Advances in Buckwheat Research

Advances in Buckwheat Research Proceedings of the 9th International Symposium on Buckwheat

held at the

Congress Centre, University of Agriculture, Prague - Suchdol 18-22 August 2004 Organised by the Research Institute of Crop Production Prague - Ruzyně under the Auspices of the International Buckwheat Research Association and Ministry of Agriculture of the Czech Republic

Editors: Iva Faberová, Václav Dvořáček, Petra Čepková, Ivan Hon, Vojtěch Holubec, Zdeněk Stehno Prague 2004

Note: Manuscripts were compiled without any stylistic or language revision. Authors are responsible for correctness of their submitted texts.

ISBN: 80-86555-46-1 © Research Institute of Crop Production, Prague 6 - Ruzyně, Drnovská 507, Czech Republic

Contents Section I. Keynote Presentations Page Ivan Kreft: The Development and Importance of IBRA, the International Buckwheat Research Association …………………………………………………………………………….. Ohmi Ohnishi: On the Origin of Cultivated Buckwheat ……………………………………………….. Taiji Adachi: Recent Advances in Overcoming Breeding Barriers in Buckwheat …………………… Clayton G. Campbell: Present State and Future Prospects for Buckwheat ……………………………………. Jiří Petr, Jana Kalinová, Jan Moudrý, Anna Michalová: Historical and Current Status of Buckwheat Culture and Use in the Czech Republic …

11 16 22 26 30

Section II. Biotechnology and Physiology Page Hiroyasu Michiyama, Keiji Tsuchimoto, Ken-ichiro Tani, Tatsuya Hirano, Hisayoshi Hayashi and Clayton Campbell: Influence of Day Length on the Growth of Stem, Flowering, the Morphology of Flower Clusters, and Seed-Set in Buckwheat (Fagopyrum esculentum Moench) …….. Jelena M. Brkljačić, Dragana B. Majić, Jovanka Miljuš-Đukić, Ana Bratić, Miroslav M. Konstantinović and Vesna R. Maksimović: Influence of Different Physiological and Stress Conditions on Buckwheat Metallothioneine Gene Expression and Structural and Functional Analysis of its Promoter Region ……………………………………………………………………….. Myung Heon Lee, Jung Sun Lee and Tae Heon Lee: Germination of Buckwheat Grain: Effects on Minerals, Rutin, Tannins and Colour …. Mateja Germ: Environmental Factors Stimulate Synthesis of Protective Substances in Buckwheat … Woo, S. H., M. Takaoka, H. S. Kim, C. H. Park, T. Adachi and S. K. Jong: Plant Regeneration via Shoot Organogenesis from Leaf Callus Culture of Common Buckwheat (Fagopyrum esculentum Moench.) ……………………………………….. Gorkova IrinaVyacheslavovna, Voroshirina Nataliya Nikolaevna, Pavlovskaya Ninel: Biotechnological Processing of Buckwheat for Obtaining Food Additions and DyeStuffs …………………………………………………………………………………… Ryo Ohsawa and Chie Nakatani: Inter Varietal Variation in Growth Inhibition to Lettuce Seedling by Water Soluble Extract in Common Buckwheat Leaves ……………………………………………….. Jelena T. Samardžić, Mira Dj. Milisavljević, Jelena M. Brkljačić, Miroslav M. Konstantinović, Vesna R. Maksimović: Cloning and Analysis of cDNA and Gene Coding for 13S Storage Polypeptide from Buckwheat ……………………………………………………………………………… Elena N. Barsukova: Utilization of Tissue Culture Method in Buckwheat Selection ....................................... Sukhovitskaja L.A., Mokhova S.V., Safronava H.V., Chernetsova I.B., Melnikova N.V.: Production of Bacterial Preparation Rhizobacterin and Efficiency of Its Application under Grain Crops ………………………………………………………………………

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41 50 55 61 66 71

76 85 91

Motoko Takaoka, Ryou Ukegawa, Nazrul Islam, Taiji Adachi, Hisashi Hirano: Changes in Proteome Patterns in Buckwheat Seed under Submergence ……………… V.I. Guliaev and L.A. Chucha: Effect of Temperature on the Shape of Growing Cells of Plants Studied by Laser Interferrence Auxanometry ............................................................................................. Binayak P. Rajbhandari: Eco-Physiological Aspects of Common Buckwheat …………………………………….. Larskaya I.A., Barisheva T.S., Zabotin A.I., Zabotina O.A.: Application of Thin-layer Explants from Buckwheat Hypocotyls for Study of Rhizogenesis …………………………………………………………………….…….. Tao Yun-Ping, Shi Qing-Liang, Zhang Xiao-Xiu, Zhou Yun-Ning: Inoculation Effect on Growth and Flavonoid Content of Tartary Buckwheat in Field Experiment ……………………………………………………………………………. T.A.Tsybina, Y. E. Dunaevsky, N.A.Popykina, N.I.Larionova and M. A. Belozersky: Kinetic Properties of Cationic Inhibitors of Serine Proteinases from Buckwheat Seeds . Vesna Maksimović, J.Brkljačić, A.Bratić, M.Konstantinović, D. Majić, J.MiljušDjukić, M.Milisavljević, S. Radović and G. Timotijević: The Genes of Buckwheat - Basic Research and Biotechnological Application ……….. N.I. Rumyantseva, V.V. Salnikov, V.V. Lebedeva: The Study of Fagopyrum esculentum Callus Surface During Induction of Morphogenesis ………………………………………………………………………… Satoshi Murayama, Yoichi Suyama, and Yukio Yanokuchi: Varietal Difference of Pre-Germination Flooding Tolerance in Buckwheat …………... Pavlova I.: Determination of Ti ((37), iaaM::Tn5)-DNA Influence on Buckwheat Plant Form …… Yang Liyan, Yang Wude: Effects of Ethephon on Flower Thinning and Yield Improvement in Buckwheat ……... Gordana S. Timotijević, Mira Đ. Milisavljević, Svetlana R. Radović, Vesna R. Maksimović: Various Forms of Aspartic Proteinases in Buckwheat Seeds ………………………….. N.V.Budagovskaya, S.L.Jiltsov and G.G.Komissarov: Registration of Buckwheat Plant Growth Rate by the Method of Laser Interference Auxanometry …………………………………………………………………………… Yao Ziqiang, Zhong Xinglian, Wu Dongchi, Zhang Jitao, Peng Jifu: Influence of CAU-2 on Plant Characters and Yield of Tartary Buckwheat …………… Olga Romanova: Northern Populations of Tartary Buckwheat with Respect to Day Length …………… Zhang Z, Zhang X, Zhao Z H, Wang Z H, Kreft I: Inhibitory Activity and Thermostability of α-Amylase-Inhibitor from Buckwheat Flour

95 99 101 109 116 124 130 136 143 146 154 158 167 170 173 179

Section III. Genetics, Genetic Resources and Breeding Page Honda Yutaka, Yuji Mukasa, Tatsuro Suzuki and Nobuyuki Abe: Stone Buckwheat, Genetic Resource of Tartary Buckwheat in Japan………………….. Mamiko Yui, Takami Hayashi, Makoto Yamamori and Masako Kato: Inter-Specific Hybridization Between Japanese Common Buckwheat ‘yabukawazairai’ and Fagopyrum cymosum Meissn………………………………………………. Friedrich J. Zeller, Heidi Weishaeupl and Sai L.K. Hsam: Identification and Genetics of Buckwheat (Fagopyrum) Seed Storage Protein ………. Dubovik E.I.: Breeding of Polyploid Buckwheat in Belarus: Results, Problems, Directions …………

185 190 195 202

Fesenko A.N., Fesenko N.V.: Use of Blocked Branching Form in Buckwheat Breeding ……………………………... Takehiko Konishi, Hiroyoshi Iwata, Yasuo Yasui, Ohmi Ohnishi, Yoshihiko Tsumura and Ryo Ohsawa: Development of Microsatellite Markers for Common Buckwheat (Fagopyrum esculentum Moench) …………………………………………………………………… Andriy V. Nikitchuk and Elena S. Alexeeva: The Features of Cotyledon Leaves of Buckwheat’s Species Fagopyrum tataricum....... Ohmi Ohnishi: Wild Buckwheat Species in the San Jiang (Three Rivers) Area of Southwestern China V.I. Kovalenko and V.K. Shumny: Homostyly in Buckwheat Fagopyrum esculentum Moench and Possibilities of Its Use A.N.Fesenko, N.V.Fesenko: Effect of Branching on Realization of Buckwheat Plant’s Productive Potential ………. Yingjie Wang, Clayton G. Campbell and Ping Jiang: Inheritance Patterns for Rutin Content, Self-compatibility, Winged Seed, and Seed Shattering in Hybrids between F. homotropicum Ohnishi and F. esculentum Moench... Lin Rufa: The Development and Utilization of Tartary Buckwheat Resources ………………….. G.E.Martynenko, N.V.Fesenko, L.N.Varlakhova: Improvement of Technologic Grain Characteristics of Determinant Buckwheat Cultivars in the Process of Breeding …………………………………………………… Lihua Wang, Fuyou Yin: The Geography Distribution of Wild Buckwheat Resources of Yunnan Province of China …………………………………………………………………………………… J.C. Rana: Buckwheat Genetic Resources Management in India …………………………………. E. S.Alekseyeva, E. I. Kascheyeva: Collection of Buckwheat (Fagopyrum Mill.) Genetic Resources in Ukraine …………. B.K. Baniya, D.M.S. Dongol, B.K. Joshi and D.R. Sharma: Indigenous Cultural Practices and Social Perception for Conservation of Buckwheat Diversity in Nepal ……………………………………………………………………… Ludmila M. Moiseenko, Alexey A. Moiseenko, Alexey G. Klykov: Buckwheat Selection Work for Large Grain …………………………………………… Sabine Scheucher: Buckwheat in Tibet (TAR) ……………………………………………………………... Tatiana N. Lazareva, Ivan N. Fesenko: Electrophoresis Spectra of Total Seed Proteins of Artificial Amphidiploid Fagopyrum giganteum Krotov and its Parental Species F. tataricum Gaertn and F. cymosum Meisn. …………………………………………………………………………………... M. Dutta: Buckwheat Improvement in India: Current Status and Future Prospects ……………… Anohina T.A., Luzhinskaya N.A.: About Attributes Linking Described Crop Capacity and Buckwheat Plant Habitus ……….. Kyoko Yamane, Koji Tsuji, and Ohmi Ohnishi: Speciation of Fagopyrum tataricum Inferred from Molecular Data …………………… Ludmila M. Moiseenko, Valentina F. Pedochenko: Utilization of un Common Methods in Selection for Development of New Initial Buckwheat Material ......................................................................................................... Nikhil K. Chrungoo and Anusuya: Genetic Diversity in Accessions of Himalayan Buckwheats Revealed by SDS PAGE of Soluble Proteins Extracted from Single Seeds and RAPD Based DNA Fingerprinting …………………………………………………………………………..

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Vilchinskaya L.A., Malina M.M, Alexeeva E.S.: The Inheritance of Quantitative Sings in Case of Saturating Crossing ………………… Byoung Jae Park, Jong In Park, Kwang Jin Chang and Cheol Ho Park: Characteristics of Genetic Resources in Tartary Buckwheat (Fagopyrum tataricum) ………………………………………………………………… Tristan Funk, Sai L.K. Hsam, Friedrich J.Zeller, Volker Mohler: Development and Characterization of RFLP Markers from Buckwheat (Fagopyrum esculentum Moench) …………………………………………………………………… Woo S. H., T. Omoto, H. S. Kim1, C.H. Park, C. Campbell, T. Adachi and S. K. Jong1: Breeding Improvement of Processing Buckwheat: Prospects and Problems of Interspecific Hybridization …………………………………………………………….. Hisayoshi Hayashi, Yingjie Wang, and Clayton Campbell: Gene Flow in Self-Pollinating Buckwheat …………………………………………….. A.P. Lakhanov: Morphophysiologic Status of Plants and its Change during Evolutionary Development of Some Species of Genus Fagopyrum Mill …………………………………………… Junyi Ma, Shuqing Yang, Jianyu Wang: Seed Selection of New Buckwheat Breed and Reviews of the Effects ………………… G.N. Suvorova, A.N. Fesenko, M.A. Fesenko: Possibilities of Micropropagation Technique Application in Buckwheat Breeding …… Qing-Fu Chen, Sai L.K. Hsam and Friedrich J. Zeller: Cytogenetical Studies on Diploid and Autotetraploid Common Buckwheat (Fagopyrum esculentum Moench) and the Production of Autotriploid and Trisomic Lines ……………………………………………………………………………………. Taranenko L.K., Yatsishen O.L., Karazhbej P.P., Taranenko P.P.: State and Prospects of Buckwheat Selection in Ukraine ………………………………. Fesenko A.N., Fesenko N.V.: Effect of Allele Lsb on the Metameric Architectonics and Productive Characteristics of Buckwheat Plant ……………………………………………………………………….. Alexeeva E.S., Kushnir V.P., Peluiko Z.I., Homina V.Y., Gavrilyanchik R.Y: Prospects of Green-Floral Buckwheat in Selection and Plant-Growing ………………. N.V.Fesenko, V.V.Kolomejchenko, G.E.Martynenko, V.I.Savkin: Production Process Habits of Contemporary Buckwheat Varieties of New Generation . Aleksey N. Fesenko, Ivan N. Fesenko, Leonid V. Golyshkin and Nikolay V. Fesenko: Study of Homeotic Mutation Atl in Buckwheat, and Some Reflections about Origin and Evolution of Inflorescence ………………………………………………………… V. Dvořáček, P. Čepková, A. Michalová, I. Kreft: Seed Storage Protein Polymorphism of Buckwheat Varieties (Fagopyrum esculentum Moench; Fagopyrum tataricum L.) ……………………………………………………. Nikolay N. Fesenko: Spontaneous Polyploidy and Fertilization Independent Seed Development as Manifestation of Apomixis in Common Buckwheat Fagopyrum esculentum Moench. .. Zongwen Zhang, M.P. Upadhayay, Zuocheng Zhao, Rufa Lin, Ming-De Zhou, V. Ramanatha Rao, Percy E. Sajise: Conservation and Use of Buckwheat Biodiversity for the Livelihood Development ….

336 342 346 350 355 360 368 375

381 391 397 401 404 407 412 419 422

Section IV. Cultivation and Plant Nutrition Page B. Halbrecq and J.F. Ledent: Evolution of Flowering and Ripening Within Inflorescence of Buckwheat Plants, Effect of Reduction of Sinks and Seed Setting of the Two Floral Morphs ...................... 433

D.M.S. Dongol, B.K. Baniya and B.K. Joshi: Psychosocial Basis of Cultivation ad Food Preference of Nepalese Buckwheat Growers ………………………………………………………………………………… Nechaev L.A., Kazmin V.M., Putintzev A.F.,Korotkov A.N., Lobatch V.N.: Fertilizer of Buckwheat in the Central Forest Steppe Zone of Russia ………………… Podolska Grażyna, Mazurek Jan: The Biology of Flowering and Fructification of Buckwheat in Relation to Nitrogen Fertilization Doses ……………………………………………………………………… R. Omidbaigi, G. De Mastro, K. Bahrami: Influence of Nitrogen and Phosphorus Fertilization on the Grain Characteristics of Buckwheat (Fagopyrum esculentum Moench) ………………………………………… Z.I.Glazova: Alternative Fertilizer for Buckwheat ………………………………………………….. Young Ho Yoon, Dong Chil Jang, Jin Cheol Jeong: Effect of Soil Moisture Condition on Some Growth Characteristics Related With Landscape and Yield of Buckwheat ……………………………………………………. G.V.Pirogovskaya, A.M.Rusalovitch, V.I.Soroko, O.P.Sazonenko, O.E.Shakovets: Efficiency of New Forms of Mineral Fertilizers for Field Grown Buckwheat on Light Textured Soils ………………………………………………………………………….. Jacek Kwiatkowski, Stefan Szczukowski, Józef Tworkowski: Production of Buckwheat Seeds on Soil of a Good Wheat Soil Suitability Complex .... Yongliang Wang: Performance of Buckwheat Varieties in the National Coordinated Trial at Erdos Location ………………………………………………………………………………… A. Gupta, H.S. Gupta, B. Chaudhri and D.D. Singh: Increasing Production and Productivity of Buckwheat in Uttaranchal ………………… J. Kalinová, B. Voženílková, J. Moudrý: Occurrence of Fusarium spp and Bacteria on Surface of Buckwheat Achenes (Fagopyrum esculentum Moench) ……………………………………………………… Valentyna Shevchuk: Phytopathological Monitoring Fagopyrum esculentum Moench ……………………… Markku Kontturi, Marjo Keskitalo , Elise Ketoja: Buckwheat Cultivars in the North ……………………………………………………… Zlotnikov A.K., Stefanina S.A., Kirsanova E.V., Glazova Z.I.: Influence of Biopreparation "Albit" on Sowing Characteristics and Yield Qualities of Buckwheat Seeds ……………………………………………………………………….. Bardijan T.G., Shashko M.N.: Influence of Buckwheat Straw Ploughing on Phytosanitary of Soil State and Productivity of Spring Crops……………………………………………………………. Kulikov N.I., Naumkin V.P.: Plant - Insect Relation in Buckwheat Agrocoenosis …………………………………… Podolska Grażyna, Podolski Bogusław: Yield, Yield Components and Fruit Quality of Buckwheat Response to Soils Conditions and Cultivars ………………………………………………………………. S.N. Slobodyan: Reaction of Buckwheat to Sowing Terms and Fertilizing Conditions in Northern Steppe of Ukraine …………………………………………………………. Novikov V.M., Nechaev L.A.: Agrienergetic Efficacy of Systems of the Basic Soil Cultivation at Cultivation of Buckwheat ……………………………………………………………………………… Hongmei Li, Junsheng Bian, Xia Liang, Xiaoyan Deng, Fang Shan, Rufa Lin: The Effects of Fertilization on Botanic Characteristic and Yield of Tartary Buckwheat (F. tataricum) …………………………………………………...

440 445 451 457 461 465 470 475 481 484 491 494 496 499 501 505 509 515 519 524

J. Kalinová: Influence of Common Buckwheat on Growth of Other Plant Species ............................ 529 Kadyrov R.M.: About the Opportunity of Associative Nitrogen-Fixed and Phosphate-Mobilized Bacterium Use in Buckwheat Breeding ………………………………………………... 532 Z.I.Glazova, V.I.Mazalov, A.D.Zadorin: New Methods in Technology of Buckwheat Cultivation ……………………………… 537 A. Brunori, G. Baviello, G. Di Giorgio: Enhancing the Production and the Use of Buckwheat in Food Preparation in Italy …… 541

Section V. Food Processing, Health and Functional Food Page Lin Rufa, Ren Jianzhen, Shen Wei: An Observation of the Effect of Tartary Buckwheat Tea on Lowering Blood Glucose .. 543 Masatoshi Kano, Tadashige Kizaki, and Toshiyuki Inasawa:

The State of Cultivating Tartary Buckwheat (Fagopyrum tataricum) and its Traditional Dishes along Everest Trekking Route, Nepal ……………………… 547 G. Wieslander D. Norbäck, Z.-H.Wang, , Z.-H. Zhao, Y. Mi, Y.-Y. Li, Z. Zhang:

Buckwheat Allergy among Schoolchildren in Urban and Rural Parts of Shanxi Province, China ..................................................................................................... 553 T. Maeda, K. Miyake, M. Tahara and N. Morita: Effect of Buckwheat Substitution for Wheat Flour on Pasta and Cookie ……………… 559 Roman Przybylski, Julianne M. Kawa and Carla G. Taylor:

Buckwheat Concentrate Reduces Serum Glucose in Streptozotocin-diabetic Rats ……………………………………………………………………………... 565 Simon Kwong, Guixing Ren, Alan Yeung, Rufa Lin, Fang Shan, Hongmei Li, Xiaoyan Deng and Xia Liang:

Tartary Buckwheat Cultivation According to SFDA Good Agricultural Practice (GAP) Guidelines for Traditional Chinese Medicine. I.The Environment …….. 576 Fang Shan, Hongmei Li, Junsheng Bian, Xiaoyan Deng, Qiuyan Sun, Rufa Lin, Guixing Ren, Alan Yeung, Simon Kwong: Tartary Buckwheat Cultivation According to SFDA Good Agricultural Practice (GAP) Guidelines for Traditional Chinese Medicine. II. High Quality Tartary Buckwheat Production Technology and Tartary Buckwheat Quality Management ……………….. Paulíčková I., Vyžralová K., Holasová M., Fiedlerová V., Vavreinová S.: Buckwheat as Functional Food ………………………………………………………… Colin J. Briggs, Clayton Campbell, Grant Pierce and Ping Jiang: Bioflavonoid Analysis and Antioxidant Properties of Tartary Buckwheat Accessions ... Peng Chen, Yuhong Li, Xuejun Li: Germination Improved the Nutrient Value of Buckwheat ……………………………… A.P.Lahanov, R.S.Muzalevskaja, N.V.Shelepina, I.V.Gorkova: Biochemical characteristics of some species of genus Fagopyrum Mill ………………. Hiroyuki Yoshioka, Natsuko Yasueda, Tsuyoshi Ohmoto, Atsuo Urisu, Yoshinori Mine, Rikio Yamazaki and Taiji Adachi: cDNA Isolation and Epitope Analysis of Allergenic Proteins in Autogamous Common Buckwheat .............................................................................. Dorota Dietrych-Szóstak: Flavonoids in Hulls of Different Varieties of Buckwheat and Their Antioxidant Activity …………………………………………………………………………………

581 587 593 599 605

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Byoung Jae Park, Jong In Park, Kwang Jin Chang and Cheol Ho Park: Comparison in Rutin Content in Seed and Plant of Tartary Buckwheat (Fagopyrum tataricum) ……………………………………………………………………………… Zhao Gang, Tang Yu,Wang Anhu, Hu Zhu:

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China’s Buckwheat Resources and Their Medicinal Values …………………… 631 D. Norbäck, Z.-H.Wang, Z.-H. Zhao, Y. Mi, G. Wieslander Z. Zhang: Buckwheat Allergy, Asthma, and a History of Atopic Sensitisation among Students at Shanxi University, China ................................................................... 634 Dolores Ciepielewska, Łucja Fornal: Natural Resistance of Buckwheat Seeds and Products to Storage Pests ………………. L.A. Zemnukhova, E.D.Shkorina, G.A. Fedorishcheva, S.V. Tomshich, G.A. Klykov: Buckwheat Processing Waste as a Raw Material for Chemical Industry ……………… Sayoko Ikeda, Kazue Tomura, Miyuki Miya and Ivan Kreft: Buckwheat Minerals and Their Nutritional Role ………………………………………. Silvia Melicháčová, Alena Vollmannová, Tomáš Tóth: Influence of Soil Properties Changes on Content of Some Risk Elements in Buckwheat K. Miyake, R. Morita, T. Handoyo, T. Maeda and N. Morita: Characteristics of Graded Buckwheat Flours and Functional Properties of Germinated Buckwheat ……………………………………………………………………………… Byoung Jae Park and Cheol Ho Park: Cytotoxic Activities of Tartary Buckwheat Against Human Cancer Cells…………….. Zhao Gang, Wang Anhu, Tang Yu, Hu Zhu: Research on the Nutrient Constituents and Medicinal Values of Fagopyrum cymosum Seeds ……………………………………………………………………………………. Carolin Ölschläger, Dieter Treutter, Friedrich J. Zeller: Breeding Buckwheat (Fagopyrum esculentum Moench) for Flavonoids ……………… Wit Chmielewski: Insects (Insecta) and Some Other Arthropods Found i Buckwheat Products ………….. Kiyokazu Ikeda, Yuya Asami, Rufa Lin, Rie Arai, Ivan Kreft: Characteristics Concerning the Nutrition and Palatability of Buckwheat Products ……. Chai Yan, Feng Baili, Hu Yin-gang,Gao Jinfeng, Gao Xiaoli: Analysis on the Variation of Rutin Content in Different Buckwheat Genotypes ……… Tatsuro Suzuki, Yutaka Honda and Yuji Mukasa: Effects of Lipase, Lipoxygenase and Peroxidase on quality deteriorations in buckwheat flour ………………………………………………………………………… Wang Z. H., Shi X. R., Chang W J, Jing W, Zhang Z., Wieslander G., Norbäck D.: Isolation and Functional Identification of an Allergenic Protein from Tartary Buckwheat Seeds ……………………………………………………………………….. Jeong-Lim Kim, Gunilla Wieslander, Dan Norbäck:

640 647 651 654 661 666 670 674 980 685 689 693 700

Allergy /Intolerance to Buckwheat and Other Food Products among Swedish Subjects with Celiac Disease ................................................................................ 706 Gorkova IrinaVyacheslavovna, Pavlovskaya Ninel Efimovna:

Comparative Characteristic of Buckwheat Genotype on Ontogenesis Flavonols Accumulation ........................................................................................................ 711 Junsheng Bian, Fang Shan, Zhifang Tian, Guangying Xu, Rufa Lin, Xue Chunsheng, Duan Yali, Ju Mingjie: Study on New Health Foods of Tartary Buckwheat …………………………………… 715 J. Kalinová, E. Dadáková: Varietal Differences of Rutin in Common Buckwheat (Fagopyrum esculentum Moench) Determined by Micellar Electrokinetic Capillary Chromatography …………. 720 Wang Min, Wei Yi-min, Gao Jin-ming:

Analysis of Fatty Acid and Unsaponifiable Matter from Tartary Buckwheat Oil and Buckwheat Oil by gc/ms …………………………………………………… 724

Andrej Šorgo, Blanka Vombergar: Computer Based Experiments with Buckwheat in Food Processing Vocational Education ………………………………………………………………………………. 731 Václav Dvořáček, Petra Čepková, Anna Michalová: Protein Content Evaluation of Several Buckwheat Varieties ………………………….. 735

Section I. Keynote Presentations

Proceedings of the 9th International Symposium on Buckwheat, Prague 2004

The Development and Importance of IBRA, the International Buckwheat Research Association Ivan Kreft University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, SI-1001 Ljubljana, Slovenia

ESTABLISHMENT AND ACTIVITIES OF IBRA The first International symposium on Buckwheat (ISB) was organized in Ljubljana, Slovenia in September 1-3, 1980. Authors of papers presented at the first ISB were from Poland (M. Ruszkowski, K. Komenda, B. Komenda, J. Komenda-Ronka, R. Kubiczek, M. Dabrowski), Japan (T. Adachi, T. Yabuya, T. Nagatomo, O. Ohnishi, N. Katayama, T. Matano, A. Ujihara, and E. Tsuzuki), Russia (N.V. Fesenko), Belarus (E. Gorina), Denmark (B. O. Eggum), Bosnia-Herzegovina (M. Bogdanović) and Slovenia (B. Vombergar, B. Javornik, I. Kreft); although all mentioned scientists were not able to travel to Ljubljana, some of authors just send their papers. Foreign participants from Denmark (Björn O. Eggum), Poland (Barbara Ruszkowska and Marek Ruszkowski) and Japan (Toshiko Matano, Takashi Nagatomo and Taiji Adachi) and I. Kreft from Slovenia discussed, by eating Slovenian buckwheat dishes at the home of the present author, the possibilities for further regular organisation of International Buckwheat Symposia. Following the idea of Prof. M. Ruszkowski was in September 1980 in Ljubljana established the International Buckwheat Research Association (IBRA), which regularly organised further international buckwheat symposia in Japan (Miyazaki, 1983), Poland (Pulawy, 1986), Russia (Orel, 1989), China (Taiyuan, 1992), Japan (Ina, 1995), Canada (Winnipeg, 1998), and Korea (Chunchon, 2001; see Table 1). For the 2nd ISB there have been two candidate countries: Japan (Miyazaki) and Poland (Pulawy). However, due to the turbulences in Poland in that time, 2nd ISB was organised in Japan, and then the 3rd in Poland. There have been always several countries candidates to organise ISB, and the decisions were not easy, it was decided by voting at the end of each ISB about the place of the next one. There were some countries or regions which were officially discussed to organize International Symposia under auspices of IBRA, but had, from different reasons, or simply because they did not get at that time yet enough high priority on IBRA assembly, to postpone the possibility to organize official symposia at some other time. Such countries candidates were Scandinavia (Denmark or Sweden), Italy, Ukraine, India, Australia and Germany. The present author hope that most of them will sooner or later get the possibility to organize the official Symposia. Voting body for the next IBS, for election of President, and on some ISB as well for electing new members of IBRA board or Fagopyrum editor, is Assembly General of IBRA, which is consisted of all buckwheat scientists, paying regularly their subscription fee for Fagopyrum Journal; the Assembly take place normally just before the end of each ISB. In meantime between official ISBs there have been in Europe as well some regional buckwheat symposia with international participation, like in Italy (Teglio, 1995), Norway (Larvik, 1996), Czech Republic (Prague, 1997), Luxembourg (1999), again in Italy (Teglio, March 2000; and in Sondrio and Teglio in October 2000 - a bigger Congress with lecturers and other participants from many European countries, and from China, Japan and Korea). Interim international symposia dedicated to buckwheat, but not officially under the auspices of IBRA, are newly organized in several countries, like in Italy as a part of

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post-symposium excursion of this ISB (in August 27-28, 2004 in Teglio), in August 2004 in Sichuan (China, on Buckwheat Ethnobotany), and in October 2004 in Tianjin (China). Buckwheat Botany and Utilization Symposium is organized in Maribor (Slovenia, Aug. 23-26, 2004, as well as a part of the post-symposium tour). Several regional buckwheat symposia were organized as well in Poland, Ukraine, Russia and in China. In 1981 started in Ljubljana, Slovenia the international scientific journal on buckwheat »Fagopyrum«, 14 volumes were published in Ljubljana (started as a bulletin), and in 1995 the editorial office was moved to Ina, Japan, where the 15th volume was published in 1998 (editor Prof. T. Matano), and further volumes are published in Kyoto (editor Prof. Ohmi Ohnishi). The financial situation of Fagopyrum Journal is generally a big problem, however usually it is slightly improved just after each ISB, while for several members or country representatives it is at ISB a suitable opportunity to pay to the editorial office their delayed payments. Slovenian international buckwheat web page was established (http://www.bf.uni-lj.si/Fagopyrum/), covering as well the contents of some issues of Fagopyrum Journal, and several links. Up to now, IBRA had no other list of members or other membership-fee than the list of addresses of scientists who are subscribed to Fagoyprum Journal and pay regularly their individual annual subscription fee, either through their regional or country representative or directly to the Editorial office in Kyoto. It is a paradox situation, that some small countries pay their annual subscription fee for Fagopyrum Journal regularly, but from some big countries it is received less regularly. It is understandable that finances are collected only through the Fagopyrum, as this is the most important IBRA activity between ISBs. The current administration expenditure, which is the only expenditure of IBRA besides the Fagopyrum Journal, has always been covered by the domestic institution of the President and/or Secretary General, when this function existed. At the end of each ISB, the new president of IBRA was elected, it was always one of the organisers of the current symposium. In some symposia (Orel and Winnipeg) the new board was elected. In Orel, the secretary general was elected (I. Kreft), who served as additional help to the president in the coordination between ISB. In Winnipeg completely new board was elected, besides Prof. O. Ohnishi as the editor of Fagopyrum journal, members of the board were representatives of different countries or regions. Among other, Europe was represented by G. Wieslander (for Northern Europe, including Poland and Russia) and I. Kreft (For Southern Europe, including Luxembourg, Czech Republic and Ukraine). INTERNATIONAL COOPERATION IN BUCKWHEAT RESEARCH AND TECHNOLOGY Buckwheat breeding is quite complicated because of complicated system of selfincompatibility. This is a possible explanation for relatively slow progress in achievements of higher yield in buckwheat. So it is understandable that an important part of IBRA activities was devoted to genetics (KREFT, 1983; OHNISHI, 1990; RUSZKOWSKI, 1990). It has been some attempts, especially in Poland by Professor Marek Ruszkowski, to breed hommostyle buckwheat varieties, which could be less dependent on pollination, and easier to obtain inbreed lines. But in hommozygote selfcompatible buckwheat varieties it would be lost the heterosis, present in such type of populations. The countries of most intense scientifically based buckwheat breeding, which started prior to establishing IBRA, were Russia, Ukraine, Belarus, Poland, Japan, Canada and USA. In Russia, Poland and Japan were among other developed varieties as well some tetraploid buckwheat varieties, based on own original polyploidisation of diploids. Interspecific 12


crossing of buckwheat started early in Russia, but later was highly developed as well in Japan, Canada and in some other countries. A lot of buckwheat breeding was done as well in China, but it was mostly the improvement and development of domestic varieties. Some buckwheat breeding, resulting in widely known buckwheat varieties, was performed as well in France, Austria, Czech Republic, Slovenia and in some other countries. Establishment of IBRA promoted strong international communication among scientists investigating and developing buckwheat cultivars, including the international exchange of buckwheat genetic material. Internationally most known buckwheat gene banks are in Russia (Sankt Petersburg), China (Beijing), Czech Republic (Prague) and Slovenia (Ljubljana). In recent years one of the prime countries for intense buckwheat breeding is Canada; scientifically based buckwheat breeding is promising a great success, especially for buckwheat for export, mainly to Japan, but with interest to export as well to Korea and to some other countries. Quality of buckwheat is more and more important. In most countries, buckwheat is in modern time not eaten mainly to satiate the people’s hunger and to get food energy. Buckwheat is mainly eaten because of its taste, for variety in the menu, for its tradition, and mostly combined with the knowledge about the importance of buckwheat food products for human health. These demands and reasons for consuming buckwheat may be met by buckwheat products of highest possible quality, with typical and clearly pronounced taste and with the content of all nutrients, important for human health¸ and free from artificial chemicals. Buckwheat is very rich in trace elements (for example Zn, Cu, Mn and Se), however, it must be grown in unpolluted areas, to avoid accumulation of contaminating elements. Different milling fractions may have different content of minerals and proteins, dark flours being generally more rich than the light ones (IKEDA ET AL., 2000; SKRABANJA ET AL., 2004). Buckwheat has a significant content of rutin (quercetin-3-rutinosid) and other polyphenols (LUTHAR, 1992). The content of rutin is listed among one of the most important characteristics from the viewpoint of nutrition (MICHALOVA, 1998). Rutin, quercetin and some other polyphenols may be, in moderate amounts, potent anticarcinogens against colon and other cancers. The anticarcinogenic and antimutagenic potentials may be related to their antioxidative property, which is important in the protection against cellular oxidative damage. Phenolic compounds may lower the blood sugar and lipid levels and contribute to the hypocholesterolaemic effect. Rutin is mainly located in flowers and in green parts of buckwheat plant. In seeds there is less rutin than in leaves, but there could be some rutin as well in buckwheat flours, more in the dark than in the light ones. Buckwheat samples may differ according to rutin content in regard to the genotype, growing conditions and technology (PARK ET AL., 2000). HAGELS (1998) published many interesting new data on rutin and other secondary metabolites of buckwheat. Buckwheat has a very low content of prolamins and, based on chemical and immunological studies, it may be a valuable source of dietary protein for gluten-sensitive individuals (SKERRITT, 1986). Buckwheat groats are very popular in Slovenia, Poland, Ukraine and Russia. Groats are obtained by husking of hydrothermaly treated (pre-cooked) buckwheat grain. Technology of some buckwheat products could be based on buckwheat groats obtained by traditional technology, namely husking buckwheat grains after the hydrothermal pretreatment (soaking or cooking in boiling water), to obtain hard buckwheat groats (or kasha), which is then further cooked and prepared for consumption. It is as well a need 13


for a husking device, suitable for husking untreated, raw buckwheat grains. In buckwheat groats, it is less digestible starch (than in white wheat bread (SKRABANJA ET AL., 2001). The slowly digested or non-digested starch of buckwheat groats could exhibit effects similar to those of dietary fibre. It could be nutritionally important to diabetics, as it helps to flatten the glycaemic response curve. A slow release of glucose from the starch could prolong endurance during physical activities and the duration of satiety is prolonged as well. Scientific institutions in Slovenia developed cooperation through IBRA, and directly, around the world. Buckwheat research is in Slovenia performed by the Biotechnical faculty, University of Ljubljana, by Faculty of Agriculture, University of Maribor, the College of Food Technology, Maribor; Jožef Stefan Institute, Ljubljana and the National Institute of Biology, Ljubljana. We cooperate with the scientists in Italy (The Cereal Research Institute in S. Angelo Lodigiano near Milano and the National Institute of Nutrition, Rome) on buckwheat growing in the Alpine region, respectively on the development of new buckwheat products of high nutritional and functional quality, like buckwheat pasta, extruded products etc. Important international cooperation of University in Ljubljana on buckwheat genetic polymorphism and nutritional quality is performed with the Japanese scientists at Kyoto University and Kobe Gakuen University. Applied research of Slovenian scientists contributed to the development in buckwheat growing and the new products in Slovenia, Croatia (buckwheat groats), Denmark (extruded buckwheat products), Japan and Australia (»Tasmania soba« export for Japan). Slovenian scientists have established close research contacts as well with scientists and buckwheat experts in Austria, Czech Republic, Germany, Luxembourg, France, Sweden (Uppsala University), Norway, Poland, Ukraine, Russia, India, China, Korea and in some other countries. Tab. 1 The list of official international symposia on buckwheat. No. 1st 2nd 3rd 4th 5th 6th 7th 8th 9 th 10

Year 1980 1983 1986 1989 1992 1995 1998 2001 2004 2007

Place Ljubljana Miyazaki Pulawy Orel Taiyuan Ina Winnipeg Chunchon Prague ?

Country Slovenia Japan Poland Russia China Japan Canada Korea Czech R. ?

Main organisers I. Kreft, B. Javornik, B. Vombergar (Dolinšek) et al. T. Nagatomo, T. Adachi et al. M. Ruszkowski et al. N. V. Fesenko et al. Lin Rufa et al. T. Matano, A. Ujihara et al. C. Campbell et al. C. H. Park, S. S. Ham, Y.S. Choi, N.S. Kim et al. A. Michalova, Z. Stehno et al. ?

…

…

…

…

…

th

ACKNOWLEDGEMENTS The research reported in this paper was partly supported by the Ministry of Education, Science and Sports of Slovenia and the Ministry of Agriculture, Food and Forestry of Slovenia.

REFERENCES HAGELS, H. (1998): Sekundäre Pflanzeninhaltsstoffe des Buchweizens; Die Wirkungen des Rutins; Gewinnung von Rutin aus Buchweizenblättern; In: Das Buchweizenbuch. Editions Saint-Paul, Luxembourg.

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IKEDA, S., YAMASHITA, Y., KREFT, I. (2000): Essential mineral composition of buckwheat flour fractions. Fagopyrum, 17: 57-61. KREFT, I. (1983): Buckwheat breeding perspectives. Proc. 2nd Int. Symp. Buckwheat., Miyazaki. Buckwheat Research, 3-12. LUTHAR, Z. (1992): Polyphenol classification and tannin content of buckwheat seeds (Fagopyrum esculentum Moench).- Fagopyrum 12, 36-42. MICHALOVÁ, A. (1998): Study of relationships between yield and quality characters of common buckwheat (Fagopyrum esculentum Moench.). In: Advances in Buckwheat Research 7, IBRA, Winnipeg, Manitoba, Canada, I - 88-96. MICHALOVÁ, A. (1998): Formation and study of collections of selected alternative crops. Res. Reports 71, 109-113. MICHALOVÁ, A. (1998): Variability of selected characteristics in sets of buckwheat, millet and amaranth; Selection of perspective genotypes and comparison of their nutritive value. Res. Reports 71, 115-125. OHNISHI, O. (1990): Analyses of genetic variants in common buckwheat, Fagopyrum esculentum Moench: A review. Fagopyrum 10: 12-22. PARK, C.H., KIM, Y.B., CHOI, Y.S., HEO, K., KIM, S.L.,LEE, K.C., CHANG, K.J., LEE, H.B. (2000): Rutin content in food products processed from groats, leaves and flowers of buckwheat. Fagopyrum, 17: 63-66. RUSZKOWSKI, M. (1990): The phenomenon of dominance, competition, compensation and overcompensation in buckwheat productivity. Fagopyrum, 10: 5-6. SKERRITT, J.H. (1986): Molecular comparison of alcohol-soluble wheat and buckwheat proteins. Cereal Chem. 63, 365-369. SKRABANJA, V., LILJEBERG ELMSTAHL, H.G.M., KREFT, I., BJÖRCK, I.M.E. (2001): Nutritional properties of starch in buckwheat products: studies in vitro and in vivo. J. Agric. Food Chem., 49, 490-496. SKRABANJA, V., KREFT, I., GOLOB, T., MODIC, M., IKEDA, S., IKEDA, K., KREFT, S., BONAFACCIA, G., KNAPP, M., KOSMELJ, K. (2004): Nutrient content in buckwheat milling fractions. Cereal Chem., 81, 172-176.

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On the Origin of Cultivated Buckwheat Ohmi Ohnishi Plant Germ Plasm Institute, Graduate School of Agriculture, Kyoto University, Nakajo 1, Mozume-cho, Mukoh 617-0001, Japan ABSTRACT After OHNISHI (1998b)’s assertion for the origin of cultivated buckwheat, major progress on this subject has come from the discovery of new natural populations of the wild ancestor of common buckwheat, F. esculentum ssp. ancestrale. They were found in the Muli district of Sichuan province, in several spots in the Deqin district of Yunnan province and in many places in the Mangkang districts of Eastern Tibet. Allozyme and AFLP analyses on these natural populations along with those of cultivated populations in Southwestern China revealed that the natural populations from Eastern Tibet and from several populations from the Deqin district of Yunnan province are genetically the closest related to the cultivated populations. This strongly suggested that Eastern Tibet was the most probable place for the origin of cultivated common buckwheat. Meanwhile, a series of RAPD and AFLP analyses of wild and cultivated Tartary buckwheat (TSUJI AND OHNISHI, 2000,2001a,b) revealed that natural populations from central Tibet are the most closely related to cultivated Tartary buckwheat. However, several populations in Eastern Tibet and in the border areas between Sichuan, Yunnan and Eastern Tibet are also very closely related to cultivated Tartary buckwheat. TSUJI AND OHNISHI (2001a) therefore concluded that these Eastern Tibetan wild Tartary buckwheats which are genetically similar to the cultivated populations are strong candidates for being the wild ancestor of cultivated Tartary buckwheat. Keywords: AFLP, allozymes, common buckwheat, F. esculentum ssp. ancestrale, F. tataricum ssp. potanini, Sanjiang area, Tartary buckwheat

INTRODUCTION Two domesticated buckwheat species, common buckwheat (Fagopyrum esculentum) and Tartary buckwheat (F. tataricum), are mainly cultivated in the temperate zones of the Northern hemisphere. Common and Tartary buckwheat are not usually major crops in any region, but they are occasionally a staple food in some regions in China and in the Himalayan hills. DE CANDOLLE (1883) considered that common buckwheat originated in Siberia or in the area of the Amur River. His conjecture was mainly based on philological considerations and on the reported existence of wild common buckwheat by Russian taxonomists (LEDEBOUR 1841, MAXIMOWICZ 1859). No proper name of buckwheat, however, has been found in ancient Chinese nor in Sanskrit which this implies that buckwheat did not originate in China nor in India. In the second half of the 19th century, botanical expeditions to China by European scientists revealed that wild buckwheat species were found growing only in Southern China (for a historical account of this botanical discovery in China, see BREDTSHNEIDER, 1898). STEWARD (1930) summarized the classification and distribution of the Polygoneae species in eastern Asia. Based on Steward’s enumeration of wild buckwheat species in Southern China, NAKAO (1957) asserted the Southern China hypothesis for the origin of common buckwheat. In Russia, many Russian scientists insisted that Tibet or the Himalayn hills were the original birthplace of buckwheat (see KOMAROV, 1938; KROTOV, 1960) as they had already decided that the Northern China-Siberian hypothesis was no longer valid. When I began to study the origin of cultivated buckwheat, which was around 1980, the wild ancestral species of cultivated buckwheat was entirely unknown. No candidate for the wild ancestor of cultivated buckwheat could be found in the list of wild species belonging to the genus Fagopyrum as enumerated by STEWARD (1930). Only F. cymosum, which is morphologically similar to common buckwheat, was a well known wild species of Fagopyrum. 16


Since my discovery of the wild ancestor of common buckwheat, F. esculentum ssp. ancestrale, in the Yongsheng district of Yunnan province in 1990 (OHNISHI, 1991), the issue of the origin of cultivated common buckwheat has rapidly progressed, mainly by isozyme analysis and by molecular phylogenetic analyses of the wild species in Fagopyrum. OHNISHI (1998b) summarized these studies which clarifies the wild ancestor of common buckwheat (F. esculentum ssp. ancestrale) and of Tartary buckwheat (F. tataricum ssp. potanini) and their distribution areas. In this article I will report on the recent progress on the issue of the origin of cultivated buckwheat which has mainly come from the discovery of new natural populations of wild ancestors of buckwheat and from the allozyme, RAPD and AFLP analyses on these newly found populations. WILD SPECIES OF FAGOPYRUM STEWARD (1930) described only eight wild species of Fagopyrum in his taxonomic studies on east Asiatic Polygoneae species (at that time, all Fagopyrum species were assigned under the genus of Polygonum). These wild species did not contain any strong candidate for being the wild ancestor of cultivated common buckwheat. Hence, F. cymosum, which had been a well known wild species, was thought to be the wild ancestor of common buckwheat (NAKAO, 1957). Later, OHNISHI (1998a) added four new species, F. pleioramosum, F. callianthum, F. capillatum, F. homotropicum and one subspecies F. esculentum ssp. ancestrale which were found in the Sichuan and Yunnan provinces of China. OHSAKO AND OHNISHI (1998) added two new species, F. rubifolium and F. macrocarpum from Sichuan province. Finally OHSAKO ET AL. (2002) added two more new species, F. jinshaense and F. gracilipedoides from Yunnan province. Among these new species and new subspecies, F. esculentum ssp. ancestrale was proposed to be the wild ancestor of common buckwheat judging from its morphological similarity, except for several cultivated plant specific characters such as it’s seed shattering habit and small achenes (OHNISHI, 1991). As for the wild ancestor of Tartary buckwheat, OHNISHI (1998b) proposed that the less-known F. tataricum ssp. potanini was the wild ancestor of cultivated Tartary buckwheat from comparative studies on allozyme variations, and he also reported on the exact distribution of this wild ancestor. During the last ten years, the molecular phylogenetic relationships between the species were extensively studied and the taxonomic relationships among the wild species are now well established (OHNISHI AND MATSUOKA, 1996; YASUI AND OHNISHI, 1998; OHSAKO AND OHNISHI, 2000, NISHIMOTO ET AL., 2003), although several new controversial issues have arisen. There now remains no ambiguity on the wild ancestral species of common and Tartary buckwheat with them being F. esculentum ssp. ancestrale and F. tataricum ssp. potanini, respectively. DISCOVERY OF NEW POPULATIONS OF THE WILD ANCESTORS The subspecies, F. esculentum ssp. ancestrale, was first discovered in the Yongsheng district of Yunnan province (OHNISHI, 1991). However, this subspecies was later also discovered in the Yanyuan and Muli districts of Sichuan province, in the Lijiang and Deqing districts of Yunnan province, and in the Mangkang district of Eastern Tibet (OHNISHI AND KONISHI, 2001; OHNISHI, 2002). This subspeceis grows on rocky mountain slopes, on stony barren lands, on barren slopes after a landslide, which is where most weed plants rarely invade. Plants of wild common buckwheat usually form large natural populations, usually consisting of more than 10,000 individuals. As for the wild ancestor of cultivated Tartary buckwheat, OHNISHI (1998b) proposed that the wild subspecies of Tartary buckwheat F. tataricum ssp. potanini, which was first found by the Russian botanist G. N. Potanin in Gansi province of

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China, was the wild ancestor of cultivated Tartary buckwheat. OHNISHI (1998a) clarified the distribution of this wild Tartary buckwheat. It was found to grow in the Gansi, Sichuan, Qinhai, Yunnan and Tibet provinces of China. It was frequently found in Northern Pakistan and in Kashmir, but wild Tartary buckwheat was quite rare in Nepal, Shikkim and Bhutan. F. tataricum ssp. potanini usually forms small populations consisting of 30-500 individuals scattered over mountain slopes, on natural barren lands, by roadsides and on the margins of farmers’ field or even in cultivated fields. The locations where the wild ancestors have recently been found are shown in Fig. 1. Fig. 1 Distribution of the wild ancestor of common and Tartary buckwheat in Sichuan and Yunnan provinces and eastern Tibet. F. esculentum ssp. ancestrale, F. tataricum ssp. potanini Cultivated populations of F. esculentum used for the allozyme study shown in Fig. 2.

DETAILED PHYLOGENETIC ANALYSES OF THE POPULATIONS OF WILD COMMON AND TARTARY BUCKWHEAT AND THEIR CULTIVATED COUNTERPARTS Genetic relationships among cultivated local populations and natural populations of the wild ancestor of cultivated buckwheat were analyzed using allozyme variability and RAPD and AFLP markers. The starch gel electrophoresis analyses of allozymes on cultivated and wild common buckwheat populations and on cultivated common buckwheat populations in northwestern Yunnan were the most variable and were found to keep both the F and S alleles at the Adh locus. The allelic frequencies of the F and S alleles gradually declined as the distance from 18


Northwestern Yunnan increased (OHNISHI unpublished). The Yunnan hypothesis for the origin of common buckwheat was strengthened by the RAPD analysis of cultivated common buckwheat (MURAI AND OHNISHI, 1996). Now the question was which natural population of the wild ancestor was the most closely related to cultivated common buckwheat. This question was tackled by allozyme and AFLP analysis (OHNISHI ET AL. unpublished; KONISHI ET AL. unpublished). Fig. 2 shows the N-J tree based on the Nei’s genetic distance calculated from the allelic frequencies at 19 loci for 12 enzymes. Among the natural populations of the wild ancestor, the populations from Eastern Tibet and the Adong population of northwestern Yunnan province are the most closely related to cultivated common buckwheat. This leads to the hypothesis of the origin of common buckwheat being in Eastern Tibet. AFLP analyses also gave the same results (data not shown). Fig. 2 The neighbourjoining (N-J) tree of the populations of common buckwheat and its wild ancestor, reconstructed from the genetic distance between populations based on 19 loci of 12 enzymes. See Fig. 1 for the location of populations.

Similarly, the issue of the origin of cultivated Tartary buckwheat was tackled by analyzing wild and cultivated populations using both RAPD and AFLP markers (TSUJI AND OHNISHI, 2000, 2001a,b). The original birthplace of cultivated Tartary buckwheat can not be estimated from the distribution of wild Tartary buckwheat alone, because its distribution extends from the Gansi province of China in the north, to the Yunnan province in the south, and from China in the east to Northern Pakistan in the west. This is in sharp contrast to the case of common buckwheat. Natural populations from Sichuan province are more variable, but the populations from central Tibet and from Northern Pakistan were most closely related with cultivated Tartary buckwheat. However, TSUJI AND OHNISHI (2001a) did not consider central Tibet to be the original birthplace of cultivated Tartary buckwheat. Rather, they noticed that wild Tartary buckwheat, with the same genotype as cultivated Tartary buckwheat, had been found in Eastern Tibet and in the border areas of Yunnan, Sichuan and Eastern Tibet. They thus arrived at the Eastern Tibetan hypothesis for the origin of cultivated Tartary buckwheat (TSUJI AND OHNISHI, 2001a).

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An allozyme survey of cultivated and wild Tartary buckwheat populations (OHNISHI 2000, 2002) also showed the same geographical distribution of the allozymes as did the RAPD and AFLP markers. Hence he reached the same conclusion for an Eastern Tibetan origin of Tartary buckwheat. SUPPLEMENTAL DATA ON THE EASTERN TIBET HYPOTHESIS OF ORIGIN OF COMMON AND TARTARY BUCWKEHAT Is the Eastern Tibet hypothesis for the origin of cultivated buckwheat supported by other evidence other than biological ones (distribution of wild species, genetic relationship between cultivated and wild buckwheat)? Who were involved in the domestication of buckwheat? At the present time the minority tribe in southern China who has most extensively utilized buckwheat is the Yi tribe in Sichuan province. They belong to the Tibet-Burmy linguistic group and are believed to have migrated from northern Sichuan to Yunnan and then went from west to east counterclockwise, first entering Quishou province from where they proceeded to Sichuan province (ZHANG, 1991; see also OHNISHI, 1998b). If this migrant tribe adopted buckwheat as a cultivated crop in Eastern Tibet during their migration then the southwestern part of China appears to be the primary center of domestication of buckwheat. The minority tribes in Southwestern China, such as Yi tribe, have their own hieroglyphic letters which mean buckwheat and Tartary buckwheat and they have inherited many folk tales and religious ceremonies that are related to buckwheat (LI AND YANG, 1992). Any archeological evidence for past buckwheat cultivation is very poor in China. However, WANG (1989) reported on the presence of archeological remains at a village near Chamdu of Eastern Tibet, which can be dated to 2600 BC. ACKNOWLEDGEMENTS The author is grateful to Dr. Clayton Campbell, Kade Research Ltd, Morden, Canada for his reading manuscript, correcting English and making numerous usefull suggestions.

REFERENCES BREDTSHNEIDER, E. (1898): History of European botanical discovery in China. Press Imp. Russ. Acad. Sci. Petersburg. DE CANDOLLE, A. (1893): L’Origine des plantes cultivees. Japanese translation by G. Kamo, 1941. Kaizosha, Tokyo. KOMAROV, V. L. (1938): Origin of cultivated plants. USSR. Lenin academy of agricultural Science, Leningrad. (in Russian) KROTOV, A. S. (1960): Historical accounts of buckwheat in Russia. Pp414-456 in Financial research of village economy and peasants in Russia. AHCCP. Moscow (in Russian) LEDEBOUR, C. F. (1841): Flora Rossica. Suntibus Librial E. Schweizerbart, Stuttgart. LI, Q. Y. AND M. X. YANG (1992): Preliminary investigation on buckwheat origin in Yunnan, China. Proc. 5th Intl. Symp. Buckwheat at Taiyuan: 44-46. MAXIMOWICZ, C. (1859): Primitiae Florae Amuruensis. Pp. 1-505 in Memoirs L’Academic Imperiale des Sciences par Divers Savants, Impriemrie de L’Academie Imperiale des Sciences, St. Petersburg. MURAI, M. AND O. OHNISHI (1996): Population genetics of cultivated common buckwheat, Fagopyrum esculentum Moench. X. Diffusion routes revealed by RAPD markers. Genes & Genet. Syst. 71: 211-218. NAKAO, S. (1957): Transmittance of cultivated plants through Sino-Himalayan rout. Pp. 397-420 in H. Kihara ed. Peoples of Nepal Himalaya. Fauna and Flora Research Society, Kyoto. NISHIMOTO, Y., O. OHNISHI AND M. HASEGAWA (2003): Topological incongruence between nuclear and chloroplast DNA trees suggesting hybridization in theurophyllum group of the genus Fagopyrum (Polygonaceae). Genes & Genet. Syst. 78: 139-153. OHNISHI, O. (1991): Discovery of wild ancestor of common buckwheat. Fagopyrum 11: 5-10.

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OHNISHI, O. (1998a): Search for the wild ancestor of buckwheat. I. Description of new Fagopyrum species and their distribution in China. Fagopyrum 18: 18-28. OHNISHI, O. (1998b): Search for the wild ancestor of buckwheat. III. The wild ancestor of cultivated common buckwheat, and of Tatary buckwheat. Economic Botany 52: 123-133. OHNISHI, O. (2000): Geographical distribution of allozymes in natural populations of wild Tartary buckwheat. Fagopyrum 17: 29-34. OHNISHI, O. (2002): Wild buckwheat species in the border area of Sichuan, Yunnan and Tibet and allozyme diversity of wild Tartary buckwheat in this area. Fagopyrum 19: 3-9. OHNISHI, O. AND T. KONISHI (2001): Cultivated and wild buckwehat in eastern Tibet. Fagopyrum 18: 3-8. OHNISHI, O. AND Y. MATSUOKA (1996): Search for the wild ancestor of buckwheat. II. Taxonomy of Fagopyrum (Polygonaceae) species based on morphology, isozymes and cpDNA variability. Genes & Genet. Syst. 71: 383-390. OHSAKO, T. AND O. OHNISHI (1998): New Fagopyrum species revealed by morphological and molecular analyses. Genes & Genet. Syst. 73: 85-94. OHSAKO, T. AND O. OHNISHI (2000): Intra- and interspecific phylogeny of wild Fagopyrum (Polygonaceae) species based on nucleotide sequences of noncoding regions in chloroplast DNA. Amer. J. Bot. 87: 573-582. OHSAKO, T., K. YAMANE AND O. OHNISHI (2002): Two new Fagopyrum (Polygonaceae) species F. gracilipedoides and F. jinshaense from Yunnan, China. Genes & Genet. Syst. 77: 399-408. STEWAD, A. N. (1930): The Polygoneae of eastern Asia. Cont. Gray Herb. Harvard Univ. 88: 1-129. TSUJI, K. AND O. OHNISHI (2000): Origin ofcultivated Tartary buckwheat (Fagopyrum tataricum Gaertn.) revealed by RAPD analyses. Genet. Resour. Crop Evol. 47: 431-438. TSUJI, K. AND O. OHNISHI (2001a): Pylogenetic position of east Tibetan natural populations in Tartary buckwheat (Fagopyrum tataricum Gaertn.) reveased by RAPD analyses. Genet. Resour. Crop Evol. 48: 63-67. TSUJI, K. AND O. OHNISHI (2001b): Phylogenetic relationships among wild and cultivated Tartary buckwheat (Fagopyrum tataricum Gaertn.) populations revealed by AFLP analyses. Genes & Genet. Syst. 76: 47-52. YASUI, Y. AND O. OHNISHI (1998): Interspecific relationships in Fagopyrum (Polygonaceae) revealed by the nucleotide sequences of the rbcL and accD genes and their intergenic region. Amer. J. Bot. 85: 1134-1142. WANG, T. Y. (1989): Buckwheat germplasm resources in Tibet. Pp. 49-51. in Buckwheat Research Association in China ed. A collection of scientific treatises on buckwheat in China. Scientific Publisher, Beijing (in Chinese) ZHANG, M. (1991): Minority tribes in Quizhou province. Quizhou Minzi Publisher, Quiyang. (in Chinese)

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Recent Advances in Overcoming Breeding Barriers in Buckwheat Taiji Adachi Osaka Prefecture University, Graduate School of Agriculture and Biological Sciences 1-1, Gakuen cho, Sakai, Osaka 599-8531, Japan ABSTRACT Three distinguished progresses concerning with reproductive system in buckwheat are described. Abnormalities were recognized progressively in some components of the cells which consisted of reproductive organs after cross-hybridization among Fagopyrum species. By using HEGS (High Efficiency Genome Scanning) system, molecular characterization of the heterostylar gene locus was performed in recent years. It might be a great breakthrough for the understanding of the nature of self-incompatibility caused from heterostylism since Darwin’s description. A homomorphic autogamous strain has developed by introgressive hybridization between F. esculentum and F. homotropicum. Keywords: Fagopyrum, inter-specific hybridization, proembryonic abortion, heterostylism, breeding barriers, autogamous strain

INTRODUCTION Buckwheat is grown in many parts of the world. But it has never attained the status of major cereal crop from the production and yield point of view. From these reasons we have made great efforts to investigate overcoming breeding barriers in genus Fagopyrum. As mentioned previously (ADACHI, 1990, ADACHI 1994), major causes of low productivity come from the following categories; 1) self-incompatibility caused by dimorphic heterostylism, 2) incompletion of the reproductive organs mainly in the female, 3) failure of fertilization and 4) seed collapse in the early developmental stage of embryo. We devoted ourselves to investigate the phenomena and to set up strategies to break through such disadvantages. Here I would like to elucidate recent advances in the following three items which are closely connected with overcoming breeding barriers, focusing mainly on attempts in my own group. Ultrastructural aspects of cross-incompatibility among genus Fagopyrum. Recent years some progresses have made in inter-specific hybridization among some major species in Fagopyrum with some help of in vitro techniques (HIROSE ET AL. 1994; LEE ET AL. 1994; CAMPBELL 1995; SAMIMY 1996). In spite of successes in such inter-specific hybridizations, it remains still for further investigation to break through breeding barriers for practical use. For this reason, ultra-structural characteristics of the fertilized ovule and another reproductive organ should be analyzed in detail. Ultra-structural aspects on degeneration of embryo, endosperm and suspensor cells were observed in some combination of the inter-specific hybridization (SHAIKH ET AL. 2001, 2002a, 2002b). To determine the nature of the post-fertilization barriers in buckwheat the comparison of TEM configurations were very effective, namely the abnormalities in cytoplasmic components were detected in different time course among different cross combinations. The deficiency and degeneration of endosperm that were observed at 2 to 3 DAP may lead to the degeneration of hybrid embryo in the case of F. esculentum x F. tataricum. Therefore, rescue of hybrid embryos at this critical stage may overcome one of the main post-fertilization barriers by ovule culture. Otherwise, in vitro fertilization technique should be operated as a non-conventional method for inter-specific hybridization.

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Recent advances in molecular biology of heterostylism Some of Fagopyrum species, including common buckwheat have a sporophytic heteromorphic self-incompatibility system which is controlled by a single multi-allelic S-locus. As DARWIN (1877) has already described that some of the self-incompatible plant have evolved independently an elegant system promoting insect mediated outcrosses, i.e. heterostyly. Heterostylism is a sexual reproduction system, which has two reciprocal floral morphs, pin and thrum in distylar species like as Fagopyrum. Genetic study of distylous species indicated that dimorphic character and self-incompatibility (SI) system appear to be determined by a single diallelic locus (S locus), where short styled morph usually heterozygous (Ss), and long styled morph are recessive homozygous (ss) (LEWIS AND JONES 1992). SHARMA AND BOYES (1961) postulated that S-locus of Fagopyrum may contain five sub-genes: G, style length; Is, stylar incompatibility; Ip, pollen incompatibility; P, pollen size; and A, anther height as S-super system. As the life cycle of buckwheat is short, a simple efficient transformation system was established (KOJIMA ET AL. 2000) and genome size is about 1 GB, this could be realized a good model plant to analyze the mechanism of heterostylism and its of self-incompatibility. To understand of the molecular basis of the heterostyly in buckwheat, positional cloning of the S locus was attempted by using the cross combination between F. esculentum and F. homotropicum. A saturated linkage map with 38 markers in 3cM was developed by a bulked segregant analysis of 200F2s with the HEGS (High Efficiency Genome Scanning) system (KAWASAKI ET AL. 2003), using 4096 AFLP primer pairs. By increasing F2 individuals to 3112, and analyzing additional 512 Anchor-SAMPL primers with HEGS, 5AFLP and 3 Anchor-SAMPL markers were found to be tightly linked to the S locus. One AFLP and one Anchor-SAMPL marker have co-segregated with the S locus. By using these molecular markers and a BAC library we have developed, we have constracted a physical map with intermittent contigs around the S locus. A BAC clone (87kb), having the longest S region, and encompassing the two co-segregating markers and a 0.03 cM marker, was sequenced. This revealed several repetitive sequences in the region, and some of which showed high homology to those in the centromeric region of Arabidopsis. Thus, the S locus of buckwheat may be located also very close to the centromere as the cases in other S-RNase mediated self-incompatible species, and this will be a major cause of the suppressed recombination between the complex components. We found also 5 putative ORFs, and 4 overlapping retro-transposons, and the amino acid sequences of the former showed homology with suggestive proteins in the databases. We are now conducting to develop a systematic complementation analyzing system to verify the functions of these genes. Establishment of autogamous buckwheat and prospect for breeding strategy Common buckwheat is an outcrossing species with distylic incompatibility as mentioned above. Due to allogamy, the individuals in a population are highly heterogeneous even in their seed storage proteins. As a strategy to overcome breeding barrier among Fagopyrum species, development of self-pollinating (autogamous) type buckwheat will be reliable by interspecific hybridization between common buckwheat and a wild homomorphic relative, Fagopyrum homotoropicum (CAMPBELL, 1995; WOO AND ADACHI 1997). The hybrids were successfully produced through embryo rescue and forwarded to successive nine generations by recurrent backcrossing and selection to homostylar morphs. Recently we have established some introgressive strains from original cultivar Miyazaki-zairai. In order to develop hypoallergenic strain from autogamous common buckwheat, we have 23


conducted molecular-genetic approaches in recent years (NAIR ET AL. 1999a; 2002). The maximum IgE binding activity was detected with protein bands of 22, 36, 39-40 and 70-72kDa (NAIR AND ADACHI 1999b). We isolated the respective cDNA, coding for a 22kDa protein, from a recent developed autogamous strain of common buckwheat and confirmed its IgE-binding activity using recombinant Fag e1 and sera of allergic patients. And the determination of the Fag e 1 epitope was performed by amino acid sequencing from Fag e 1 cDNA and then by overlapping peptide library (YOSHIOKA ET AL. 2004). Detail data will be described in the proceedings in detail. Transgenic techniques will be shown to have the potential of modifying, decreasing or even removing allergenic substances in buckwheat. We are trying to develop a transgenic hypoallergenic crop from our autogamous strain of common buckwheat soon. CONCLUSION The complementary application of conventional but standard breeding techniques and molecular biotechnology tools for buckwheat improve program appears to be indispensable for the achievement of effective practical goals in near future. This is evident from the results so far obtained from studies on sub-cellular components of reproductive organs and on molecular understanding of dimorphic heterostylism. Introgressive hybridization between F. esculentum and F. homotropicum will offer beneficial traits for further strategy of buckwheat breeding. ACKNOWLEGEMENTS Grateful appreciation is extended to all of my colleagues for their collaborations and kind supports in every respects. And I am also thankful for all of my students both in the Laboratory of Plant Biotechnology and Breeding, Applied Genetics and Biotechnology Division, Faculty of Agriculture, Miyazaki University and in the Laboratory of Plant Genes and Physiology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University.

REFERENCES ADACHI, T. (1990): How to combine the reproductive system with biotechnology in order to overcome the breeding barrier in buckwheat.? Fagopyrum 10:7-11. ADACHI, T. (1994): Recent advances in overcoming breeding barriers in crops-focused on buckwheat research in Miyazaki.Proc. IPBA, Rogla. Dec., 5-7, 1994:1-8. AII, J., M. NAGANO, C. CAMPBELL, A. SHIMIZU, T. ADACHI AND S. KAWASAKI (2003): Molecular characterization of the S locus in buckwheat. Abstract of the Int’l. Symp. on Plant SI., Sept. 17-18.:18. CAMPBELL, C. (1995): Interspecific hybridization in genus Fagopyrum. Proc. of 6th Int’l. Symp. on Buckwheat, Ina, Japan :255-263 DARWIN, C. (1877 ): The different forms of flowers on plants of the same species. Murray, London HIROSE, T., A. UJIHARA, H. KITABAYASHI AND M. MINAMI (1994): Interspecific cross compatibility in Fagopyrum according pollen tube growth. Breed. Sci. 44: 307-314. KOJIMA, M., Y. ARAI, N. IWASE, K. SHIROTORI AND M. NOZUE (2000): Development of a simple and efficient method for transformation of buckwheat plants (Fagopyrum esculentum) using Agrobacterium tumefaciens. BBB 64:845-7. LEE, B.S., A. UJIHARA, M. MINAMI AND T. HIROSE (1994): Breeding of interspecific hybrids in genus Fagopyrum. (4) Production of interspecific hybrids through ovule culture among F. esculentum, F. tataricum and F. cymosum. Jpn. Jour. Breed. 44(Suppl. 1): 183 (in Japanese) NAIR, A., T. OHMOTO, S.H. WOO AND T. ADACHI (1999): A molecular-genetic approach for hypoallergenic buckwheat. Fagopyrum 16:29-36.

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NAIR, A. AND T. ADACHI (1999a): Protein at 69 kDa expressed during the initial maturation stage of buckwheat(Fagopyrum esculentum) seed development. Cereal Chem. 76:321-322. NAIR, A. AND T. ADACHI (2002): Screening and selection of hypoallergenic buckwheat species. The Scientific World Jour. 2:818-826. SAMIMY, C., T. BJORKMAN, D. SIRITUNGA AND L. BLANCHARD (1996): Overcoming the barriers to interspecific hybridization of Fagopyrum esculentum with F. tataricum. Euphytica 91:323-330. SHAIKH, N. Y., L.M.GUAN AND T. ADACHI (2001): Ultrastructural analysis on breeding barriers of interspecific hybridization in genus Fagopyrum. Kinki Jour. of Crop and Breed. 46:37-45. SHAIKH, N. Y., L.M.GUAN AND T. ADACHI (2001): Failure of fertilization associated with absence of zygote development in the interspecific cross of Fagopyrum tataricum x F. esculentum. Breed. Sci. 52: 9-13. SHAIKH, N. Y., L.M.GUAN AND T. ADACHI (2002): Ultrastructural aspect on degeneration of embryo, endosperm and suspensor cells following interspecific crosses in genus Fagopyrum. Breed. Sci 52: 171-176. WOO, S.H. AND T. ADACHI (1997): Production of interspecific hybrids between Fagopyrum esculentum and F. homotropicum through embryo rescue. SABRAO Jour. 29:89-95. YOSHIOKA, H., T. OHMOTO, A. URISU, Y. MINE AND T. ADACHI Expression and epitope analysis of the major allergenic protein Fag e 1 from buckwheat. Jour. Plant Physiol. 161: 761-767.

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Present State and Future Prospects for Buckwheat Clayton G. Campbell Kade Research Ltd. 135 13 Street, Morden, Manitoba, Canada R6M 1E9

Buckwheat is a very unusual and unique crop. It is usually treated as a cereal but it does not resemble the cereals in growth habit, seed quality or seed composition. It also has many very desirable health components which make it a valuable part of the human diet. Buckwheat has a unique growth habit as compared to the cereals and thus fits into a much different area of crop production due to its rapid growth and long flowering period. I think that many of us are here today, not because of the similarity of buckwheat to the cereal crops, but because of the difference between buckwheat and the cereal crops and the many unique traits that this very interesting crop contains. Crop improvement programs on buckwheat in the past have concentrated on common buckwheat, Fagopyrum esculentum. However, there has been recent interest in the improvement of Tartary buckwheat, especially in the areas of ease of dehulling and in some of its nutraceutical components such as rutin and quercitin. There also has been increasing utilization of the wild species Fagopyrum homotropicum in crop improvement programs due to its self compatible characteristic which is being introgressed into common buckwheat. Although crop improvement programs have been working on the development of common buckwheat for more than one hundred years, the increased yielding ability of this crop have not kept pace with the larger acreage cereal crops. This has resulted in the replacement of buckwheat with other crops in many areas of the world and continues to put pressure on the crop as a viable long term part of any farming system. (For a more detailed report on the improvement of buckwheat please refer to “Buckwheat Crop Improvement” in the twentieth issue of Fagopyrum) So what is the present state of crop improvement programs on buckwheat and how should we proceed to ensure that this crop can better compete against the many other alternative crops that growers can produce? PRESENT STATE The first crop improvement programs on common buckwheat resulted in the development of some famous and well known varieties. The variety Bogatyr was developed in Russia from 1901 to 1909. The Japanese variety Hashigamiwase was bred in 1919 and Botan Soba was released in 1930. It is very interesting to observe that some of these original varieties are still being grown to a limited extent. This clearly demonstrates that progress on crop improvement in common buckwheat has been slow due to its’ out-crossing nature and may also be due to limited variation that is present in this species. The development of new buckwheat varieties has been successful in the production of larger seeded varieties which give better groat yields. There also has been development of lines with other desirable characteristics such as earliness or with shorter plant habits which are more resistant to lodging. These include the determinate flowering habit types as well as semi-dwarf plant habits. Tetraploid varieties were produced in the USSR in the 1940s with Miyazakiootsoba being released in Japan in 1972 followed by Shinshuoosoba in 1974. These varieties were developed for increased lodging resistance but had problems with decreased fertility and increased hull

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thickness. Therefore most crop improvement programs have abandoned this method of crop improvement. Mutational breeding in buckwheat has been utilized by many crop improvement programs. This has resulted in types such as the determinant type of plant habit which has limited growth, lodging resistance and a narrow leaf and is therefore was suited to regions with a short hot summer. The determinant character was first reported in the USSR 1940 but breeding did not begin until 1954. The determinant type has also received attention in crop improvement programs in Japan. Self-compatible common buckwheat has also received attention in several breeding programs. They were developed from both natural mutations and by ion exposure. A comprehensive review of the published literature on buckwheat , however, shows that a great deal of work has been concentrated on determining the growth , flowering and seed set of buckwheat and how it is affected by environmental factors. This has supplied us with very valuable information and aids us in having a very good knowledge basis for the crop. But unless we can apply this knowledge to the development of new varieties with superior yield characteristics as well as improved quality traits the crop will still remain under pressure in the competition against other crops. PRESENT AREAS OF INTEREST Tartary buckwheat is grown in many countries of the world. Plants of F. tataricum are more branched and the leaves are more arrow shaped than in common buckwheat. The flowers are smaller, have inconspicuous greenish-white sepals, are homomorphic, self-fertile and are cleistogamous, with pollination occurring before the flower opens. The plants are also more tolerant to environmental stresses than is common buckwheat. Although Tartary buckwheat is grown in many countries much of the production is utilized in the producing country. Only a small amount enters into international trade, however, this amount is now increasing. The discovery of ‘rice’ Tartary buckwheat which has a non-adhering hull and therefore dehulls very readily, has been reported in several countries. It is surprising that this interesting trait, although reported as desirable, has not resulted in increased production of this type of Tartary buckwheat. Crosses between Rice buckwheat and normal Tartary buckwheat have been extremely difficult to accomplish which may account for the lack of progress in this area. Tartary buckwheat is self-pollinating and therefore much of the work on it has been by single plant selections. This has been successful in increasing components, such as rutin, however, only a few crop improvement programs world wide are working on Tartary buckwheat. Increased interest has recently been shown for human consumption of Tartary buckwheat due to some of its’ components that are very beneficial to human health. This has created increased crop improvement programs in China, Japan and Canada. This increased breeding effort on this species would be expected to increase in the future and result in a second buckwheat species that will become increasingly commercially important on an international scale. Over the past decade there has been increased interest in finding new species of buckwheat that is closely related to Tartary buckwheat. A putative progenitor species that would have a self-incompatible pollination mechanism, However, to date no such species has been found. The finding of such a progenitor species would be a great benefit to any crop improvement program working on Tartary buckwheat, or on any program desiring to transfer traits between Tartary buckwheat and other economically important species. The first successful inter-specific hybridization of buckwheat, at the diploid level, was 27


reported in 1995. Using conventional pollination at the diploid level, successfully hybridization of Fagopyrum esculentum Moench. and F. homotropicum Ohnishi (both diploid, 2n=16) was accomplished. The resulting progeny were fertile and further back-crosses to common buckwheat have been carried out to introgress the self compatible character into common buckwheat. This successful inter-specific hybridization of Fagopyrum esculentum and F. homotropicum at the diploid level has opened a new methodology for the improvement of common buckwheat. Traits such as self-pollination and seed and plant characteristics can now been transferred to common buckwheat. F. homotropicum, being a self-pollinating species, sets all of its seeds before flower opening, in a similar manner as Tartary buckwheat. Common buckwheat has a seed abortion problem so that approximately 10 to12 % of the flowers it produces develop into seeds. Therefore, the transfer of the self-pollinating mechanism into common buckwheat allows for increased yield. However, the high number of flowers produced by Fagopyrum esculentum must be drastically reduced to allow for the nutrients now being utilized for their growth to be diverted into filling the increased number of seeds being formed. The development of haploids for utilization in crop improvement programs is now receiving attention in several countries. These can be utilized for the production of double haploid homozygous lines. They also can be used for producing homozygous lines for use in the development of buckwheat hybrids. FUTURE PROSPECTS Tartary buckwheat has been utilized in many countries as a subsistence food or has entered into commercial trade, mainly at a local level. With increased interest in this species due to it’s health benefits for human consumption this species has now entered into international trade. This development should promote the inclusion of this species into crop improvement programs in several countries. Although Tartary buckwheat has greater tolerance to environmental stresses than common buckwheat it also has some negative traits which must also be addressed. These include such traits as ease of seed shattering, adhering seed coat which makes dehulling very difficult, a bitter taste in the flour that is produced and a yellow foam that is produced from cooking noodles made from this species. Although the bitter taste can be modified through product development this would appear to be the area where the greatest need exists for this species. A much more complete understanding of the cause and possible elimination of this taste would greatly benefit the acceptance of this species in many products. The ease of dehulling of this species must also be addressed so that it can be handled in much the same way as common buckwheat. As there now exists a great deal of variation in seed shape and size it would appear that there is also a need to develop larger seeded lines or varieties with a seed shape that will maximize groat or flour recovery. As the milling characteristics of the species depends to a large extent on both of these traits the simultaneous inclusion of them into any crop improvement program would be very feasible. And what direction should we take if we want to help change common buckwheat’s competitive advantage against other crops? Based on the progress, or really the slow progress that has been made during the past 50 years in increasing buckwheat yield or diversity, it would appear that we should strongly consider new approaches. These new approaches for crop improvement must be closely related to both quality and health aspects of buckwheat. The needs of the end user of buckwheat products should be ascertained and should be a major influence in determining the emphasis that each crop improvement program places on the development of new varieties. While superior agronomic traits of buckwheat are very important to the grower and must remain a major portion of each program, development of new and improved quality or 28


health compounds must receive increased emphasis. This in turn requires that plant breeders must be in close contact with food researchers and nutritionists. This is very important, not only for the development of new varieties with new or improved quality aspects but also for the development of new and improved products from these new varieties after they have been developed. This is indeed an area were close collaboration not only helps in defining the direction of the research but can also directly benefit the consumer through the development of new products or the improvement of already existing ones. At the present time the end user of buckwheat really only has one type of buckwheat flour to work with which contain a fairly narrow range in its’ quality components. Studies have shown that some of these components can be increased or decreased in buckwheat. However, we have not yet seen any large effort in the development of new buckwheat varieties with increased quality characteristics. In contrast if we look at some of the larger commercial crops, such as wheat or rice, we see an extensive range of components that affect the end use of the product. I believe that if we can develop and offer new varieties with improved quality aspects we can increase the use of this underutilized crop. At the same time, however, we must also be able to develop and promote these products in the market place. Crop improvement programs on buckwheat must now consider both supply and demand of buckwheat as it affects their development of new varieties. To ensure supply we must first consider the economic return the growers can receive from this crop as it compares to other crops they can produce. Thus yield and tolerance to environmental stresses remain the major desirable characteristics of any new variety. The development of self pollinating buckwheat has allowed some programs to rapidly develop characteristics that effect yield in several ways. The initial increase is from the increased yield potential due to the self compatibility of the lines. However, characteristics such as large leaf size, weed control leaves, chrysanthemum plant shape and flowering habit may also greatly affect the yielding ability of these new types. Increased demand for buckwheat products from end users can be increased by the development of new varieties with quality components that either increases the quality of existing products or that allow for the development of new products. While the improvement of existing products is important it will be the development of new products that can be made from buckwheat that will ultimately create increased demand. With the small number of crop improvement programs that are presently working on buckwheat it would be very desirable as well as beneficial to have increased cooperation or collaboration between the programs. This is especially important if we consider the future of buckwheat and its competitive place against the many other commercial crops now being grown. While many crops that are produced on a larger scale than buckwheat generally have a large number of plant breeders that are developing new varieties we should utilize all the advantages we have which will allow us to make the most rapid progress we can. Perhaps the largest advantage we have is that almost all the buckwheat breeders world wide know each other, in many cases due to the International Buckwheat Symposiums. As other researchers, whose work directly affects crop improvement programs, are also present at these meetings it presents a very valuable opportunity to develop discussions, exchanges of genetic material as well as future collaborations which will benefit not only the collaborators but also the entire buckwheat industry.

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Historical and Current Status of Buckwheat Culture and Use in the Czech Republic Jiří Petr¹, Jana Kalinová,² Jan Moudrý², Anna Michalová³ Czech University of Agriculture in Prague¹ South-Bohemian University in České Budějovice² Research Institute of Crop Production Prague-Ruzyně³

The buckwheat is the youngest grain crop in Europe. In the Middle Ages it was not known at all here. The buckwheat was introduced into the European continent at the latest in the 13th century from the northern China via the so-called “Silky Route”. It was spread into the central Europe and Bohemian lands probably by two ways – either outside the Carpathian ridge through the Polish territory or through the Danubian region, here the latest with the arrival of Hungarians. Stronger introduction was in the 13th century during invasions of Mongolian, Turkish and other troops. These soldiers could transport and prepare buckwheat as a nutritional food very easy. Owing to the fact that this crop was introduced into Europe by the troops that were not followers of the Christian religion, this crop was called in Bohemia “pohanka” (the Czech word “pohan” means in English “the pagan”). It was the same in Italy (grano saracen), in France (le blé sarrrasin) and in Germany (Heidenkorn). According to Mongol Tartars it had a popular name “tatarka”. The most ancient macro-remains of buckwheat in the Bohemian territory come from the 12th century and they were found during archaeological excavations in Uherský Brod. The oldest written reference to buckwheat was in Klaret’s Versed Glossary that originated of around the year 1365. Italian physician Matthioli also mentions buckwheat in 1596 in his herbal and among other things he recorded that buckwheat grows plentifully in Bohemia and elsewhere and it is domestic, popular and ordinary food of the folk. In Bohemia and Moravia it was domesticated above all in mountain and submontane regions. In Slovakia its cultivation spread almost to the whole territory, however, here it was gradually transferred particularly to mountain and submontane areas and belonged mainly a to the food of the poor. Historical sources mentioned it as a spring crop, i.e. it was cultivated as a major crop. In southern Slovakia buckwheat was the second crop in a year (the so-called stubble crop). Other different documents refer that the importance of buckwheat was not lower even in modern time. It was used to prepare grouts mash, better digestible than the other cereal kinds of mash. In the 16th century buckwheat was cultivated in Bohemia, but it was not spread as a market crop. In wartime only, e.g. in 30 years’ war, it was more cultivated. In Moravia it was domesticated in Walachia. An obligation of natural levies testifies an ordinary cultivation of buckwheat in Slovakia in the middle of the 17th century. However, since the 18th century buckwheat has been receded from cultivation, what was associated also with the change in composition of food, recession from mashed meals. The consumption of bread and bakery products was falling, the people started to cultivate and eat potatoes, and also imported rice. In Slovakia millet was an important competitive crop of warmer regions. In 1896 in Slovakia buckwheat was cultivated on about 1200 hectares with the yield ranging between 0.31 and 1.18 tons per 1 hectare depending on the region of cultivation. In the 19th century cultivation of buckwheat was mentioned in the Chrudim and Poděbrady regions. In the Těšín region and in the Beskydy Mountains buckwheat in those times was almost a popular meal. In Walachia region it was much popular right to the 20th 30


century. It was cooked in milk or in water and was prepared baked in with pork scrap, other times only poured over with milk, mingled with potatoes, sweet or as a garnish in soup. In Czechoslovakia areas sown with buckwheat took 3046 hectares in 1920 and gradually they were reducing. In 1935 these areas took 2110 hectares and in 1945 they took 1406 ha. Yields were ranging about 0.7 t per 1 hectare. After the Second World War cultivation of buckwheat was somewhat higher in Bohemia with the arrival of the Czechs from the Volyně region from the former Soviet Union. Acreage was fluctuating around 1000 hectares. Since 1962 further diminishing of areas has been continued in Czechoslovakia (500 hectares and less). In 1980 as little as 2 hectares were recorded in the statistics. At the beginning of the 20th century the buckwheat varieties Česká krajová, Vígľašská and Lipnovská were cultivated in mountain and submontane regions. In the 1950s two buckwheat varieties were certified - Moravská krajová and Slovenská krajová. They were cultivated in Walachia, in the central and the upper Váh river basin, in the Central and Eastern Slovakia. In the 1960s the variety Doksanská was approved, that was bred from the varieties imported into Bohemia by the Czechs originating from the Volyně region after the Second World War from the former USSR. However, their small areas led to its restriction. Graph 1 The development of areas sown with buckwheat in the Czechoslovak Republic according to the FAO statistics ha 1000 800 600 400 200 1989

1987

1985

1983

1981

1979

1977

1975

1973

1971

1969

1967

1965

1963

0

Graph 2 The development of yields in the Czechoslovak Republic according to the FAO statistics

1989

1987

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1983

1981

1979

1977

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1973

1971

1969

1967

1965

1963

1961

Hg .ha -1 16 14 12 10 8 6 4 2 0

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RENAISSANCE OF BUCKWHEAT AFTER 1990 The renaissance of buckwheat cultivation and its consumption started in the 1990s in connection with growing concern in rational healthy nutrition and ecological agriculture. In 1993 it was estimated that buckwheat is cultivated on area of 800 hectares in the Czech Republic. There it became an interesting crop mainly for ecological farmers. A complex buckwheat program was provided here, including the sale by the company Pro-Bio Staré Město pod Sněžníkem, which tries to develop the co-operation of farmers, processors, traders, research institutes, physicians and consumers. Tab. 1 The development of buckwheat production in ecological farming Year Yield (t)

1994 108

1995 208

1996 263

1997 325

1998 400

1999 600

2000 840

2001 1000

The concern in buckwheat cultivation conditioned also its breeding. In 1990 one of the first new cultivar was bred at the breeding station Horní Moštěnice, semi-early cultivar Pyra. It originated from Moravian (the Beskydy Mountains) and Slovak land races. This cultivar is relatively early and is marked by a slow starting growth. Previously the view on the buckwheat varieties was rather influenced by the possibility of its cultivation as the second crop in a year, i.e. tolerant to late sowing. The present concern is directed to grain varieties, cultivated as main crops to reach higher yield and particularly high weight of achenes. In 1997 another cultivar was approved in the Czech Republic, bred from Russian varieties - Jana. In 2001 the Ukrainian variety was added to them - Kara-Dag and in 2002 as the fourth buckwheat variety - the registered Slovak cultivar- Špačinská 1, bred in 1998 in Slovakia (Borovce - Špačince). It is highly yielding cultivar with a good resistance to fungal diseases. At present buckwheat grows on 3000 hectares, 900 hectares of it are in ecological farming, however, an exact area under crops is not recorded in the Czech Bureau of Statistics. So, the size of area is on the level of 1920 and is reaching the greatest range of buckwheat cultivated on the territory of the independent Czech state. The prices of buckwheat are based upon the world prices in the Czech Republic that are influenced particularly by producers in Poland, USA, China and the states of the former Soviet Union and those in the South America. According to an offer and demand the price is fluctuating over the years between 5500 and 10000 CZK per ton. The price of bio-buckwheat ranges from 8500 to 9000 CZK. The price of scoured buckwheat is between 16000 (import from Russia) to 30000 CZK per 1 ton. The research of buckwheat in the Czech Republic is directed to the accumulation, characteristics and selection of prospective genotypes to be used in practice, evaluation of nutritive quality of buckwheat products (basic composition, amino acids, minerals, vitamins, fatty acids, polymorphism of reserve substances, rutin and other phenolic antioxidants, the content of hazardous elements), technological parameters of quality (bread baking, testifying of suitability of the line for buckwheat processing in view of production of gluten-free products), monitoring of the factors affecting the yield and yield formation, determination of photoperiodic reaction, cold-resistance and evaluation of health conditions. In 2000 buckwheat together with millet were chosen as priority crops for ignored cereals and pseudocereals in Europe within the working and co-ordinating groups for lowvolume and underestimated crops ECP/GR Minor crops. The Czech Republic is a coordinator for these crops. At present the main processor of buckwheat in the Czech Republic is the company PRO-BIO, where buckwheat is scoured under cold conditions, mechanically abraded, to

32


prevent influencing of the quality of enzymes, vitamins of amino acids etc. Lower yielding capacity is a disadvantage, which is caused by processing segment for 45-55%. This type of processing is a follow-up to tradition preserved in Walachia. Hostašovice was a centre of buckwheat processing. Mills worked here full time as late as to the World War I. Buckwheat imported from Austria, Poland and Halič were processed here predominantly. Special scourers were used to scour buckwheat achenes on many places neighbouring the mills. In majority of households in towns and in the countryside the people had the so-called manual scourers, in which the pericarp of buckwheat, millet etc. was chipped away with a mallet. Farmers used millstones for manual scouring. Picking out by hand then purified the mixture obtained. Buckwheat is scouring in this way in the regions of East Slovakia to prepare the socalled vol-au-vents. To the beginning of the 20th century in the region of Nový Jičín a special handicraft was preserved – mash preparation, domestic production of buckwheat mash. Nowadays, the present market offers over 40 different products, prevailingly in bioquality (non-peeled buckwheat, peeled buckwheat – grout), semolina, dark and light flour, buckwheat-spelt and only buckwheat pastes, mixtures for omelettes, raised pancakes, instant mash with rice, potato pancakes with buckwheat, puffed buckwheat, flakes, buckwheat drink, buckwheat tea with hip, husks, buckwheat cushions etc.). The market offers different baking (buckwheat bread, crisp bread with buckwheat, buckwheat toast) and confectioner’s products (e.g. biscuits) and special products for patients with the coeliac disease. Extruded maize slice with buckwheat and garlic was awarded on the international food fair SALIMA Brno 2002. It is the product containing buckwheat haulm, product of ecological farming. Innovation on the market is a semi-product BIOHARMONY – chopped buckwheat. The sales of buckwheat are increasing from year to year. It is caused by a wide assortment and high quality of products that are distributed above all in shops with healthy food as well as in supermarkets of the whole Czech Republic. Buckwheat may become an important alternative crop and starting material for further new and interesting foods. Renewed concern in buckwheat is based upon e.g. nutritive composition and fact that buckwheat is an ideal natural product. Nutritive quality of proteins, extremely low content of prolamines, presence of rutin and other polyphenolic composition, fibre and starch with controllable rate of digestion. This all gives to buckwheat products suitability for healthy nutrition, for diabetics as well as for the patients with coeliac disease. Consumption of buckwheat may play an important role in prevention and treatment of hypertension and hypercholesterolemy as cardiovascular risk factors.

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34

Section II. Biotechnology and Physiology


Influence of Day Length on the Growth of Stem, Flowering, the Morphology of Flower Clusters, and Seed-Set in Buckwheat (Fagopyrum esculentum Moench) Hiroyasu Michiyama, Keiji Tsuchimoto, Ken-ichiro Tani, Tatsuya Hirano, Hisayoshi Hayashi1 and Clayton Campbell2 Meijo University, Nagoya 468-8502, Japan Univetsity of Tsukuba, Ibaraki 305-8572, Japan 2 Kade Research Ltd., Morden, Manitoba, R6M 1E9, Canada 1

ABSTRACT The purpose of this study was to demonstrate the changes to main stem growth, flowering, morphology of flower clusters, and seed-set as affected by day length and to clarify the difference among the cultivars and the growth parameters. For this study we used Shinanonatsusoba (summer eco-type) and Miyazakizairai (autumn eco-type) and BLO 1999 (a long cluster line which usually develops DM clusters in Kade research Ltd., Canada). A long day period influenced main stem growth, flowering, and seed-set as was found in previous studies. Furthermore, it was shown that a long day period increased the frequency of DM clusters, the length of flower clusters and the number of sub-flower-clusters per cluster in Shinanonatsusoba and Miyazakizairai as well as in BLO 1999. It is confirmed that the effects of day-length period varied among the growth parameters and that there were three types of responses to day length. The varietal difference between the summer and autumn eco-type cultivars was elucidated, and was shown to be involved in the responses to day length in four groups of parameters; the main stem elongation; the first flowering node and the first flowering day; the elevating rate of flowering cluster position; and the number of seeds and seed-set ratio. Keywords: buckwheat, day length, eco-type, flower cluster, flowering, growth, seed-set

INTRODUCTION Growth, flowering and seed-set in buckwheat have been shown to be influenced according to the sowing date (YAMAZAKI, 1947; UEHARA AND TAGUCHI, 1955; 1956; NAGATOMO, 1961; SUGAWARA, 1973; MICHIYAMA AND HAYASHI, 1998; MICHIYAMA ET AL., 1998). It appears that the changes are mostly caused by varied day length (TABATA ET AL., 1931; Xu, 1938; NAGATOMO, 1961; SUGAWARA, 1958; 1973; LACHMANN AND ADACHI, 1990; HAYASHI ET AL., 1997; HAGIWARA ET AL. 1998; MICHIYAMA ET AL., 2003). But it is not yet clear as to how the day length affected each of the growth parameters, as the experiments in most of these studies were conducted under only two or three day-length conditions. It is well known that the days to flowering in rice plants do not show linear correlation with day length (SUGE 1976). So a further study was necessary to determine the day-length conditions over a wide range with short interval times, for example of 30 minutes. In our previous study (MICHIYAMA ET AL. 2003), it appeared that the critical day length period varied with the cultivars and the growth parameters in buckwheat. The purpose of the present study was to demonstrate the changes of the growth parameters according to the day length and to clarify the difference among the cultivars and the growth parameters, using summer and autumn eco-type clutivars from Japan. We also observed that the long-day conditions increased the length of flower clusters and branched flower clusters which were named double and multiple clusters (DM clusters) as was found in our previous study (MICHIYAMA ET AL. 2003). The long cluster lines and the DM cluster lines were developed by Kade research Ltd. in Canada (HAYASHI, 1999). We also investigated the length and branching of the flower clusters under different day-length conditions in this study, using one of the long cluster lines which usually developed DM clusters in Canada, in addition to the two Japanese cultivars. MATERIALS AND METHODS The experiments were conducted at Meijo University in 2003 using two common buckwheat cultivars and a line (Fagopyrum esculentum Moench), Shinanonatsusoba (summer eco-type cultivar), Miyazakizairai (autumn eco-type cultivar), and BLO 1999 (from Kade Research Ltd. in Canada) which 35


is a long cluster line and usually develops double and multiple clusters. Thirty-two pots (1/5000 a) each containing 3 kg soil were used for growing each cultivar. Five seeds per pot were sown on August 28 and fertilized with 5 g fertilizer (15N-15P-10K). The seedlings were not thinned. Plants were watered as needed to ensure that the soil was sufficiently moist, and side dressing was not applied. The plants were divided into eight groups and subjected to the following day length conditions immediately after sowing and throughout the experimental period by supplementing incandescent light before sunrise and after sunset to produce day length periods of 13, 13.5, 14, 14.5, 15, 15.5, and 16 hours (h). The natural day length (NDL) decreased from 13 hours and 5 minutes during the growth period, which began with the times of sunrise and sunset on the sowing day of 5:21 and 18:26 respectively. In the 13-h plot, the day length period was the same as NDL plot during the very early stage. The main stem length was measured every fifth day from the emergence until the end of elongation. The day of first flower opening in each flower cluster on the main stem was recorded. As all plants did not complete stem elongation and flowering in the 14.5-h ~ 16-h plots of Miyazakizairai, the measurements of the main stem length and flowering were completed on November 26 (90 days after sowing (DAS)) and on December 1 (95 DAS), respectively. The plants were harvested at the full ripe stage, which was from October 21 to November 29, except for 14.5-h ~ 16-h plots of Miyazakizairai. In 14.5-h ~ 16-h plots of Miyazakizairai the plants were harvested on December 1 to 3, even though stem growth and flowering on the main stem was not completed at harvest time. The length of the flower-developing region, the number of branching, and the number of sub-flower-clusters in each flower cluster on the main stem were measured. In the DM clusters, these parameters were measured individually on each branch and the values of the longest branch in a cluster were used. The average of the 3rd to 5th cluster from the first flowering node was used for the expression of the length of the flower clusters and the number of sub-flower-clusters per cluster. The number of flowers and seeds in each flower cluster on the main stem were measured by counting the withered flowers and the mature seeds. The sum of the branches in a DM cluster was used for the recording of the number of flowers, seeds and the seed-set ratio. The numbers of flowers and seeds per cluster were expressed by the average of the flower clusters on the main stem. The developing seeds were included in the total number of seeds in 14.5-h ~ 16-h plots of Miyazakizairai. In this study, the cotyledonary node on the main stem was used as the zero node and the node positions were numbered from the base to the apex. The cotyledonary node was not included in the number of nodes on the main stem. RESULTS AND DISCUSSION A long day length period prolonged the stem elongation period (Fig. 1), elevated the first flowering node (Fig. 2), delayed the first flowering day (Fig. 2), decreased the elevating rate of flowering cluster position (Fig. 3), increased the number of nodes and flower clusters (Fig. 4), increased the number of flowers (Fig. 7), the number of seeds (Fig. 8), and decreased the seed-set ratio (Fig. 9) in this study as well as was found in previous studies (TABATA ET AL., 1931; XU, 1938; NAGATOMO, 1961; SUGAWARA, 1958; 1973; LACHMANN AND ADACHI, 1990; HAYASHI ET AL., 1997; HAGIWARA ET AL. 1998; MICHIYAMA ET AL., 2003). This study showed that a long day period increased the frequency of DM clusters (Fig. 5). In general, the results of this study indicated that a long day period also increased the length of the flower-developing region of the flower clusters and the number of sub-flower-clusters per flower cluster, although they were often decreased for the DM clusters (Fig. 6). The response of BLO 1999 to day length was characteristic of summer eco-type in Japan, such as Shinanonatsusoba in this study. It appears that the Canadian long cluster lines exhibit their abilities under long day conditions which are approximately 16-h during the summer in Canada, but do not exhibit them under short day conditions which are around 13.5-h in autumn cultivation in Japan. These results indicate that buckwheat grown in high latitudes has a high potential for yield owing to the increase in the number of flower clusters per stem, the branching of flower clusters, and the number of sub-flower-clusters and flowers per flower cluster. However, the day length period during the seed-setting stage in Canada (August) is below 14-h.

36


As the seed-set ratio is affected by day length after the start of flowering (MICHIYAMA ET AL., 2003), the seed-set ratio appears to be higher in Canada. Therefore it is possible that high latitudes seem to be adequate for buckwheat production in obtaining high yields. It was confirmed in this study that the details of day-length period effects varied among the growth parameters. Three types of responses to the day length period were observed. Firstly, some of the parameters changed with an increasing day length period and the specific day length bringing about considerable change of the parameters was not observed, for example the length of the flower cluster and the number of sub-flower-clusters (Fig. 6) in all cultivars. The first type included the number of nodes and flower clusters in BLO 1999 (Fig. 4), the frequency of DM clusters and the number of flowers in the autumn eco-type cultivar (Fig. 5 and 7), the number of seeds and seed-set ratio in the summer eco-type cultivars (Fig. 8 and 9). Secondly, some parameters changed with an increasing day length period and the specific day length period bringing about considerable change of the parameters was observed, for example stem elongation (Fig. 1) and the elevating rate of flowering cluster position (Fig. 3) in all cultivars. The second type included the first flowering day (Fig. 2), the first flowering node (Fig. 2), the number of seeds (Fig. 8), and the seed-set ratio (Fig. 9) in the autumn eco-type cultivar. Thirdly, the parameters were invariable with an increasing day length period up to 13.5-h or 14-h, but changed with an increasing day length period after that, for example the first flowering day and the first flowering node in the summer eco-type clutivars (Fig. 2), the number of nodes and flower clusters in Shinanonatsusoba and Miyazakizairai (Fig. 4), and frequency of DM clusters and the number of flowers in the summer eco-type cultivars (Fig. 5 and 7). It was shown that the varietal differences between the summer and autumn eco-type cultivars were involved in the responses to day length in the four groups of growth parameters; main stem elongation (Fig. 1); the first flowering node and the first flowering day (Fig. 2); the elevating rate of flowering cluster position (Fig.3); and the number of seeds and seed-set ratio (Fig. 8 and 9). These parameters appeared to be the basis of classification of the eco-types in buckwheat. The stem elongation period was prolonged and the final stem length was increased with an increasing day length period from NDL to 16-h, and they changed markedly during the day length period from 14-h to 14.5-h in both eco-type cultivars. However, they were found to change more in the autumn eco-type cultivar than in the summer eco-type cultivars during the day length periods from NDL to 16-h, and a more marked change was observed in the autumn eco-type cultivar during the 14-h to 14.5-h day length periods. The first flowering node was elevated with an increasing day length period above 14-h and invariable from NDL to 14-h day length period in the summer eco-type cultivars. However, in the autumn eco-type cultivar it was elevated with an increasing day length period from NDL to 16-h. The first flowering day showed the same response to varied day length periods. An elevating rate of the flowering cluster position was decreased with an increasing day length period from NDL to 16-h. That of the summer eco-type cultivars was higher than that of the autumn eco-type cultivar under the same day length period. This was decreased considerably during the day length period between 14-h and 14.5-h in the summer eco-type cultivars, although that was similar to that between 13-h and 13.5-h day length periods in the autumn eco-type cultivar. The number of seeds and the seed-set ratio were found to decrease with an increasing day length period in both eco-type cultivars. They were also considerably decreased during the day length periods from 13.5-h to 14-h in the autumn eco-type cultivar, although the result was unclear in the summer eco-type cultivars.

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Proceedings of the 9th International Symposium on Buckwheat, Prague 2004 70

110 100 90 80 70 60 50 40 30 20 10 0

First flowering day

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Fig. 1.

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Effect of differnt day length on elongation of the main stem in three buckwheat cultivars sown on Aug 28. 13

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Shinanonatsusoba BLO1999 Miyazakizairai

0.8 0.6

Number of flower clusters

1.2 1.0

Effect of different day length on the first flowering day and the first flowering node in three buckwheat cultivars sown on Aug 28. Vertical bars in the figures indicate standard errors. *:Cotyledonary node was not included in the first flowering node.

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The elevating rate of flowering cluster position

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Effect of different day length on the elevating rate of flowering cluster position on the main stem in three buckwheat cultivars sown on Aug 28. Vertical bars in the figure indicate standard errors. The data expressed by open circles were the average of the plants which were halfway through growth and flowering.

Number of nodes

Daylength Fig. 3.

18 16 14 12

Fig. 4.

38

Effect of different day length on the number of flower clusters and the number of nodes on the main stem in three buckwheat cultivars sown on Aug 28. See the notes of Fig. 2 and 3.

10 NDL

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Daylength Fig. 5. Effect of different day length on the frequency of the plants which have DM clusters on the main stem and the frequency of DM clusters on the main stem in three buckwheat cultivars sown on Aug 28. See the notes of Fig. 2 and 3. *:DM cluster means Double and Multiple cluster.

Fig. 6.

Effect of different day length on the average of the number of sub-flower-clusters and the average of cluster length of the 3rd to 5th flower clusters acropetally from the first flowering node on the main stem in three buckwheat cultivars sown on Aug 28. In DM clusters, the number and thelength of the longest branch were measured. See the notes of Fig. 2 and 3.

6

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Fig. 7.

Effect of different day length on the number of flowers per flower cluster in three buckwheat cultivars sown on Aug 28. See the notes of Fig. 2 and 3.

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Daylength

Daylength Fig. 8.

Effect of different day length on the number of seeds per flower cluster in three buckwheat cultivars sown on Aug 28. See the notes of Fig. 2 and 3.

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Proceedings of the 9th International Symposium on Buckwheat, Prague 2004 20

Seed-set ratio in the flower clusters on the main stem (%)

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Seed-set ratio (%)

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Daylength Fig. 9.

Effect of different day length on the seed-set ratio in the flower clusters on the main stem in three cultivars sown on Aug 28. See the notes of Fig. 2 and 3.

REFERENCES HAGIWARA, M., INOUE, N. AND MATANO, T. (1998): Variability in the length of flower bud differentiation period of common buckwheat. Fagopyrum 15: 55-64. HAYASHI, H., OOTAKE, K. AND MICHIYAMA, H. (1997): Effect of daylength on growth and development of different agroecotype cultivars in buckwheat (Fagopyrum esculentum Moench). Jpn. J. Crop Sci. 66 (extra issue 2): 269-270.*** HAYASHI, H. (1999): Present status of buckwheat cultivars in Japan and buckwheat breeding in Canada. Japan. J. Farm Work Res. 34: 129-135.*** LACHMANN, L. AND ADACHI, T. (1990): Studies on the Influence of photoperiod and temperature on floral traits in buckwheat (Fagopyrum esculentum Moench) under controlled stress conditions. Plant Breeding 105: 248-253. MICHIYAMA, H. AND HAYASHI, H. (1998): Differences of growth and development between summer and autumn-type cultivars in common buckwheat (Fagopyrum esculentum Moench). Jpn. J. Crop Sci. 67: 323-330.* MICHIYAMA, H., FUKUI, A. AND HAYASHI, H. (1998): Differences in the progression of successive flowering between summer and autumn eco-type cultivars in common buckwheat (Fagopyrum esculentum Moench). Jpn. J. Crop Sci. 67: 498-504.* MICHIYAMA, H., ARIKUNI, M., HIRANO, T., AND HAYASHI, H. (2003): Influence of day length before and after the start of anthesis on the growth, flowering and seed-setting in common buckwheat (Fagopyrum esculentum Moench). Plant Production Sci. 6: 235-242.* NAGATOMO, T. (1961): Studies on physiology of reproduction and some cases of inheritance in buckwheat. Report of Breeding Science Laboratory, Faculty of Agriculture, Miyazaki University. 1: 1-212.** SUGAWARA, K. (1958): On the injury of buckwheat pistil. Retardation of pistil growth as influenced by day-length. Proc. Crop Sci. Soc. Jpn. 26: 269-270.** SUGAWARA, K. (1973): Studies on buckwheat. Koryoshuppan, Morioka. 1-96.*** SUGE, H. (1976): Floral initiation in plants. Crops, its morphology and function. Hojo, Y. and Hoshikawa, K. ed., Nogyo Gijutsu Kyokai, Tokyo. pp. 95-111. TABATA, K., OGATA, T. AND OGATA, K. (1931): Effect of day length on growth, flowering and fructification in buckwheat and soybean. Proc. Crop Sci. Soc. Jpn. 3: 188-202.*** UEHARA, S. AND TAGUCHI, R. (1955): Infliences of different day-lengths upon the growth and reproduction of the buckwheat grown in different seasons. Bull. Fac. Text. Seric., Shinshu Univ. 5: 31-35.** UEHARA, S. AND TAGUCHI, R. (1956): The growth and reproduction of the buckwheat of summer and autumn types grown in various seasons. Bull. Fac. Text. Seric., Shinshu Univ. 6: 32-36.** YAMAZAKI, Y. (1947): Buckwheat. Nogyo. 778: 16-32.*** XU, Q. (1938): Studies on the effects of seasonal change of day length and temperature on reproductive period in crop. 2. Flowering time and its uniformity in buckwheat. Nogyo oyobi Engei 13: 1901-1602.*** * In Japanese with English abstract. ** In Japanese with English summary. *** In Japanese.

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Influence of Different Physiological and Stress Conditions on Buckwheat Metallothioneine Gene Expression and Structural and Functional Analysis of its Promoter Region Jelena M. Brkljačić, Dragana B. Majić, Jovanka Miljuš-Đukić, Ana Bratić, Miroslav M. Konstantinović and Vesna R. Maksimović Laboratory for Plant Molecular Biology, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, P.O. box 446, 11000 Belgrade, Serbia and Montenegro ABSTRACT The buckwheat metallothionein (MT3) gene expression was studied throughout seed and leaf development, as well as under the influence of different external stimuli. It was found that Cu and Zn ions had stimulatory effect on expression in leaf. MT3 expression was significantly enhanced in the early stage of seed development in response to Zn ions. In addition, the buckwheat metallothionein MT3 gene was cloned and its promoter region was examined. Computer analysis of the promoter region revealed several putative regulatory sequences which could be involved in responses to different hormonal and external stimuli, as well as the presence of putative binding sites for plantspecific transcription factors. In this analysis the specificity of putative Dof1- and Athb1-binding sites was confirmed, but more importantly, it was found that Dof1- protein factor is capable to interact with some of the protein(s) present in nuclear extracts of buckwheat leaf. In order to functionally analyze the putative promoter, 533bp long fragment was cloned upstream of the gus-reporter gene, within pRD410 binary vector, which was used for Agrobacterium-mediated transformation of buckwheat, tobacco and Arabidopsis. Activity of the defined region was examined by qualitative and quantitative GUS assay. Keywords: buckwheat, metallothioneine promoter, gene expression, stress

INTRODUCTION Metallothioneins (MT) are a group of low molecular weight, Cys-rich proteins, with high affinity for metal ions. MT genes can be expressed at different levels in various tissues and developmental stages, still being able to response to environmental and hormonal factors. In spite of wide distribution and importance of MT, little is known about their function. Pproposed functions of MT are heavy metal detoxification, oxidative stress protection and involvement in cellular metal homeostasis, which is essential for normal plant growth and development. Buckwheat (Fagopyrum esculentum Moench) cDNA clone pBM 290 (GenBank accession number AF056203) coding for a 59 amino acid metallothionein-like protein was isolated from developing buckwheat seed cDNA library (BRKLJAČIĆ et al. 1999). According to Cys arrangements, it was classified as a type 4 MT (RAUSER 1999), but as most authors use the previous classification (ROBINSON et al. 1993), in the following text we specify buckwheat MT-like protein as MT3. Deduced amino acid sequence showed the highest homology with the MT3-like protein from Arabidopsis (MURPHY et al. 1997) and from a number of MT cDNA clones isolated from ripening fruits. Expression analysis showed that buckwheat MT3 mRNAs can be detected in buckwheat root, leaf and throughout seed development in normal conditions (BRKLJAČIĆ et al. 1999). In this paper we present investigation of MT3 gene expression in different physiological and stress conditions by Northern analysis followed by cloning of the buckwheat MT3 gene and structural and functional analysis of its promoter region. The presented data should make a contribution to the understanding of the complex regulation of plant MT gene expression and their function in general.

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MATERIALS AND METHODS Isolation of the buckwheat MT3 gene A total genomic DNA was isolated from mature buckwheat seeds, according to the method of DELLAPORTA (1983). After digestion with EcoRV it was ligated to Marathon cDNA Adaptor (Clontech). Adaptor ligated genomic DNA was used as the template for touchdown PCR amplification using Adaptor Primer 1 (Clontech) and the specific primer WS1 (5'-TCCGCATTTGCACTTGCCATCGTT-3') derived from the MT3 cDNA sequence. Nested PCR amplification was performed with Nested Adaptor Primer 2 (Clontech) and WS1 primer. The PCR products obtained were subjected to Southern blot analysis using the BioPrime® DNA Labeling System (Gibco BRL) and BluGene® Nonradioactive Nucleic Acid Detection System (Gibco BRL), with buckwheat MT3 cDNA as a probe. PCR products that hybridized with the MT3 cDNA probe were eluted and cloned into the pUC18/SmaI/BAP vector, using the SureClone® Ligation Kit (Amersham Pharmacia). The obtained clones were subjected to Southern blot analysis, using MT3 cDNA as the probe to identify the MT3 genomic clone. Final identification was done by sequencing using the ALFexpress™ and ALFexpress™ AutoRead™ Sequencing Kit (Amersham Pharmacia). The complete nucleotide sequence of the 979 bp long clone gFeMT 4.1, representing the buckwheat MT3 gene is available from the GenBank database under accession number AY361956. Preparation of nuclear extracts Nuclear extracts were prepared from leaves or seeds of buckwheat, as described previously (BUSK AND PAGÈS, 1997), with few changes (BRKLJAČIĆ, 2003). Purification of GST-HD-Zip-1 and GST-Dof1∆C proteins GST-HD-Zip-1, containing the homeodomain-leucine zipper part of the Athb-1 protein, fused with the GST of the expression vector pGEX-2T (SESSA et al., 1993) was isolated from E.coli JM 109 strain, kindly provided by Ida Ruberti. The pGST-Dof1∆C, kindly provided by Shuichi Yanagisawa (YANAGISAWA, 1997) was used to transform E.coli XL1Blue strain. Isolation of both GST-HD-Zip-1 and GST-Dof1∆C was done as described in Doctoral thesis of J. Brkljačić. Electrophoretic mobility shift assays (EMSA) Purified proteins or nuclear extracts were incubated in a solution containing 10 mM HEPES pH 7.8, 10% glycerol, 66 mM KCl, 2 mM MgCl2, 5 mM DTT, 0.1 mM EDTA, 100 µM ZnSO4, 1 µg poly (dI-dC)•poly (dI-dC) (Amersham Pharmacia), 10 µg BSA (BioLabs) and 1-3 ng (5000 cpm) probe DNA at room temperature for 30 min. The competition assays were performed in the same reaction conditions, with addition of a 100-fold molar excess of competitor probes and incubation on ice for 30 min before adding the labeled probe. The amounts of proteins used are indicated in the legends of the figures. Electrophoresis was carried out using 5% or 3.5% polyacrylamide gels (acrylamide:bis-acrylamide 29:1), in 0.5 x TBE at 200 V at 4 °C. RNA isolation and Northern blot Total RNA was isolated from 1g of buckwheat leaves or seeds using CTAB method, followed by CsCl gradient purification, as described in Taylor and Powell 1982. For Northern blot analysis, 10µg of the total RNA from buckwheat tissues were blotted on Hybond-N+ membrane and hybridized with 32P-labeled cDNA clone pBM 290 (coding for buckwheat metallothionein-like protein-AF056203) or part of the pFeLEG643 (coding for buckwheat 13S legumin-like storage protein-AY256960) at 65°C in NaPi/SDS buffer without formamide (CHURCH AND GILBERT 1984). Transformation and selection For Arabidopsis transformation, seeds (cv. Columbia) were vernalised for a week at 4º C, then surface sterilized and grown on fitohormones-free MS (Murashige-Skoog) medium. Explants of three weeks old seedlings were used for transformation. For tobacco, leaves from 42


micropropagated plants were transformed, while for buckwheat transformation, isolated cotyledons (cv. Darja) were used. Transformation was performed according to modified leaf disc method (Horsch et al, 1984). (Plant growth regulators added: 1 mg/l IAA and 1 mg/l KIN for tobacco, 1mg/l BAP and 0.1mg/l NAA for Arabidopsis, 2.25mg/ BAP and 0.175mg/l IAA for buckwheat), supplemented with 100mg/l cefotaxime and 100mg/l kanamycin for selection. Regenerated shoot tissue were used for further propagation, and for GUS assays. Buckwheat treatments for Northern blot analysis: Field-grown buckwheat plants (Fagopyrum esculentum Moench, cv. Darja) were “cultivated” in defined water solutions (only roots were submerged). For metal iontreatments, plants were submerged for 36h in solutions with defined rising concentration of ZnSO4 or CuSO4. Control plants were submerged in water or MS medium (Murashige and Skoog 1962). Transgenic plants treatments for quantitative GUS-assay: For hydrogen peroxide (H2O2) young leaves from micropropagated tobacco plants were floated on 0.4M sorbitol solution containing 1mM H2O2, in Petri dish under continuous light at 25°C for 48h. For UV treatment, leaves in a Petri dish were exposed to UV-C light in air-flow cabinet for 2h, and GUS activity was measured after keeping samples in a growth chamber for 24h. All data is a mean ± SE of three replicates. Qulitative and quantitative GUS-assay was performed according to Jefferson et al., 1987. RESULTS AND DISCUSSION Influence of metal ions on MT3-like buckwheat gene expression in leaf Analysis of regulation of MT gene expression was one of the approaches used to get closer to the elusive function of metallothioneins. High constitutive MT3 expression in buckwheat leaf was noticed previously by Northern blot analysis (BRKLJAČIĆ et al. 1999). In order to investigate influence of different metal ions (Cu, Zn) on buckwheat MT3 gene expression in leaf, Northern experiments have been performed, as described in Materials and Methods. The effect of Cu and Zn ions on MT3 gene expression was shown in Figure 1A. The most obvious influence was noticed for Cu ions. The six- and eight-fold increase of MT3 mRNA level was observed when 10µM and 50µM Cu were applied respectively, compared to the level detected for control water-submerged plants. The observed stimulatory effect of Cu ions on buckwheat MT3 expression in the leaf could be considered as a part of a general detoxification system. While in 100µM ZnSO4 two-fold increase of MT3 mRNA level was noticed, 30µM ZnSO4 caused no effect per se, but the same concentration in MS medium caused three-fold increase, which could be explained by synergistic effects with other ions present in MS (especially Co). Observed influence could be related to the proposed MT function in binding and release of Zn as a delivery system for Zn-dependent proteins and could be also linked to cellular redox state (CHEN AND MARET 2001, MARET AND VALLEE 1998, MARET 1995). MT3 buckwheat gene expression throughout seed development and influence of Zn ions. It was shown for the field-grown plants (Figure 1B), that MT3 mRNA was detected already in the early stage of seed development. The expression was continued through seed development, reaching the peak level in mid-maturation stage, where 13S storage protein expression started. The influence of Zn ions on MT3 gene expression was obvious only in the early stage of seed development, where corresponding mRNA levels were increased in proportion with raising concentrations of Zn ions. The proposed function of MT3 in a buckwheat seed could be discussed regarding the stability of storage proteins, which have to

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be maintained in protein bodies of developing seeds. Buckwheat MT3 could chelate excess cations (especially Zn), thus preventing the premature activation of metalloproteinase present in the protein bodies in the same developmental stages (ELPIDINA et al. 1991). Analysis of the buckwheat MT3 gene structure The buckwheat MT3 gene was cloned by a modified 5' RACE method, using one specific primer derived from the buckwheat MT cDNA sequence, as described in Materials and Methods. The entire 979 bp long cloned fragment gFeMT 4.1 was sequenced. The organization of the genomic clone structure is shown in Scheme 1. It was found that genomic fragment gFeMT 4.1 consisted of 3 exons and 2 introns of the buckwheat MT3 gene, preceded by a 640 bp sequence upstream of the first ATG codon. The third exon (58 bp long) was partially represented in the gFeMT 4.1 clone, due to the position of the WS1 primer used for isolation of the gene. Although four different transcriptional start sites were predicted by computer analysis, primer extension results (data not shown) defined a single transcription start site and that position was marked as +1. According to primer extension results, 71 bp of the sequence downstream of +1 represented 5' untranslated region, while 569 bp upstream corresponded to the 5' regulatory region of the buckwheat MT3 gene. The complete sequence of the 5' regulatory region of the buckwheat MT3 is shown in Figure 2. The putative TATA box was placed at position –29 relative to the transcriptional start site. Computer assisted analysis using three different databases revealed regulatory sequences involved in different responses: hormonal and secondary messenger (ERE, TCA element, CGTCA motif, heat shock (HSE), light and stress (GT-1, I-box, GATA, G-box), metal (MRE), as well as putative binding sites for plant-specific or plant-abundant transcription factors (Dof1, NtBBF1, Athb-1, MYB). It should be noticed that distal part of the 5' regulatory region contains two direct repeats (34 bp long), common for plant metallothionein promoters. In order to study the buckwheat MT3 promoter in more detail, we focused on the region M0/P30. The studies included analysis of the interaction of that DNA fragment with the purified protein factors Dof1∆C and HD-Zip-1 domain, as well as with buckwheat nuclear extracts. Binding of Dof1 and Athb-1 and buckwheat leaf nuclear extract to the M0P30 buckwheat putative promoter fragment The studies included analysis of interactions of M0P30 buckwheat putative promoter fragment with the purified Dof1∆C domain of Dof1 and the HD-Zip-1 domain of Athb-1 transcription factors, as well as with buckwheat nuclear extracts. In this analysis the specificity of putative Dof1- and Athb1-binding sites was confirmed ( Figure 3A), but more importantly, competition for complex formation of Dof1∆C domain protein factors with protein(s) present in buckwheat leaf nuclear extracts was noticed ( Figure 3B) Functional analysis of MT3 promoter region using transgenic plants Our next goal in analyzing MT3 promoter region, following gel shift experiments, was to confirm it's function as a gene promoter in vivo. Putative promoter fragment flanked by M0 and P0 primers, located between positions - 114 and – 647 (taking the ATG as +1 ) and including all proposed regulatory elements, was chosen to be analyzed. In order to determine its capability to drive GUS gene expression in transgenic plants, it was cloned upstream of the GUS reporter gene, within pRD410 binary vector, giving the pRDM0P0. After electrophoration to A.tumefaciens LBA4404, resulting strain was used for transformation of tobacco, Arabidopsis and buckwheat explants. Efficiency of transformation process was summarized on Table 1. Putative transformants were further selected for the presence of M0P0-GUS cassette and absence of CaMV35S:GUS cassette, by PCR analysis. Control plants were transformed with pRD410 binary vector carrying CaMV35S:GUS cassette as a positive control.

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Tab. 1 LBA4404/pRD410/MoPo LBA4404/pRD410 Arabidopsis Tobacco Arabidopsis Tobacco Buckwheat Buckwheat No.transformed explants Kanamycin resistant GUS positive eplants Explants regenerated No. of nptII/gus positive transformants

120

100

80

120

100

80

100

92

65

91

88

66

96 (100%)

92 (100%)

64 (87%)

83 (65%)

56 (50%)

66 (86%)

89

76

5

68

70

5

15

10

nt

9

10

nt

The activity of MT buckwheat promoter was firstly examined by qualitative GUS assay in regenerated tissues of To transgenic plants (Figure 4). Quantitative GUS assay was performed for transformed tobacco only. Quantitative GUS assay and stress treatments We measured the activity of β-glucuronidase fluorimetrically, in crude protein extracts of different tobacco M0P0 transformants. M0P0 transformants showed significantly higher GUS expression level regarding to untransformed control plants, although much lower than 35S:GUS transformants, witch was expected keeping in mind that 35S is an extremely strong promoter. In the next step we determined buckwheat MT promoter activity in response to stress influence of hydrogen peroxide, and UV light (Figure 5). In the majority of independent transformation lines, UV irradiation and H2O2 treatment increased GUS expression levels. Considering these results we can assume that MT3 promoter is an oxidative stress regulated promoter. That would be in agreement with one of the proposed MT functions – involvement in oxidative stress protection network. Our further experiments will be focused on the analysis of specific protein-DNA interactions responsible for the regulation of MT gene expression in different stress and physiological conditions.

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Fig. 1 A. Northern analysis of buckwheat MT3 mRNA expression in leaf under influence of Cu and Zn ions. Total RNA was isolated from leaves of buckwheat treated with defined concentrations of ZnSO4/CuSO4, or MS medium and 10µg was loaded on each lane. Hybridization was done with labeled MT3 cDNA clone (pBM 290). Middle panel represents a part of ethidium bromide stained gel showing 25S rRNA to confirm equal loading. Bottom panel represents quantitative evaluation of hybridization signals performed using BioDocAnalyze software package (Biometra). The results of three independent experiments were statistically processed. B. Comparative Northern analysis of buckwheat MT3 and buckwheat 13S legumin mRNA expression through seed development and influence of Zn ions.Total RNA was isolated from buckwheat seeds in different developmental stages either from field grown plants (field), or from plants treated with raising concentration of ZnSO4, including water control. Labels above the first panel correspond to all panels. Blots were hybridized with labeled pBM290 (MT3) and re-hybridized with 13S probe (13S). Ethidium bromide stained 25S rRNA is shown at the bottom of each panel to confirm equal loading. A

B

Scheme 1 Schematic presentation of the gFeMT 4.1 clone. Exon/intron sequences are marked as E/I, respectively. The transcription start site, determined by primer extension, is marked as +1. WS1- primer was used in the cloning procedure.

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Fig 2 Nucleotide sequence of the 569 bp long 5' regulatory region of the buckwheat MT3 gene. The sequence represents part of the gFeMT 4.1 clone. Numbering was done with respect to the transcription start site (+1). The putative TATA box; binding sites for Athb-1, Dof1, NtBBF1 and MYB-like transcription factors; GT-1 motif, I-box; HSE; GATA; TCA-element and CGTCA-motif; G-box; ERE; MRE; SBF-1 binding sites and octamer motif are underlined. The regulatory elements were predicted using the MatInspector professional program with 0.75 as the value for core similarity and optimized matrix similarity, except for MRE prediction (0.70/0.70). G-box, HSE, GT1, TCA and CGTCA motifs were predicted using the PlantCARE database.

Fig. 3 Analysis of DNA-binding specificity of Dof1∆C and HD-Zip-1 by EMSA. A. Radiolabelled M0/P30 probe was incubated in the absence of proteins (lane 1), with 50ng of purified Dof1∆C (lane 2) or 50ng of purified HD-Zip1 (lane 4). In the competition experiments 100-fold molar excess of unlabeled probe was used either with Dof1∆C (lane 3) or with HD-Zip1 (lane 5). B. Analysis of DNA-binding pattern of Dof1∆C in the presence of buckwheat leaf nuclear extract by EMSA.Radiolabelled M0/P30 probe was incubated in the absence of proteins (lane 1), leaf nuclear extract (lane 2), 5 ng of purified Dof1∆C applied with leaf nuclear extract (lane 4), 10 ng of purified Dof1∆C applied with leaf nuclear extract (lane 6), 50 ng of purified Dof1∆C applied with leaf nuclear extract (lane 8), with 10 ng of purified Dof1∆C (lane 10). In the competition experiments 100-fold molar excess of unlabeled probe was used (lanes 3,5, 7, 9 ) The amount of proteins in the nuclear extract was approximately 10 µg per reaction.

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Fig. 4 Histochemical GUS assay showing positive GUS staining in (B) buckwheat, (C) Arabidopsis and (D) tobacco tissue transformed with M0P0/GUS construct. (A) untransformed buckwheat as a control A

B

C

D

Fig. 5 Quantitative GUS activity in UV and H2O2 treated tobacco leafs harbouring M0P0/GUS construct 4000 3500 3000 2500

sorbitol

2000

H2O2 UV

1500 1000 500 0 MoPo- MoPo- MoPo- MoPo- MoPo- MoPo- MoPo- MoPo- MoPo1 2 3 4 10 12 51 55 59

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REFERENCES BRKLJAČIĆ J.M., MAKSIMOVIĆ, V.R., RADOVIĆ, S.R., SAVIĆ A.P.: (1999): Isolation of metallothionein-like cDNA clone from buckwheat. J. Plant Physiol. 154: 802-804 BRKLJAČIĆ J. (2003): Doctoral thesis. University of Belgrade BUCHANAN-WOLLASTON V. (1997): The molecular biology of leaf senescence. J. Exp. Bot. 48 (307): 181-199 BUSK P.K., PAGÈS M. (1997): Microextraction of nuclear proteins from single maize embryos. Plant Mol. Biol. Rep. 15: 371-376 CHAN R.L., GAGO G.M., PALENA C.M., GONZALES D.H. (1998): Homeoboxes in plant development. Biochim. Biophys. Acta 1442: 1-19 CHEN Y., MARET W. (2001): Catalytic selenols couple the redox cycles of metallothionein and glutathione. Eur. J. Biochem. 268: 3346-3353 CHURCH G.M., GILBERT W. (1984): Genomic sequencing. Proc. Natl. Acad. Sci. 81(7): 1991-1995 DELLAPORTA S.L., WOOD J., HICKS J.B. (1983): Maize DNA minipreps. Maize Genet. Coop. Newsl. 57: 26-29 ELPIDINA E.N., VOSKOBOYNIKOVA N.E., BELOZERSKY M.A., DUNAEVSKY Y.E. (1991): Localization of a metalloproteinase and its inhibitor in the protein bodies of buckwheat seeds. Planta 185: 46-52 HORCH R.B., FRY J.E. HOFFMANN N.L., EICHOLTZ D., ROGERS S.G., FRALEY R.T. (1985): A simple and general method for transferring genes into plants. Science, 227: 1229-1231 JEFFERSON R.A., KAVANAGH T. A., BEVAN M. W. (1987): GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-3907 MARET W., VALLEE B.L. (1998): Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc. Natl. Acad. Sci. 95: 3478-3482 MARET W. (1995): Metallothionein disulfide interactions, oxidative stress, and the mobilization of cellular zinc. Neurochem. Int. 27: 111-117 MURASHIGE T., SKOOG F. (1962): A Revised medium for rapid growth and bioassays with tobacco tissue culture. Hort Sci 9: 175-180 MURPHY A., ZHOU J., GOLDSBROUGH P.B., TAIZ L. (1997): Purification and immunological identification of metallothioneins 1 and 2 from Arabidopsis thaliana. Plant Physiol 113: 12931301 RAUSER E.W. (1999): Structure and Function of Metal Chelators Produced by Plants. Cell Biochem. Biophys. 31: 19-48 ROBINSON N.J., TOMMEY A.M., KUSKE C., JACKSON P.J. (1993): Plant metallothioneins. Biochem. J. 295: 1-10 SESSA G., MORELLI G., RUBERTI I. (1993): The Athb-1 and –2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities. EMBO J. 12: 3507-3517 YANAGISAWA S. (1997): Dof DNA-binding domains of plant transcription factors contribute to multiple protein-protein interactions. Eur. J. Biochem. 250: 403-410 YANAGISAWA S. (2002): The Dof family of plant transcription factors. Trends Plant Sci. 7: 555-560

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Germination of Buckwheat Grain: Effects on Minerals, Rutin, Tannins and Colour Myung Heon Lee, Jung Sun Lee and Tae Heon Lee1 Department of Biofood, Hallym College, Chunchen, 200-711, Korea 1 R&D Center, Dongwon F&B Co., Ltd., Songnam, 462-120, Korea ABSTRACT The effect of germination on proximate composition, minerals, rutin, tannins and colour of buckwheat grains was studied. Buckwheat grains were germinated at 25°C for 7 days, and the buckwheat grain (BW), germinated-buckwheat grains for 3 days (G3-BW) and 7days(G7-BW) were examined. In proximate composition of BW and germinated buckwheat grains (GBWs), the crude protein and fat contents of GBWs increased as compared to those of BW, whereas the digestible sugars decreased, on dry basis. The total dietary fiber, insoluble dietary fiber and soluble dietary fiber contents of GBWs appeared higher than those of BW, on dry basis. The contents of calcium, phosphorus, magnesium, potassium, sodium, zinc, copper and manganese in GBWs increased but the iron content of G3-BW decreased compared to those of BW, on dry basis. The rutin contents on dry basis sharply increased in GBWs in compared with BW, especially G7-BW. The tannin contents on dry basis markedly increased in G7-BW compared to BW. The colour values for redness and yellowness markedly increased in GBWs. Keywords: buckwheat, germination, minerals, rutin, tannins, colour

INTRODUCTION Buckwheat (Fagopyrum esculentum Moench) is a health food because it is rich in essential nutrients including protein and mineral. It is known to contain various antioxidative compounds such vitamins B1, B2, and E, and phenolic compounds, such as rutin, quercetin, and proanthocyanidines (condensed tannins) (WATANABE et al. 1995, WATANABE et al. 1997). Increasing attention to buckwheat as a functional food is currently occurring, but it’s utilization and the applied products are limited in Korea. The biochemical composition and utilization of the buckwheat sprouts and germinated buckwheat grain as a functional vegetable and grain has been reported (KIM et al. 2001, LEE 1999). So buckwheat sprouts and germinated buckwheat grain were considered to be an attractive food. The purpose of this study was to provide the more information about the nutritional properties of the germinated buckwheat grain under different environmental condition. MATERIALS AND METHODS The grains used in this study were cultivars Yangjulmaemil of common buckwheat and cultivated in Pyeongchang, Kangwon-Do of Korea. Buckwheat grains were soaked in water and germinated at 25°C for 7 days. Samples of the germinated-buckwheat grains were taken at 3, 7 days. The germinated buckwheat grains reached a root length from 10.2mm after 3 days to 39.3mm after 7 days of germination. The germinated samples were freeze-dried and natural-dried, milled and stored in refrigerator. The moisture, ash, protein, lipid (AOAC 1990), total dietary fiber and insoluble dietary fiber (AOAC 1995) were determined. Minerals such as Ca, P, Mg, K, Na, Fe, Zn, Cu and Mn were determined with inductively coupled spectrometer (ICP, Lactam 8440, Australia) and Se was assayed using atomic absorption spectrometer (Spectra AA 40, U.S.A). The tannin was assayed by vanillin-HCl method of Burns (1971). The rutin was analyzed by the HPLC analysis with µ-Bondapak C18 (Jasco 851-AS, Japan). The Colour was measured using a colour meter (colour & colour difference meter, No. UC 600-IV, Japan). Statistical analysis was performed using ANOVA. Differences were considered to be significant when p