FAGOPYRUM Volume 36(1), June 2019 ISSN 0352 – 3020 Scientific Journal on Buckwheat Research International Buckwheat Research Association Slovenian Academy of Sciences and Arts FAGOPYRUM volume 36(1), 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. Front page photo: Buckwheat pasta with 1.7% addition of seaweed, see Asami et al., 5-11. FAGOPYRUM volume 36(1) (2019) CONTENTS ORIGINAL PAPERS Mechanical characterization of buckwheat noodles mixed with seaweed (fu-nori) Yuya ASAMI, Sena OOTO, Masanori KITAMURA, Kenta SAKANASHI, Tesshu TAMAI, Tsuyoshi FURUMOTO, Sayoko IKEDA and Kiyokazu IKEDA ................................................................................ 5 Non-destructive methodology in comparative physiology of buckwheat genotypes within the different origin Oksana SYTAR, Klaudia BRUCKOVA, Alyona PLOTNITSKAYA, Marek ZIVCAK, Marian BRESTIC ................ 11 Research results of local buckwheat varieties and forms of Ukrainian origin Oleh TRYHUB ....................................................................................................................................................... 23 INFORMATION Information for authors ...................................................................................................................................... 30 Fagopyrum 36(1):5-9 (2019) 5 Research paper Mechanical characterization of buckwheat noodles mixed with seaweed (fu-nori) Yuya ASAMI1, Sena OOTO1, Masanori KITAMURA1, Kenta SAKANASHI1, Tesshu TAMAI1, Tsuyoshi FURUMOTO1, Sayoko IKEDA2 and Kiyokazu IKEDA*2 1 Faculty of Agriculture, Ryukoku University, Seta oe-cho, Otsu, 520-2194, Japan 2 Faculty of Nutrition, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan * Corresponding author: Prof. Kiyokazu Ikeda, Faculty of Nutrition, Kobe Gakuin University, Nishi-ku Kobe 651-2180, Japan, Fax +81 78 974 5689 E-mail addresses of authors: Yuya ASAMI: yuya_asami@agr.ryukoku.ac.jp, Sena OOTO: n150404@mail.ryukoku.ac.jp, Masanori KITAMURA: n150413@mail.ryukoku.ac.jp, Kenta SAKANASHI: sakanashi@agr.ryukoku.ac.jp, Tesshu TAMAI: ttamai@agr.ryukoku.ac.jp, Tsuyoshi FURUMOTO: tfurumoto@agr.ryukoku.ac.jp, Sayoko IKEDA: ikeda-k@nutr.kobegakuin.ac.jp, Kiyokazu IKEDA: ikeda-k@nutr.kobegakuin.ac.jp DOI https://doi.org/10.3986/fag0006 Received: December 5, 2018; accepted: January 30, 2019 Keywords: common buckwheat, mechanical characteristics, noodles, seaweed ABSTRACT The present study was conducted to clarify the effect of incorporation of seaweed, i.e. funori (Gloiopeltis tenax (Turner) J. Agardh) into buckwheat noodles on their mechanical characteristics. Mechanical analysis of buckwheat noodles with funori showed that incorporation of funori into buckwheat noodles enhanced breaking stress and energy. On the other hand, incorporation of funori into buckwheat noodles enhanced decreased solubility of the albumin plus globulin frac- tion. The present study findings suggest that the endogenous protein may be an important factor responsible for the mechanical characteristics of buckwheat noodles with seaweed. Assami et al., (2019): Mechanical characterization of buckwheat noodles with seaweed 6 (A) (B) (A) (B) called “hegi-soba”. These buckwheat noodles are prepared by incorporating into buckwheat dough a kind of sea- weed, i.e. funori, (Gloiopeltis tenax (Turner) J. Agardh) as a dough-binder (Zen-men-kyo, 2014). Before ingestion, the noodles prepared with funori are usually put on a unique wooden-tray which is called “hegi”; so these buck- wheat noodles are called “hegi-soba”. Although this buck- wheat dish is traditionally utilized only in Niigata region, many Japanese people currently often enjoy these local buckwheat noodles. Hegi-soba noodles have a unique masticatory sense with refreshing sense on ingestion. Mechanical characterization of “hegi-soba” noodles is an interesting subject in view of buckwheat research. In this background, the present study was conducted to charac- terize noodles made from buckwheat flour with seaweed. MATERIALS AND METHODS Materials Buckwheat flour (Fagopyrum esculentum Moench, var. Kitawase-soba), which was harvested in Hokkaido (in 2017), was used in this research. Buckwheat flour was kindly provided prepared from Terao Milling Co. (Hyogo, Japan) and stored at -80oC until use. Ground seaweed, i.e. fu-nori in Japanese, Gloiopeltis tenax J. Agardh) used in this study was a commercial product (Oowaki-man- zou-shoten Co., Fukui, Japan). Fig. 1. Buckwheat noodles. (A), non added seaweed; and (B) added seaweed (1.7% addition). INTRODUCTION Buckwheat (Fagopyrum spp.) is an important crop in some regions of the world (Kreft et al., 2003; Ikeda, 2002). Buckwheat flour contains various beneficial com- ponents for human health such as protein, polyphenolics, rutin and minerals at high levels (Ikeda 2002; Ikeda and Yamashita 1994). Thus, buckwheat can contribute as an important dietary source of such beneficial components. There is a large variety of buckwheat products pro- duced on a global basis (Ikeda, 2002). Attention has been currently paid to the palatability and acceptability of buckwheat products from the perspective of their cook- ing and processing. However, there are still unanswered questions on the palatability and acceptability of buck- wheat products. As buckwheat flour has low cohesiveness, dough-binders, such as wheat flour, egg, seaweed, Japa- nese yam flour, are often added in preparing buckwheat noodles (ZMCS, 2004). A variety of buckwheat noodles with various dough-binders has been traditionally avail- able in Japan. We reported mechanical effects by addi- tion of various dough-binders to common and Tartary buckwheat noodles in view of two analysis, i.e., tensile analysis and breaking analysis (Ikeda, et al., 2005). How- ever, further systematic analysis is needed to understand the exact mechanical effects of various dough-binders to buckwheat products. In Niigata district, located in the middle region of Japan, there is a famous buckwheat dish. This dish is Fagopyrum 36(1):5-9 (2019) 7 0 20000 40000 60000 80000 100000 120000 0.0% 0.3% 0.7% 1.0% 1.4% 1.7% a a a ab b c 0 5000 10000 15000 20000 25000 0.0% 0.3% 0.7% 1.0% 1.4% 1.7% a a a a ab b Seaweed blending ratio of buckwheat flour to total Seaweed blending ratio of buckwheat flour to total 1.2 1.0 0.8 0.6 0.4 0.2 0 2.5 2.0 1.5 1.0 0.5 (A) (B) Br ea ki ng st re ss (P a X1 05 ) Br ea ki ng e ne rg y (J/ m 3 X 10 4 ) Mechanical measurements For the study of the effects of the seaweed on the mechanical characteristics of buckwheat noodles, buck- wheat noodles were prepared by hand. The mechanical characteristics of buckwheat noodles were evaluated by breaking analysis. Prior to the mechanical analysis, the buckwheat flour which had been stored at -80oC was placed in a desiccator at room temperature until the flour exhibited a constant moisture content. The moisture of the flour was measured with a moisture analyzer (ML- 50, A&D Co. Ltd., Japan). Seaweed was boiled, and sticky seaweed was added to buckwheat flour. The buckwheat dough was prepared just prior to mechanical analysis to have a moisture content of 42% by adding the appropri- ate amount of distilled water. Then the buckwheat noo- dles were made from the buckwheat dough using a hand- made pasta machine (SP-150, Imperta Co., Torino, Italy). Figure 1 shows buckwheat noodles prepared in this study. The buckwheat noodles obtained were subjected to me- chanical analysis. Before the mechanical analysis, buck- wheat noodles prepared were heated in boiling water for 150 seconds and subsequently were cooled for 150 sec- onds at 4oC. Immediately after cooling, mechanical meas- urements of the noodles were performed. The breaking analysis of the buckwheat noodles was performed with Rheoner RE2-3305C (Yamaden Co. Ltd., Japan). Meas- urements of breaking analysis were performed with a load cell of 200N and measurement speed of 0.50 mm/ sec. A wedge-style plunger (No.49: W 13mm, D 30mm, H 25mm) was used in measurements with the Rheoner RE2-3305C. Mechanical measurements were replicated twenty times for each sample. Protein determination For chemical analysis of the combined fractions of buckwheat albumin plus globulin (AG) in the heated noo- dle samples which had been subjected to the mechanical measurements, the noodle samples were lyophilized and then ground into flour. The flours obtained were extract- ed with a ten-fold (v/w) volume of 0.2M NaCl for 1hr at Fig. 2. Breaking characteristics of buckwheat noodles made with seaweed. (A), breaking stress; and (B), breaking energy. Vertical bars in the figure show the standard deviations. Values that within the same row that are not followed by the same letter are significantly different at P<0.05. Assami et al., (2019): Mechanical characterization of buckwheat noodles with seaweed 8 N aC l s ol ub ul e pr ot ei n co nt en t (g /1 00 g flo ur ) Seaweed blending ratio of buckwheat flour to total 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 0.0% 0.3% 0.7% 1.0% 1.4% 1.7% 4oC. After extraction, the suspensions were centrifuged at 17,000 Xg for 20 min. Protein concentration was de- termined using the Bradford method with bovine serum albumin as a standard protein. Statistical analysis Statistical analysis was conducted using a personal computer with the program Excel (Microsoft Co., USA), Ekuseru-Toukei 2015 (Social Survey Research Informa- tion Co., Japan) and SPSS Ver.23.0 (IBM, USA). RESULTS AND DISCUSSION Mechanical characteristics of buckwheat noodles made with seaweed Figure 2 shows the breaking characteristics of hegi-so- ba buckwheat noodles prepared with funori-seaweed. The breaking stress and energy of the hegi-soba noodles gradually increased as the added concentration of funori seaweed increased (Fig. 2 (A and B)). A significant high breaking stress (Fig. 2 (A)) was found with hegi-soba buck- wheat noodles with a concentration of funori seaweed with 1.4% or over as compared the buckwheat without funori seaweed (P<0.05). Similarly, a significant high breaking energy (Fig. 2 (B)) was found with buckwheat noodles with a concentration of funori seaweed with 1.7% as compared the buckwheat without funori seaweed (P<0.05). These findings characterize showed the unique mastication characteristics of hegi-soba noodles. Protein compositions of buckwheat noodles made with seaweed Figure 3 shows the NaCl-soluble protein content of buckwheat noodles made with seaweed. The NaCl-solu- ble protein exhibits the combined fraction of the major buckwheat proteins, i.e., albumin plus globulin (Ikeda, 2002), designated as the AG fraction below. Changes by the addition of the seaweed in solubility of the AG fraction were found (Fig. 3). Incorporation of seaweed Fig. 3. NaCl-soluble protein content of buckwheat noodles made with seaweed. Vertical bars in the figure show the standard deviations. Fagopyrum 36(1):5-9 (2019) 9 into buckwheat dough was found to reduce the solubil- ity of the AG fraction in buckwheat dough as the funori seaweed added increased (Fig. 3). The seaweed contains dietary fiber at high levels (Ooishi, 1993). Judged from our previous findings (Ikeda and Kusano 1983), this phenomenon may be due to in-solubilization of proteins arisen by dietary fiber in seaweed. Interest in the nutri- tional function of dietary fiber for humans is currently increasing. Dietary fiber has many beneficial effects on human such as blood glucose increase suppression and antihypertensive (Mori and Tsuji, 1997). Considering in view of current nutritional science concerning the bene- ficial effects of dietary fiber, the intake of buckwheat noo- dles with seaweed with high level of dietary fiber, should be recommended as a key source of dietary fiber. Relationships of the observed breaking characteris- tics (Fig. 2) to the protein components (Fig. 3) was ana- lyzed. The AG fraction content (Fig. 3) negatively corre- lated to their observed breaking stress (Fig. 2 (A)) with r = -0.934 (P<0.01), breaking energy (Fig. 2 (B)) with r = -0.942 (P<0.01). These findings suggest that proteins in the AG fraction may be an important factor involved in the observed changes in mechanical characteristics aris- en by the addition of funori seaweed. Finally, the present study shows changes in mechan- ical characteristics of buckwheat noodles made with sea- weed. The present study suggests that changes in the pro- tein of AG fraction in buckwheat noodles with seaweed may be an important factor affecting the mechanical characteristics of buckwheat noodles, although the exact mechanism remains uncertain. The present findings pro- vide a scientific basis in the understanding of palatability and acceptability of buckwheat noodles. ACKNOWLEDGMENT This work was supported by the Research Institute for Food and Agriculture of Ryukoku University, Japan. REFERENCES Ikeda, K. and T. Kusano., 1983. In vitro inhibition of digestive enzymes by indigestive polysaccharides. Cereal Chem., 60: 260-263. Ikeda, K., 2002. Buckwheat: composition, chemistry and processing. In: S.L. Taylor (ed.), Advances in Food and Nutrition Research, Academic Press, Nebraska, USA, pp.395-434. Ikeda, K., Y. Asami, R. Lin, Y. Honda, T. Suzuki, R. Arai and K. Yasumoto, 2005. Characterization of buckwheat noodles with various dough-binders with respect to mechanical characteristics. Fagopyrum, 22: 63-69. Ikeda, S. and Y. Yamashita. 1994. Buckwheat as a dietary source of zinc, copper and manganese. Fagopyrum, 14: 29-34. Kreft, I., L.J. Chang, Y.S. Choi and C.H. Park (eds), 2003. Ethnobotany of buckwheat, Jinsol Publishing Co., Seoul. Mori, B. and Tsuji, K. 1997. Shokumotsu-seni no kagaku (Science of Dietary Fiber), Asakura Shoten Co., Ltd, Tokyo. Ooishi. 1993. Kaisou no kagaku (Science of Seaweed), Asakura Shoten Co., Ltd, Tokyo. Zenkoku Menrui-bunka Chiikikan-kouryu Suishin-kyougikai (ZMCS). 2004. Soba-uti kyouhon (Textbook of buckwheat noodles preparation techniques), Shibata Shoten Co., Ltd, Tokyo. Zen-men-kyo. 2014. Kaitei Soba-Uti Kyouhon (Revision, Textbook of buckwheat noodle making). Shibata Shoten Co., Ltd, Tokyo. IZVLEČEK Mehanska karakterizacija ajdovih rezancev z dodatkom morskih alg funori. Namen raziskave je bil ugotoviti kako vpliva dodatek morskih alg funori (Gloiopeltis tenax (Turner) J. Agardh) na me- hanske lastnosti ajdovih rezancev. Ugotovljeno je, da dodatek alg poveča občutljivost na lomljenje in energijo. Dodatek alg funori pospeši zmanjšanje topnosti albuminske in globulinske frakcije beljakovin. Rezultati raziskave kažejo, da so proteini testenin pomemben dejavnik, ki vpliva na mehanske lastnosti rezancev z dodatkom alg. Fagopyrum 36(1):11-21 (2019) 11 Research paper Non-destructive methodology in comparative physiology of buckwheat genotypes within the different origin Oksana SYTAR1,2, Klaudia BRUCKOVA1, Alyona PLOTNITSKAYA3, Marek ZIVCAK1, Marian BRESTIC1 1 Department of Plant Physiology, Slovak University of Agriculture, Nitra, A. Hlinku 2, 94976 Nitra, Slovak Republic 2 Plant Biology Department, Taras Shevchenko National University of Kyiv, Institute of Biology, Volodymyrska str., 64, Kyiv 01033, Ukraine 3 Laboratory of Quality and Safety of Products of Agro Industrial Complex. Mashynobudivnykiv Street Building 7, Chabany village, Kyiv-Svyatoshinsky district, Kyiv region, 08162, Ukraine E-mail address of corresponding author: Oksana SYTAR: oksana.sytar@gmail.com Coauthors: Marek ZIVCAK, email: marek.zivcak@uniag.sk, Alyona PLOTNITSKAYA, email: pl_av@ukr.net, Klaudia BRUCKOVA, email: klaudia.bruckova@gmail.com, Marian BRESTIC, email: marian.brestic@uniag.sk DOI https://doi.org/10.3986/fag0007 Received: March 14, 2019; accepted: May 25, 2019 Keywords: chlorophyll fluorescence, phenolic acid, non-destructive measurement, flavonoids, anthocyanins ABSTRACT In the presented study has been used non-destructive method for prescreening of flavonoids, anthocyanins and pig- ments from early stage of growth till flowering period of buckwheat genotypes of different origin. The similar increasing tendency in the changes of FLAV, ANTH and MFI indexes of Chinese genotypes compared to the tendency of Ukrainian genotypes has been observed. Genotypes of F. tataricum compared to the genotypes of F. esculentum have been shown to lower ANTH index during seedling growth. SFR index which relates to chlorophyll concentration shown different dynamic between Ukrainian and Chinese genotypes. In the middle phase 30 days after sowing (DAS) of growth has been discovered significant increase of SFR index almost in all experimental genotypes. On 36 DAS just two Ukrainian genotypes which characterized also high ANTH and MFI indexes has been kept tendency to increase SFR index. HPLC analysis of experimental samples found that presence of p-anisic acid was typical for F. tataricum species compared to the experimental cultivars of F. esculentum of both origin. Buckwheat genotypes of different origin can vary in flavonoid, anthocyanins and pigments content during stages of growth, but changes in their contents can be similar for represent- atives of the same origin. Sytar et al., (2019): Methodology in comparative physiology of buckwheat 12 INTRODUCTION The Food and Agriculture Organization of the United Nations (FAO) supposes that the world’s population in 2050 will be 34% greater than it is today. Currently, 49% of the world’s population lives in urban areas, while in 2050 this will be closer to 70% (Alexandratos and Bruins- ma 2012). During this time period, climate change and the development of biofuel production will present ma- jor risks to long-term food security. Population growth, urban civilization and climate change can effectively sup- port high crop concurrence as possible sources of food, bioenergy, fiber and other industrial needs. Going for- ward, these problems will require innovative approaches to the genetic and agronomic components of systems of crop production and developing concurrent food market. While we are entering a period of increasingly rapid climate change, the overall objective of the project initi- ative is to design new strategies to maintain high yield and qualitative parameters of crop plants produced un- der changed environmental conditions, using novel crop plants which is promising for development in food mar- ket. Crop yield is a complex trait depending on the suc- cessful completion of different steps of vegetation (phase of reproductive organ development), which can be sensi- tive to environmental factors. Among innovative approaches is high interest in plant biotechnology and non-destructive methods of prescreening agriculture plants regarding content of spe- cific secondary metabolites, regarding development of some diseases, stress reaction for the missing of nitrogen supply or to discover plant species biodiversity. In target- ed plant metabolites, the main goal is to achieve a high throughput. Therefore, there is often an initial desire for a rapid pre-screening of the samples. This is especially the case when dealing with many sample numbers, where only a limited number of individuals might be expected to be different (Verhoeven et al. 2006). This is the case when searching for valuable genetic resources (e.g. those having a high content of desired compounds) within nat- ural populations or population obtained by crosses or mutagenesis (Sytar et al. 2015; 2017). Nowadays many scientific groups in the functional food area are looking for plants with high content of phy- tochemicals as an important source of active pharmaceu- ticals or with valuable nutritional properties (Abuajah et al. 2015; Varzakas et al. 2016). Buckwheat among these plants got a prime position as potential source of gluten free food products as well as rich source of antioxidant compounds (Saturni et al. 2010). The various types of bi- oactive compounds presented in different buckwheat va- rieties provide basic background needed for the efficient production of buckwheat foods with added value (Sytar et al. 2016). Two main buckwheat species have been commonly produced and consumed: common buckwheat (Fagopyrum esculentum Moench) and Tartary buckwheat (Fagopyrum tataricum Gaertn). The biodiversity of buck- wheat plant species which can be used farther are wide. Therefore, its is important to develop study difference be- tween buckwheat species of different origin. The non-invasive fluorescence-based phenomics method for determination of plant phenolics based on the strictly UV-absorbing properties, the effects of phe- nolic compounds on visible light-induced chlorophyll flu- orescence is negligible, whereas their presences strongly suppress the chlorophyll fluorescence emission under UV excitation (Cerovic et al. 2002). This phenomenon has been successfully applied for estimation of transmittance of UV radiation by chlorophyll fluorescence (Burchard et al. 2000; Ounis et al. 2001). In the previous decades, the numerous studies examined and confirmed possibility to use the chlorophyll fluorescence signal in the estimation of phenolics and anthocyanins. In addition to self-con- structed devices or standard fluorometers combined with external light sources and filters, which were used in the majority of studies, the factory-made special devices for this purpose were also introduced (Sytar et al. 2015). In this scientific work we would like to combine the non-invasive fluorescence-based, and traditional bio- chemical methods (HPLC, spectrophotometry etc.), typi- cally used for plant phenomics studies to discover growth characteristics of buckwheat species of different origin. MATERIAL AND METHODS Plant material Plants of 7 buckwheat samples from Ukraina (Fa- gopyrum tataricum himalaicum, Faropyrum tataricum ro- tundatum – red, Fagopyrum esculentum cv. Rubra - red, Fagopyrum esculentum cv. Karadag) and Chinese cultivars (Fagopyrum esculentum cv. SuQiao 1, Fagopyrum esculen- tum cv. YuQiao 4, Fagopyrum esculentum cv. NingQiao 1) were exposed immediately from sowing to direct sunlight in open field conditions for 52 days. The non-destructive measurements were started from early growth stage (stage of vegetation). First measurements were done at Fagopyrum 36(1):11-21 (2019) 13 stage of two leaves (22 days after sowing (DAS)), other measurements were carried out at stage of 4-5 leaves (30 DAS), at the beginning of flowering stage when buck- wheat plants had 5-6 leaves (36 DAS) and last measure- ments were done in flowering stage (51 DAS). The leaves for biochemical analysis were collected in the flowering phase. Cultivar Rubra with high anthocyanins content (3.87−4.41 mg/100 g DW) in the vegetative organs has been received by family selection method from chemo mutants. Cultivar Karadag is received from the Scientific Research Institute of Groat Crops in Ukraine. F. tatari- cum G. is a one-year plant which, among the species re- searched, has a better pollination of flowers and a higher grain production. F. giganteum Krot. is amphidiploid ob- tained after crossing F. tataricum G. with the perennial plant F. cymosum Meissn. (Krotov and Dranenko 1973). F. cymosum Meissn. is a polyploid with 32 chromosomes. The collection of buckwheat germplasm, which is main- tained at the Scientific Research Institute of Groat Crops in Ukraine comprises nearly 1000 samples which are readily available for breeding research. Chlorophyll fluorescence records and analyses using fluorescence excitation ratio method The chlorophyll fluorescence analysis was done using the portable optical fluorescence sensor Multiplex-3® (Force-A, Paris, France). Multiplex-3® is a hand-operated, multi-parametric sensor based on light-emitting-diode excitation and filtered photodiode determination that is arranged to work in the field, greenhouse and laboratory conditions. The sensor of Multiplex-3® has three, red- blue-green LED-matrices emitting light at 470 nm (blue), 516 nm (green) 153 and 635 nm (red). There are three in- tegrated photodiode detectors for fluorescence recording: far-red, red and yellow (Ghozlen et al. 2010). We used val- ues of fluorescence measured at UV (375 nm), green light (516 nm), red light (635 nm) and 735 nm (FRF). The evaluation of phenolic compounds contents in plants was done via calculation of fluorescence values de- tected after excitation by light of the defined wavelengths (details are below). In analogy to the spectrophotometric method for assessing leaf absorbance, the parameters were based on the Beer-Lambert’s law and calculated as logarithm of the fluorescence ratio values. UV absorbing compounds (mostly flavonols) content described by flavonoid (FLAV) index (Cerovic et al. 2002; Agati et al. 2011) was estimated using the modified for- mula of Zivcak et al. (2017), as the logarithm of the ratio of the red-light induced far-red fluorescence (FRFR) and the UV-induced far- red fluorescence (FRFUV): FLAV = log[ FRFR/ (kUV*FRFUV)] Similarly, the ANTH Index that provides estimates of green-light absorbing compounds (logFERR/G), mostly red-colored flavonoids and anthocyanins, was calculat- ed as the logarithm of the ratio of the red- light induced fluorescence (FRFR) and the green light-induced fluores- cence (FRFG): ANTH = log[FRFR/(kG*FRFG)] The correction coefficients kUV or kG was applied to measurements of fluorescence to avoid negative values (Zivcak et al. 2017). The constant values of the coeffi- cients were used as the minimum values of the FRFUV/ FRFR and FRFG/FRFR ratios found in the database that contains several thousand records from over three hun- dred plant species grown in diverse environments (Zivcak et al. unpublished results). The same constants have been used when processing data across all experiment and cul- tivars. We also calculated the modified Flavonoid Index (MFI) that provides a better estimate of total flavonoid content when plants with different colors are compared (Zivcak et al. 2017). The MFI was calculated as the loga- rithm of the ratio of the red-light induced fluorescence (FRFR) and the green light-induced fluorescence (FRFG). MFI = log[2*FRFR/(kG*FRFG + kUV*FRFUV)] The values of correction coefficients (kG, kUV) for MFI were the same as for ANTH and FLAV. Chlorophyll content was estimated from values of fluorescence measured at 735 nm (FRF) and at 685 nm (RF) after excitation by red light (635 nm). The Simple fluorescence ratio (SFR) was calculated as: SFR = FRFR/RFR Because the diameter of the measuring area was only 50 mm, 6-7 measurements were taken on each plant in different position to account for heterogeneity in leaf color and structure. This number of measurements from the top view provides sufficient data to characterize the entire plant. Anthocyanins estimation 0.1 - 0.5 g plant material was homogenized on ice with 3 ml of acidified methanol (1% HCl) and then incubated at 4 °C for 12 h with moderate shaking. The mixture was centrifuged for 10 min at 14 000 rpm at 4 °C. Absorb- tion of the extracts at 530 and 657 nm wavelengths was determined spectrophotometrically. The blank was acid- Sytar et al., (2019): Methodology in comparative physiology of buckwheat 14 ified methanol. The concentration of the anthocyanins was expressed as mg.g-1 dry weight and was calculated by formula: anthocyanins=[A530-(0.25*A657]*V/(W*1000), where A is absorbance; V is total volume of the extract (ml) and W is weight of the dry leaf tissue (g). Total phenolics estimation Total phenolic content in the buckwheat leaves ex- tracts was determined by standard spectrophotometric method of Lachman et al. (2003) by using Folin–Ciocal- teu reagent (Singleton and Rossi, 1965). 0.25 g powdered samples (freeze-dried) was extracted for 16-18 hours with 20 ml of 80% ethanol. After the time of extraction a volume of 100 µl of the plant extract was pipetted into 50 ml volumetric flask. 2.5 ml of Folin-Ciocalteau reagent was added to the extract. Then after 3 minutes (agitation) 5 ml 20% Na2CO3 solution was mixed. After two hours at 25 °C the absorbance was measured on the spectro- photometer Jenway UV/Vis 6405 (Jenway, UK) at wave- length λ = 765 nm against blank. Gallic acid was used as a reference standard for plotting calibration curve. Total phenolic content was expressed as mg.kg-1 gallic acid equivalent of dry matter. Analysis of hydroxycinnamic acid derivatives Analysis of hydroxycinnamic acid derivatives has been previously developed (Mewis et al. 2010). Sam- ples were taken after finishing the freeze-drying process where the material was ground by a flint mill (20 000 g for 2 min). A total of 20 mg ground samples from leaf suspension were extracted for 15 min using 0.75 mL 70% methanol (v/v, pH 4.0, phosphoric acid) in an ultrasonic water bath on ice. Then samples were centrifuged for 5 min at 6000 rpm. The supernatants were collected and the pellets were re-extracted twice more with 0.5 mL of 70% methanol (HPLC-Gradient grade, VWR chemicals). Coumaric acid or cinnamic acid (Sigma–Aldrich Chemie GmbH) (40 µL of 3 mM solution) was added as internal standard to the first extraction. The combined superna- tants from each sample were reduced to near dryness in a centrifugation evaporator (Speed Vac., SC 110) at 25 °C. Samples were added up to 1 mL with 40% acetoni- trile (HPLC Ultra Gradient Grade, Roth). The samples were filtrated using 0.22-mm filters and then analyzed with HPLC. The chromatography was performed using a DionexUltiMate 3000 HPLC System with a diode array detector (DAD-3000) with a WPS-3000 SL auto sampler, LPG-3400SD pump and a TCC-3000RS Column Com- partment (Dionex Corp., Sunnyvale, CA, USA). Extracts (1 mL) were analyzed at a flow rate of 0.4 mL/ min and a column temperature of 35 °C. The column used is Narrow-Bore Acclaim PA C16-column (3 mm, 120A, 2.1 _ 150 mm, Dionex). A 49-min gradient program was used with 0.1% v/v phosphoric acid in ultrapure water (eluent A) and 40% v/v acetonitrile in ultra-pure water (eluent B) as follows: 0–5 min: 0.5% B, 5–9 min: 0–40% B, 9–12 min: 40% B, 12–17 min: 40–80% B, 17–20 min: 80% B, 20–24 min: 80–99% B, 24–32 min: 99–100% B, 32–36 min: 100–40% B, 36–49 min: 40–1% B. The gradient pro- gram was followed by a 4-min period to return to 0.5% B and a 5-min equilibration period resulting in a total dura- tion of 39 min. Peaks were monitored at 290, 330 and 254 nm respectively. The phenolic acid quantity was calculated from HPLC peak areas at 290 nm. The retention times in the HPLC for the experiments were 12.13 min for vanillic acid, 12.72 min for chlorogenic acid, 13.29 min for caffeic acid, 15.98 min for the internal standard p-coumaric acid and 21.59 min for cinnamic acid. For the identification of unknown phenolic compounds, a semiquantitative analy- sis was performed using HPLC coupled with mass spectro- metric detection (LC/MS) and NMR (Mewis et al., 2010). Statistical analysis Means and standard deviations were calculated by the Microsoft Office Excel 2013. Significant differences of these data were calculated using analysis of variance ANOVA Duncan’s multiple test (STATISTICA 10, Stat- Soft, Tulsa, USA). All results were expressed as mean+ standard deviations from replications n = 50. RESULTS AND DISCUSSION Chlorophyll fluorescence records and analyses of FLAV, ANTH, MFI and SFR indices using fluorescence excitation ratio method Chlorophyll fluorescence records and analyses using fluorescence excitation ratio method of Ukrainian and Chinese genotypes during growth periods of 51 DAS shown significant increasing of FLAV index from begin- ning of seedlings stage to the of flowering stage (Fig.1). The highest increase of FLAV index which is con- nected with flavonols content has been observed in the Chinese genotypes. The high FLAV index at the flower- ing stage was not depended from flavonols content on Fagopyrum 36(1):11-21 (2019) 15 the beginning of seedlings stage (22 DAS). For example, Ukrainian genotype F. esculentum cv. Rubra on 22 DAS has a highest FLAV index (0.64 RU) compared to other experimental genotypes. At the flowering stage on 51 DAS FLAV index in this experimental genotype was 0.95 RU which was lower compared to the other experimental genotypes. Plus other Ukrainian genotype F. esculentum cv. Karadag on the beginning of seedlings stage (22 DAS) got FLAV index 0.35 RU which was similar to the level of FLAV index of Ukrainian F. tataricum rotundatum and Chinese F. esculentum cv. SuQiao 1, 6 – F. esculentum cv. YuQiao 4, 7 – F. esculentum cv. NingQiao 1. At the flow- ering stage on 51 DAS FLAV index in these experimental genotypes was in range 1.06-1.13 RU. The different tendency of ANTH index increasing from beginning of seedlings stage to the flowering stage Figure 1. Process of flavonols accumulation (logFERR/UV – FLAV) in the leaves of investigated buckwheat plant species exposed to direct sunlight during 51 days after seedlings (numbers indicate individual cultivars of buckwheat as follow: 1 – F. tataricum rotundatum, 2 – F. tataricum himalaicum, 3 – F. esculentum cv. Rubra, 4 – F. esculentum cv. Karadag, 5 – F. esculentum cv. SuQiao 1, 6 – F. esculentum cv. YuQiao 4, 7 – F. esculentum cv. NingQiao 1). Figure 2. Process of anthocyanins accumulation (logFERR/G – ANTH) in the leaves of investigated buckwheat plant species exposed to direct sunlight during 51 days after seedlings (numbers indicate individual cultivars of buckwheat as follow: 1 – F. tataricum rotundatum, 2 – F. tataricum himalaicum, 3 – F. esculentum cv. Rubra, 4 – F. esculentum cv. Karadag, 5 – F. esculentum cv. SuQiao 1, 6 – F. esculentum cv. YuQiao 4, 7 – F. esculentum cv. NingQiao 1) Sytar et al., (2019): Methodology in comparative physiology of buckwheat 16 has been observed for Ukrainian genotype F. esculentum cv. Rubra compared to other investigated buckwheat plant genotypes (Fig. 2). At the beginning of seedlings stage this genotype has highest ANTH index (0.50 RU) and at the flowering stage on 51 DAS (0.57 RU) too. At the flowering stage came to significant increase of ANTH index in F. esculentum cv. Rubra, on the contrary to other Ukrainian genotypes F. tataricum rotundatum and F. ta- taricum himalaicum, in which has ANTH index decreas- ing character from beginning of flowering stage. On the 51 DAS at the flowering stage in F. tataricum genotypes ANTH index was almost on the same level as at the be- ginning of seedlings growth after significant increasing at the 30 and 36 DAS. ANTH index for other investigat- ed genotypes (was) ranged from 0.39 to 0.43 RU at the beginning of seedlings stage. Genotypes of F. tataricum compared to the genotypes of F. esculentum of both or- igin shown decreasing of ANTH index during seedling growth. All F. esculentum genotypes of both orgin has in- ceasing on ANTH index on 51 DAS (Fig.2). MFI, the parameter that takes into consideration the accumulation of both flavonols and anthocyanins, is a better estimate of flavonoids than FLAV (Zivcak et al., 2017). Results of statistical analyses using MFI are high- ly similar to those that use data of biochemical analysis (Zivcak et al., 2017). MFI index did not shown significant difference between F. tataricum and F. esculentum geno- types of both origin but highest values has been found for F. esculentum cv. Rubra during all growth stages. Based on the obtained prescreening results during 51 DAS of growing period is possible to conclude that non-destruc- tive methodology can be used to choose genotypes with high antioxidant content. The majority of published vegetation indices for non-invasive remote sensing techniques are not sensitive to rapid changes in plant photosynthetic status brought on by common environmental stressors. The SFR index is connected with chlorophyl concentration in the leaves. In presented experiment with buckwheat genotypes of dif- ferent origin the tendency of SFR index changes was dif- ferent between genotypes of Ukrainian and Chinese ori- gin (Fig.4). In the middle phase (30 DAS) of growth has been observed significant increase of SFR index almost in all experimental genotypes. On 36 DAS just two Ukrain- ian genotypes which characterized also high ANTH and MFI indexes has been kept tendency to increase SFR in- dex - F. esculentum cv. Rubra and F. esculentum cv. Karadag. The Chinese genotypes shown decreasing of SFR index on 36 DAS with farther deacresing at 51 DAS of flow- ering stage with parallel increasing of ANTH, FLAV and MFI indexes. Total phenolics and anthocyanins estimation The biochemical analysis among experimental buck- wheat genotypes of different origin has been shown that highest total phenolic contents was found in Ukrainian Figure 3. Values of MFI index in the leaves of investigated buckwheat plant species exposed to direct sunlight during 51 days after seedlings (numbers indicate individual cultivars of buckwheat as follow: 1 – F. tataricum rotundatum, 2 – F. tataricum himalaicum, 3 – F. escu- lentum cv. Rubra, 4 – F. esculentum cv. Karadag, 5 – F. esculentum cv. SuQiao 1, 6 – F. esculentum cv. YuQiao 4, 7 – F. esculentum cv. NingQiao 1). Fagopyrum 36(1):11-21 (2019) 17 buckwheat Fagopyrum esculentum cv. Rubra and Chinese genotypes (Figure 5). At the same time leaves of Ukraini- an Fagopyrum esculentum cv. Rubra has been characterized by highest anthocyanins content compared to the other experimental buckwheat genotypes of different origin. This data is connected with previous research where was studied role of anthocyanins as marker for selection of buckwheat plants with high rutin content in Fagopyrum esculentum cv. Rubra (Sytar et al. 2014). Analysis of hydroxycinnamic acid derivatives HPLC prescreening buckwheat genotypes of differ- ent origin on (51 DAS) has been identified chlorogenic, p-coumaric, p-anisic, cinnamic, methoxycinnamic, fer- ulic and vanilic acids. It was found highest content of chlorogenic and p-coumaric acids in the Chinese geno- types F. esculentum cv. NingQiao 1 and F. esculentum cv. YuQiao 4. The highest p-anisic acid content was found for buckwheat genotypes of F. tataricum himalaicum Figure 4. Values of SFR (simple fluorescence ratio) in the leaves of investigated buckwheat plant species exposed to direct sunlight during 51 days after seedlings (numbers indicate individual cultivars of buckwheat as follow: 1 – F. tataricum rotundatum, 2 – F. tataricum hima- laicum, 3 – F. esculentum cv. Rubra, 4 – F. esculentum cv. Karadag, 5 – F. esculentum cv. SuQiao 1, 6 – F. esculentum cv. YuQiao 4, 7 – F. esculentum cv. NingQiao 1). Figure 5. Content of total phenolics (5a) and anthocynanins (5b) in the buckwheat leaves determined by HPLC method (numbers indicate individual cultivars of buckwheat as follow: 1 – F. tataricum rotundatum, 2 – F. tataricum himalaicum, 3 – F. esculentum cv. Rubra, 4 – F. esculentum cv. Karadag, 5 – F. esculentum cv. SuQiao 1, 6 – F. esculentum cv. YuQiao 4, 7 – F. esculentum cv. NingQiao 1) Sytar et al., (2019): Methodology in comparative physiology of buckwheat 18 (Table 1). It is important to admit that genotypes of F. tataricum characterized by higher p-anisic acid content compared to the experimental genotypes of F. esculen- tum of both origins. The nondestructive technique of infrared spectrosco- py is recommended as alternative technique for routine analysis of main flavonoids like rutin, quercetin and quer- citrin in aerial parts of buckwheat (Ladan et al. 2017). It can be pointed out that individual bioactive compounds compositions are suitable indicators of the physiological stage of crop plants. The phenotype of a plant is the result of a complex interaction between morphological, ontogenetical, phys- iological, and biochemical factors (Gratani 2014). The highest increasing of FLAV index has been observed on 51 DAS of flowering stage which is connected with flavo- noids content in the Chinese genotypes. Rutin content of the grain of 22 buckwheat genotypes (F. esculentum and F. tataricum) grown in same region of origin had variation (Bai et al. 2009), so its important to use non-destructive methods for prescrening of flavonoids content for dif- ferent genotypes. The high FLAV index at the flowering stage was not depended on flavonoids content on the be- ginning of seedlings stage (22 DAS). Other investigated genotypes for ANTH index in range from 0.39 to 0.43 RU at the beginning of seedlings stage. Genotypes of F. tataricum compared to the geno- types of F. esculentum of both origin shown decreasing of ANTH index during seedling growth. Liu et al. (2008) have shown that ethanol extracts of Tartary buckwheat sprouts had higher free radical scavenging activity and superoxide anion scavenging activity than those of com- mon buckwheat sprouts (Liu et al. 2008). Total phenolics and rutin in tested samples were related to the antioxi- dant activities (Holasova et al. 2002).  The SFR index is linked to the Chl concentration of leaves (Diago et al. 2016). Leaf Chl and FLAV concen- tration on a surface basis depends on leaf age and the amount of light radiation received during their devel- opment. Both increase with leaf expansion and light ex- posure until veraison, while afterwards, leaf Chl usually decreases (Louis et al. 2009) while FLAV remain unvary- ingly high (Downey et al. 2003). Such tendency has been confirmed in the our experiments with buckwheat geno- types of different origin just development of SFR index changes was different between Ukrainian and Chinese genotypes origin. The antioxidant capacity can be connected with total phenolic and anthocyanin contents and variety of plant species plus maturity (Prior et al. 1998; Kim et al. 2003). Flavonoids and phenolic acids have relevant antioxidant properties (Barriada-Bernal et al. 2014). The concentra- tion is affected by environmental conditions, age, and phenological stage (Almaraz-Abarca et al. 2013), while the qualitative phenolic profiles are more stable and vary among different groups of plants with a species-specific tendency (Emerenciano et al. 2001). It was observed that presence of p-anisic acid was typical for F. tataricum genotypes compared to the ex- perimental genotypes of F. esculentum of both origins – Ukrainian and Chinese. p-anisic acid is one of the isomers of anisic acid which has antiseptic properties (Bhimba et Table 1. Content of phenolic acids identified via HPLC analysis in the experimental buckwheat samples Leaves of buckwheat chl. acid p-coum. acid p-anis. acid cinn. acid Methox. acid fer. acid van. acid F. tataricum rotundatum 0.4±0.1 8.4±0.2 0.6±0.0 0.0±0.0 0.8±0.2 0.2±0.0 0.1±0.0 F. tataricum himalaicum 0.3±0.0 0.2±0.1 0.8±0.1 0.0±0.0 2.6±0.1 0.2±0.0 0.1±0.0 F. esculentum cv. Rubra 0.1±0.0 9.4±0.3 0.0±0.0 0.4±0.1 11.2±2.8 0.5±0.1 0.5±0.0 F. esculentum cv. Karadag 0.2±0.0 9.4±0.8 0.0±0.0 0.1±0.1 4.8±0.6 2.2±0.3 0.2±0.0 F. esculentum cv. SuQiao 1 0.3±0.1 20.1±8.3 0.1±0.0 0.0±0.0 3.0±0.3 0.2±0.1 0.3±0.0 F. esculentum cv. YuQiao 4 0.4±0.1 21.2±5.0 0.0±0.0 0.0±0.0 2.4±0.1 0.2±0.0 0.2±0.0 F. esculentum cv. NingQiao 1 0.5±0.0 25.1±0.6 0.1±0.1 0.0±0.0 4.0±0.3 0.1±0.0 0.3±0.0 Fagopyrum 36(1):11-21 (2019) 19 al. 2010). It is also used as an intermediate in the prepa- ration of more complex organic compounds. Cinnamic acid has low toxicity and in the search for novel pharmacologically active compounds, cinnamic acid derivatives are important and promising compounds with high potential for development into drugs (Sova 2012). The high content of cinnamic acid at 52 d of flowering period found in Ukrainian F. esculentum cv. Rubra, which is charac- terized by high anthocyanins content. In plants, flavanone biosynthesis begins with the hydroxylation of cinnamic acid to p-coumaric acid by a membrane-bound P450 monooxy- genase, cinnamate 4-hydroxylase (C4H) (Yan et al. 2005). At 52 day of flowering period the p-coumaric acid content was higher more than 2 times in Chinese genotypes com- pared to the Ukrainian genotypes of F. esculentum. CONCLUSION: The screening of biological active compounds of phe- nolic nature in the early stages of growth with non-de- structive chlorophyll fluorescence techniques can be used for qualitative traits analysis of plant sprouts, especial- ly buckwheat. The high flavonoids level at the flowering stage is not dependent on flavonoids content on the begging of seedlings stage. Plant genotypes of different origin can vary in flavonoid, anthocyanins and pigments content during stages of growth but changes in their contents can be similar for representatives of the same origin. The presence of some phenolic acid can be typical for genotypes of F. tataricum compared to the genotypes of F. esculentum. To support natural plant biodiversity re- search it would be good to develop use of fast pre-screen- ing methods of plants during all stages of development what can be helpful in applied food plant research. 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Plant metabolomics strategies based upon quadrupole time of flight mass spectrometry (QTOF-MS). In Saito K, Willmitzer L, editors. Biotechnology in agriculture and forestry. Berlin: Springer. p. 33–48. Yan, Y., Kohli, A., Koffas Mattheos, A.G. 2005. Biosynthesis of Natural Flavanones in Saccharomyces cerevisiae. Appl Environ Microbiol 71: 5610–5613. doi: 10.1128/AEM.71.9.5610-5613.2005 Zivcak, M., Brückova, K., Sytar, O., Brestic, M., Olsovska, K., Allakhverdiev, S.I. 2017. Lettuce flavonoids screening and phenotyping by chlorophyll fluorescence excitation ratio. Planta 245: 1215–1229. doi: 10.1007/s00425-017-2676-x IZVLEČEK Predstavljeni so nedestruktivni načini ugotavljanja flavonoidov, antocianinov in pigmentov tekom osebkovega ra- zvoja pri genotipih ajde različnega izvora, do faze cvetenja. Podobno naraščanje indeksov FLAV, ANTH in MFI je bilo ugotovljeno pri kitajskih in ukrajinskih genotipih. Genotipi Fagopyrum tataricum so v primerjavi z genotipi F. esculentum imeli tekom rasti kalic nižji indeks ANTH. Indeks SFR, povezan s koncentracijo klorofila je kazal različno dinamiko v primerjavi genotipov iz Ukrajine in Kitajske. V srednji fazi (30 dni po setvi) je bil pri večini genotipov ugotovljen povečan indeks SFR. Na 36 dan po setvi sta dva ukrajinska genotipa z visokima indeksoma ANTH in MFI imela naraščajoč in- deks SFR. HPLC analize raziskanih vzorcev so pokazale vsebnost p-anisinske kisline, značilne za F. tataricum v primerjavi s kultivarji F. esculentum. Rastline genotipov ajde lahko imajo različno vsebnost flavonoidov, antocianinov in pigmentov tekom faz rasti in razvoja, vendar podobne vsebnosti pri vzorcih enakega porekla. Fagopyrum 36(1):23-29 (2019) 23 Research paper Research results of local buckwheat varieties and forms of Ukrainian origin Oleh TRYHUB Ustymivska Experimental Station of Plant Production of Plant Production Institute nd. V.Ya. Yuryev of NAAS of Ukraine, Ustymivka, Poltavs’ka region, Ukraine Tel. +38 066 7261363, E-mail: Trygub_oleg@ukr.net DOI https://doi.org/10.3986/fag0008 Received: March 27, 2019; accepted: May 25, 2019 Keywords: accesses, buckwheat, collection, local varieties and forms ABSTRACT The national collection of buckwheat in Ukraine consists of 578 local samples and forms of Ukrainian origin added into the collection during 1929 to 2012. In 1994-2000 and 2014-2018, the material has been studied and described, the common characteristics of samples peculiar of a certain area of origin, feature manifestation peculiarities, material flex- ibility and stability of the quantitative and qualitative characteristics depending on the weather in growing years have been identified. The range of studies included sample performance indicators, architectonic values, grain characteristics, and so on. A common characteristic of local buckwheat varieties is a significant sensitivity to changes in growing con- ditions, which is reflected in the change of growth processes (increase in the plant height by lengthening of internodes, the number of branches of the second and higher orders, the number of leaves per plant) and adjustment of the growth duration (lengthening the duration of the growing season after the onset of more favorable conditions for growth and development). The performance indicators related to the grain characteristics are quite stable. However, the general trends characteristic of plants from a certain origin of the collection material remained unchanged. Tryhub, (2019): Local buckwheat varieties of Ukrainian origin 24 INTRODUCTION Buckwheat has not become one of the most demand- ed crops by the international community yet. The main reason is its limited distribution around the world and low yields. However, the conclusions made in recent years concerning the maintenance of full-fledged human life, health and outstanding buckwheat plant properties indicate a significant need in buckwheat products (Kreft, 2010, Alekseeva at al., 2005). Gradually, the buckwheat products are distributed from the major regions of its traditional consumption to the new areas, where it gains the status of the core component of a healthy diet and becomes the foundation for a healthy lifestyle. As a result of in-depth study of biochemical compo- sition of buckwheat grant and the whole plant, involv- ing new types of crops into the research, the scientists around the world have greatly expanded the application areas of buckwheat products from traditional to exotic. To carry out this work, the genetic material with ex- tensive properties and characteristics, most of which is concentrated in banks of plant genetic resources, is used (Alekseeva at al., 2004). Genetic diversity preservation has long been an im- perative of global importance. The work to create the banks of genetic plant resources, launched more than a hundred years ago, not only validated the feasibility of this direction development, but also confirmed an un- valued contribution to ensuring food security worldwide (Alexanian, 2003). To solve scientific problems, the collection material of various eco-geographical origin and biological status is critical. However, given the importance of addressing the issue of the plant productive potential realization in contrasting environmental conditions, the study of local varieties and forms, the research of adaptive mechanisms and biochemical plant components became of paramount importance. MATERIALS AND METHODS A part of the National Buckwheat Collection in Ukraine, which is stored in Ustymivska Experimental Station of Plant Production (Poltavs’ka region), includes over 1,600 authentic samples, of which 1.1 thousand samples are the local varieties and forms of the common buckwheat (Fagopyrum esculentum Moench.) (Tryhub at al., 2015). 578 samples in the collection have Ukrainian origin. The material included in the collection is the result of its collection by employees of the N. I. Vavilov All-Un- ion Institute of Plant Genetic Resources (VIR, Leningrad) during the missions (Fesenko at al., 2006), by researchers of Ternopil Breeding and Research Station, the Research Institute of Agriculture and Livestock of the Western Regions of Ukraine led by Olena Alekseeva (Alekseeva, 1967), as well as employees of the National Center for Plant Genetic Resources of Ukraine, assisted by the Usty- mivska Experimental Station of Plant Production (Kir- jan at al. 2014) in the territory of Ukraine from 1929 to 2012. As regards the ecological and geographical origin, there are samples from 18 regions. These are the repre- sentatives of the Carpathian region, Woodlands, Forest Steppe and Steppe zones. The overwhelming majority is the samples from the North, Central and Western parts of Ukraine, and a small number comes from the Eastern and Southern regions (see Table 1 and Pic. 1). Natural-climatic zones and regions of Ukraine Number of accesses Forest Steppe 294 Kyivs’ka (part) 12 Sums’ka (part) 60 Khmel’nyts’ka 11 Ternopil’s’ka 34 Cherkas’ka 9 Poltavs’ka 70 Vinnyts’ka 58 Kharkivs’ka 40 Steppe 20 Kirovohrads’ka 8 Dnipropetrovs’ka 4 Odes’ka 8 Polissya and Carpathian region 264 Zakarpats’ka 8 Ivano-Frankivs’ka 19 L’vivs’ka 36 Rivnens’ka 11 Volyns’ka 8 Chernihivs’ka 128 Kyivs’ka (part) 8 Sums’ka (part) 24 Zhytomyrs’ka 22 Total 578 Table 1. Distribution of origin of the local sample collection by climatic zone of Ukraine Fagopyrum 36(1):23-29 (2019) 25 The research of the collection material of buckwheat varieties and forms was carried out as required by the “Complete unified classifier of the genus Buckwheat (Fa- gopyrum Mill.)” (Tryhub at al., 2013), “Descriptors for buckwheat (Fagopyrum spp.)” (Descriptors..., 1994) , “Guidelines for the study of collection samples of corn, sorghum and groats” (Krotov, 1968), “Analysis of the structure of buckwheat plants (Methodical recommenda- tions)” (Bochkareva, 1994). The material study and description was carried out in several stages during 1994-2000 and 2014-2018, and the results obtained were compared to the standard variety of Ukrainka. The common characteristics of samples pe- culiar of a certain area of origin, feature manifestation peculiarities, material flexibility and stability of the quan- titative and qualitative characteristics depending on the weather in growing years have been identified. The range of studies included the sample performance indicators (the number of seeds and buds per plant, inflorescence yield), architectonic parameters (plant height, number of branches and inflorescences, height of attachment of the inflorescences and branches, the length of the branching and grain production zone), grain characteristics (grain size, evenness and husk content), the length of the grow- ing season and its components, and so on. RESULTS AND DISCUSSION To obtain high and stable buckwheat yields, it is re- quired to continue creating new varieties combining high performance, friendly maturation, resistance to drought, temperatures below zero, lodging, grain shattering, pests and diseases, as well as a high grain quality. For this pur- pose, in selection, as noted by N.I. Vavilov, we need to use the local material subjected to prolonged exposure to natural selection and adapted to specific conditions. This material has great value, and is widely used in selection (Korynyak at al., 2017). It should be noted that in terms of soil and climat- ic conditions, the research region, namely the south of Poltavs’ka region (central part of Ukraine, Forest Steppe zone) is one of the most favorable for growing buck- wheat. Still, it has recently been affected by the ongoing significant climate change towards higher temperatures during flowering and insufficient precipitation during in- itial growth, which is typical for most areas of buckwheat cultivation in Ukraine. These trends are typical for most regions of the world where the buckwheat is sown. The features of sign manifestation were identified based on a set of researches conducted in two stages. The samples were studied following a three-year cycle during 1994- 2000 and 2014-2018. The first cycle of study (1994- 2000) was remarkable for more favorable weather con- ditions with precipitation and temperature regime of the growing season close to the average long-term data. The second cycle of study (2014-2018) was characterized by a high temperature in summer and a significant lack of moisture in the spring. The data obtained in the course of study indicate a considerable variety of feature manifestations by the local plant varieties and forms. There is a considerable dependence in the levels of feature manifestation on climatic zones of the sample origin. A significant impact on the plant characteristics was exercised by the climatic factors, precipitation and temperature regime during the years of cultivation. However, a set of the studied traits showed the characteristic, genetically determined fea- tures of samples and allowed describing the quantitative and qualitative material characteristics. Given the considerable heterogeneity of the climat- ic zones of Ukraine in terms of soil conditions, heat and precipitation, the entire buckwheat gene pool of these areas has been further divided into smaller, but more similar regions. To characterize the samples from the western regions of Ukraine, the classification pro- posed of Olena Alekseeva (Alekseeva, 1999) was ap- plied. For other areas, the author’s classification was applied. Fig. 1. Territorial distribution of origin of the local buckwheat samples (name of district and number of samples). Tryhub, (2019): Local buckwheat varieties of Ukrainian origin 26 Samples from Forest Steppe of Ukraine In terms of soil and climatic conditions, the Forest Steppe Zone was subdivided into western, central and eastern parts, each with its own characteristics, signifi- cantly differing among themselves, but with rather sim- ilar buckwheat growing conditions within each part. The western part includes the samples from Khmel’nyts’ka, Ternopil’s’ka and Vinnyts’ka regions, having more pre- cipitation as compared to the other parts of the Forest Steppe Zone, and their more even distribution during the vegetation period, lower average daily temperatures and fertile soils. The central part of the Forest Steppe (Cherkas’ka, Poltavs’ka, Sums’ka and Kyivs’ka regions) is characterized by harsher weather conditions in terms of the temperature and humidity. Here is sufficient pre- cipitation during the growing season, but its distribution is very uneven. The cover in most of the area is present- ed with fertile soil with excellent quality characteristics. The eastern part of the Forest Steppe zone includes Kharkivs’ka region and the south of Sums’ka region. This part shows the most extreme weather conditions with lit- tle precipitation and high temperatures, especially during the buckwheat flowering and graining. In general, the gene pool of this region is represented by 294 samples. The samples of this group are the most malleable ge- netic material grown in the area with conditions, which are the most suitable for the buckwheat cultivation. Such a genepool was the source material for most of the modern high-tech varieties, and today the scientists are searching for the forms remarkable for their performance indicators combined with resistance to abiotic environ- mental factors, grain quality, and so on primarily among these samples. The samples from the western part of the Forest Steppe Zone of Ukraine show an extensive diversity in terms of the key parameters of the plant organism structure, which requires their subdivision into two sub- groups by the growing season duration, i.e. mid-late-rip- ening (78-85 days) and early-ripening (70-77 days). Mid-late-ripening samples form tall plants (1.5 m high) with a large number of internodes (9-13 pcs.), a large number of leaves and branches (9-16 pcs.). Lower inter- stitials are thickened, forming resistance to lodging. The samples’ yield is 180-220 g/m2, and the productivity is 1.8-2.6 g/plant. They have medium-sized grain, up to 25.6 g/1000 grain with uniformity up to 82% and husk content 22.7-23.5%. The early-ripening samples have the plants 85-110 cm high with 10 internodes and the ratio of grain formation zone vs. branching zone as 1.0-1.3. The plants form a large number of inflorescences, up to 55 pieces per plant. Samples have large dark brown grain as for the local forms; the weight of 1,000 grains is 25.8- 26.9 g, the husk content increased to 23.8%, and a good inflorescences uniformity, i.e. 85%. The yields of these samples is within 165-200 g/m2, and the productivity is 1.6-2.2 g/plant. Plants have a significant number of leaves and the medium number of branches (5-8 pcs.), are resistant to lodging and moderately resistant to grain shattering after ripening. The central part of the Forest Steppe Zone has a more form genepool in terms of manifestation of quantitative and qualitative traits of the buckwheat plant organism. Local varieties from this part feature the medium-term ripening (70-80 days), medium and large plant height (100-150 cm), 7 to 16 internodes on the main stem and the ratio of the graining zone vs the branching zone as 1.1-1.3. The plants from this group are resistant to lodg- ing, have many leaves and 8-14 branches (including 5-8 first-order branches) and 85 buds per plant. The grain of these samples is gray and dark brown with an average weight of 1,000 grains as 24.2-27.3 g, grain husk content as 22.1-23.5%, and 90% uniformity. The average grain yield varies between 185-236 g/m2, and the productivity – within 1.8-2.8 g/plant. The plants from the local samples of eastern part of the Forest Steppe of Ukraine are potential sources of the variety resistant to abiotic environmental factors (high temperatures and drought). Samples from this part form the medium-term ripening (75 days) plant up to 120 cm high with 5-9 internodes, 5-12 branches and 65 buds per plant. These samples are characterized by high resistance to lodging and medium resistance to the grain shattering. Grain has a dark brown color with a clear or blurry pat- tern in the form of dots or dashes, the weight of 1,000 grains is 23.5-26.7 g, the medium husk content (up to 23.1%) and evenness (75%). The yield of samples from this group is 168-218 g/m2 with the plant performance at 1.45-2.05 g/plant. Samples from Polissya and Carpathian region The material from this zone also features extensive di- versity due to a significant difference between the weath- er, climate and soil conditions within the region. The samples from this area were subdivided into 3 groups: the north-western, western, northern and Carpathian. Fagopyrum 36(1):23-29 (2019) 27 The north-western part includes the samples originating from Volyns’ka and Rivnens’ka regions, the climate in which is characterized by excessive precipitation, moder- ate temperature conditions and poor nutritional compo- sition of their soils. The western part (Ivano-Frankivs’ka, L’vivs’ka and Zhytomyrs’ka region) is characterized by a moderate precipitation and temperature conditions fa- vorable for plant growth and development, with soils of varying fertility. The northern part of the Polissia zone (Chernihivs’ka and a part of Kyivs’ka and Sums’ka re- gions) has the most fertile soils in this region, sufficient precipitation and heat. The genepool of the Carpathian region (Zakarpats’ka and a part of Ivano-Frankivs’ka re- gion) includes representatives of foothill and mountain areas, featuring moderate and sometimes low tempera- ture during the growing season, often excessive precipi- tation and poor soils. The collection of local varieties and forms from this region includes 264 samples. The samples of the north-western part of the region are characterized by a short growing season, low (1.2 m) stem with 4-5 internodes; slight branching (3-7 branch- es, of which 2-3 are the first-order branches) with a ratio of the graining zone vs the branching zone as 0.8, the me- dium number of leaves, the sample yield at 120-180 g/ m2 per plant and the productivity as 0.8-1.0 g/plant; the medium grain particle size (up to 23.5 g/1000 grains), uniformity (80%) and husk content (up 23.5%), brown grain with a clear patter; resistant to lodging and having a medium degree of grain shattering after ripening. The samples from the western part feature a signifi- cant heterogeneity by the growing season duration; the samples vegetation varies from early-ripening (70 days) to late-ripening (90 days), and respectively, the plant height and the number of internodes ranged from 80- 100 cm and 5-7 pieces. to 125-130 cm and 9-12 pieces. In general, all samples were dark brown and gray, with me- dium and large grain size (23.7-26.8 g/1000 seeds), me- dium evenness (85%) and high husk content (24%); the plants have many leaves with a medium degree of grain subsidence and lodging. The samples’ yield was 175-210 g/m2, and the plant productivity – 1.6-2.2 g/plant. The samples from the northern part feature a high yield (up to 250 g/m2) and plant productivity (up to 3.0 g/plant); large grain (24.5-26.4 g/1000 grain), good evenness (up to 85%), and medium husk content (22.6- 23.1%), brown and dark brown color. The plants of these samples are tall (1.2-1.5 m) with a large number of branches (13-18 pcs.) and buds (70 pcs.) per plant, as well as many leaves. The stem is thick and resistant to lodging. The samples from the Carpathian region (foothill ar- eas of Zakarpats’ka and Ivano-Frankivs’ka regions) are characterized by a considerable length of the growing pe- riod (80-90 days) and tall plants (150 cm) with a signifi- cant number of branches (15 pcs.) and internodes (10-13 pcs.); the ratio of the graining zone vs the branching zone is 0.9-1.1; the plants have a large number of leaves and buds (80 pcs.); yield at 180-200 g/m2 and productivity up to 2.2 g/plant; the medium grain size (up to 25.1 g/ plant), medium husk content (23%) and evenness (85%). The plants are remarkable for a low resistance to plant lodging and grain shattering. Samples from the Carpathi- an mountain areas have a medium length of the grow- ing period (75 days), the plants are lodging heavily due to thin stems and lots of leaves, the medium number of branches per plant (7-10 pcs.) and internodes per stem (6-9 pcs.); the ratio of the graining zone vs the branching zone is 0.6-0.9. The sample yield is medium, 160 g/m2 per plant, and the productivity is 0.9-1.5 g/plant, while the number of buds is - 50 pcs. The grain features a medi- um size 21.3-24.6 g/1000, low evenness (70%) and high husk content (23.4-24.6%); gray or brown color with a distinct pattern. Samples from Steppe of Ukraine This is the least numerous group of samples available in the National Collection of Ukraine. The total gene pool of the region comprises 20 samples. However, in terms of its climatic conditions, each of the regions included in this group has the contrasting features and requires sub- division into two parts, namely the genepool of Odes’ka region, which was formed in conditions of unstable hu- midity, relatively poor soils and high temperature during the vegetation period, as well as samples originating from Kirovohrads’ka and Dnipropetrovs’ka regions, where the sample forming conditions are remarkable for unstable, but more moderate precipitation, more favorable temper- ature conditions and availability of fertile soils. The samples originating from Odes’ka region feature the early ripening (68-75 days), low height (110 cm), a small number of internodes per stem (48 pcs.), low re- sistance to lodging due to a thin stem, a small number of branches (5-9 pcs.) and a moderate number of leaves per plant. The graining zone vs the branching zone ratio is 1.1-1.3. The sample productivity level varies considera- bly, several times, depending on the weather conditions. Tryhub, (2019): Local buckwheat varieties of Ukrainian origin 28 The average yield in this group of samples ranged from 68.5 to 174 g/m2, and the plant productivity was 0.4- 2.3 g. Samples form a quite fine grain, 21.6-23.2 g/1,000 grain, with a medium husk content – 22.8-23.4%, and low evenness (75%). The samples originating from Kirovohrads’ka and Dnipropetrovs’ka regions are more grainful (179-236 g/ m2) with the productivity of 1.9-2.6 g/plant. They also form larger (24.7-26.5 g/1,000 grain), even (85%) grain with thinner husk (up 23.1%). The samples of these plants have more leaves, branches (7-13 pcs.) and buds (80 pcs.) per plant. The plant height is medium, 125 cm, with 9-13 internodes. The plants are more resistant to the grain shattering after ripening and resistant to lodg- ing due to the thickened lower internodes. A common characteristic of all local buckwheat va- rieties of Ukrainian origin is a significant sensitivity to changes in growing conditions, which is reflected in the change of the growth processes (increase in the plant height by lengthening of internodes, the number of branches of the second and higher orders, as well as the number of leaves per plant) and adjustment of the growth process duration (extension of the growing season length after the onset of more favorable conditions for growth and development). A quite stable performance is relat- ed to grain characteristics (size, husk content and uni- formity, color, grain, etc.). However, the general trends, characteristic of the collection material origin, remained unchanged. Extensive variety options are important for selection, because enables finding a material with specific charac- teristics among varieties and forms, identifying and ex- amining the same according to a set of indicators. While working to study and describe the sample collection, some contrasting forms in comparison with the origi- nal populations were discovered (in terms of the shape of plants and buds, flower color and size, availability and varying degrees of anthocyanin color, initial growth rate, a lack of branching, ability to counter or avoid the ex- treme environmental factors, etc.). This is the most val- uable genepool, because it allows expanding the variety polymorphism, extending the traditional and starting the new areas of buckwheat selection or use of products obtained from its cultivation. REFERENCES 1. Alekseeva, E., 1967. Ecological groups of local buckwheat varieties of the western regions of the UkrSSR // Selection and seed-growing. Vol. 7, Ukraine, pp. 13-25. (in Russian) 2. Alekseeva, E., 1999. Breeding of Podolsk varieties of buckwheat, Chernovcy: Ruta, Ukraine, 120 p. (in Russian) 3. Alekseeva, E., Taranenko, L., Malyna, M., 2004. Genetics, selection and seedling of buckwheat, Kyiv: Vyscha shkola, Ukraine, 216 p. (in Ukrainian) 4. Alekseeva, E., Elagin, I., Taranenko, L., Bochkareva, L., Malina, M., Rarok, V., Yacishin, O., 2005. Culture of buck- wheat. Vol. 1. Historical of culture, botanical and biological feature. Kamenetz Podolsky, Moshak, Ukraine, pp. 66-75. (in Russian) 5. Alexanian S.M., 2003. State and Bioresources, St. Peterburg: VIR, Russia, 180 p. (in Russian) 6. Bochkareva, L., 1994. Analysis of the structure of buckwheat plants (Methodical recommendations), Chernovcy, Ukraine, 45 p. (in Russian) 7. Descriptors for buckwheat (Fagopyrum spp.), 1994. IPGRI. 48 p. 8. Kirjan, V., Boguslavs’kyy, R., Smekalova, T., Bagmet, L., 2014. Collection of samples of the gene pool of plants for re- plenishment of the National genebank of plants of Ukraine (results of the expedition 20-30 August 2013) // Theoret- ical and applied aspects of the development of natural sciences: mater. of Intern. scient.-pract. conf. (20-21 November 2014), Poltava: PNPU, Ukraine, pp. 73-75. (in Ukraine) 9. Kreft, I., Ikeda, K., Ikeda, S., Vombergar, B., 2010. Development of functionally new food products based on buck- wheat ordinary and tartary // Vesnik OrelSAU, No4 (25), Russia, pp.15-17. (in Russian) 10. Krotov, A., 1968. Guidelines for the study of collection samples of corn, sorghum and groats. Leninhrad: VIR, Russia, pp. 37-44. (in Russian) 11. Korunyak, O., Burdyha, V., Rarok, A., Rarok, V., 2017. Collection of Fagopyrum Mill world genepool: formation, stud- ing and use of the samples. Podilian Bulletin: agriculture, engineering, economics. Kamianets-Podilskyi, Ukraine, pp. 87-93. (in Ukrainian) Fagopyrum 36(1):23-29 (2019) 29 12. Tryhub, O., Burdyga, V., 2015. Formation of the collection of the world genepool of buckwheat in Ukraine and direc- tions its use. The manual of Ukrainian grain growers, pp. 118-123. (in Ukrainian) 13. Fesenko, N., Fesenko, N., Romanova, O., Alekseeva, E., Suvorova, G., 2006. Buckwheat. Genepool and breeding groat cultures (Theoretical basis of plant breeding) (edited by Drahavcev V.), Vol.5. St. Peterburg: VIR, Russia, 196 p. (in Russian) 14. Tryhub, O., Kharchenko, Yu., Ryabchun, V., Hryhoraschenko, L., Dokukina, K., 2013. Complete unified classifier of the genus Buckwheat (Fagopyrum Mill.), Ustymivka, Ukraine, 54 p. (in Ukrainian) IZVLEČEK Zbirko ajde Ukrajine tvori 578 lokalnih vzorcev, ki so bili nabrani in vključeni v zbirko v letih 1929 do 2012. V letih 1994-2000 in 2014-2018 so potekale raziskave in opisovanje vzorcev, zlasti običajnih lastnosti, značilnih za posamezna območja izvora, posebnosti vzorcev, variabilnost in stalnost kvantitativnih in kvalitativnih lastnosti v odvisnosti od vre- mena in leta pridelovanja. Raziskave so med drugim vključevale kazalce rodnosti, opis zgradbe rastlin in lastnosti zrn. Običajne lastnosti lokalnih varietet ajde so odvisne od sprememb v razmerah pridelovanja, kar se kaže v spremembah poteka rasti (večanje višine rastlin z daljšanjem internodijev, števila stranskih vej drugega in višjega reda, števila listov na rastlino). Kazalci povezani z lastnostmi zrn so precej stabilni. Vseeno pa ostajajo lastnosti rastlin glede na izvor v znatni meri nespremenjene. 30 INSTRUCTIONS TO 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. 31 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 file shall start with the family name of the first author, following for example: Kranjc Fig. 1. Authors will be contacted by the Editor-in-Chief when the initial review process is completed. Correspondence with authors will be by e-mail. When preparing final versions of revised manuscript, the revised manuscripts including figures should be prepared and forwarded to the Editor-in-Chief. A manuscript describing research on human subjects should contain a statement on the manner in which the research complied with the code of ethics of the World Medical Association (Declaration of Helsinki). The author(s) should also submit a letter of approval with the research. 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