch transparency decreased significantly (P < 0.05), which may be due to the starch retrogradation, rearrangement of starch molecules and Note: D10, D50 and D90 represented the critical particle size values when the minimum particle size was added up to 10%, 50% and 90% of the sample. Germination time/d D10 D50 D90 Average sphericity Average aspect ratio 0 4.97 7.86 8.89 0.84 1.45 2 4.68 6.64 7.98 0.76 1.43 4 3.29 5.17 6.97 0.68 1.40 6 2.36 4.05 5.84 0.57 1.37 Table 2 Starch particle size distribution during the germination of common buckwheat d. Starch granules in 6d (×2000). Fig.1. Scanning electron microscopy of starch granules of common buckwheat at different germination stages a. Starch granules in 0d (×2000); b. Starch granules in 2d (×2000); c. Starch granules in 4d (×2000); vious differences. Chang found that the corn starch size ranged from 5.76 to 8.64 μm (Chang et al. 2004). Com- mon buckwheat starch particles between 4.00 to 9.00 microns in diameter, which was smaller than the corn starch granules. After germination, the diameter, aver- age sphericity and average aspect ratio of starch granules decreased significantly with the increase of germination time. In 6 d, the average diameter of starch granules de- creased from 7.24 μm to 4.08 μm, a decrease of 43.65%. The average sphericity and the average aspect ratio de- creased by 32.14% and 5.52%, respectively. Starch transparency Transparency is one of the important external char- acteristics of starch, which is directly related to the ap- pearance and use of starch products (Wang et al. 2017). Fig.2. Changes of the transparency of common buckwheat starch at different germination stages Fagopyrum 36(2):43-50 (2019) 47 scattering of light, thus reducing light transmission and starch transparency (Zhou et al. 2017). Starch aging value The essence of starch aging is that gelatinized starch molecules re-form hydrogen bonds during the cooling process(Jiamjariyatam et al. 2014). The aging process of starch can be regarded as the reverse process of gelatini- zation, but the degree of starch crystallization decreases after aging (Verma et al. 2018). It could be seen from Fig. 3 that there were significant differences in the aging val- ue of common buckwheat starch in different germination stages (P < 0.05). The maximum aging value was 71.20% in mature grains. Subsequently, the aging value decreased gradually with the extension of germination time. Both 4d and 6d, the aging value decreased by 28.84%, 39.35%, respectively, which might be related to the weakened ability of buckwheat starch molecules to form hydrogen bonds again after germination (Liu et al. 2006). Starch aging not only makes food taste worse, but also reduces the digestibility (Verma et al. 2018). However, the aging value of common buckwheat gradually decreased during germination, indicating that common buckwheat sprouts were good in taste, easy to digest and had broad market development value. Pasting viscosity Starch granules rapidly absorb water in aqueous solu- tion due to thermal expansion, resulting in the fracture of intramolecular and intermolecular hydrogen bonds, and the process of gradual diffusion of starch granules is called starch paste (Jane et al. 1992). The pasting temper- ature was different in different germination stages due to the size of starch granules. The starch pasting proper- ties of common buckwheat in the process of germination were shown in table 3, the results showed that starch pasting viscosities gradually decreased and significantly different (P < 0.05) in different period. After germina- tion, peak viscosity, through viscosity, breakdown, final viscosity, setback, peak time and pasting temperature were lower than those of the mature grain starch. Peak viscosity refers to the increase in the viscosity of starch paste caused by the friction between starch par- ticles after full water absorption and expansion, which can reflect the expansion capacity of starch (Xiao-Li et al. 2008). As shown in table 3, the peak viscosity was 1394 - 2982 cp, the average peak viscosity was 2242 cp, and the difference was great. Through viscosity is caused by the sharp decrease in the viscosity of starch paste due to the fact that starch particles no longer friction with each other after they have expanded to the limit (Xiao-Li et al. 2008). The through viscosity was between 1335 and 2819 cp, and the average was 2131 cp. The breakdown is the difference between peak viscosity and through viscosity, which can reflect the thermal stability of starch paste. The smaller the breakdown is, the better the thermal stability is (Karim et al. 2000). The average breakdown was 111 cp, and the breakdown in mature grains was 2.76 times high- er than that after germination, indicating that the starch Fig.3. Changes of the aging value of common buckwheat starch at different germination stages Germination time/d peak viscosity /cp Through viscosity /cp breakdown/cp final viscosity /cp setback/cp peak time /min pasting temperature /°C 0 2982±37.64 a 2819±32.67 a 163±4.98 a 4765±34.43 a 1946±3.53 a 6.41±0.08 a 74.29±0.03 a 2 2587±18.50 b 2455±16.50 b 132±2.03 b 3982±41.51 b 1527±25.01 b 5.36±0.05 b 68.50±0.06 b 4 2005±7.84 c 1914±6.69 c 91±1.16 c 2933±17.53 c 1019±11.24 c 4.06±0.06 c 60.63±0.40 c 6 1394±11.29 d 1335±10.41 d 59±0.88 d 1988±18.02 d 654±7.62 d 2.24±0.04 d 50.73±0.34 d Table 3 Characteristic values of gelatinization of common buckwheat starch during germination Gao et al. (2019): Common buckwheat starch during germination 48 had good thermal stability after germination and was suitable for the development of noodles and thickening agents. The final viscosity can reflect the retrogradation property of starch (Xiao-Li et al. 2008). After germina- tion, the final viscosity decreased significantly, reaching 58.28% in 6d. The setback is the difference between final viscosity and through viscosity, which can reflect the sta- bility of cold paste of starch. It can be seen from table 3 that the starch was not easy to age after germination and was suitable for making food such as common buckwheat instant noodles. Correlation analysis of pasting properties and starch composition Starch is the main component of common buckwheat grain, accounting for 60 -75% of the grain. The contents, composition and properties of buckwheat starch directly affect the processing technology of buckwheat food (Xin- Hua et al. 2009). The table 4 showed that peak viscosity, through viscosity and final viscosity of germinated com- mon buckwheat was significantly positive correlated with amylopectin content (P < 0.05), which was the same as the relationship between the pasting viscosity and starch composition of the rice starch or germinated brown rice starch reported in previous studies, that was, the higher the content of amylopectin was, the higher the peak vis- cosity, through viscosity and final viscosity were. Break- down, final viscosity and setback were significantly nega- tively correlated with amylose content (P < 0.05). Studies have shown that the short-term retrogradation of starch is mainly caused by the gelation order and dehydration crystallization of amylose molecules. However, setback was significantly negatively correlated with amylose con- tent (P < 0.05), indicating that germinated common buck- wheat with low amylose content was easy to retrogradate. Correlation analysis of pasting properties and other nutrients The correlation analysis of starch pasting properties and other nutrients was shown in table 5. In 6d, past- ing properties was positively correlated and negatively correlated with the main nutrients. Breakdown and set- back were significantly negatively correlated with crude fat content (P < 0.01), indicating that the thermal sta- bility and cold paste stability gradually increased after germination with the decrease of crude fat content. Peak viscosity, through viscosity and final viscosity were nega- tively correlated with crude fat content (P < 0.05). There was no significant relationship between pasting viscosity and water content, which may be due to the fast growth rate and the need to consume more water for growth, re- sulting in low water content. Similarly, pasting viscosi- ty had no significant relationship with ash content and crude protein content. Varieties peak viscosity /cp Through viscosity /cp breakdown/cp final viscosity /cp setback /cp peak time /min pasting temperature /°C Total starch 0.973/0.149 0.974/0.147 0.960/0.180 0.967/0.164 0.956/0.189 0.910/0.273 0.872/0.326 Amylose -0.996/0.059 -0.995/0.062 -0.999/0.028* -0.998/0.044* -1.000/0.019* -0.995/0.064 -0.983/0.118 Amylopectin 1.000/0.017* 1.000/0.014* 0.997/0.048* 0.999/0.032* 0.996/0.057 0.976/0.141 0.954/0.194 Varieties peak viscosity /cp Through viscosity /cp breakdown/cp final viscosity /cp setback /cp peak time /min pasting temperature /°C Water 0.769/0.442 0.771/0.439 0.737/0.472 0.753/0.457 0.727/0.482 0.631/0.565 0.564/0.618 Ash -0.798/0.412 -0.795/0.415 -0.826/0.381 -0.812/0.397 -0.834/0.372 -0.899/0.288 -0.933/0.235 Crude fat -0.998/0.039* -0.998/0.042* -1.000/0.009** -0.999/0.024* -1.000/0.000** -0.991/0.084 -0.977/0.137 Crude protein -0.772/0.438 -0.770/0.441 -0.802/0.407 -0.787/0.423 -0.811/0.398 -0.880/0.315 -0.917/0.261 Table 4 Correlation coefficient between pasting properties and starch composition (r/p) Note: Linear correlation coefficient between r. **: Significant correlation at the level of 0.01, *: Significant correlation at the level of 0.05, the same below. Table 5 Correlation coefficient between pasting properties and main nutrients (r/p) Fagopyrum 36(2):43-50 (2019) 49 CONCLUSIONS The results showed that in comparison to non-germi- nated grain, after germination, the concentration of main nutrients of common buckwheat were significantly differ- ent, where the content of crude fat, total starch, amylose and amylopectin decreased significantly while the content of ash and crude protein increased significantly. Starch granules were arranged in a disorderly manner, most of which were irregular in shape, few of which were spherical in shape. Moreover, the crystal structure of most starch granules was destroyed, and obvious cracks and voids ap- peared on the surface. In addition, starch size of mature common buckwheat was 4-9 μm. Pasting properties were closely related to the starch composition, and peak viscos- ity, through viscosity, final viscosity and setback were sig- nificantly positively correlated with amylopectin content, while breakdown, final viscosity and setback were signifi- cantly negatively correlated with amylose content. In ad- dition, breakdown, setback and fat content were signifi- cantly negatively correlated, and peak viscosity, through viscosity and final viscosity were significantly negatively correlated with fat content, while pasting properties were not significantly correlated with other nutrients. ACKNOWLEDGEMENT This study was sponsored by the National Natural Science Foundation of China (Grant No.31671631), Mi- nor Grain Crop Research and Development System of Shaanxi Province (2016-2019). REFERENCES Chang Y H, Lin J H, Li C Y, 2004. Effect of ethanol concentration on the physicochemical properties of waxy corn starch treated by hydrochloric acid. 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Yoshimoto Y, Egashira T, Hanashiro I, Ohinata H, Takase Y, & Takeda Y, 2004. Molecular structure and some physico- chemical properties of buckwheat starches. Cereal Chemistry, 81(4), 515–520. Yanli M, Yifeng R, Ruijin Y, et al, 2018. Effect of white kidney bean extracts on estimated glycemic index of different kinds of porridge. LWT, 96:576-582. Zhou H, Wang C, Shi L, et al, 2014. Effects of salts on physicochemical, microstructural and thermal properties of potato starch. Food Chemistry, 156(2):137-143. Zhou Y, Wang H, Cui L, et al, 2017. Physicochemical Properties and Digestibility of Germination of Buckwheat Starch. Journal of the Chinese Cereals & Oils Association, 32(3):25-29 and 35. IZVLEČEK Za raziskavo fizikalno kemijskih lastnosti škroba tekom kalitve navadne ajde je bil izbran kultivar ‘Xinong9976’. Raziskava je vključevala glavna hranila, zgradbo delcev, razporeditev velikosti delcev, prosojnost, vrednost staranja, last- nosti gnetenja ter korelacijo med lastnostmi gnetenja, sestavo škroba in glavnimi nutrienti. Pri glavnih nutrientih so bile tekom kalitve značilne razlike. Premer škrobnih zrn je bil od 2.36 do 8.89μm, škrobna zrna so bila nepravilnih oblik in na površini z vidnimi odprtinami in razpokami. V različnih fazah kalitve so bile razlike glede prosojnosti, vrednosti staranja in končne viskoznosti. Najvišja vrednost viskoznosti, prava viskoznost in končna viskoznost so bile pozitivno povezane z vsebnostjo amilopektina (P < 0.05), več parametrov viskoznosti je bilo negativno povezanih z vsebnostjo amiloze (P < 0.05). Lastnost gnetljivosti škroba ni bila značilno povezana z vsebnostjo hranil. Fagopyrum 36(2):51-65 (2019) 51 Research paper Variation of rutin and quercetin contents in Tartary buckwheat germplasm Je Hyeok YU1, Soo Jeong KWON1, Ju Young CHOI1, Yong Hwan JU1, Swapan Kumar ROY1, Dong-Gyu LEE1, Cheol Ho PARK2, and Sun-Hee WOO1* 1 Department of Crop Science, Chungbuk National University, Cheong-ju, Korea 2 College of Agricultural and Life Sciences, Kangwon National University, Chunchon, Korea * Corresponding author: Sun-Hee Woo, Chungbuk National University, 1, Chungdae-ro, Seowon-gu, Cheongju-si, Chungbuk 28644, Korea, Tel: +82-43-261-2515, Fax: +82-43-273-2242, E-mail: shwoo@chungbuk.ac.kr E-mail addresses: Je Hyeok YU- urbanhip@naver.com, Soo Jeong KWON- kwonsj1220@naver.com, Ju Young CHOI- jychoi8519@gmail.com. Yong Hwan JU- joh7598@naver.com, Swapan Kumar ROY- swapankhulna@gmail.com, Dong-Gyu LEE- dongguu00@naver.com, Cheol Ho PARK- chpark@kangwon.ac.kr DOI https://doi.org/10.3986/fag0011 Received: April 29, 2019; accepted: June 15, 2019 Keywords: Tartary buckwheat; seed; sprout; germplasm; rutin; quercetin ABSTRACT This study was carried out to investigate the regional variation and effects of shape and color of seeds and sprouts on the content of rutin and quercetin in Tartary buckwheat germplasm. A total of 44 foreign Tartary buckwheat ger- mplasms were examined and compared their rutin and quercetin contents based on the collected countries, seed shape and seed color using high-performance liquid chromatography and spectrophotometry. The results revealed that rutin and quercetin content varied at different regions. Rutin content in seed (1326.5 mg/100 g) and sprouts (5440.4 mg/ 100 g) of the accession collected from Nepal area was higher than any accession collected from other regions. In seeds, the quercetin content showed the highest value (22.5 mg/100 g) from Pakistan whereas sprouts showed the highest quercetin content (392.0 mg/100 g) from China. However, the quercetin content in sprout was 4~90 times higher than that of seeds. Taken together, the present study suggests that sprouts could be used more effectively than seeds in the case of quercetin, and strains from Nepal, Bhutan, China, and Japan have a high potential material to use seed and sprouts for buckwheat industry in making functional food and medicine. Yu et al. (2019): Rutin and quercetin in Tartary buckwheat germplasm 52 INTRODUCTION Buckwheat, which belongs to the family Polygonace- ae, genus Fagopyrum, has been a popular health food in Asian and European countries for a long time (Kreft, 2003). It has been widely cultivated in Korea since it has abundant nutrients and medicinal efficacy. Buckwheat sprouts are higher in nutritional value than other crops regarding fiber, anthocyanin, and rutin contents (Shin, 2010). Unlike traditional buckwheat, Tartary buckwheat remains in the wild and is most widely grown buckwheat species in the Himalaya region (Golob et al., 2015). The main cultivation areas are Tibet or Chinese mountainous regions, such as India, Bhutan and Nepal (Kreft et al., 2003). However, they are abundant in various nutrients and high in protein (12%) and fat (3.9%) content. Also, oleic acid and linoleic acid account for 80% of total fatty acids (Park et al., 2004). Buckwheat contains many important bioactive com- pounds especially flavonoids that have been shown to exert excessive stress via activating antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and glutathione reductase, (Park et al., 2000; Zhu et al., 1999). These compounds are com- plementary to the lack of enough activity due to envi- ronmental factors, eating habits, smoking and lifestyle habits, and by reactive oxygen species (ROS), and DNA damage (Lee et al., 2011; Park et al., 2011), organ and tissue damage (Shin et al., 1990; Graf et al., 2005). Fla- vonoids found in buckwheat, include rutin, c-glycosylfla- vones (orientin, isoorientin, vitexin, isovitexin), querce- tin, and phenolic acid as chlorogenic acid (Margna et al., 1978; Watanabe et al., 2002), however, the content of rutin in Tartary buckwheat is much higher than that of common buckwheat and other crops (Wang et al., 1995; Park et al., 2005a; Park et al., 2005b). However, it has been only a few years since the culti- vation of buckwheat in Korea has started, and basic eco- logical studies have not been systematically established yet (Park et al., 2004; Lim et al., 2009). With growing de- mand for food and functional foods, interest in sprouting vegetables is increasing. In the United States, Europe and Australia, sprouting vegetables account for about 30% of the vegetable stores. In Asia, interest in sprouting vegeta- bles is increasing, mainly in Japan. Sprouting vegetables account for 10 to 20% of the market. It is estimated that the sprout vegetable market in Korea is about 2 billion won in 2005 (Lee, 2007). In recent years, buckwheat sprouts have been developed in Korea and Japan (Kim et al., 2004; Kim et al., 2007), and sprouting vegetables (Hokkai T9, Hokkai T10) were registered especially in Japan in 2007 (Suzuki, 2008). In addition, the content of quercetin, as well as rutin, was also higher in Tartary buckwheat sprouts than in common buckwheat sprouts. Thus, the consumption of buckwheat sprouts as a source of rutin is increasing (Jeon, 2012). Rutin, which is more abundant in sprouts than seeds in Tartary buckwheat, and compared than common buckwheat and has been reported to be effective against various diseases, including antioxidant activity (Lee et al., 2000), diabetes (Lee et al., 1994), antioxidative effects (Kwon et al., 1995) and the prevention of cardiovascular disease (He et al., 1995; Wojcicki et al., 1995). Previous study was conducted with buckwheat genetic resources for rutin, the comparison was made by the color of seed coat, seed type, and country of collection (Park et al., 2005). Quercetin is a flavonoid substance belonging to the flavonol family. Quercetin is mainly found in fruits and minerals, especially in onions (Formical et al., 1995). In the United States, quercetin is also known to be a rep- resentative flavonoid (Park et al., 1991), as it is known to consume 25 mg per person per day (Lamson et al., 2000). The pharmacological effects of quercetin have been extensively studied both in vivo and in vitro models and have been shown to be associated with reduced lipid peroxidation (Cavallini et al., 1978) and decreased activ- ity of carcinogens (Edenhader et al., 1996), hypotension, (Daniel et al., 2003) and antimicrobial effects (Kimura et al., 1984). In recent years, attempts to search for natural anti- oxidants harmless to the human body have been stud- ied in various ways (Halliwell et al., 1992; Lee, 2007). Recently, Korean people have much interest in using of Tartary buckwheat because of its higher rutin content and bio-active compounds than common buckwheat that has been traditionally utilized in Korea. However, there are no cultivars of Tartary buckwheat in Korea. So, it is urgent to develop the promising cultivars with high yield of seeds and herbs and good quality with high rutin con- centration. MATERIALS AND METHODS 1. Materials collection and plant growth condition The experimental materials were collected from 7 countries, including China, Nepal, Bhutan, India, Japan, Fagopyrum 36(2):51-65 (2019) 53 Pakistan and Slovenia. A total of 44 kinds of Tartary buckwheat genetic resources were stored at Chungbuk National University (4°C, 30-40% RH). The genotypes used in the present study were 28 specimens from Chi- na, 5 specimens from Nepal, 3 specimens from Bhutan, 2 specimens from India, 2 specimens from Japan, 2 spec- imens from Pakistan, 2 specimens from Slovenia. How- ever, the seed size of the genotypes were 6 specimens of notched, 22 specimens of round, 16 specimens of slen- der, and seed color were identified as 8 brown, 12 dark- brown, 15 dark-gray, and 9 gray-brown (Table 1). The above-mentioned genetic resources were cultivat- ed in Chungbuk National University Farm in 2015 and planted on July 31, 2015, maintaining planting density at 30×10 cm. Other cultivation management was pro- vided in accordance with the crop cultivation guideline from Rural Development Administration. A total of 44 seedlings were transferred to a seedling tray (5.1 cm × 4.7 cm), and grown for 7-days in a controlled (25°C, 14 h day/10 h night, 150 μmol.m-2.s-1 light intensity) growth chamber (GC-300 TLH, JEIO TECH). Shoot cotyledons and hypocotyls of buds which were watered twice daily and grown under conditions of 10 hours of dark condi- tions. Prior to analysis, the collected buds were drying using freeze-drier (FDU-1200, EYELA). After freeze-dry- ing for 3 days, the samples were ground into fine powder with a pestle in liquid nitrogen, and stored in a cryogenic freezer (DF8517S, Ilshinbiobase, 4°C, 30-40% RH) for rutin and quercetin analysis. 2. Rutin and quercetin analysis methods 2.1. Pretreatment for rutin and quercetin analysis A portion (0.5 g) of Tartary buckwheat seeds and buds powder stored in a cryogenic freezer (DF8517S, Il- shinbiobase) was weighed into 15 ml tubes (BD, Falcon™). 10 ml of ethanol (96% Germany, MERCK) was added to each of the weighed tubes, and vortexed vigorously. The tubes were sonicated using an ultrasonic clearer (SD- D300H, Seongdong) for 60 minutes at 80°C and then cooled in a refrigerated shell (IBK-1400RFD, Infobiotech) for 60 minutes at 4°C. The mixture was centrifuged at 5000 rpm for 5 min at 4ºC. 2 ml of the extracted sam- ple was filtered using a 0.45 μm PVDF membrane syringe filter (Whatman, USA) and analyzed by HPLC (Agilent 1200 series) instrument with 3 repetitions. The contents of rutin and quercetin were expressed on dry weight ba- sis. The content of both investigated compounds was ex- pressed in mg/100 g DW. 2.2. HPLC analysis method Methanol, acetonitrile, and water (Honeywell B & J) used in HPLC analysis were HPLC grade. For the solvent A, 0.05% TFA buffer (Sigma) was added to a total volume of 100% water. The solvent B was prepared with 60% of methanol, 40% of acetonitrile, 0.05% of TFA buffer re- spectively. All analyses for rutin and quercetin were performed on a HPLC system (HPLC 1200 series manufactured Origin Seed shape Seed color Notched Round Slender Total Brown Dark-brown Dark-gray Gray-brown Total China 5 14 9 28 5 7 8 8 28 Nepal N/A 4 1 5 N/A 2 3 N/A 5 Bhutan N/A N/A 3 3 2 N/A 1 N/A 3 India N/A 1 1 2 N/A 1 N/A 1 2 Japan N/A 2 N/A 2 N/A N/A 2 N/A 2 Pakistan 1 N/A 1 2 N/A 1 1 N/A 2 Slovenia N/A 1 1 2 1 1 N/A N/A 2 Total 6 22 16 44 8 12 15 9 44 *N/A=Not applicable Table 1. Country-wise number of Tartary buckwheat germplasm based on seed shape and seed color used in evaluation of rutin and quercetin contents Yu et al. (2019): Rutin and quercetin in Tartary buckwheat germplasm 54 by Agilent), equipped with a column used YMC-pack OSD-AC18 (4.6 mm ID × 250 mm, S-5 μm, YMC Co., LTD., Japan). The flow rate was 1.0 ml/min, the column temperature was 25°C, the injection volume was 10 μl, and the detection wavelength was set at 359 nm. The detailed HPLC analysis conditions are shown in Table 2. For the quantitative analysis of rutin and quercetin, the standards were made according to the protocol of Sig- ma, and the concentrations used in this analysis were 1, 2, 5, 10, 20, 50, 100 and 200 ppm. The pattern and retention time of rutin and quercetin observed in HPLC apparatus are shown in Fig. 1, and the equation of line- ar regression of rutin and quercetin y=14.993x-62.119 y=32.943x-19.09 respectively while the coefficient of determination (R2) was 0.9989 and 0.9999 in rutin and quercetin respectively. 3. Statistical processing analysis Analysis of variance was performed using SAS soft- ware (SAS Institute Inc., ver. 9.2). The significance test for the rutin and quercetin content of each country, seed shape, and seed color was performed using Duncan’s multiple range tests. Also, the correlation between rutin and quercetin content was investigated within the seeds, within the shoots and between the seeds and sprouts. RESULTS AND DISCUSSION 4. Variation of rutin and quercetin content in seed 4.1. Content, distribution and resource selection of rutin and quercetin in seed Among the 44 kinds of Tartary buckwheat genet- ic analyses, the average content of the two components (sum of rutin and quercetin) was 815.4 mg/100 g, and the all investigated genetic samples ranged from 308.3 to 1337.0 mg /100 g. In terms of each component analysis, the average content of rutin was 808.4 mg /100 g, the in all genetic samples ranged from 304.5 to 1326.5 mg/ 100 g. On the other hand, the average content of querce- tin was 7.0 mg /100 g, and in all genetic samples ranged from 2.0 to 22.5 mg /100 g. However, the results obtained from the present study revealed that the content of rutin was higher compared to quercetin regarding all investigat- ed samples. The distribution of rutin and quercetin contents in the tested buckwheat samples is shown in Fig. 2. The highest distribution of rutin (16 germplasms) was observed in the range of 600~800 mg/100 g followed by 12 germplasms in the range of 800~1000 mg/100 g, 7 germplasms in the range of 600 mg/100 g and 6 ger- Gradient Time (min) Mobile phase condition A (%): water + TFA (0.05%) B (%): MeOH (60%) + ACN (40%) + TFA (0.05%) 0 90.0 10.0 5 85.0 15.0 50 60.0 40.0 60 50.0 50.0 61 90.0 10.0 Table 2. Analytical conditions of HPLC Figure 1. HPLC chromatogram patterns and retention time of rutin & quercetin Fagopyrum 36(2):51-65 (2019) 55 mplasms in the range of 1000~1200 mg/100 g respec- tively. However, the lowest distribution (3 germplasms) of rutin was observed in the range of 1200 mg/100 g. In the case of quercetin, the highest (19 germplasms) was observed in the range of 5~10 mg / 100 g, followed by 15 germplasms with less than 5 mg/100 g, and 9 ger- mplasms with the range of 10~15 mg/100 g. Table 3 summarizes the rutin and quercetin content, seed shape and seed color properties among the test- ed resources. The highest content of rutin (1326.0 mg/ 100 g) was found in CBU408 (Collected from Nepal), whereas the highest content of quercetin (22.5 mg/ 100 g) was observed in CBU456 (Collected from Paki- stan). Taken together, the results obtained from the present study revealed that the quercetin exhibited the highest CV among the component tested in this study. 4.2. Country-wise variation of rutin and quercetin content of seeds The total amount of the two components in seeds was measured and compared in this study. The high- est total amount of the tested components (sum of ru- tin and quercetin) was 1002.6 mg / 100 g, followed by 854.0 mg/100 g, 838.9 mg/100 g, 813.6 mg/100 g, 802.6 mg/100 g, 715.0 mg in the order of the following man- ner Nepal> Bhutan> Japan> China> Pakistan> Slovenia> India. But no significant difference was observed among the germplasms (Table 4). Rutin was also found to be in the same order as the total amount of the two compo- nents. However, the total amount of rutin was in the or- der of 995.1 mg/100 g in Nepal, 845.2 mg / 100 g in Bhu- tan, 835.7 mg/100 g in Japan, 806.9 mg/100 g in China, 788.3 mg/100 g in Pakistan, 396.0 mg/100g in India. The data shown in Table 4 showed that there was no sig- nificant difference among the data examined. Quercetin showed different results compared than the rutin find- ings; however, the quercetin contents were 14.3 mg/100 g, 8.7 mg/100g, 7.5 mg/100 g, 7.4 mg/100 g, 6.7 mg/100 g, 3.12 mg/100 g in the order of the following manner; Pakistan> Bhutan> Nepal> India> China> Japan> Slove- nia (Table 5). However, the quercetin exhibited the high- est CV among the components tested in this study. Park et al. (2005) reported that rutin content was in the order of Bhutan> Slovenia> China> Pakistan> Nepal> Japan> India. Taken together, the obtained results sug- gest that the various concentrations of components may provide important insights regarding the development of functional components in buckwheat. 4.3. The content of rutin and quercetin in seeds regarding seed shape Table 5 summarizes the rutin and quercetin contents in seeds regarding seed shape. The highest total amount Figure 2. Frequency distributions of rutin (A) and quercetin (B) contents in seed of Tartary buckwheat germplasm Statistics Rutin (mg/100g) Quercetin (mg/100g) Total (mg/100g) Tartary buckwheat germplasm (Seed) Max. 1326.5 22.5 1337 Min. 304.5 2 308.3 Mean 808.4 7 815.4 ±SD 243.4 3.9 245.2 CV (%) 30.1 55.9 30.1 Table 3. Statistical analysis of rutin and quercetin content in seed of Tartary buckwheat germplasm Yu et al. (2019): Rutin and quercetin in Tartary buckwheat germplasm 56 Location Statistics Rutin (mg/100g) Quercetin (mg/100g) Total (mg/100g) Bhutan (n=3) Max. 911.5 10.2 920.3 Min. 753.8 7.2 764 Mean 845.2 8.7 854 ±SD 81.8 1.5 80.8 CV (%) 9.7 17.2 9.5 China (n=28) Max. 1247.5 13.7 1259 Min. 364.2 2 374.3 Mean 806.9 6.7 813.6 ±SD 236.9 3 238.6 CV (%) 29.4 45.2 29.3 India (n=2) Max. 487.5 10.8 498.4 Min. 304.5 3.9 308.3 Mean 396 7.4 403.4 ±SD 129.4 4.9 134.4 CV (%) 32.7 67.2 33.3 Japan (n=2) Max. 902.4 3.77 906.2 Min. 769.1 2.47 771.5 Mean 835.7 3.12 838.9 ±SD 94.3 0.92 95.2 CV (%) 11.3 29.4 11.3 Nepal (n=5) Max. 1326.5 10.5 1337 Min. 583 3 586.3 Mean 995.1 7.5 1002.6 ±SD 321.5 4 325.1 CV (%) 32.3 53 32.4 Pakistan (n=2) Max. 901.3 22.5 923.8 Min. 675.2 6.2 681.4 Mean 788.3 14.3 802.6 ±SD 159.9 11.5 171.4 CV (%) 20.3 80.3 21.4 Slovenia (n=2) Max. 798.6 3.5 802.1 Min. 625.4 2.6 628 Mean 712 3.1 715 ±SD 122.5 0.6 123.1 CV (%) 17.2 20.7 17.2 p-value 0.1593 0.0508 0.1659 Table 4. Statistical analysis of rutin and quercetin content in seed of Tartary buckwheat germplasm based on country of origin Fagopyrum 36(2):51-65 (2019) 57 of the sum of these two components were 831.2 mg/ 100 g, followed by 822.8 mg/100 g, and 745.9 mg/ 100 g in the order of Slender> Round> Notched. The rutin contents were the similar as the total amount. However, the highest rutin (824.8 mg/100 g) was observed from slender shape seed followed by round shape (816.0 mg/ 100 g), and notched shape (736.8 mg/100 g). In the case of quercetin, pronounced results were observed from notched shaped seed (9.1 mg/100 g) followed by round (6.8 mg/100 g), and slender (6.4 mg/100 g) respectively in the order of Notched> Round> Slender. However, the quercetin exhibited the highest CV among the compo- nent tested in this study. A previous study reported that seed shape played a crucial role in the variation of rutin content in the Tar- tary buckwheat genetic resources (Park et al., 2005). However, they found that the rutin contents were in the order of Slender> Notched> Round, wherein the results showed differences compared than that of the present study. 4.4. Variation of rutin and quercetin content regarding seed color Table 6 summarizes the rutin, quercetin and total amount of these two components in grain seeds consider- ing seed color. The total contents of the two components were measured according to their seed color. However, the highest content was exhibited from the dark-gray (865.3 mg/100 g), followed by dark-brown (806.3 mg/100 g), gray-brown (778.7 mg/100 g), and brown (776.5 mg/ 100 g) respectively. Rutin content showed the simi- lar results as the total amount of the two components. However, the pronounced rutin content was observed from the dark-gray (858.6 mg /100 g), followed by dark-brown (799.0 mg/100 g), gray-brown (771.5 mg/ 100 g) and brown (769.7 mg/100 g). The highest content of quercetin (7.4 mg/100 g) was observed from dark- brown, followed by gray-brown (7.2 mg/100 g), gray- brown and brown (6.7 mg/100 g) respectively. However, the quercetin exhibited the highest CV among the com- ponents tested in this study. Seed shape Statistics Rutin (mg/100g) Quercetin (mg/100g) Total (mg/100g) Notched (n=6) Max. 1028.7 22.5 1038.7 Min. 420.1 2.6 422.7 Mean 736.8 9.1 745.9 ±SD 216 7 220.6 CV (%) 29.3 76.8 29.6 Round (n=22) Max. 1326.5 13.7 1337 Min. 364.2 2.5 374.3 Mean 816 6.8 822.8 ±SD 258.9 3.3 260.4 CV (%) 31.7 48.2 31.6 Slender (n=16) Max. 1247.5 11.5 1259 Min. 304.5 2 308.3 Mean 824.8 6.4 831.2 ±SD 240.8 3.2 242.8 CV (%) 29.2 49.8 29.2 p-value 0.7447 0.3587 0.7608 Table 5. Statistical analysis of rutin and quercetin content in seed of Tartary buckwheat germplasm based on seed shape Yu et al. (2019): Rutin and quercetin in Tartary buckwheat germplasm 58 A previous study reported that seed color played an essential role in the variation of rutin and quercetin con- tent in the Tartary buckwheat genetic resources (Park et al., 2005). Taken together, the results obtained from the present study revealed that seed color may provide insights in the variation of rutin and quercetin content in Tartary buckwheat. The variation of the content of ru- tin in Tartary buckwheat samples showed in the order of Dark-gray> Dark-brown (Dark)> Gray-brown> Brown. 5. Variation of rutin and quercetin content in sprout 5.1. Contents, distribution and resource selection of rutin and quercetin in sprout The amount of rutin (3362.9 mg/100 g) was observed from the sprout with the total genetic resources ranges from 328.8 to 5440.4 mg/100 g whereas the average content of quercetin was 143.2 mg/100 g with the total genetic resources ranged from 53.5 to 392.0 mg/100 g. The results revealed that the content of rutin was over- whelmingly higher than that of quercetin. Considering the genetic resources between seed and sprout, rutin was increased by 0.5~10.5 times and quercetin was increased by 3.7~90.7 times. The distribution of rutin and quercetin content in the sprouts of 44 buckwheat genetic resources is shown in Fig. 3. The highest distribution of rutin (16 germplasms) was observed in the range of 3000~4000 mg/100 g fol- Seed color Statistics Rutin (mg/100g) Quercetin (mg/100g) Total (mg/100g) Brown (n=8) Max. 1184.9 11.3 1196.2 Min. 364.2 2.6 374.3 Mean 769.7 6.7 776.5 ±SD 308.4 3.3 309.4 CV (%) 40.1 49 39.8 Dark-brown (n=12) Max. 1326.5 22.5 1337 Min. 304.5 2 308.3 Mean 799 7.4 806.3 ±SD 239.6 5.5 242.1 CV (%) 30 75.1 30 Dark-gray (n=15) Max. 1275.1 13.7 1285.3 Min. 583 2.5 586.3 Mean 858.6 6.7 865.3 ±SD 180.1 3.1 181.9 CV (%) 21 46.8 21 Gray-brown (n=9) Max. 1247.5 11.5 1259 Min. 419 2.3 421.5 Mean 771.5 7.2 778.7 ±SD 302.6 3.6 305.1 CV (%) 39.2 49.5 39.2 p-value 0.7974 0.9704 0.8029 Table 6. Statistical analysis of rutin and quercetin content in seed of Tartary buckwheat germplasm based on seed color Fagopyrum 36(2):51-65 (2019) 59 lowed by 8 germplasms in the range of 2000~3000 mg/ 100 g, 7 germplasms in the range of 4000~5000 mg/ 100 g and 6 germplasms in the range of 1000~1200 mg/100 g, and 3 germplasm in the range of 1000~2000 mg/100 g respectively. However, the lowest rutin fre- quency distribution (1 germplasm) was observed from less than 1000 mg/100 g. In the case of quercetin, the range of 100 ~ 200 mg/100 g showed the highest fre- quency (30 germplasms), followed by less than 100 mg/100 g (9 germplasms), and more than 300 mg/100 g (3 germplasms) respectively, while the lowest frequency (2 germplasms) was observed in the range of 200~300 mg/100 g. Table 7 summarizes the rutin quercetin contents of the sprouts regarding location, seed shape and seed color. The highest rutin amount (5440.4 mg/100 g) was obtained from CBU460 in Nepal with round shaped and dark-gray color seeds while the lowest amount (1462.4 mg/100 g) was obtained from CBU302 in China with round shaped brown color seeds. On the other hand, the highest quercetin contents (392.0 mg/100 g) was ob- tained from CBU302 in China with round shaped brown color seeds CBU302 and the lowest amount of quercetin (268.0 mg/100 g) was obtained from CBU460 in Nepal. 5.2. Country-wise variation of rutin and quercetin content in sprouts Table 8 shows the rutin and quercetin amount in sprouts and their statistical analysis regarding different countries of the world. Nepal showed the highest amount of rutin (4205.0 mg/100 g), followed by Slovenia (3684.3 mg/100 g), Japan (3662.9 mg/100 g), Pakistan (3402.3 mg/100 g), China (3337.7 mg/100 g), Bhutan (3108.8 mg/1682.3 mg/100 g). In the case of quercetin con- tent, Slovenia showed the highest amount (191.1 mg/ 100 g), followed by Nepal (165.4 mg/100 g), Japan (161.5 mg/100 g), India (145.1 mg/100 g), Bhutan (130.9 mg/100 g), Pakistan (130.9 mg/100 g) in the order of Nepal> Japan> India> China> Bhutan> Pakistan. How- ever, no significant differences were observed among the components. 5.3. Variation of rutin and quercetin contents in sprouts regarding seed shape Table 9 shows the rutin and quercetin contents from sprouts according to their various seed shape. The slen- der shaped seeds showed the highest amount of rutin (3890.2 mg/100 g), followed by round (3399.8 mg/ 100 g), and notched (2871.9 mg/100 g) in the order of Slender> Round> Notched. In the case of quercetin, the highest amount of quercetin was observed from the round shape seed (158.1 mg/100 g), whereas the lowest amount of quercetin (125.8 mg/100 g) was observed from the slender shaped seed. However, the amount of quercetin was in the order of Round> Notched> Slender. 5.4. Variation of rutin and quercetin contents in sprouts regarding seed color Table 10 shows the rutin and quercetin contents of the seeds in the sprouts according to seed color. The highest rutin content (3565.0 mg/100 g) was obtained from the dark-gray color seed, followed by dark-brown Figure 3. Frequency distributions of rutin (A) and quercetin (B) contents in sprout of Tartary buckwheat germplasm Line number Location Seed shape Seed color Rutin (mg/100g) Quercetin (mg/100g) Total (mg/100g) CBU 460 Nepal Round Dark-gray 5440.4 268 5708.4 CBU 302 China Round Brown 1462.4 392 1854.4 Table 7. Accessions with highest total content of rutin and quercetin in sprout Yu et al. (2019): Rutin and quercetin in Tartary buckwheat germplasm 60 Location Statistics Rutin (mg/100g) Quercetin (mg/100g) Total (mg/100g) Bhutan (n=3) Max. 3160.2 140.3 3300.5 Min. 3039.3 121.6 3170.1 Mean 3108.8 130.9 3239.8 ±SD 62.5 9.3 65.6 CV (%) 2 7.1 2 China (n=28) Max. 5214 392 5273.5 Min. 328.8 53.5 633.9 Mean 3337.7 139.3 3477 ±SD 1048.5 70.1 998.2 CV (%) 31.4 50.3 28.7 India (n=2) Max. 1822.7 176 1998.7 Min. 1542 114.1 1656.1 Mean 1682.3 145.1 1827.4 ±SD 198.5 43.8 242.3 CV (%) 11.8 30.2 13.3 Japan (n=2) Max. 3825.7 228.7 3919.9 Min. 3177.2 94.2 3405.9 Mean 3501.4 161.5 3662.9 ±SD 458.5 95.1 363.5 CV (%) 13.1 58.9 9.9 Nepal (n=5) Max. 5440.4 268 5708.4 Min. 3313 122.8 3435.9 Mean 4205 165.4 4370.4 ±SD 841.2 59.6 893 CV (%) 20 36.1 20.4 Pakistan (n=2) Max. 4088.6 102.6 4191.3 Min. 2716.1 83.6 2799.7 Mean 3402.3 93.1 3495.5 ±SD 970.6 13.4 984 CV (%) 28.5 14.4 28.2 Slovenia (n=2) Max. 3603.8 317.9 3700.5 Min. 3382.6 64.3 3668.1 Mean 3493.2 191.1 3684.3 ±SD 156.4 179.3 22.9 CV (%) 4.5 93.9 0.6 p-value 0.1406 0.8467 0.1038 Table 8. Statistical analysis of rutin and quercetin content in sprout of Tartary buckwheat germplasm according to country of origin Fagopyrum 36(2):51-65 (2019) 61 (3558.1 mg/100 g), brown (3141.6 mg/100 g) and gray- brown (2962.3 mg/100 g) in the order of Dark-gray> Dark-brown (Dark)> Brown> Gray-brown. On the other hand, brown color seeds exhibited the highest amount of quercetin (156.0 mg/100 g), followed by gray-brown (147.4 mg/100 g), dark-gray (140.9 mg/100 g), and dark- brown (134.5 mg/100 g) in the order of Brown> Gray- brown> Dark-gray> Dark-brown. Seed shape Statistics Rutin (mg/100g) Quercetin (mg/100g) Total (mg/100g) Notched (n=6) Max. 3781.6 179.4 3919.4 Min. 2242.1 83.6 2378.5 Mean 2736.6 135.2 2871.9 ±SD 537.6 37.2 536.5 CV (%) 19.6 27.5 18.7 Round (n=22) Max. 5440.4 392 5708.4 Min. 328.8 64.3 633.9 Mean 3241.7 158.1 3399.8 ±SD 1058.7 79.2 1024.1 CV (%) 32.7 50.1 30.1 Slender (n=16) Max. 5214 317.9 5273.5 Min. 1542 53.5 1656.1 Mean 3764.4 125.8 3890.2 ±SD 930.2 60.7 912.7 CV (%) 24.7 48.3 23.5 p-value 0.0701 0.3532 0.0686 Table 9. Statistical analysis of rutin and quercetin content in sprout of Tartary buckwheat germplasm based on seed shape The correlation between rutin and quercetin in seeds is shown in Fig. 4. The obtained results demonstrated that the content of quercetin was positively correlated (r = 0.4530, p-value = 0.002) with the content of rutin. As a result, the results suggest that both rutin and querce- tin selection criteria may provide crucial insights for de- veloping varieties. Fig. 5 shows the correlation between rutin and quercetin in sprouts. The content of quercetin Figure 4. Correlation between rutin and quercetin contents in seed of Tartary buckwheat germplasm Figure 5. Correlation between rutin and quercetin contents in sprout of Tartary buckwheat germplasm Yu et al. (2019): Rutin and quercetin in Tartary buckwheat germplasm 62 Seed color Statistics Rutin (mg/100g) Quercetin (mg/100g) Total (mg/100g) Brown (n=8) Max. 4660.4 392 4714 Min. 1462.4 53.5 1854.4 Mean 3141.6 156 3297.6 ±SD 925.4 105.2 830.1 CV (%) 29.5 67.5 25.2 Dark-brown (n=12) Max. 5214 317.9 5273.5 Min. 1542 59.5 1656.1 Mean 3558.1 134.5 3692.7 ±SD 1118.4 65 1112.1 CV (%) 31.4 48.3 30.1 Dark-gray (n=15) Max. 5440.4 268 5708.4 Min. 2245.9 91.7 2355.5 Mean 3565 140.9 3705.9 ±SD 708.1 51.7 730.9 CV (%) 19.9 36.7 19.7 Gray-brown (n=9) Max. 4414.9 305.1 4517.2 Min. 328.8 60.9 633.9 Mean 2962.3 147.4 3109.6 ±SD 1300.9 70.8 1239.8 CV (%) 43.9 48.1 39.9 p-value 0.4204 0.9213 0.4165 Table 10. Statistical analysis of rutin and quercetin content in sprout of Tartary buckwheat germplasm based on seed color in the sprout was high (r = -4110, p-value = 0.0056) com- pared to rutin. The amount of quercetin is decreased as the amount of rutin produced when the seeds germinate, and sprouts come out as a typical quercetin glycoside (Lee et al., 2013). As a result, it would be useful to devel- op a variety containing high content of each component based on the criteria of rutin or quercetin using sprouts. To this end, the present study postulated that sprout would be a great choice to develop a cultivar with a high rutin and quercetin content. Fig. 6 shows the correlation between rutin content in seed and sprouts of Tartary buckwheat germplasm. The rutin content in the seeds was found to be increased with increasing the rutin content in the sprouts (R = 0.3552, p-value = 0.018). However, the content of quercetin in the seeds and the sprouts showed no correlation (R = 0.0169, p-value = 0.9134). However, the use of quercetin would be a potential choice for utilization because it increases 3.7~90.7 times in sprout compared to seeds (Fig. 7). CONCLUSION The present study was carried out to investigate the variation of rutin and quercetin contents in seeds and sprouts of 44 buckwheat genetic resources for the de- velopment of high-quality Tartary buckwheat. Rutin and quercetin content varied at different regions from which each accession was collected. Rutin content in seed and sprouts of the accession collected from Nepal area was higher than any accession collected from other regions. Fagopyrum 36(2):51-65 (2019) 63 Accession collected from Pakistan showed lower rutin content in seeds compared to the accession collected from other regions. On the other hand, quercetin con- tent showed the opposite results in the seeds. 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Ito. 2002. Changes in antioxidative activity and flavonoids composition of the extracts from aerial parts of buckwheat during growth period. Nippon Shokuhin Kagaku Kogaku Kaishi. 49: 119-125. Wojcicki, J., B. Barcew-Wiszniewska, L. Samochowiec, and L. Rozwsicka. 1995. Extractum fagopyri reduces atherosclerosis in high fat diet fed rabbits. Die Pharmazie. 50: 560-562. Zhu, M., Y. Gong, Z. Yang, G. Ge, C. Han, J. Chen. 1999. Green tea and its major components ameliorate immune dysfunc- tion in mice bearing lewis lung carcinoma and treated with the carcinogen NNK. Nutrition and cancer. 35(1): 64-72. Fagopyrum 36(2):51-65 (2019) 65 IZVLEČEK Namen raziskave je bil proučiti geografsko pogojeno raznolikost in povezavo oblike in barve zrn in kalic z vsebnostjo rutina in kvercetina, z uporabo zbirke semen tatarske ajde. Raziskano je bilo 44 tujih vzorcev glede na obliko in barvo zrn in in glede na rezultate analiz s HPLC. Vsebnost rutina in kvercetina je bila različna pri vzorcih z različnih obmo- čij. Pri vzorcih iz Nepala sta bili najvišji vsebnosti rutina v zrnih (1326,5 mg/100 g) in v kalicah (5440,4 mg/100 g). Pri vzorcih zrn iz Pakistana je bilo rutina največ 22,5 mg/100 g in v kalicah iz Kitajske največ kvercetina (392,0 mg/100 g). V primerjavi z zrni je bilo v kalicah 4~90-krat več kvercetina. Ta raziskava kaže, da so kalice v primerjavi z zrni lahko pomembnejši vir kvercetina, vzorci iz Nepala, Butana, Kitajske in Japonske so obetavni vir pri pridelavi ajde za funkcij- sko hrano in zdravila. 66 INSTRUCTIONS FOR AUTHORS FAGOPYRUM accepts scientific papers, and information and bibliographies on buckwheat. SCIENTIFIC PAPERS Manuscript should be written in standard English and submitted to the Editorial office as a .doc document. Figures (photographs) should be IN SEPARATE FILE each in jpg or other original file, not imbeded in word .doc document. Submission for the 2020 issue shall be sent latest on November 30, 2019 to the email ivan.kreft@guest.arnes.si. After accepting the paper, the editorial office will ask the authors to provide the original figures if the first submission will not be adequate. Your manuscript should be sent to the Editor-in-Chief (Prof. Ivan Kreft). E-mail: ivan.kreft@guest.arnes.si For the time beeing, it is no charge for editing and publishing the papers. Papers are available as open access. Complete recent issues of FAGOPYRUM journal are available on web page: www.sazu.si/uploads/files/57fb8d9c9de14adc9ded154d/Fagopyrum%2035.pdf or www.sazu.si/uploads/files/57fb8d9c9de14adc9ded154d/Fagopyrum%2034.pdf or www.sazu.si/publikacije-sazu Separate papers (PDFs) of recent issues of FAGOPYRUM journal are available on web page: https://ojs.zrc-sazu.si/fagopyrum/index or »archives« on the same web page (https://ojs.zrc-sazu.si/fagopyrum/issue/archive). Manuscripts should be typed double-spaced on DIN A4 format (21x29cm or 8.5x11 inch) with sufficiently wide margins (2.5-3cm), in one column (we will transfer later the text to two paralell columns). All pages, including the tables, legends and references, should be numbered consecutively. The manuscript should be arranged in the following order, or other suitable similar order: 1. Title page (page 1) • Title (the title should be as short as possible, but should contain adequate information to indicate the contents) • Author´s full name(s) • Affilation(s)/Adress(es), including e-mail addresses of all authors (coauthors). 2. Key words/Running head (not to exceed 50 letters including spaces) (page 2) • Key words (maximum of 8, in alphabetical order, suitable for indexing) 3. Abstract (brief and informative, not to exceed 250 words). 4. Main text • Introduction, Materials and Methods, Results, Discussion • The relative importance of headings and subheadings should be clear. 5. The approximate location of figures and tables could be indicated in the margin or in the text. • The use of footnotes is to be avoided. 6. After the main text • Acknowledgements (also grants, support etc., if any) should follow the text and precede the references. 7. References Abstract in Slovenian will be for foreign authors made by the editors. Review papers are wellcome, main text has to be organised according to authors‘ suggestion. The literature references should be arranged alphabetically, typed double spaced and in the text referred to as: author and year of publication, e.g., Budagovskaya (1998), (Inoue et al. 1998). Citation of personal communications and unpublished data should be avoided, unless absolutely necesarry. Such citations should be in text, appear only as (R. Brown, personal communication), and not in the reference list. To abbreviate titles of periodicals, refer to recent issues of FAGOPYRUM. 67 Each literature source listed in the list of references should contain at the end the doi number, if such number exists, like: If the cited poaper has a doi number, folow the case: Chettry, U., L. Dohtdong and N.K. Chrungoo, 2018. Analysing structural diversity of seed storage protein gene promoters: Buckwheat a case study. Fagopyrum 35: 5-17. https://doi.org/10.3986/fag0004 If the cited paper has no doi number: Budagoskaya, N., 1998. Changes in the state of photoautotrophic and heterotrophic organs of buckwheat plants at iron deficiency and low pH. Fagopyrum 15: 1-7. Inoue, N., M. Hagiwara, H. Y. Kim and T. Matano, 1998. A preliminary study for modeling seed production in common buckwheat. Fagopyrum 15: 35-41. Books (edited by someone other than author of article) Hattermer, H. and H. G. Gregorius, 1990. Is gene conservation under gloval climate meaningful? In: Jackson, M. T., B. V. Ford-Lloyd and M. L. Parry (Eds.), Climatic Change and Plant Genetic Resources, pp. 158-166, Bellhaven Press, London. Books (identical author and editor) Campbell, C.G., 1997. Buckwheat. IPGRI, Rome. 8. Tables • Each table should be mentioned in the text. • Each table should be typed on a separate page. • Tables should be numbered by Arabic numerals. • Horizontal rules should be indicated; vertical rules should not be used. • Tables may be re-edited by the editor to permit more compact typesetting. 9. Figures • Each figure should be mentioned in the text. • Each figure should be numered by Arabic numerals. • Line drawings should be in a form suitable for reproduction without modification. • Extremely small type should be avoided as figures are often reduced in size. • Photographs should be supplied as black-and-white or colour, high. contrast, original jpg or other, in a separate file. The name of figure file shall start with the family name of the first author, following for example: Kranjc Fig. 1. 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The protocol of research on animals should also be approved by an appropriate commitee in line with the Guide for the Care and Use of Laboratory Animals. Information (including short preliminary reports) and bibliographical contributions should be two printed pages or shorter, and will be published without being refereed. Ivan Kreft, Nutrition Institute, Tržaška cesta 40, SI-1000, Ljubljana, Slovenia