ISSN 0352-3020 FAGOPYRUM NOVOSTI O AJDI - NAJNOVVSZE SNFORMACJE O GRYKE BUCKWHEAT NEWSLETTER - БЮЛЛЕТЕНЬ "ГРЕЧИХА" NOVOSTI O HELJDI - LES ANNALES DE SARRASIN
Os
FAGOPYRUM (LJUBLJANA), Vol. 13, 1993, 1-44 FAG OP Υ RU M (NOVOSTI O AJDI), zvezek 13, Ljubljana 1993. Izdajatelj: Biotehniška fakulteta, Oddelek za agronomijo, Center za rastlinsko biotehnologijo in žlahtnjenje, Jamnikarjeva 101, 61111 Ljubljana Za publikacijo odgovarja: Ivan Kreft Oblikovanje teksta in ilustracij: Borut Bohanec. Tekst urejen s programom za namizno založništvo STEVE avtorja Primoža Jakopina, Trnovska 2, Ljubljana Lektoriranje angleščine: Martin Cregeen Tisk: Tiskarna Planprint d.o.o. Ljubljana, Rožna dolina c. IV/32-36, cena izvoda 100 SIT. V publikacijo so vključena znanstvena dela (izvirna dela in pregledni članki), vsako pred objavo pregledata vsaj dva mednarodno priznana strokovnjaka. Bibliografske in druge informacije niso recenzirane. Po mnenju Ministrstva za znanost in tehnologijo št. 2839/92 z dne 5. 3. 1992 šteje revija Fagopyrum med proizvode, za katere se plačuje 5 % davek od prometa proizvodov na osnovi 13. točke tarifne številke 3 tarife davka od prometa proizvodov in storitev. Pri finansiranju revije Fagopyrum sodeluje Ministrstvo za znanost in tehnologijo Republike Slovenije.
FAGOPYRUM (BUCKWHEAT NEWSLETTER), Vol. 13, Ljubljana 1993. Published under the auspices of the International Buckwheat Research Association (IBRA). Editor: Ivan KREFT (Biotechnical Faculty, Agronomy Department, Jamnikarjeva 101, 61111 Ljubljana, Slovenia) Advisory committee: Taiji ADACHI (Fac. of Agriculture, Miyazaki University, Gakuen Kibanadai Nishi 1-1, Miyazaki 889-21, Japan), N.V. FESENKO (All-Union Research Institute of Legume and Groat Crops, Orel, Russian Federation), Toshiko MATANO (Fac. of Agriculture, Shinshu University, Ina, Nagano-Ken, Japan), Takashi NAGATOMO (7075 Funahiki, Kiyotake-cho, Miyazaki, 889-16, Japan), Marek RUSZKOWSKI (Institute of Soil Science and Plant Cultivation, 24-100 Pulawy, Poland) V.P. SINGH (School of Studies in Botany, Vikram University, Ujjain 456 010, India). Language editor: Martin Cregeen Managing editor: Borut Bohanec Published by: Biotechnical Faculty, Agronomy Department, 61111 Ljubljana, Jamnikarjeva 101, Slovenia Subscription Information: One volume per year (one issue in 1993). Subscription prices for 1993 are 30.00 USD for individuals and scientific institutions.
FAGOPYRUM (Buckvvheat Newsletter) is open to everyone interested in buckwheat and will cover ali aspects of buckwheat research: genetics, cytology, breeding, cultivation, nutrition, utilization, biochemistry and other. No special priority is given to any language. Scientific papers, reviews, research notes on work in progress or on results not yet published: comments and speculations related to buckvvheat; list of stock materials wanted or available; lists of names, addresses and field of work of scientists who have expressed the desire to receive the Newsletter; lists of publications which are related to buckwheat and which have appeared during preceding years; announcements concerning the promotion of research on buckwheat (workshop, symposia, and so on); abstracts and/or contents of published papers/books on buckwheat; bibliographies and other information related to buckwheat or buckwheat research will be published. In order to facilitate the elaboration of the bibliography scientists are asked to send reprints of their own publications to the editor of Fagopyrum.
Front page photo: Cultivation of buckwheat in Tibet: harvest of common buckvvheat at Shigatse and flowering of common buckvvheat, at lOkm east of Shigatse (See paper of O. Ohnishi, pp. 3-10) X L«" J 771,0
KAZALO - CONTENTS
3 OHMI OHNISHI: A memorandum on the distribution of buckwheat species in Tibet and the Himalayan hills: has buckwheat crossed the Himalayas?
11 SAYOKO IKEDA and YOSHIHISA YAMAGUCHI: Zine contents in various samples and products of buckwheat
15 SAYOKO IKEDA and TAKAKO SHIMIZU: Changes in zine of buckwheat on processing into noodles and cooking
21 KIYOKAZU IKEDA and MASAYO KISHIDA: Digestibility of proteins in buckwheat seed
25 TAMAKI HIROSE, AKIO UJIHARA, HIROMI KITABAYASHI and MINEO MINAMI: Morphology and identification by isozyme analysis of interspecific hybrids in buckwheats
31 WANG ZHUANHUA, LIN RUFA, ZHANG ZHENG, ZHOU MINGDE: Purification and characterization of superoxide dismutase from tartary buckwheat leaves
35 BRANKA JAVORNIK and BOJKA KUMP: Random amplified polymorphic
DNA (RAPD) markers in buckwheat
40 Sixth international symposium on buckvvheat: First announcement
41 PROFESSOR DR. MAREK RUSZKOWSKI, 1925-1993 The publication of this issue of Fagopyrum was supported by:
The Ministry of Science and Technology of Slovenia; Japanese members of IBRA (International Buckivheat Research Association) and by Mr. Satsuma, of Mimiu, Osaka and Tokyo; The Centre of Plant Biotechnology and Breeding, Agronomy Department, Biotechnical Faculty, University of Ljubljana Fagopyrum 13 (1993): 3 - 10
A memorandum on the distribution of buckwheat species in Tibet and the Himalayan hills: has buckwheat crossed the Himalayas?
Ohmi Ohnishi Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan
Key words: Fagopyrum cymosum, F. gracilipes, F. tataricum, allozyme variability, buckwheat cultivation in Tibet, wild form of tartary buckwheat.
Abstract
Cultivation of common and Tartary buckwheats is rare in central and western parts of Tibet, while it is relatively extensive in eastern parts of Tibet. As for the wild species, wild Tartary is widely distributed throughout Tibet, mainly along the Yalutsangpu river. The distribution of F. cymosum is limited only in eastern parts of Tibet. No other wild species can be seen. Cultivated buckwheats, both common and Tartary, diffused from southern China, their original birth plače, along the southern slopes of the Himalayas and two wild species, F. cymosum and F. gracilipes, took the same route in their diffusion. Wild Tartary has spread westward along the Yalutsangpu river in Tibet and arrived at Kashmir and Karakoram. Transmittance of buckwheat species across the Himalayas has been extremely limited, if any.
Raziskava razširjenosti vrst ajde v Tibetu in na območju Himalaje: ali je ajda prečkala Himalajo?
V osrednjih in zahodnih predelih Tibeta le redko pridelujejo navadno in tatarsko ajdo, medtem, ko jo relativno pogosto pridelujejo v vzhodnih predelih. Od divjih vrst je divja tatarska ajda razširjena po vsem Tibetu, predvsem ob reki Yalutsangpu. F. cymosum je le v vzhodnih predelih Tibeta. Druge divje vrste niso našli. Gojena ajda, tako navadna kot tatarska, sta se sem razširili iz južne Kitajske, od koder izvirata, po južnih pobočjih Himalaje. Tudi divji vrsti F. cymosum in F. gracilipes sta se širili po isti poti. Divja tatarska ajda se je razširjala ob reki Yalutsangpu vse do Kašmirja in Karakorama. Prehoda vrst ajde preko Himalaje ni bilo, ali pa je bil le v zelo majhnem obsegu. (Prevod uredništva).
Introduction the distribution of wild buckwheat species. Wang (1989) has already reported on the Travelling in Tibet is not an easy task for cultivation and distribution of buckwheat in foreigners, because of the poor Tibet, mainly the eastern parts of Tibet, transportation system and of the and commented on the rich buckwheat governmental restriction policy against genetic resources in this area. foreigners. There is, therefore, no recent This article overviews buckwheat report on any cultivated crops in Tibet by cultivation and the distribution of wild European scientists. I had opportunities to species in the central parts of Tibet. I travel in Tibet in 1990 and 1992, and had entirely rely on Wang (1989) for chances to зее buck\vheat cultivation and information on the distribution in eastern parts of Tibet. Nothing is reliably known as noodles. Wild Tartary buckvvheat could for western parts of Tibet. By comparing be also seen in Shigatse, primarily in wild species distribution in Tibet and the barley or buckwheat fields. No other wild Himalayan hills, and by a comparative buckwheat species was found in Lhasa and isozyme study of cultivated common and Shigatse districts. Tartary buckwheat in these two regions, I Wang (1989) reported cultivation of concluded that buckvvheat species, both common buckwheat along the Yalutsangpu cultivated and wild ones, have never river from Mangkan in the east to Zhada crossed the Himalayas; cultivated common in the west. However, the distribution and Tartary buckwheat diffused along the along the river is apparently discontinuous, southern slopes of the Himalaya (see, there being a major disruption in the Ohnishi 1992b), while wild Tartary Lhasa area. Furthermore, I can not believe buckwheat travelled westward along the common cultivation in western parts of Yalutsangpu (Brahmaputra) river in Tibet Tibet, around Zhada in the Ali district. Не and arrived in Kashmir and Karakoram, also mentioned that Tartary buckwheat is where we can now see it. cultivated in almost the same regions as common buckvvheat; i.e. along the Cultivated and wild buckwheat Yalutsangpu river and more extensively in species in Tibet eastern parts of Tibet. As for wild Tartary, he mentioned a My travel route in Tibet is given in Figure distribution up to 4900 m above sea level 1. At present, foreigners are completely at Mt. Mila and it forms populations at excluded from eastern parts of Tibet, lower altitudes, below 3000 m. Plant height Changdu and Linzhi districts; so I quote is lower, several tens of cm in the high Wang (1989) on the distribution of altitude zone, but at a lower altitude, it buckwheat in this region. Fig. 1 also gives may be higher than 100 cm, as I saw in the distribution of buckwheat species in Sichuan province. Не distinguished several Tibet and the Himalayan hills. grain types, grey, black, notched winged, I saw no buckwheat cultivation in the but I believe that they ali belong to the Lhasa district in 1990 and 1992; wheat same subspecies, F. tataricum ssp. Potanini and barley supplemented by vegetables such Batalin, the wild form of tartary as potatoes, beans, radish and rape are the buckwheat. main crops in this area (maize is lacked in Не also reported the distribution of F. Tibet) . The same is also true in the cymosum Meisner in south eastern parts of Shannan district (Zedang, Gongga; see Fig. Tibet; he described large leafed and small 1), where the climate is slightly warmer leafed F. cymosum. I cannot decide which and drier than Lhasa. Wild Tartary (F. scientific subspecies or variety correspond tataricum ssp. Potanini Batalin), however, to each, or whether the difference in leaf can be seen commonly on road sides and size is due only to soil or climatic farmer's fields both in Lhasa and Shannan. conditions. F. cymosum is seen mainly in In contrast, in Shigatse I saw cultivation warmer districts as a cultivated buckwheat of common buckvvheat (F. esculentum species. He noticed the rich genetic Moench) and occasionally Tartary resources of cultivated buckwheat and F. buckwheat {F. tataricum Gaertn.), too (see cymosum in Tibet and mentioned that the Fig. 2). Chinese farmers rather than history of cultivation in eastern Tibet is Tibetans mainly cultivated them. Because very old, more than 4600 years in the of the cool climatic conditions, the plant is Lancang river valley near Changdu (The only 30-60 cm tali. It is sown in July and Lancang river is the upper stream of the harvested in late September. It is Mekon river, see Fig. 1). consumed as a tsampa like food and never Fig.l: Distribution of Fagopyrum species in Tibet and the Himalayan hills. • • • • Travelling route o: F. esculentum • : F. tataricum (cultivated) ®: F. tataricum ssp. Potanini (wild form) •: F. cymosum э Fig. 2: Cultivation of buckwheat in Tibet: A: Harvest of common buckvvheat at Shigatse, В Flovvering of common buckvvheat, at lOkm east of Shigatse. го o со го ко • I • • • • o о о о о o • o· к. к. «к к. к. k. k. «— гм го rr m ι я • « • • « Η 2 00 σ σι σ СГ\СГ\ σ • σ \· ο σι σι σι σ СГ\№ σ σ\ CU к. о. •к «к •к »k к. -H w О ГО о о 1 CN (Ν Γ- -С с η со го in о ίη σ\ νοι Λ со • • • а • • Ι • И 0 • • • • • « • о о о О о О О О -н -н и m 00 о τ— t— (Ν 1Л• о· о С P с »— tu U") m LA LO •4· ГО rtг о Л (0 о I ъ Sb к. ч «к к. о о о о о "Ч< О (Ν О гЧ •H ж г-
Table 2. Allele frequency (%) at polymorphic loci in vvild and cultivated tartary buckwheat populations
Vild population Cultivated population Locus Allele Central Northern Southern 1 2 3 4 Lixian Markan Tibet Pakistan China Bhutan Nepal China
PGM-2 N 0 0 100 100 100 100 100 100 F 100 34 .6 0 0 0 0 0 0 G 0 65 .4 0 0 0 0 0 0 ADH N 0 9 .1 100 100 100 100 100 100 F 100 90 .9 0 0 0 0 0 0 LAP F 0 0 0 0 0 69.2 36.8 0 N 0 0 100 100 100 30.8 63.2 100 S 46.2 92., 3 0 0 0 0 0 0 U 53.8 7., 7 0 0 0 0 0 0 IDH N 100 100 100 100 44.1 100 100 100 F 0 0 0 0 55.9 0 0 0
1, 2, 3 and 4 : average of 8, 19, 19 and 10 populations, respectively Discussion river, Nakao (1957)'s Tibetan are. This is in sharp contrast vvith the diffusion of Commercial, cultural and political contact cultivated tartary mentioned above. Thus, between Tibet and the Himalayan hills are in spite of a high possibility of wellknown to have been historically transmittance of buckvvheat species across extensive (see for example Bhatt 1977). the Himalayas, no buckvvheat species seems Among crops, barley, Tibetan's major food to have crossed. The assertion that tartary as tsampa, is believed to have been buckvvheat cultivated in the Himalayan transported across the Himalayas. Naked hills came from Tibet, by Matsuoka (1956), six-rowed barley cultivated in high may be misleading. mountain hills in Nepal and India comes F. cymosum found at Zhangmu, in Tibet, from Tibet (Nakao 1956, Takahashi et al. located just on the border of Tibet and 1968). Nepal (see Fig. 1) is the only sample which Buckvvheat, both common and tartary, crossed the Himalayas. Apparently, this are extensively cultivated in the high hills sample came from Nepal. Wang(1989) of the Himalayas. They are commonly deseribed a notehed vving tartary in Jilong cultivated in eastern parts of Tibet (Wang xian in central Tibet. Contamination of 1989), Linzhi and Changdu districts, but notehed vving tartary is a characteristic in cultivation is relatively rare in central and the high hills of the Kaligandaki- Mustang western parts of Tibet. Today, it is well region (Hirose et al. 1992); since Jilong is recognized that common buckwheat not so far from Kaligandaki, this tartary originated in Yunnan province of China may also be a migrant from Nepal, rather (Ohnishi 1991, Li and Yang 1992) and than from eastern Tibet. diffused along the southern slopes of the Why did buckvvheat species not cross Himalayas in one direction (Ohnishi 1992b), the Himalayas? As far as wild tartary in while Tartary buckwheat was born in the Tibet is concerned, climatic conditions at western part of Sichuan province and several passes over 4000 m a.s.l. betvveen travelled westward following the same the Himalayas and the Yalutsangpu valley route as common buckwheat (Ohnishi seems too severe for natural settlement. F. 1992a). Two wild weedy species, F. cymosum generally prefers a relatively cymosum and F. gracilipes Hermsl. took vvarmer climate. It arrived at Zhangmu in the same route as cultivated buckwheat Tibet from Nepal, but it could not proceed (see Ohnishi 1992b). to the Tibetan plateau, probably for the As can be seen in Figure 1, F. same reason mentioned above. cymosum is distributed over the eastern Cultivated buckwheat may have parts of Tibet, but completely lacking in overcome high passes or plateaus vvith central and western parts of Tibet. In human assistance, but actually it didn't. contrast, wild tartary buckvvheat {F. Migration of peoples and of the tataricum ssp. Potanini Batalin) is widely accompanying crops seem mainly from distributed in Tibet, and further vvest up Tibet to the Himalayan hills; rice and to Kashmir and northern Pakistan. Wild sugarcane of Nepal and India vvere tartary has never been reported from the bartered for salt and vvool from Tibet (see, southern slopes of the Himalayas (see for example, Hagen 1980). Hooker 1886, Hara 1966, Polunin and Cultivation of buckvvheat in central Stainton 1984). Among vvild tartary Tibet was rare, so buckvvheat has no populations, only the vvild population from chance to migrate from Tibet to the Sichuan province has genetic variability in Himalayan hills. Buckvvheat cultivation in isozymes(see Table 2, Ohnishi 1992a) that the Himalayan hills vvas vvidespread, and clearly indicates diffusion of vvild tartary probably came from southern China or from east to vvest along the Yalutsangpu eastern Tibet as discussed in Ohnishi (1992b). It never spread to the Tibetan Himalaya. (ed. H. Kihara) pp.313-343. Fauna and Flora plateau and the Yalutsangpu valley. At Res. Soc., Kyoto Nakao S. 1957. Transmittance of cultivated plants through present, I have no reasonable explanation of Sino-Himalayan route. In Peoples of Nepal Himalaya. this, but barren plateaus and high passes (ed. H. Kihara) pp.397-420. Fauna and Flora Res. Soc., in Tibet rather than the Himalayan massif Kyoto itself may have been a barrier against the Ohnishi O. 1988. Population genetics of cultivated common diffusion of buckvvheat. buckvvheat, Fagopyrum esculentum Moench. VII. Allozyme variability in Japan, Korea and China. Jpn. J. Genet. 63:507-522. Ohnishi O. 1990. Analyses of genetic variants in common buckvvheat Fagopyrum esculentum Moench: A revievv. References Fagopyrum 10:12-22. Ohnishi O. 1991. Discovery of vvild ancestor of common Bhatt D. D. 1977. Natura1 History and Economic Botany of buckvvheat. Fagopyrum 11:5-10. Nepal. Orient Longman, Calcutta Ohnishi O. 1992a. Search for the vvild ancestor of tartary Hagen T. 1980. Nepal-Kingdom in the Himalaya, 3rd ed. buckvvheat. Jpn. J. Breed. 42. Suppl. 1:276-279. (in Kumerly & Frey, Berne Japanese) Hara H. 1966. The Flora of East Himalaya. Токуо Univ. Ohnishi O. 1992b. Buckvvheat in Bhutan. Fagopyrum 12:5-13. Press Токуо Ohnishi O. and Nishimoto T. 1986. Population genetics of Hirose Т., Yoshida M., Matsuhira N., Ujihara A. and cultivated common buckvvheat, Fagopyrum esculentum Minami M. 1992. Studies on the differentiation of Moench. V. Further studies on allozyme variability in ecotypes of genus Fagopyrum in Nepal. IV. Variation of the Indian and Nepali Himalaya. Jpn. J. Genet. grain type of tartary buckvvheat in Mustang region. Jpn. 62:397-414. J. Breed. 42. suppl. 1:280-281. (in Japanese) Polunin O. and Stainton A. 1984. Flowers of the Himalaya. Hooker J. D. 1866. Flora of British India. v. Polygonaceae Oxford Univ. Press, Delhi pp.22-61. Reeve & Со., London Takahashi R., Hayashi J., Hiura U. and Yasuda S. 1968. A Li Q. Y. and Yang M. X. 1992. Preliminary investigation on study of cultivated barleys from Nepal Himalaya and buckvvheat origin in Yunnan, China. Proc. 5th Intl. Symp. northern India, vvith special reference to their Buckvvheat at Taiyuan(eds. Lin R., Zhou M., Tao Y., Li phylogeneti differentiation. Ber. Ohara Inst. Landvv. Biol. J. and Zhang Z.) pp.44-46. Agr. Publ. House, Beijin Окауата Univ. 14:65-122. Matsuoka M. 1956. Buckvvheat. In Land and Crops of Wang T. Y. 1989. Buckvvheat germplasm resources in Tibet, Nepal Himalaya. (ed. H. Kihara) pp.125-134. Fauna and pp. 49-51. In Collection of Scientific Treatises on Flora Res. Soc., Kyoto Buckwheat in China. (ed. Chinese Buckvvheat Research Nakao S. 1956. Barley. In Land and Crops of Nepal Assoc.) Academic Journal Publ. Со., Beijin (in Chinese) Fagopyrum 13 (1993): И -14
Zine contents in various samples and products of buckwheat
Sayoko Ikeda and Yoshihisa Yamaguchi
Faculty of Nutrition, Kobe-Gakuin University Nishi-ku, Kobe 651-21, JAPAN
Abstract The contents of zine in various samples of buckvvheat were analyzed. A variation in the zine content of various cultivated buckvvheat seeds, including tetraploid, was found. On the other hand, there was less or substantially no difference in zine content betvveen common buckvvheat and tartary buckvvheat. The content of zine in commercial buckvvheat flours available in Japan was in a range from 0.63 mg to 2.38 mg per 100 g flour; and the content of zine in buckvvheat flours available in Slovenia was relatively higher than those in Japan. The nutritional implications of these findings are discussed. Vsebnost cinka v različnih vzorcih in izdelkih iz ajde
Avtorja sta raziskovala vsebnost cinka v različnih vzorcih ajde. Ugotovljene so različne vsebnosti v različnih vzorcih. Med vsebnostjo cinka navadne ajde in tatarske ajde praktično ni razlike. Vsebnost cinka mok ajde, ki so na prodaj na Japonskem je od 0,63 mg do 2,38 mg na 100 g moke; vsebnost cinka v mokah kupljenih v Sloveniji je razmeroma višja. Prediskutiran je prehranski pomen teh ugotovitev. (Prevod uredništva).
Introduction variety of buckvvheat species, including common and tartary species, is available Buckvvheat is an important crop in the for human consumption around the vvorld. vvorld. Although buckvvheat is taxinomically There are also many kinds of buckvvheat distant from true cereals, the seed has products, both flour products and groats; many chemical characteristics in common but little information is available about the vvith cereal grains and thus is usually zine content of these buckvvheat species classified vvith cereals. Cereals, including and products. buckvvheat, serve as an important source This study vvas designed to analyze the for supplying some essential nutrients due zine content in various kinds of buckvvheat to their large daily consumption. species and products. Zine is an essential micronutrient for humans. Nutritional deficiency of zine is Materials and methods fairly common throughout the vvorld and is a major nutritional problem (Prasad 1982, MATERIALS. Twenty-seven samples of Sandstead 1973, Moynahan and Barnes buckvvheat vvere selected for this 1973). Interest in zine in food and its investigation: eight kinds of common nutritional funetion is grovving rapidly. In buckvvheat seed (F. esculentum); two kinds this connection, buckvvheat contains a of tartary buckvvheat seed (F. tataricum); relatively high amount of zine (Ikeda et al. tvvelve kinds of commercial common 1990, Pomeranz 1984). It appears that buckvvheat flour; and five kinds of common buckvvheat may be a valuable source of buckvvheat products dietary zine. On the other hand, a wide Two different kinds of buckvvheat seeds, i. е., Miyazakiootsubu (tetraploid), and a ashed vvith sulphuric acid and 30% tartary species, which were cultivated in hydrogen peroxide prior to atomic Miyazaki, Japan, were kindly provided by absorption spectrophotometry Dr. Takashi Nagatomo, Professor Emeritus STATISTICAL ANALYSIS. Data vvere of Miyazaki University. Four different subjeeted to analysis of variance and the species of buckvvheat seeds, i е., Shinano significance of difference vvas determined No. 1, Kisozairai, Shinsyunatsusoba and by the t-test. Shinsyuoosoba (tetraploid), which were cultivated at the Nagano Chushin Results and discussion Agricultural Experiment Station, Japan were kindly provided by Dr. Hisayoshi Table 1 shovvs the contents of zine in Hayashi of the Station. Two different various samples of commercial buckvvheat species of buckvvheat seeds, i.е., Kitawase flour. The content of zine of buckvvheat and Hokkai No 3 (tetraploid), grown in flours available in Japan vvas in a range Hokkaido, were kindly provided by Japan from 0.63 mg to 2.38 mg per 100 g flour, Agricultural Cooperatives, Hokkaido Shikaoi vvith an average of approximately 1.57 mg. Branch. Two different species of buckwheat On the other hand, the content of zine of seeds, i.е., Dolenjska Temna and a tartary buckvvheat flours available in Slovenia vvas species, grown in Slovenia, were kindly in a range from 1.98 mg to 3.47 mg per provided by Dr. Ivan Kreft, Professor at 100 g flour, vvith an average of Ljubljana University, Slovenia. These approximately 2.80 mg. The buckvvheat buckvvheat seeds were milled in an electric flours of Slovenia contained a relatively mili before analysis. higher level of zine as compared vvith those Nine different kinds of commercial of Japan (Table 1). buckvvheat flour vvere purchased from local markets at various places in Japan: Table 1: Zine contents in various buckvvheat flour A vvas purchased in Ivvate commercial buckvvheat flours. prefecture; flours В and C, in Ehime pref.; flour D, in Gifu pref.; flour E, in Kouchi pref.; flour F, in Tokushima pref.; flour G, Buckvvheat Zine content in in Nagano pref.; and flours H and I, in samples 100 g food (mg) Hyogo pref. Three different kinds of commercial buckvvheat flour sold in Buckvvheat flour available in Japan Slovenia vvere kindly provided by Dr. Ivan A 1.81 ± 0.01 Kreft, Professor at University of Ljubljana, В 1.36 i 0.01 Slovenia. C 2.07 ± 0.02 Five different kinds of commercial D 0.63 ± 0.01 buckvvheat products in Japan, i.е., four Ε 1.69 ± 0.01 kinds of dried noodles and one kind of F 1.92 ± 0.03 groat, vvere purchased in Japan: the four G 1.54 ± 0.02 buckvvheat noodles А, В, C and D vvere H 2.38 ± 0.06 produced in factories in Nagano pref.; I 0.76 t 0.02 buckvvheat groats, in a factory in Buckvvheat flour available in Slovenia Tokushima pref. Flour A 2.96 ± 0.02 DETERMINATION OF ZINC. The zine В 1.98 ± 0.04 content of samples vvas determined vvith a C 3.47 ± 0.03 Hitachi 208 atomic absorption spectrophotometer. In determining zine in solid food samples, the samples vvere wet Values are means ± S.D. (n=4). Table 2 shows the contents of zine in Table 3: Zine contents in various species of commercial buckvvheat products. The zine buckvvheat seeds. content in the dried noodles vvas in a range from 1.32 mg to 1.84 mg per 100 g flour on a fresh vveight basis, vvith an Buckvvheat Zine content average of approximately 1.60 mg. samples in 100 g food Buckvvheat groats contained a lower level (mg) of zine as compared vvith the dried Buckvvheat seeds grovvn in Japan noodles. Although there are a variety of е buckvvheat products in Japan, buckvvheat Shinano No.l 2.05 ± 0.06 noodles are most popular. On the other Kisozairai 1.84 ± 0.18cd hand, buckwheat groats, "Soba-gome" in Shinsyunatsusoba 1.68 ± 0.01° Japanese, have been traditionally eaten in Kitavvase 1.60 ± 0.03f only a fevv regions of Japan, sueh as Hokkai No.3 1.78 ± 0.07d Tokushima and Yamagata. In Japan, Miyazakiootsubu 1.29 ± 0.08K buckvvheat groats are usually prepared by Shinsyuoosoba 1.77 ± 0.04d boiling buckvvheat seeds, follovved by Tatary sp. 1.97 ± 0.19cd dehulling. One reason for a lovver level of Buckvvheat seeds grovvn in Slovenia zine in buckvvheat groats (Table 2) may be, Dolenjska Temna 4.88 ± 0.11a at least in part, aseribable to leakage of Tatary sp. 2.26 ± 0.13b zine into the boiling soak on preparing groats. Values are means ± S.D. (n=4) Values that do not share a common Table 2: Zine contents in commercial superseript are significantly different at buckvvheat products. p < 0.05.
grovvn in Slovenia, Dolenjska temna, vvas Buckvvheat Zine content significantly (p<0.05) higher than those samples in 100 g food grovvn in Japan. A similar finding appears (mg) in Table 1. These findings are interesting. Although the exact reason for the difference in zine content in buckvvheat Buckvvheat noodles A 1.69 ± 0.02 samples (Tables 1 to 3) is stili unclear, the В 1.32 ± 0.02 content of zine in buckvvheat seeds may be C 1.55 ± 0.05 generally influenced by some factors sueh D 1.84 ± 0.04 as buckvvheat species, soil conditions, Buckvvheat groats 1.20 t 0.08 milling conditions and so on. On the other hand, there was less or Values are means ± S.D. (n=4). substantially no difference in zine content between tartary species and common Table 3 shovvs the contents of zine in species (Table 3), although Dolenjska temna various species of buckvvheat seeds (F. esculentum) contained a significantly cultivated. A variation in zine content of higher level of zine than a tartary species cultivated buckvvheat species, including grovvn in Slovenia (Table 3). There is a tetraploid, vvas found (Table 3). The content suggestion that tartary buckvvheat has a of zine in common species (F. esculentum) beneficial effect on the therapy of diabetes cultivated in Japan vvas in a range from mellitus in China (Lu Chun jing 1992), 1.29 to 2.05 mg per 100 g flour. The although the factors responsible for its content of zine in a common buckvvheat effect have been not clarified. We have shown that zine may be closely associated Japan, to Dr. Ivan Kreft, Professor of with diabetes mellitus (Ikeda and Kotake Ljubljana University, Slovenia and to Dr. 1984 and 1986). Research should be Hisayoshi Hayashi, of the Nagano Chushin performed to reveal endogenous factors, Agricultural Experiment Station, Japan for perhaps including zine as at least one kindly providing buckvvheat seeds. The factor, involved in sueh a beneficial effect. authors are also sincerely grateful to Japan Recommended dietary allowances for Agricultural Cooperatives, Hokkaido Shikaoi zine were established in several countries Branch for providing buckvvheat seeds. during 1978 to 1991. The allowances of dietary zine for adults ranges from 7 to 15 Reference s mg per day around the world. Recently, the Ministry of Health and Welfare of Ikeda S. and Kotake Y. 1984. Urinary excretion of Japan has reported a suggested level of xanthurenic acid and zine in diabetes: 1) Separation of daily dietary intake of zine as an xanthurenic acid-Zn2"1" complex by ion-exchange additional item to the Recommended chromatography. Acta Vitaminol. Enzymol. 6: 23-28. Ikeda S. and Kotake Y. 1986. Urinary excretion of Dietary Allowances for Japanese (1984), xanthurenic acid and zine in diabetes: 3) Occurrence of although the adequacy of zine intake for xanthurenic acid-Zn2_b complex in urine of diabetic the Japanese people is stili not established. patients and of experimentally-diabetic rats. Italian J. In a report of the Ministry of Health and Biochem. 35: 232-241. Welfare of Japan (1984), it is suggested Ikeda S., Edotani M., Naito S. 1990. Zine in buckvvheat. that adults need about 10 mg zine per day. Fagopyrum 10: 51-56. We have estimated the contribution of Lu Chun-jing, Xu Jia-sheng, Zhao Ping, Ma Hui-jin, Tong dietary zine from buckvvheat noodles on a Hui-jun, Jin Yan-zhen, Li Shu-qing 1992. Clinical application and therapeutic effect of composite tatary normal consumption level (ca. 80g noodle buckvvheat flour on hyperglycemia and hyperiipidemia. per one dish) based on both the data of Proc. 5th Intl.Symp. Buckvvheat China, pp. 484-486. the present study and the above report Moynahan E.J. and Barnes P.M. 1973. Zine deficiency and (1984); our estimate shovvs that one dish of a synthetic diet for lactose intolerance. Lancet 1: 676- buckvvheat noodle may provide 677. approximately 10% of the daily need for Pomeranz Y. 1984. Buckvvheat: strueture, composition, and dietary zine for adults. In conclusion, these utilization. CRC Critical Revievvs in Food Science and findings suggests that buckvvheat may be Nutrition 19: 213-258. Prasad A.S. 1982. Clinical and biochemical spectrum of zine an important source of dietary zine. deficiency in human subjeets. in: Clinical, biological, and nutritional aspects of trace elements. Vol. 6, Current Topics in Nutrition and Disease, Prasad A.S. (Ed.) Alan Ack no wle dgement s R. Liss, Inc., Nevv York, pp 3-62. Sandstead, H.H. 1973. Zine nutrition in the United States. Am. J. Clin. Nutr. 26: 1251-1260. The authors vvish to express their sincere The Recommended Dietary Allovvance for Japanese (in gratitude to Dr. Takashi Nagatomo, Japanese) 1984. (ed.) The Ministry of Health and Professor Emeritus of Miyazaki University, Welfare, Dai-ichi Shuppan Pub. Fagopyrum 13 (1993): 15 - 20
Changes in zine of buckwheat on processing into noodles and eooking
Sayoko Ikeda and Takako Shimizu
Faculty of Nutrition, Kobe-Gakuin University Nishi-ku, Kobe 651-21, JAPAN
Key words: Zine, buckwheat, chromatography, digestion, gel filtration, soluble zine.
Abstract
Changes in zine of buckwheat on processing into noodles and eooking were studied. When raw noodles prepared from buckwheat flour were cooked, approximately 20% of the zine in the raw noodles was found to leak into the water soak during eooking. There was a significant difference in the proportion of zine released on in vitro digestion, between raw noodles and cooked noodles. Chromatographic analysis indicated that most of the soluble zine occurring on digestion of both raw and cooked noodles was found in substances with relatively low molecular weight. The nutritional implications of the present findings are discussed.
Spremembe vsebnosti cinka v ajdovem testu med izdelavo rezancev in kuhanjem
Avtorici sta raziskovali vsebnost cinka v ajdovem testu med izdelavo rezancev in kuhanjem. Med kuhanjem približno 20% cinka ajdovega testa rezancev preide v vodo. Značilna je razlika v deležu sproščenega iz testa med in vitro prebavljanjem surovih in kuhanih rezancev. Kromatografska analiza kaže, da večina cinka, dobljenega pri prebavljanju, izvira iz snovi z razmeroma majhno molekulsko težo. Avtorici sta prediskutirali prehranski pomen teh ugotovitev. (Prevod uredništva).
Introduction availability, but the exact mechanism involved is stili obscure. Zine is an essential micronutrient for Buckvvheat (Fagopyrum esculentum humans. Nutritional deficiency of zine is Moench) is an important source for humans today fairly prevalent throughout the vvorld of some essential nutrients sueh as proteins and is one of the major nutritional and vitamins. Buckvvheat contains a problems (Prasad 1982). Evaluation of diets relatively high amount of zine (Freeland for zine adequacy requires knovvledge of and Cousins 1976, Pomeranz 1984, Ikeda et both the amount and the availability of al. 1990a, Ikeda et al. 1991). The authors the zine present. Although information on have recently shovvn that a high level of the zine contents of foods has become zine vvas released on the in vitro digestion reasonably adeguate (Murphy et al. 1975, of buckwheat flour (Ikeda et al. 1990a). In Freeland and Cousins 1976) , knovvledge of this connection, processing and eooking may availability for the intestinal absorption of have any influence upon the chemical form dietary zine is largely incomplete. Current of zine in buckwheat. Hovvever, detailed evidence suggests that the ehemical form information is not available. of dietary zine prior to the absorption may The present study vvas undertaken to have a profound influence on its clarify changes in the chemical form of zine in buekwheat on processing and 3hr at 37 °C. The enzyme-to-protein ratio eooking. vvas 1:100. Immediately after peptic digestion, the ineubates vvere adjusted to Materials and analytical methods pH 8.0 vvith 2M Tris - HC1 buffer. Panereatin solution vvas then added to the MATERIALS. Fresh buckvvheat flour was digestion mixtures (enzyme-to-protein ratio obtained loeally and used immediately. 1:20) and ineubated for an additional 20hr Buekwheat noodles were prepared in our at 37 °C under 0.2M Tris - HC1 buffer (pH laboratory in a normal manner. Figure 1 8.0). Sodium azide vvas added to the provides a diagram of the preparation and digestion medium to a final concentration eooking of buckvvheat noodles. About 100 g of 0.025% to prevent grovvth of of buckwheat flour was mixed with about microorganisms. Immediately after 45 ml of hot distilled water, and digestion, the suspensions vvere placed in buckvvheat noodles vvere formed by hand. an ice eold vessel to diminish enzymatic The prepared noodles vvere cooked in about aetion and then clarified by centrifusion 500 ml of boiling vvater for 3 min. (10,000 χ g, 20 min). Blank tests of the Subsequently, additional vvater (about 100 digestion vvere performed vvith the above ml) vvas added to the suspension of the two enzymes in the absence of the foods. noodles. The noodles vvere further cooked The content of zine in the soluble digesta for 4 min. After eooking, the noodles vvere obtained vvas determined by substraetion of separated from the vvater soak. The the food-free blank. Aliquots of the soluble noodles vvere fully vvashed in eold vvater. digesta were applied to a Sephadex G - 50 Figure 1 also shovvs changes in vveight column (1.6 χ 95 cm) vvhich had been pre- during the preparation of buckvvheat equilibrated against 0.1M Tris - HC1 buffer noodles. Figure 2 shovvs buckvvheat noodles (pH 8.0). prepared in this study. Crystalline pepsin (ЕС 3.4.23.1, from ANALYTICAL METHODS. Protein (Ν χ porcine stomach mucosa, 2 χ cryst.) vvas 6.31) vvas assayed by the micro-Kjeldahl obtained from Sigma Chemicals Со.; and method (AOAC 1984). The distribution of panereatin NF, from Difco Laboratories. protein in column effluents vvas determined
Sephadex G - 50 vvas a produet of by A2SO measurements. The peptide Pharmacia LKB Biotechnology. Ali other content vvas assayed by the colorimetric chemicals used vvere of analytičal grade. procedure vvith 2,4,6-trinitrobenzene sulfonic acid (TNBS) (Goldharb 1966) and DETERMINATION OF ZINC. The zine phosphorus vvas assayed by the method of content of samples vvas determined vvith a Bartlett(1959). Data vvere subjeeted to Hitachi 208 atomic absorption spectro analysis of variance and the significance of photometer. In determining zine in solid means vvas tested by the t- test. food samples, the samples vvere vvet-ashed vvith sulphuric acid and 30% hydrogen Results and discussion peroxide prior to atomic absorption spectrophotometry. Table 1 shovvs the contents of zine and protein in raw noodles, cooked noodles and IN VITRO PROTEOLYTIC DIGESTION. water soak on eooking. The raw noodles Proteolytic digestion vvas performed examined contained approximately 1.51mg according to the method of Akeson and per 136g of noodles (Table 1). A decrease Stahmann (1964) vvith a modification in the content of zine in buckvvheat noodles (Ikeda 1984). Peptic digestion vvas vvas observed on eooking (Table 1). A performed in 0.06N hydrochloric acid for considerable amount of zine vvas (Weight 260 g) (Weight 203 g)
Fig. 1: Scheme on the preparation and eooking of buckwheat noodles.
Fig. 2: Buckvvheat noodles prepared substantially found in the vvater soak: that the vvater soak may contain some about 18% of zine in ravv buckvvheat nutrients (Nagatomo 1984), although the noodles leaked into the vvater soak on scientific basis for sueh a habit has been eooking. Japanese people often drink the yet not fully elucidated (Ikeda et al. hot vvater soak from the eooking of 1990a). The vvater soak of noodles vvas buckvvheat noodles, "Soba-yu" in Japanese, shovvn to contain a considerable amount of after eating the noodles. It vvas believed zine after eooking (Table 1).
Table 1: Zine and protein contents in buckvvheat noodles
Foods Weight1 Zine content Protein content examineed (g) (mg/each vveight1) (g/each vveight1)
Ravv noodles 136±3 1.51±0.29 (100%) 14.1±0.3 Cooked noodles 260±18 1.20±0.10 (80%) 11,5±0.7 Water soak 203±45 0.27±0.04 (18%) 2.1±0.1
Values are means S.D. (n=5)
A decrease in the content of protein in Table 2: Zine released on in vitro digestion buckvvheat noodles was also found on of buckvvheat noodles eooking (Table 1). It is knovvn that buckvvheat flour contains a high level of vvater- soluble protein (Javornik and Kreft Foods Per cent of zine released 1984). The present finding indicates that a examined on digestion to total zine part of the vvater soluble protein in ravv buckvvheat noodles may also leak into the Ravv noodles 87.1±8 ,9a vvater soak. Cooked noodles 71.5±10.1b Table 2 shovvs changes on eooking in the soluble zine released on the peptic and Values are means ± S.D. (n=5) pancreatic digestion of buckwheat noodles. Values that do not share a common The majority of zine in the ravv and cooked superseript are significantly different at noodles vvas released in the digesta on p<0.05. peptic and pancreatic digestion (Table 2). Soluble zine arising on digestion may aseribable to the fact that the cooked emerge as a soluble complex bound vvith noodles contained less vvater-soluble zine, some component, sueh as peptide, in the since a considerable quantity of vvater- digesta, vvhereas insoluble zine may emerge soluble zine in the ravv noodles leaked into on digestion as an insoluble complex bound the vvater soak on eooking (Table 1). vvith some component, sueh as phytic acid, Figure 3 shovvs the chromatographic in the digesta. elution profiles on Sephadex G-50 of the There vvas a significant (P < 0.05) soluble digesta from buckvvheat flour, ravv difference in the proportion of zine released buckvvheat noodles, cooked noodles and on digestion betvveen ravv noodles and vvater soak. The soluble zine in the digesta cooked noodles (Table 2). This may be Fraction nurnber (A.OrnUtube) Fraction number (4j0ml/tube)
Fig. 3: Gel filtration chromatographic elution profiles of the soluble zine components oeeurring on in vitro digestion of buckwheat flour, raw buckwheat noodles, cooked noodles and water soak. (A) Buckwheat flour; (B) raw buckwheat noodles; (C) cooked noodles; (D) vvater soak. -<>—Zine; , phosphorus; , absorbance at 280 nm; and , absorbance at 420 nm after ineubation vvith TNBS. consisted of one or two components. Zine in buckvvheat may be available for present in the soluble digesta of both intestinal absorption in vievv of the buckvvheat flour and raw noodles emerged chemical form of zine after digestion (Ikeda as a single peak, vvith a molecular vveight et al. 1990a). The present study further of around 1500 dalton. Zine in cooked supports our suggestion. Research is noodles and the vvater soak consisted of currently underway in our laboratory tvvo components: a major component vvith further to characterize the zine in molecular vveight of around 1500 dalton; buckvvheat. and a minor one vvith around 5000 dalton. Generally, soluble zine formed on digestion References is not necessarily easily available for absorption (Ikeda 1990b). Current evidence Akeson W.R. and Stahmann M.A. 1964. A pepsin suggests that solubilization of dietary zine, panereatin digest index of protein quality evaluation. J. through its binding vvith some low- Nutr. 83: 257-261. molecular-vveight soluble-component on AOAC 1984. In: Official Methods of Analysis, 14th Association of Official Ana!ytical Chemists (Ed.), digestion, may be essential for the Washington, DC. gastrointestinal ahsorption of dietary zine Bartlett G.R. 1959. Phosphorus assay in column (Ikeda 1990b). We have suggested that zine chromatography. J. Biol. Chem. 234: 466-468. Freeland J.H. and Cousins R.J. 1976. Zine content of Javornik B. and Kreft I. 1984. Characterization of selected foods. J. Ara. Dietet. Assoc. 68: 526-529. buckvvheat proteins. Fagopyrum 4: 30-38. Goldfarb A.R. 1966. A kinetic study of the reactions of Murphy E.W., Willis B.W, Watt B.K. 1975. Provisional amino acids and peptides with trinitrobenzenesulfonic tables on the zine content of foods. J. Am. Dietet. acid. Biochem. 5: 2570-2574. Assoc. 66: 345-355. Ikeda S. 1984. Characterization of zine components on in Nagatomo T. 1984. Soba no Kagaku, Nagatomo T. (Ed.), vitro enzymatic digestion of foods. J. Food Sci. 49: Shincho-shya, Japan. 1297-1300. Pomeranz Y. 1984. Buckvvheat: strueture, composition, and Ikeda S., Edotani M., Naito S. 1990a. Zine in buckvvheat. utilization. CRC Critical Revievvs in Food Science and Fagopyrum 10: 51-55. Nutrition. 19: 213-258. Ikeda S. 1990b. Dietary zine and the zine components in Prasad A.S. 1982. Clinical and biochemical spectrum of zine various foods subjeeted to in-vitro enzymatic digestion. deficiency in human subjeets. In: Clinical, Biological, and J. Sci. Food Agric. 53: 229-234. Nutritional Aspects of Trace Elements, Vol.6, Current Ikeda S., Matsui N., Shimizu Т., Murakami T. 1991. Zine in Topics in Nutrition and Disease, Prasad A.S. (Ed.) Alan cereals. In: Cereals Intl., Martin D.J. and Wrigley C.W. R. Liss, Inc., Nevv York, pp 3-62. (Eds), pp.248-250. Fagopyrum 13 (1993): 21 - 24
Digestibility of proteins in buckwheat seed
Kiyokazu Ikeda and Masayo Kishida
Faculty of Nutrition, Kobe-Gakuin University, Nishi-ku, Kobe 651-21, Japan
Abstract The digestibility of proteins in buckvvheat seed vvas studied. There vvas a difference in susceptibility to proteolytic action betvveen buckwheat proteins and some other proteins, vvith buckvvheat proteins being less digestible by pepsin than hemoglobin and ovalbumin. Electrophoretic analysis indicated that the major component of the albumin in buckvvheat seed vvas less susceptible to pepsin action. The relationship betvveen the digestibility of buckvvheat proteins and their molecular structure is discussed. Prebavljivost beljakovin semen ajde
Avtorja sta raziskovala prebavljivost beljakovin zrn ajde. Beljakovine ajde so slabše razgradljive s pepsinom kot hemoglobin in ovalbumin. Elektroforetska analiza nakazuje, da je glavni del albuminov ajde manj dovzeten za razgradnjo s pepsinom. Avtorja sta prediskutirala odnos med prebavljivostjo beljakovin ajde in njihovo molekulsko zgradbo. (Prevod uredništva).
Introduction buckvvheat are stili not fully clarified. We have suggested that the digestibility of Buckvvheat (Fagopyrum esculentum Moench) buckvvheat may be defined by two factors: is an important crop in some areas of the the inhibitory potency of endogenous vvorld. Buckvvheat seed contains a relatively antinutrients, sueh as protease inhibitor high level of protein, and thus has and tannin, and the susceptibility of the potential value as a dietary source of protein to proteolytic action (Ikeda et al. protein. The protein in buckvvheat seed 1991). Hovvever, detailed information about consists of vvell-balanced amino acids the digestibility of the protein per se is (Pomeranz and Robbins 1972) vvith high stili obscure. biological value (Sure 1955). Hovvever, The present study vvas designed to experiments vvith animals have shovvn that clarify the digestibility of the protein in the availability of buckvvheat protein for buckvvheat seed. gastrointestinal absorption is lovv (Farrell 1978, Eggum et al. 1981, Thacker et al. 1983). The revised Japanese Standard Materials and analytical methods Tables of Food Composition shovv the protein digestibility of buckvvheat by Materials humans to be lovver than that of other edible seeds sueh as soybean and vvheat Samples of fresh buckvvheat seed vvere (Resources Council, Science and Technology obtained from Takii Со. (Kyoto, Japan). The Agency, Japan 1981). Despite the albumin and globulin from buckvvheat vvere importance of buckvvheat as a dietary isolated from the seeds aecording to the source of protein, factors responsible for procedure of Javornik and Kreft (1984). the poor digestibility of the protein in Hemoglobin, ovalbumin, bovine serum albumin, β-lactoglobulin, trypsinogen, and ovalbumin (Fig. 1). On the other hand, lyzozyme were obtained from Sigma buckwheat globulin vvas more digestible by pepsin than buckvvheat albumin (Fig. 1), Chemical Со. Ali other chemicals used agreeing vvith the previous report vvith vvere analytičal grade. pepsin and trypsin (Ikeda et al. 1991).
Analytical methods
The digestibility of proteins vvas assayed vvith pepsin: proteins examined (0-0.75 mg) 600 /O vvere incubated at 37 °C vvith 0.3 mg of pepsin in 0.1 M HC1-KC1 buffer in a total volume of 2.5 ml. After incubation for 15 min, 1.0 ml of 15% trichloroacetic acid solution vvas added to each reaction H mixture and allovved to stand for 10 min at room temperature. The suspensions vvere centrifuged at 3,000 rpm for 15 min, and the supernatants obtained vvere then assayed for peptide. Peptide and free amino group of protein vvere assayed by the colorimetric procedure vvith 2,4,6- trinitrobenzene sulfonate (Goldfarb 1966, Habeeb 1967), respectively. The free sulfhydryl group of proteins vvas determined by the procedure of Ellman (1959). The hydrophobicity of proteins vvas analyzed vvith l-anilino-8-naphthalene sulfonate (Stryer 1965). Electrophoresis in the presence of sodium dodecylsulfate and mercaptoethanol vvas performed by the procedure of Laemmli (1970); and the marker proteins used vvere bovine serum albumin, trypsinogen, p lactoglobulin, and J ' lysozyme. 0 0.2 0.4 0.6 0.8 Substrate (mg/ml) Results and discussion
Figure 1 shovvs the susceptibility of buckvvheat albumin, buckvvheat globulin, Fig 1: Susceptibility of various proteins to hemoglobin, and ovalbumin to pepsin action. pepsin action. H denotes hemoglobin; O, There vvas a striking difference in the ovalbumin; G, buckvvheat globulin; and susceptibility to pepsin action among the A, buckwheat albumin. proteins examined; vvith hemoglobin being the most digestible by pepsin. Some reports (Kato et al 1985, Porter et al 1984) have Figure 2 shovvs changes in buckvvheat shovvn that ovalbumin is relatively less albumin during incubation vvith pepsin. digestible by proteases as compared vvith Some minor components, vvhich are some other proteins. The two buckwheat distributed in the range of molecular proteins vvere less digestible than vveight among 24,000 dalton to 67,000 мь jr aS9 S ^»Jbi "''ШШШЁШ! O / ™ ™
24kD—»
18.4kD 143kD
MP P A 0 t 2 5 Ю20 60 Fig 2: Changes in buckvvheat albumin during incubation vvith pepsin. Pepsin digestion vvas performed vvith an enzyme-to-protein ratio of 1:5. MP denotes marker proteins; P, pepsin alone; A, buckvvheat albumin alone; and 0-60, incubation periods (min) of buckvvheat albumin vvith pepsin. dalton (Fig. 2), disappeared rapidly as the the peptic digestibility of the four proteins enzymic reaction proceeded. On the other (Fig. 1) to their free amino group vvas hand, a major component of the buckvvheat albumin, vvhich vvas located at a molecular Table 1 Free amino groups and free vveight of around 14,300 dalton, vvas less sulfhydryl (SH) groups of various digestible vvith pepsin: a considerable proteins amount of this albumin component persisted even after incubation vvith pepsin for 60 min (Fig. 2). Proteins Free amino Free SH Table 1 shovvs the free amino groups group group and free sulfhydryl groups of the four examined (jjmol/mg (nmol/mg proteins. The free SH group of hemoglobin protein) protein) could not be determined, since the intrinsic colour of this protein interfered vvith its Buckwheat albumin 3.09 1.86 determination. A higher level of free amino Buckvvheat globulin 4.35 0.938 group characterized the two buckvvheat Ovalbumin 1.88 0.296 proteins as compared vvith hemoglobin and Hemoglobin 1.43 - ovalbumin. The correlation coefficient of -0.822. In addition, the two buekwheat Ellman, G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. proteins had a relatively higher level of Biophys. 82:70-77. free SH group (Table 1). There was also a Farrell, D.J. 1978. A nutritive evaluation of buckvvheat (Fagopyrum esculentum). Anim. Feed Sci. Technol. 3:95- high correlation between the digestibility of 108. the proteins and their free SH groups, Goldfarb, A.R. 1966. A kinetic study of the reaction of although hemoglobin data was laeking. On amino acids and peptides vvith trinitrobenzenesulfonic the other hand, there was no correlation acid. Biochem. 5:2570-2574. between the digestibility of these four Habeeb, A.F.S.A. 1966. Determination of free amino groups proteins (Fig. 1) and their hydrophobicity in proteins by trinitrobenzenesulfonic acid. Anal. Biochem. 14:328-336. (data not shown). These findings suggest Ikeda, K., Sakaguchi, Т., Kusano, T. and Yasumoto, K. 1991. that the surface state on the protein Endogenous factors affecting protein digestibility in molecule in buckwheat seed may be closely buckvvheat. Cereal Chem. 68:424-427. associated with its digestibility. Javornik, B. and Kreft, I. 1984. Characterization of Buckwheat seed serves as an important buckvvheat proteins. Fagopyrum 4:30-38. source of dietary protein around the world. Kato, Α., Komatsu, K., Fujimoto, K., and Kobayashi, K. The nutritional function of the proteins in 1985. Relationship betvveen surface functional properties and flexibility of proteins detected by the protease buckvvheat is a major concern in the susceptibility. J. Agric. Food Chem. 33:931-934. utilization of buckvvheat seed. Laemmli, U.K. 1970. Cleavage of structural proteins during Characterization of buckvvheat protein in the assembly of the head of bacteriophage T4. Nature relation to bioavailability is in progress 227:680-685. (Ikeda et al., unpublished data, 1993). In Pomeranz, Y. and Robbins,G.S. 1972. Amino acid particular, our above suggestion on a composition of buckvvheat. J. Agric. Food Chem. 20:270- relationship betvveen digestibility and 274. Porter, D., Svvaisgood, H.E. and Catignani, G.L. 1984. molecular structure should be confirmed. Characterization of an immobilized digestive enzyme system for determination of protein digestibility. J. Acknovvledgement Agric. Food Chem. 32:334-339. Resources Council. 1981. Data on the Revision of the 4th The authors vvish to thank Professor K. Standard Tables of Food Composition in Japan. Data Yasumoto, Research Institute for Food No.92. Resources Council, Science and Technology Science, Kyoto University, for his Agency, Japan. Stryer, L. 1965. The interaction of a naphthalene dye vvith suggestions during the course of this apomyoglobin and apohemoglobin - A fluorescent probe study. of non-polar binding sites. J. Mol. Biol. 13:482-495. Sure, B. 1955. Nutritive value of proteins in buckvvheat and References their role as supplements to proteins in cereal grains. J. Agric. Food Chem. 3:793-795. Eggum, B.O., Kreft, I. and Javornik, B. 1981. Chemical Thacker, P.A., Anderson, D.M., and Bovvland, J.P. 1983. composition and protein quality of buckvvheat Nutritive value of common buckvvheat as a supplement (Fagopyrum esculentum Moench). Qual. Plant. Plant to cereal grains when fed to laboratory rats. Can. J. Foods Hum. Nutr. 30:175-179. Anim. Sci. 63:213-219. Fagopyrum 13 (1993): 25 - 30
Morphology and identification by isozyme analysis of interspecific hybrids in buckwheats
Tamaki Hirose1, Akio Ujihara2, Hiromi Kitabayashi1 and Mineo Minami2 1 The United Graduate Sehool of Agricultural Sciences, Shinshu University, Minamiminowa, Nagano 399-45, Japan 2 Laboratory of Plant Breeding, Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano 399-45, Japan
Key words: hackcross, esterase, Fagopyrum esculentum, Fagopyrum cymosum, interspecific hybrid, isozyme analysis, morphology, ovule culture, tetraploid
Abstract
Fourteen interspecific hybrids were obtained through ovule culture betvveen cultivated tetraploid common buckvvheat (Fagopyrum esculentum, 2n=32) and the vvild perennial species (Fagopyrum cymosum, 2n=32) originated in Nepal. The morphological characteristics of the hybrids and parent species are described, and the results of identification and estimation of genetic variation of the hybrids and backcrossing progenies by esterase isozyme analysis vvere reported. The hybrids exhibited polymorphism in their morphological characteristics. They had common band to either of their parents in zymograms. They had, at least, a common band to male parent and a common band to female parent.
Morfologija in identifikacija medvrstnih križancev ajde z esteraznimi izoencimi
S pomočjo kulture ovul so avtorji dobili štirinajst medvrstnih križancev med tetraploidno navadno ajdo (Fagopyrum esculentum, 2n=32) in divjo trpežno vrsto (Fagopyrum cymosum, 2n=32) iz Nepala. Opisane so morfološke lastnosti križancev in izhodiščnih vrst ter rezultati identifikacije. Z analizo esteraznih encimov je ocenjena genetska variabilnost križancev in povratnih križancev. Po morfoloških lastnostih so križanci polimorfni, cimogrami pa kažejo identičnost črt z vsaj enim od staršev. (Prevod uredništva).
Introduction characteristics of the vvild species, sueh as tolerance to environmental stress and A large number of studies using the resistance to disease. embryo or ovule culture system have been In addition to Fagopyrum cymosum reported, in relation to the potential of Meissner, the typical and vvidespread vvild interspecific hybrids to overcome cross species of the genus Fagopyrum, several incompatibility in many crops including other species have been identified in China their vvild species (Hu et al. 1986). (Wu et al. 1984, Ohnishi 1989). Accordingly, our research shovved that the Prior to the study, only tvvo attempts at utilization of interspecific hybrids seems to interspecific hybridization vvithin the genus be one of desirable methods for the Fagopyrum had been made, by Nagatomo promotion of breeding in common (1961) and Krotov (1975). Nagatomo's buckvvheat, Fagopyrum esculentum Moench. attempt vvith F. esculentum and F. Through interspecific hybridization, it is cymosum vvas unsuccessful. In the USSR, possible to introduce into common Krotov vvas successful in producing F. buckwheat varieties, valuable giganteum, vvhich is an interspecific hybrid betvveen F. tataricum and F. cymosum. WL In 1987, vve obtained fourteen interspecific hybrids through ovule culture betvveen cultivated tetraploid common buckvvheat (F. esculentum, 2n=32) and the LL vvild perennial species (F. cymosum, 2n=32) originated in Nepal (Ujihara 1989, Ujihara et al. 1992). In this paper, vve will describe the WP morphological characteristics of the above hybrids and parent species, and also report on the results of identification and estimation of genetic variation of the Leaf Blade Floret hybrids by esterase isozyme analysis.
Fig. 1: Determination of the leaf and the Materials and methods floret characteristics.
Plant materials: Six clonal hybrid strains and two backcross progenies (hybrid No. 9 Isozyme analysis: In order to identify the χ Shinshu-Osoba) vvere used for the genetic variation of hybrid plants, we experiments. In order to compare the attempted to develop a simple analytical hybrids to their parents, Shinshu-Osoba {F. procedure by applying isoelectric electro esculentum, tetraploid registered variety, focusing (IEF) for the analysis of esterase 2n=32) and vvild buckvvheat introduced isozyme. from north-vvest Nepal (F. cymosum, vvhich Florets for the analysis vvere collected has perennial grovvth habits and is also from plants at the fully flovvering stage. tetraploid, 2n=32) vvere also used. Samples from individual plants vvere In 1988-1989, ali hybrid plants vvere treated separately throughout the analysis. grovvn in greenhouses after being Each sample vvas homogenized by a transplanted from in vitro culture into soil microhomogenizer vvith 40 yl of chilled pots. extraction buffer (0.2% carrier ampholyte, 1% 2-mercaptoethanol) per floret. The IEF Morphological observation: Morphologi- gel contained 5.82% (w/v) acrylamide, cal observation of the hybrids and parent 0.18% (w/v) N,N'-methylene-bis-acrylamide, species vvas carried out in 1989-1991. The 4. 8% glyceroland 3.2% carrier ampholyte nature of the hybrids vvas determined by "LKB Ampholine pH 3.5-10". Isoelectric comparing their morphological characteris- focusing vvas carried out under constant tics, sueh as the shape of leaves and voltage conditions in a stepped-up fashion. flovvers, and the degree of starch accumula- Electrophoresis vvas run at 100V for 15 tion in the stem or rhizome, vvith those of minutes, then at 200V for 15 minutes, and the parents. finally, at 450V for an additional 60 The leaf shape of the largest 2-3 leaves minutes. After each electrophoretic run, vvas measured at the fully flovvering stage gels vvere immediately stained to detect of each plant, and at the same time, the isozyme bands according to the staining leaves vvere prepared for microscopic solution in Table 1. Gels vvere ineubated at observation. Measurements of leaf parame- 30° C until the isozyme bands developed ters and floret shapes vvere taken as indica- sufficient intensity to permit seoring. ted in Fig. 1. Table 1. Staining solution for esterase
Stain ingredients Quantity of ingredient
α -Naphthyl acetate 20mg β -Naphthyl acetate 20mg Aceton lml Distilled water lml 1/15M Phosphate buffer,pH 7.0 50ml Fast blue BB salt 50mg
1/15M Phosphate buffer,pH 7.0: Na 2ΗΡ04·12Η20 24g/l,KHiP04 9g/l.
Results and discussion Generally, in flower shape, number of stomata, shape of epidermal cells and the Morphological characteristics: The development of the xylem and aerial roots growth of the hybrids was the same or of the stem, the hybrid plants exhibited less than that of the parent species. intermediate characteristics of the parents. Comparisons of several traits were made Hovvever, leaf shape, development of between the hybrids and their parent veins, and density of hair were variable species. among hybrid strains.
I. leaf blade and vein
II. upper epidermis
III. lovver epidermis
IV. cross section: A. F. esculentum, B. hybrid No. 9, C. hybrid No. 18, 1 ЮОдт D. F. cymosum. А в с D Fig. 2. Comparative representation of morphological characteristics of the leaf in interspeci- fic hybrids (Fagopyrum esculentum χ F. cymosum) and parent species. Table 2. Morphological characteristics of interspecific hybrids Fagopyrum esculentum χ F. cymosum) and parents.
Characteristics F.esculottum Hybrids F.cymosum No.l No.2 No.5 No. 9 No. 12 No.18
Leaf index 1 1.33 0.96 1.01 1.01 1.71 1.34 1.27 0.91 Leaf index 2 0.19 0.12 0.09 0.16 0.24 0.18 0.16 0.13 Leaf index 3 0.8 0.98 1 0.91 0.49 1 0.9 1 Margin undulate entire entire entire entire entire undulate entire Ocrea truncate triangular triangular truncate triangular triangular triangular acuminale Ocrea index 1 1 2 2 2-3 2-3 2 3< Leaf hair + - - + + - ++ ++ Hair + - - - ++ + + +++ Bundle sheath - - - - + + + ++
Palisade parenchyma ++ ++ ++ ++ + + + - Starch - + + + ++ ++ ++ ++
Xylem - + + + + + + ++ Rhizome - + + + + + + ++ Inflorescence index 1 2 2 2 3 3 3 4< • • Flower index 1.07 • 1.13 1.13 1.18 1.56
Leaf index 1: LIVWL, Leaf index 2: DH/LL, Leaf index 3: DC/V/L, F)ower indei: LP/WP; See Fig. 1. Ocrea index: length of ocrea/diameter of direct]y under internode, Inflorescence index: maximum number of raceme in inflorescence.
In the shape and size of the cotyledons vvith pin type flovvers. and hypocotyls, the seedlings which When vve attempted interspecific hybridi- emerged from ovules of the hybrids zation betvveen common buckvvheat (F. resembled the wild species, F. cymosum. esculentum) and vvild buckvvheat {F. cymosum), vve vvere able to observe fertili- Furthermore, the hybrids exhibited root zation, but vve could not obtain mature characteristics of the wild species, seeds. We therefore rescued the ovules particularly in the substantial development after the experiment and, through in vitro of the rhizome, the accumulation of starch, culture, vve succeeded in obtaining fourteen and an elongated development of the tap hybrid plants. root. These three root features are ali Then vve began backcrossing of the present in perennials. It vvas not, therefore, hybrids vvith common buckvvheat for about surprising that ali of the hybrids exhibited tvvo months, from August through so many other perennial characteristics. September, 1989. We vvere quite successful Fig. 2 and Table 2 shovv the in obtaining vvhole fertilized ovules, morphological characteristics of interspecific although full maturation vvas almost hybrids and parent species. impossible to achieve. Hovvever, by again carrying out ovule culture, vve succeeded in Inheritance of flovver type and producing tvvo vvhole progenies of back- backcrossing: The hybrids exhibited crossed plants that achieved the flovvering dimorphic flovvers. The long-styled (pin) stage. type and short-styled (thrum) type Through improving the seed fertility of appeared at a ratio of 5:4 respectively. the hybrids by this type of repeated back- Table 3 shovvs hybrid combinations in the crossing process, we believe there are great production of interspecific hybrids and each possibilities of successful results. plant's flovver type. None of the hybrids vvith thrum type Isozyme anaiysis: We preliminarily flovvers grevv as vigorously as the hybrids examined five enzymes, namely peroxidase, Table 3: Hybrid combinations and flower type of the interspecific hybrids in genus Fagopyrum
Hybrid combination Strain Flower tjTfje
F.esculentum (thrum) X F.cymosum (pin) No.l thrum F.esculentum (thrum)xF.cymosum (pin) No.2 thrum F.esculentum (thrum) X F.cymosum (pin) No.5 thrum F.esculentum (pin) X F.cymosum (thrum) No. 6 (not f]owering) F.esculentum (thrum) X F.cymosum (pin) No.8 pin F.esculentum (pin) X F.cymosum (thrum) No. 9 pin F.esculentum (pin) X F.cymosum (thrum) No. 10 (not flowering) F.esculentum (thrum) X F.cymosum (pin) No. 12 pin F.esculentum (thrum) X F.cymosum (pin) No. 13 (not flowering) F.esculentum (thrum) X F.cymosum (pin) No. 17 (not flowering) F.esculentum (pin) X F.cymosum (thrum) No. 18 pin Hybrid No.9(pin) χ F.esculentum (thrum) No.9-1 thrum Hybrid No.9(pin) X F.esculentum (thrum) No.9-5 pin
acid phosphatase, malate dehydrogenase, differences. Two major isozyme bands were 6-phosphogluconate dehydrogenase and found in F. esculentum. In F. cymosum, esterase, and found that only the esterase two or three major bands and two minor zymogram gave distinct interspecific bands were found. The esterase zymograms differences in every growth stage. exhibited three to five bands in the Accordingly, we used esterase zymograms hybrids, and each zymogram showed an to identify interspecific hybrids. Magenta identical pattern to at least one of its and deep-green bands were developed with parents (Fig. 3). staining solution containing alpha- and The esterase zymograms revealed beta-naphthyl acetate in the range of pH apparent interspecific differences. Hence, 8-10. the esterase zymogram was considered to The combination of magenta and be a useful marker in the identification of deep-green bands showed interspecific interspecific hybrids.
Fig. 3. Esterase zymograms of interspecific hybrids and parents.
*: Progeny of backcross. References Ohnishi O. 1989. Cultivated buckvvheat species and their relatives in the Himalayas and southern China. Proc. 4th Intl. Symp. on Buckwheat, pp. 562-571. Hirose Т., Ujihara Α., Ivvasaki H. and Horiuchi A. 1991. Ujihara A. 1989. A preliminary report on the geographical Morphological characteristics of interspecific hybrids distributiion and characteristics of cultivated buckwheat, betvveen tetraploid buckwheat (Fagopyrum esculentum genus Fagopyrum in Nepal. Exp. & collect. Plant genetic Moench) and perennial buckwheat (F. cymosum resources, Part I, pp. 161-170, JICA, Токуо. Meissner). Jour. Fac. Agric. Shinshu Univ. 28: 53-62 (in Japanese with English summary). Ujihara Α., Nakamura Y. and Minami M. 1992. Interspecific hybridization in genus Fagopyrum - Properties of Hu C. and Wang P. 1986. Embryo culture: Technique and hybrids ( F. esculentum Moench χ F. cymosum applications. In "Handbook of plant ceil culture", Evans Meissner) through ovule culture. Gamma field symposia D.A., Sharp W.R. and Ammirato P.V. (eds.), Vol. 4, pp. 29: 45-53, 1990, Institute of radiation breeding, NIAR, 43-96, Macmillan publishing сотрапу, New York. MAFF, Japan. Krotov A.S. 1975. Fagopyrum Mili. Flora of cultivated Wu C.Y, Yin W.Q., Rao S.Y, Tao D.D., Yuan S.H, Deng plants III. Groat crops. Kolos, Leningrad, pp. 7-118 (in X.F., Yuan S.X., You H.Z. and Lin Q. (eds.). 1984. Russian). Index Florae Yunnanensis. Tomus I, pp. 265-285. The Nagatomo T. 1961. Studies on physiology of reproduction People's Pub. House, Yunnan, China (in Chinese). and some cases of inheritance in buckvvheat. Miyazaki University, Japan (in Japanese vvith English summary). Fagopyrum 13 (1993): 31 - 34
Purification and characterization of superoxide dismutase from tartary buckwheat leaves
Wang Zhuanhua1, Lin Rufa^, Zhang Zheng1, Zhou Mingde3 1 Department of Biology, Shanxi University, Taiyuan, 030006, China 2 Small Grain Programme, Shanxi Academy of Agricultural Sciences, Taiyuan, 030031, China 3 IBPGR Office for East Asia c/o CAAS, Beijing, 100081, China
Key words: Fagopyrum tataricum, amino acid composition, buckwheat leaves, isozyme, purified, superoxide dismutase
Abstract
Superoxide dismutase (SOD) was isolated and purified from tartary buckwheat leaves by the modified method of Asada et al. (1974). The results of the isozyme zymogram of PAGE indicate that there are 3 bands. The SOD has a molecular weight of 31000 and, by SDS-PAGE determination, is composed of two subunits of equal size. The amino acid analysis of superoxide dismutase showed 308 residues in tartary buckwheat leaves.
Čiščenje in karakterizacija encima superoksidne dizmutaze iz listov ajde
Superoksidna dizmutaza (SOD) je izolirana iz listov tatarske ajde po modificirani metodi Asada et al. (1974). S PAGE so ugotovljene 3 elektroforetske črte. SOD ima molekulsko maso 31000 in ga sestavljata, kot je ugotovljeno z SDS-PAGE, dve podenoti. Z analizo amino kislin je ugotovljeno, da encim sestavlja 308 amino kislin. (Prevod uredništva).
Introduction properties of SOD from tartary buckvvheat leaves. The molecular weight, amino acid Superoxide dismutase (ЕС 1.15.1.1.) composition, isozyme, and inhibition by catalyzes the dismutation of superoxide cyanide and by chloroform plus ethanol radicals to oxygen and hydrogen peroxide. were also investigated. This enzyme has been isolated from a wide range of living organisms. With respect to Material and methods the prosthetic metals, three types of SOD have been found: CuZn-, Mn- and Material. The experimental material was Fe-containing enzymes. In prokaryotes, leaves of tartary buckwheat ('Wutai' algae and protozoa, Fe- and Mn- SODS are tartary buckvvheat) obtained from Shanxi the major enzymes. However, in animals Academy of Agricultural Sciences. Sephadex and funghi, as well as plants, the major G-75, DEAE-32 and standard proteins for enzyme is CuZn-SOD. purification of SOD and determination of The occurence of chloroplast and cytosol molecular weight were from Shanghai types of CuZn-SOD has been reported in Chemicals. pond, scum, horsetail (Kanematsu and Asada 1989b), rice (Kanematsu and Asada Isolation and purification. The leaves (200g) 1989a) and pea (Duck and Salin 1983). from tartary buckvvheat vvere homogenized This report describes the purification and in a Polytrom blender vvith 0.05 M potassium phosphate buffer (pH 7.8)/0.1 procedure similar to published methods mM EDTA for 1 min. After 1 h the (Asada et al. 1973, 1974). The results are homogenate was filtered through four summarized in Table 1. layers of gauze and centrifuged at 17000 χ g 15 min. The supernatant solution was Isozyme of SOD. Extracts of tartary treated with chloroform plus ethanol, and buckvvheat leaves generated three bands of ammonium sulfate was added to the SOD activity after native PAGE (Fig. 1A). extract to 40% saturation, with the pH Band a (beside cathode) vvas insensitive to maintained at 7.8 by addition of cyanide, but it vvas sensitive to chloroform ammonium hydroxide. After centrifugation, plus ethanol, being thus identifiable as ammonium sulfate was added to the Mn-SOD (Fig. 1B). The bottom tvvo bands supernatant to 90% saturation. The (b, c) vvere CuZn-SOD (Fig. 1С), vvhich precipitate obtained by centrifugation was vvere sensitive to cyanide. By contrast, dissolved in and dialyzed against 10 mM CuZn-SOD is the dominant isozyme in potassium phosphate (pH 7.8)/0.1 mM tartary buckvvheat leaves. EDTA. The dialyzed enzyme was clarified by Optical spectra. The absorption spectra of centrifugation and applied to a column of CuZn-SOD from tartary buckvvheat leaves DEAE-32 (2.3 i.d. χ 20 cm), equilibrated in the visible regions, is illustrated in Fig. with 10 mM potassium phosphate. The 2. It had absorption maximum at 665 nm. column was washed with the same buffer. The absorption spectra in the ultraviolet A linear gradient of NaCl (0-200 mM, 600 region is at 261 nm (Fig. 3). The optical ml) in 10 mM potassium phosphate (pH properties of the buckvvheat leaves enzyme 7.8)/0.1 mM EDTA, was then applied and are very similar to those already seen in fractions were collected at a flow rate of the CuZn-SOD from other sources 20 ml/h. Each fraction of SOD activity (Beauchamp et al. 1973, Steinman 1982). was pooled and concentrated by PEG-20000, and applied to a column of Molecular vveight and amino acid Sephadex G-75 (2.5 cm i.d. χ 24 cm). The composition. Subunit molecular vveight column was washed with the same buffer. determination by SDS-PAGE vvas exhibited at 15500 (Fig. 4), calculated from the Assay for SOD. One unit of SOD activity amino acid composition (Table 2), the was defined as the amount of enzyme molecular vveight of CuZn-SOD vvas required for the reduction of NBT by 50%. estimated to be 31000. Thus, it appears We used a reaction volume 3 ml (McCord CuZn-SOD from buckvvheat leaves is and Fridovich 1969). homodimer vvithout disulfide bonds betvveen the subunits, and it has the same Methods for characterization of SOD. properties as that from other sources Native PAGE, identification of CuZn-, (Kitagavva et al. 1986, 1987). Table 2 Mn-SOD by treatment of cyanide or shovvs the amino acid composition of chloroform plus ethanol were performed as CuZn-SOD purified in buckvvheat leaves. It described previously (Kanematsu and Asada did not contain tyrosine, as vvas deduced 1989a). Molecular weight determination by from the UV absorption spectra (Fig. 3). SDS-PAGE, metals analysis, amino acid Analysis indicated that the SOD in analysis and optical spectra were performed buckvvheat leaves has a molecular vveight (Kanematsu and Asada 1989a). in the range previously described for SOD from other sources though at the lovver end Results and discussion of the range. Activity of the crude extract of SOD vvas inhibited by 90% in the Purification of SOD. SOD was purified presence of cyanide, but no inhibition vvas from tartary buckwheat leaves by a detected by the chloroform-ethanol Table 1. Purification of CuZn-SOD from tartary buckwheat leaves
Pur i fication Total protein Total activity Specific activity Vield Purification step (mg) (units) (units/mg protein) % (fold)
Homogenate 1510 19250 12.7 100 1 Chloroform-ethanol 1100 18900 17.2 98.2 1.4 m (NH ) S0 600 14800 24.7 76.9 1.9 4 2 4 Ш (NH ) S0 120 7910 65.9 41 5.2 4 2 4 Sephadex G-75 11 2940 267.3 15.3 21 DEAE-32 0.7 1823 2604.3 9.5 205
Fig. 1. Isozyme scanning zymogram of the SOD from tartary buckwheat leaves. After native polyacrylamide gel electrophoresis. A. extracts, B. extracts plus cyanide, C. extracts plus chloroform and ethanol.
Table 2. Amino acid composition of treatment, indicating that SOD extracted CuZn-SOD from buckwheat leaves from buckwheat leaves is a CuZn-SOD, and copper and zine were found in the purified fraetion in amounts corresponding to 2.01 amino residues residues g-atoms of Cu and 1.87 g-atoms of Zn per acid per subunit amino acid per subunit mol of enzyme.
Lys 7 1/2 Cys 1 His 9 Val 16 Arg 4 Met 1 Asp 20 I le 5 Thr 15 Leu 13 Ser 6 Туг 0 Glu 12 Phe 2 Pro 8 Trp 0 G1 у 24 Total 154 Ala 11 V/avelength (nm) VVovetersgth (nm)
Fig. 2. Visible absorption spectrum at 1 Fig. 3. Ultraviolet absorption spectrum at mg/ml 10 mM potassium phosphate (pH 0.5 mg/ml in 10 mM potassium phosphate 7.8)/0.1 mM EDTA. (pH 7.8)/0.1 mM EDTA.
superoxide dismutase. Eur. J. Biochem. 36: 257-266. 4. 8 • Beauchamp C.O., Fridovich I. (1973): Isozymes of superoxide dismutase from wheat germ. Biochem. Biophys. Acta 317: 50-64. Duke M.V., Salin M.L. (1983): Isoenzymes of cuprozinc superoxide dismutase from Pisum sativum. ? 4.4" Phytochemistry 22: 2369-2373. Kanematsu S., Asada К. (1989а): CuZn-superoxide 4. 2 t dismutases in rice: occurrence of an active, monomeric enzyme and two types of isozyme in leaf and non-photosynthetic tissues. Plant Celi Physiol. 30: 381-391. Kanematsu S., Asada K. (1989b): CuZn-superoxide 0.2 0.4 0.6 0.8 1.0 dismutases from the fern Equisetum arvense and the green alga Spirogyra sp.: occurrence of chloroplast and Fig. 4. Determination of the subunits cytosol types of enzyme. Plant Celi Physiol. 30: 717-727. molecular weight by SDS-PAGE. Reference Kitagavva Y., Tsunasawa S., Tanaka N., Katsube Y., proteins: a. BSA 867000); b. Egg albumin Sakiyama F., Asada K. (1986): Amino acid sequence of (43000); c. Carbonic anhydrase (30000); d. copper, zinc-superoxide dismutase from spinach leaves. J. Cytochrome C (12000). Biochem 99: 1289-1298. Kitagavva Y., Tsunasawa S., Tanaka N., Katsube Y., Sakiyama F., Asada K. (1987): Structure and molecular evolution of CuZn-superoxide dismutase from spinach. References Proc. Int. Symp. Computer Anal. Life Sci pp. 65-72, Ohmusha, Kyoto. Asada K, Takahashi M., Nagate M. (1974): Assay and McCord J.M., Fridovich I. (1969): Superoxide dismutase: an inhibitors of spinach superoxide dismutase. Agric. Biol. enzymatic function for erythrocuprein. J. Biol. Chem. Chem. 38: 471-473. 244: 6049-6055. Asada K., Urano M., Takahashi M. (1973): Subcellular Steinman H.M. (1982): Copper-zinc superoxide dismutase locations of superoxide dismutase in spinach leaves and from Caulobacter crescentus CB15. J. Biol. Chem. 257: preparation and properties of crystalline spinach 10283-10292. Fagopyrum 13 (1993): 35 - 39
Random amplified polymorphic DNA (RAPD) mar ker s in buckwheat
Branka Javornik and Bojka Kump Center of Plant Biotechnology and Breeding, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, Ljubljana 61111, Slovenia
Key words: buckwheat, DNA polymorphism, RAPD
Abstract The recently developed approach to deriving genetic markers by amplification of random DNA segments (RAPD) with primers of arbitrary nucleotide sequences was tested in buckwheat. Analysis of a limited number of individuals of different cultivars and populations of Fagopyrum esculentum Moench and populations of Fagopyrum tataricum Gaertn. using three primers, showed high polymorphism within cultivars and populations and three cultivar specific RAPD were detected. RAPD analysis seems very promising in studying genetic variability in buckwheat.
Molekularni markerji (RAPD) v ajdi
V prispevku je opisana molekularna tehnika polimerazne verižne reakcije s poljubnimi pri- mer ji, katere rezultat so naključno amplificirani segmenti DNK (RAPD). Analiziranih je bilo omejeno število osebkov različnih kultivarjev in populacij Fagopyrum esculentum Moench in populacije Fagopyrum tataricum Gaertn s tremi primerji in prvi rezultati kažejo velik poli- morfizem znotraj kultivarjev oz. populacij pri navadni ajdi. Kljub omejenemu številu analiz so bili najdeni specifični RAPD markerji. Opisana molekularna tehnika, ki je relativno enostav- na, odpira možnosti za proučevanje genetske variabilnosti na molekularni ravni tudi pri ajdi.
Introduction information on the target genome and only minute quantities of DNA are needed. DNA DNA polymorphism based on polymerase sequences in the total genome DNA are chain reaction (PCR) using single primers amplified by arbitrary short primers to of an arbitrary nucleotide sequence and generate randomly amplified DNA segments. detected as amplified products of random The amplification products are separated on DNA segments was described by Williams agarose gel and visualized by ethidium et al. (1990). Polymorphism, called random bromide. Different DNA bands can be amplified polymorphic DNA (RAPD detected between individuals and markers), between two individuals can polymorphisms serve as dominant genetic arise either from chromosomal changes in markers which are inherited in Mendelian the amplified regions or base changes that fashion. alter the primer binding. The RAPD assay is relatively simple, time and cost effective This type of polymorphism makes RAPD in comparison with well established RFLP markers valuable in plant genotype (Restriction fragment length polymorphism) fingerprinting (Welsh and McClelland 1990, markers. This PCR based polymorphism Hu and Quiros 1991, Welsh et al. 1991, assay does not require any prior Wilde et al. 1992, Dweikat et al. 1993, Harada et al. 1993, Yang and Quiros (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). 1993), estimation of genetic diversity The DNA concentration was estimated by (Chalmers et al. 1992, Kresovich et al. mini DNA fluorometer (Hoefer, TKO 100). 1992) and genetic mapping (Williams et al. 1990, Martin et al. 1991, Sobral et al. Polymerase chain reaction 1993). The conditions described by Williams et al. In this paper we report a preliminary (1990) for creating RAPD markers by PCR survey of RAPD markers in buckwheat for were first optimized for use, on a further analysis of genetic diversity in buckwheat template DNA. Tests of reaction buckwheat. components included variation of DNA concentration, magnesium concentration and Materials and methods enzyme concentration. The optimum polymerase chain reaction mixtures (25 jjl Plant materials final volume) contained approximately 40 ng genomic DNA, 50 mM each dATP, The following buckwheats were included in DGTP, dCTP and dTTP, 200 nM primer the analysis: five cultivars (cvs. Siva, (Operon Technologies), 4 mM MgCl2 and Darja, Darina and Rana 60 from Slovenia; 0.5 units of Taq polymerase with 10x cv. Majskaja from the Ukraine) and three incubation buffer (Boehringer Mannheim). populations (two from Nepal and one from The reaction mixture was overlaid with Japan) of Fagopyrum esculentum Moench approximately 30 jjl of mineral oil to and four F. tataricum populations (one prevent evaporation. Amplifications were population from Slovenia and three from performed in a Perkin-Elmer/Cetus Nepal: accessions no.83-N-147, no.85-9093 Thermal Cycler 480 with the following and no.85-3044). temperature conditions: preliminary 5 min denaturation of DNA at 95°C followed by DNA isolation the addition of the enzyme and then a total of 48 cycles of 1 min at 94°C, 1 min The procedure for total genomic DNA at 37°C and 1 min 30 sec at 72°C. The isolation was based on the protocol of amplification finished with an incubation at Saghai-Maroof et al. (1984) with 72°C for 10 min, followed by a 4°C soak modifications as described by Kump et al. until recovery. (1992). 0.005 - 0.15 g of fresh leaves was homogenized in 1.5 ml microcentrifuge Primers (Operon Technologies Inc., tubes. 600 jjl of preheated (65°C) СТАВ Alameda, CA, USA) : isolation buffer (2% (w/v) СТАВ, OPA-Ol: 5'CAGGCCCTTC'3 cetyltriammonium bromide, Sigma), 1.4 M OPA-13: 5'CAGCACCCAC'3 NaCl, 20 mM EDTA, 100 mM Tris-HCl (pH OPB-Ol: 5'GTTTCGCTCC'3 8.0), 0.2% 2-mercaptoethanol) was added and incubated for 1.5 hour at 65°C. The Agarose electrophoresis mixture was extracted with chloroform : isoamylalcohol (24:1) and centrifuged at 10 jjl of amplification products were loaded 10.000 rpm for 10 min. The DNA in the on 1,4% agarose (FMC) gels containing aqueous phase was precipitated by the ethidium bromide (0,5 g/ml). Molecular addition of 0.1 vol of 3 M sodium accetate DNA marker no. 4 of Boehringer and 0.6 vol of isopropanol. The precipitates Mannheim was used. DNA fragments were were washed in 70% ethanol and dissolved revealed by illumination with UV light and in a small volume of TE buffer photographed with Polaroid MP-4. Only intensive reproducable bands were scored. among them. To eliminate polymorphism within a sample, a bulk analysis was made Results and discussion pooling the DNA of five individuals of one sample and these combined DNAs served as Three Operon primers were randomly a temple for amplification. RAPD's pattern selected to test RAPD markers produced in (Fig. 1) revealed eight bands, of which six different buckwheat samples. The number were monomorphic and only two were of individual plants was limited to 4 - 7, polymorphic. In addition, the population except for cv. Rana 60 where 15 from Nepal and cv. Darina showed unique individuals were analysed. RAPD's.
Fagopyrum esculentum Moench: Fagopyrum tatatricum Gaertn.:
Primer OPA-Ol generated 14 scorable Three primers generated 10 - 12 scorable fragments ranging in length from 2300 to fragments ranging in length from 2500 to 500 base pairs (bp). High polymorphism 500 base pairs (Fig. 2). Tartatry was detected within cultivars or buckwheat is a self-pollinated species, so populations and cv. Darina and population much lower polymorphism was expected from Nepal showed unique bands. Eleven whithin accessions. As a test, we analysed fragments amplified by primer OPA-13 only two individual plants of each accession were scored in the range from 1600 to 500 and the same fragments were generated bp in four cultivars. Three of the bands among samples with primers OPA-13 and were monomorphic; others were polymorphic OPB-Ol. However, with primer OPA-Ol a showing higher variabilty within cultivars unique fragment was detected in both than between them. Primer OPB-Ol samples of accession no.85-9093. Tartary generated 8 fragments in the range from buckwheat shares with common buckwheat 2500 to 500 bp. Polymorphism was again one fragment amplified with OPA-Ol, one higher within cultivars or populations than with OPB-Ol and none with OPA-13.
Figure 1: RAPD's of bulk DNA samples amplified by primer OPB-Ol. From left to right: a) cv. Rana 60, b) population from Nepal, c) cv. Majskaja, d) cv. Darina, e) population from Japan, d) population from Nepal, f) cv. Siva, g) tartary buckwheat, h) DNA marker. Specific RAPD's of Nepal population and cv. Darina are indicated by arrows. Figure 2: RAPDs of tartary buckwheat generated by primer OPA-Ol. From left to right: a-b) no.85-3044, c-d) no.85-9093, e), g) and j) population from Slovenia, f) DNA marker, h-i) no.83-N-147. Specific RAPD of accession no.85-9093 is indicated by an arrow.
A preliminary survey of RAPD's markers Hu J. and Quiros C.F. 1991. Identification of broccoli and in F. esculentum Moench has shown a cauliflower cultivars vvith RAPD markers. Plant Celi higher degree of polymorphism within Rep 10: 505-511. Kresovich S., Williams JG.K., McFerson J.R., Routman E.J., cultivars or populations than between Schaal B.A. 1992. Characterization of genetic identities them. With bulk analysis and the and relationships of Brassica oleracea L. via a random application of a larger number of different amplified polymorphic DNA assay. Theor Appl Genet primers, it would be possible to detect 85: 190-196. more specific RAPD's in genetically distant Kump В., Svetek S., Javornik B. 1992. Isolation of high cultivars, which could be used for molecular vveight DNA from plant tissues. Zb. identification purposes. RAPD analysis can Biotehniške fak. UL 59: 63-66 (Sn). Martin G.B., Williams J.G.K., Tanksley S.D. 1991. Rapid be used as a powerful tool because of the identification of markers linked to a Pseudomonas high number of markers for determining resistance gene in tomato by using random primers and the extent of genetic diversity among near-isogenic lines. Proč Natl Acad Sci USA 88: buckwheat genotypes. RAPD analysis of 2336-2340. buckwheat would contribute additional Ohnishi O. 1990. Analysis of genetic variants in common information to genetic variation of buckwheat, Fagopyrum eculentum Moench: A review. Fagopyrum 10: 12-22. buckwheat studied by Ohnishi (1990). Saghai-Maroof M.A., Soliman K.M., Jorgensen R.A., Aliard R.W. 1984. Ribosomal DNA spacer lenght polymorphism References in barley: Mendelian inheritance, chromosomal location and population dynamics. Proč Natl Acad Sci USA 81: Chalmers K.J., Waugh R., Sprent J.I, Simons A.J., Powell 8014-8018. W. 1992. Detection of genetic variation betvveen and Sobral B.W.S. and Honeycutt R.J. 1993. High output genetic within populations of Gliricidia sepium and G. maculata mapping of polyploids using PCR-generated markers. using RAPD markers. Heredity 69: 465-472. Theor Appl Genet 86: 105-112. Dweikat I., Mackenzie S., Levy M., Ohm H. 1993. Pedigree Welsh, J. and McClelland M. 1990. Fingerprinting genome assessment using RAPD-DGGE in cereal crop species. using PCR vvith arbitrary primers. Nucleic Acids Res Theor Appl Genet 85: 497-505. 189: 7213-7218. Welsh J., Honeycutt R.J., McClelland M., Sobral B.W.S. VVilliams J.G.K., Kubelik A.R., Livak K.J., Rafalski J.A., 1991. Parentage determination in maize hybrids using Tingey S.V. 1990. DNA polymorphisms amplified by the arbitrarily primed polymerase chain reaction arbitrary primers are useful as genetic markers. Nucleic (AP-PCR). Theor Appl Genet 82: 473-476. Acid Res 18: 6531-6535. Wilde J., Waugh R., Powell W. 1992. Genetic fingerprinting Yang X. and Quiros C. 1993. Identification and of Theobroma clones using randomly amplified classification of celery cultivars vvith RAPD markers. polymorphic DNA markers. Theor Appl Genet 83: Theor Appl Genet 86: 205-212. 871-887. FIRST ANNOUNCEMENT
8ΙΧΤΗ INTERNATIONAL SYMPOSIUM ON BUCKWHEAT
August 20-26, 1995 opportunity for discussions on a wide range of t opic s: Shinshu University, Ina, Nagano JAPAN Genetic resources Ecology and physiology of productivity Production technologies This is the first announcement of the sixth Post-harvest technologies international symposium on buckwheat. The Quality and nutrition second announcement, including more Processing technologies detailed information on this symposium, Ethnobotany will be sent in December, 1993 to those returning the preliminary registration form ΕΧΗΙΒΙΤΙΟΝ in advance. An exhibition including harvesting machines, buckwheat products and books on INTRODUCTION buckwheat is planned. List and outlines of The Sixth International Symposium on institutes for buckwheat study in Japan Buckwheat will be held in Nagano, Japan, and many other countries will be also August 20-26, 1995. The first buckwheat displayed. symposium was held in Slovenia in 1980, and thereafter in each three years, the SUBMISSION OF PAPER(S)/ second being in Japan, the third in Poland, POSTER(S) the fourth in Russia and the fifth in Prospective authors are kindly requested to China in 1992 under the auspices of the indicate the tentative title of their International Buckwheat Research paper(s)/poster(s) in the attached Association. More than one hundred preliininary registration form. Full text of buckwheat scientists participated in each paper should reach the secretary office by symposium, which provided the impetus for February 28, 1995. We recommend you to international consultation among the submit the paper written with a floppy participants. Nagano is a leading area of disk, informing us of the software and the buckwheat production in Japan and is operating system of the disk you used. famous for its high mountains and summer resorts. We have many kinds of buckwheat LANGUAGE dishes, but we have to import about 80% The official language will be English. of our buckwheat consumption from other countries. There are many problems to be SYMPOSIUM FEE solved on buckwheat production. The 300 U.S. $ organizing committee is pleased to invite you to this symposium which offers the CORRESPONDENCE opportunity to exchange the latest Ali correspondence should be addressed to: information on buckwheat research. The committee welcomes you to spend time VI ISB Organizing Committee Faculty of enjoying beautiful buckwheat flowers, Agriculture, SHINSHU UNIVERSITY Ina, buckwheat noodles and other Japanese Nagano 399-45, JAPAN traditional dishes. We have a plan to publish a booklet titled "Buckwheat in Tel: 0265-72-5255 ext. 311 or 312 Japan". Fax: 0265-72-5259 Tel&Fax: 0265-76-9331
PROGRAM The symposium will include special Dr. Toshiko Matano lectures, 20 minutes oral presentations, posters (lx2m2), workshops, and an Chairman of the Organizing Committee excursion, an exhibition and business Sixth International Symposium on meeting. The symposium will provide an Buckwheat Welsh J., Honeycutt R.J., McClelland M., Sobral B.W.S. VVilliams J.G.K, Kubelik A.R., Livak K.J., Rafalski J.A, 1991. Parentage determination in maize hybrids using Tingey S.V. 1990. DNA polymorphisms amplified by the arbitrarily primed polymerase chain reaction arbitrary primers are useful as genetic markers. Nucleic (AP-PCR). Theor Appl Genet 82: 473-476. Acid Res 18: 6531-6535. Wilde J., Waugh R., Powell W. 1992. Genetic fingerprinting Yang X. and Quiros C. 1993. Identification and of Theobroma clones using randomly amplified classification of celery cultivars vvith RAPD markers. polymorphic DNA markers. Theor Appl Genet 83: Theor Appl Genet 86: 205-212. 871-887. FIRST ANNOUNCEMENT
SIXTH INTERNATIONAL SYMPOSIUM ON BUCKWHEAT
August 20-26, 1995 opportunity for discussions on a wide range of topics: Shinshu University, Ina, Nagano JAPAN Genetic resources Ecology and physiology of productivity Production technologies This is the first announcement of the sixth Post-harvest technologies international symposium on buckwheat. The Quality and nutrition second announcement, including more Processing technologies detailed information on this symposium, Ethnobotany will be sent in December, 1993 to those returning the preliminary registration form ΕΧΗΙΒΙΤΙΟΝ in advance. An exhibition including harvesting machines, buckwheat products and books on INTRODUCTION buckwheat is planned. List and outlines of The Sixth International Symposium on institutes for buckwheat study in Japan Buckwheat will be held in Nagano, Japan, and many other countries will be also August 20-26, 1995. The first buckwheat displayed. symposium was held in Slovenia in 1980, and thereafter in each three years, the SUBMISSION OF PAPER(S)/ second being in Japan, the third in Poland, POSTER(S) the fourth in Russia and the fifth in Prospective authors are kindly requested to China in 1992 under the auspices of the indicate the tentative title of their International Buckwheat Research paper(s)/poster(s) in the attached Association. More than one hundred preliminary registration form. Full text of buckwheat scientists participated in each paper should reach the secretary office by symposium, which provided the impetus for February 28, 1995. We recommend you to international consultation among the submit the paper written with a floppy participants. Nagano is a leading area of disk, informing us of the software and the buckwheat production in Japan and is operating system of the disk you used. famous for its high mountains and summer resorts. We have many kinds of buckwheat LANGUAGE dishes, but we have to import about 80% The official language will be English. of our buckwheat consumption from other countries. There are many problems to be SYMPOSIUM FEE solved on buckwheat production. The 300 U.S. $ organizing committee is pleased to invite you to this symposium which offers the CORRESPONDENCE opportunity to exchange the latest Ali correspondence should be addressed to: information on buckwheat research. The committee welcomes you to spend time VI ISB Organizing Committee Faculty of enjoying beautiful buckwheat flowers, Agriculture, SHINSHU UNIVERSITY Ina, buckwheat noodles and other Japanese Nagano 399-45, JAPAN traditional dishes. We have a plan to publish a booklet titled "Buckwheat in Tel: 0265-72-5255 ext. 311 or 312 Japan". Fax: 0265-72-5259 Tel&Fax: 0265-76-9331
PROGRAM The symposium will include special Dr. Toshiko Matano lectures, 20 minutes oral presentations, posters (lx2m2), workshops, and an Chairman of the Organizing Committee excursion, an exhibition and business Sixth International Symposium on meeting. The symposium will provide an Buckwheat Fagopyrum 13 (1993): 41 - 44
PROFESSOR DR. MAREK RUSZKOWSKI 1925 - 1993
Professor Marek Ruszkowski passed away in the Department of Crop Husbandry in March 1993, in Poland. Не was a headed by Professor S. Lewicki, his prominent scholar who, inter alia, spent scientific and intellectual master for a few many years investigating the biology and years to come. He worked at the production technology of buckwheat. Не university until 1952 and he published his was one of the founders of the first scientific paper there on plant International Buckwheat Research vernalization. Association (IBRA), and was president of By that time already a promising this organization for three years. brilliant young scientist, Professor Prof. Ruszkowski died in Pulawy, the Ruszkowski joined the Institute of Plant oldest agricultural research center in Breeding and Acclimatization, Pulawy, Poland. Since the early 1950's he was a Poland. Within a few years, he was member of staff of the institute of Soil appointed to the Institute of Soil Science Science and Plant Cultivation, where he and Plant Cultivation, an institution passed through ali the stages of his regarded as the heir to the great tradition scientific career, from PhD to the title of of agricultural research in Pulawy. Soon professor ordinarius. Due to his constant after, in 1960, Professor Ruszkowski was diligence, quick and inquisitive mind and appointed head of the Department of organizational talents he obtained Cereal Crops and thus established his meaningful and important results mainly lifetime bond with the institute and with in the area of zoning and productivity of Pulawy, a location that suited him both various cereal crops in different habitats because of its tradition and its scenic and in cereal husbandry and production landscape and climate. He felt good here technology. Those achievements gained him and made Pulawy his home, an ideal plače the recognition of both the scientific and where he could pursue his scientific and farming communities. personal interests. The life and work of Professor By no means an armchair scientist, Ruszkowski were most closely linked with Professor Ruszkowski conducted vigorous the turbulent fate of Poland and Polish teaching, social and political activities science after World War II. alongside his research work. He was born in Torun, Poland, in 1925. He supervised more than 20 doctoral By the time the war broke out, he had theses and associate professor's finished primary school and the first grade dissertations. Young scientists stuck by of high school. During the Nazi occupation him, attracted by his kindness, willingness he stayed in Lublin where he attended a to help his junior colleagues and his broad commercial school. In the third year of the intellectual and scientific horizons. war he got a job of a workman in a Professor Ruszkowski worked for many seed-cleaning plant to earn a modest public and professional organizations and he livelihood for himself and his family. In also took an active part in the peasants 1946, after completing high school, movement. In the recent period he was a Professor Ruszkowski began to study member of Solidarity, an organization he agriculture at the newly-established Marie had much hope in. He was thus a scientist Curie-Sklodowska University in Lublin. As and an activist of no ordinary calibre. A early as 1948, he gained an assistantship recipient of many prizes and awards, he was popular and esteemed by his fellow technology of cereals and other crops, scientists and by the community. including 55 publications on buckwheat. His rather sudden and unexpected He kept introducing new topics to his decease was received in those circles with buckwheat research and finding new issues great sorrow and with a profound sense of to be solved. loss. In his last years, he studied a problem Professor Ruszkowski was remarkable that had been seldom dealt with before - for the broad scope of his interests and his the relationship between buckwheat receptiveness to new ideas, both scientific productivity and сапору architecture. and social. His diligence being almost Approached in an innovative way, the issue proverbial in the intellectual community, was the subject of his last papers those interests bore fruit in the form of published at home and abroad*. meaningful scientific, intellectual and Professor Ruszkowski was an able and organizational achievements. recognized popularizer of buckwheat Two wide fields of research, husbandry production in Poland. His output included a of cereals and development of up-to-date number of extension and dissemination cereal production technologies, took up instructions. He presented the results of most of Professor Ruszkowski's time and his buckwheat investigations at many energy during his long-time activity in the training seminars for farm advisers and institute. He was the first to develop an farmers themselves. Publications by integrated techology for cereal production Professor Ruszkowski on buckvvheat in Poland. production under Poland's conditions have Professor Ruszkowski was passionately been re-edited several times. devoted to research related to the biology, On his initiative, a so called yield potential and production technology of "buckwheat-growing commune" was buckwheat. His first paper on buckwheat organized on one of the communes of the production appeared as early as 1956. He Lublin province. The idea was to followed it up with a number of concentrate buckwheat production in one publications written jointly vvith Professor Consolidated area, to be used as a test S. Lewicki. ground for improved buckwheat growing Professor Ruszkowski's output grew technologies and their dissemination among with the passing years. He probed more farmers, owners of small and medium sized and more deeply into the principles of farms. buckwheat's breeding and husbandry*. He Professor Ruszkowski had a gift of became an unquestionable authority on getting his ideas through to his audience. buckwheat research in Poland, co-organizer He was a welcome speaker at meetings of seminars and scientific sessions on and training courses in the field and he buckwheat breeding and production never tried to shun such occasions. technology. As already mentioned earlier, Sadly, Professor Ruszkowski passed away he was also active in the international too early. The agricultural sciences have forum. A member of IBRA, he gave sustained a great loss. It will be presentations of his research results at particularly poignant in those communities various international symposia. and centers in Poland and abroad which He had several hundred publications to conduct research on wheat and buckwheat. his credit, mostly on broad-sense production
*Professor Ruszkowski prepared four presentations on the subject to be delivered at the national seminar "Buckwheat husbandry and processing" held in June 1993 in 01sztyn; he did not live to deliver them Publications of Professor Dr. Marek Ruszkowski in the area of buckwheat research
[titles in brackets are English translations of original Polish titles]
1. Ruszkovvski M- [Buckwheat production techniques on 15. Ruszkovvski MJ [Studies on buckvvheat. Relationship of farms in the Lublin and Rzeszowprovinces]. Nowe grovvth and development to yields. Parts 1 and 2]. Rolnictwo 4, 1956. Pam.Pul. no. 19: 3-76. 2. Lewicki S., Osinska K, Ruszkovvski M.: [Studies on 16. Duda G, Prochiak J, Ruszkovvski M.: [Results of buckwheat Part 1. Cultivation and fertilization problems]. buckvvheat trials performed in 1955-1964]. PWRL 1967. Biuletyn IUNG. Prace Zakladu Rošlin Zbožowych, 1957. 17. Ruszkowscy B. i MJ [Buckvvheat] PWRL. Warszawa 3. Lewicki S, Ruszkovvski M.: [Studies on buckvvheat Part 2. 1967. Some special practices that have potential to affect 18. Ruszkovvski M.: [Buckvvheat] PWRL. Warszawa 1974, buckvvheat yields]. Biuletyn IUNG. Prace Zakladu Rošlin 1977, 1981. Zbožowych, 1957. 19. Ruszkovvski M.: [Results of buckvvheat variety trials 4. Levvicki S, Ruszkovvski MJ [Studies on buckvvheat Part 1. conducted in 1965-1968]. PWRL 1970. Biology of buckvvheat germination]. Part 1, 1958, 20. Ruszkovvska B, Ruszkovvski M.: [Buckvvheat]. 2nd Hod.Rosl. Aklim. 2 (1): 67-68. Edition. PWRL 1970. 5. Levvicki S, Ruszkovvski MJ [Studies on buckvvheat Part 1. 21. Ruszkovvski M. et alj [Effects of some husbandry Biology of buckvvheat germination]. Hodovvla Rošlin elements on the yields of tetraploid buckvvheat cv. Aklimatyzacja i Nasiennictvvo 2 (2),1958. Emka]. R(120), 1977: 157-164. 6. Levvicki S, Ruszkovvski MJ [Studies on buckvvheat Part 6. 22. Ruszkovvska B, Ruszkovvski M.: [Effect of nitrogen Variation of yields vvithin different varieties and fertilization, seeding date and seeding rate on the yields populations of buckvvheat]. Hod.Rosl.Aklim., 1958, 2 of tetraploid buckvvheat cv, Emka.]. Badania nad (4):561-566. agrotechnika odmian zbož. IUNG, 1977, R(120). 7. Levvicki S., Ruszkovvski M, Ruszkovvska M: [Causes of 23. Ruszkovvski M.: The possibility of changing the yielding poor fruit set in buckvvheat. Part 3. Effect of different ability of buckvvheat by breeding homostyle varieties. cultural practices on fruit set in buckvvheat]. Buckvvheat. Ljubljana 1980: 7-16. Hod.Rosl.Aklim, 1958, 2 (5): 587-602. 24. Ruszkovvski MJ [Buckvvheat]. 3rd revised edition. PWRL 8. Ruszkovvski M- [Studies on buckvvheat. Part 7. 1981 Investigations on obtaining forms of buckvvheat and 25. Ruszkovvski MJ The buckvvheat productivity on millet less sensitive to spring chills]. Hod.Rosl.Aklim., different soils treated vvith soil conditioner Agromax 1958, 2 (6): 687-695. (17-5-5). 1982. Fagopyrum 2. 9. Ruszkovvski M.: [Studies on buckvvheat. Part 8. 26. Ruszkovvski M.: The yield ability of buckvvheat on Investigations of grovvth and development]. various soils. 1982. Fagopyrum 2. Hod.Rosl.Aklim, 1958. 27. Ruszkovvski M.: The cultivation and utilization of 10. Levvicki S, Ruszkovvski M, Kaszelej Z.: [Studies on buckvvheat in Poland. Proc. 2nd Intl.Symp.Buckwheat, buckvvheat. Part 9. Yields of grain and green matter Japan 1983: 13-23. from 10 different seeding dates]. Hod.Rosl.Aklim, 1959, 28. Ruszkovvski M.: The productivity of homostyle 3 (2): 195-207. homomorphic lines of buckvvheat. Proc. 2nd 11. Ruszkovvski MJ [Studies on buckvvheat. Part 10. Intl.Symp.Buckwheat, Japan 1983: 79-86. Comparison of the grovvth and development of different 29. Ruszkovvski M.: Studies on the agrotechnical populations]. Hod. Rosl.Aklim, 1959, 3 (2): 209-216. characteristics of buckvvheat varieties in Europe and 12. Levvicki S, Osirtska K, Ruszkovvski MJ [Studies on Japan. Proc. 2nd Intl.Symp.Buckwheat, Japan 1983: buckvvheat. Part 1. Problems of cultivation and 127-129. fertilization] Dosvviadczenia z roslinami zbozowymi. 30. Ruszkovvski M.: The influence of grovvth regulators on Warszawa 1960 no. 1: 175-228. grain yield of buckvvheat. 1984, Buckvvheat Nevvsletter 1. 13. Levvicki S, Ruszkovvski M, Szklarz Jj [Studies on 31. Ruszkovvski M.: Studies on the agrotechnical buckvvheat. Part 2. Suitability of certain special practices characteristics of buckvvheat varieties in Europe and under this country's conditions]. Dosvviadczenia z Japan. 1986, Buli. of Environmental Conservation roslinami zbozowymi. VVarszavva 1960, no. 1: 230-242. Shinshu University 10, 1986. 14. Mazurek J, Ruszkovvski MJ [Causes of poor fruit set in 32. Ruszkovvski MJ Studies on productivity of various buckvvheat. Part 5. Investigations on self pollination]. buckvvheat varieties in Japan and Europe. Japanese Pam.Pul, 1963, no. 10: 167-174. Journal of Crop Science 54: Extra Issue 1. 33. Ruszkovvski MJ Productivity of buckvvheat. Buckvvheat 45. Ruszkovvski M. et al. [Improved buckvvheat production Research 1986, IUNG Pulawy: 78-98 technology. Extension training papers]. IUNG ed, 1990: 34. Ruszkovvski M.: The productivity of homomorphic and 1-111. heteromorphic buckvvheat forms in various agrotechnical 46. Ruszkovvski M, Holubovvicz-Kliza Gj [Improved conditions. Buckvvheat Research 1986, IUNG Pulawy: buckvvheat production techno!ogy. Extension instruction 105-109. no. 162/90]. Pulawy ed, 1990. 35. Edited by M. Ruszkovvski. [Technology of buckvvheat 47. Ruszkovvski MJ [Buckvvheat productivity as affected by production (training handbook)]. IUNG Pulawy 1986. changes in the number of inflorescens in the lst order 36. Ruszkovvski MJ [Integrated buckvvheat production branches]. VII Krajovve Sympozjum Gryki. IUNG technology. Extension paper no. 126/86]. IUNG Pulawy, Pulawy, 1991: 15-22. 1986. 48. Ruszkovvski M.: [Buckvvheat productivity as affected by 37. Ruszkovvski M.: [Results of investigations on buckvvheat changes in leaf assimilation area of the main stalk]. VII production technology]. IV Krajovve Sympozjum Gryki, Krajovve Sympozjum Gryki. IUNG Pulawy, 1991: 22-30. 1987, Strzelno Pulawy: 14-25. 49. Ruszkovvski M.: [Changes in the model and vveight of 38. Ruszkovvski M, Novvorolnik KJ [Effect of different buckvvheat grain at different spacings of plants on a husbandry practices and nitrogen fertilization on unit area] . VII Krajovve Sympozjum Gryki. IUNG buckvvheat yields]. IUNG ed, 1988, R(241): 5-18. Pulawy, 1991: 30-35. 39. Ruszkovvski M, Novvorolnik KJ [Effect of nitrogen 50. Ruszkovvski M. et alj [The phenomenon of domination, fertilization on the yields of tvvo buckvvheat cultivars]. competition and compensation in the productivity of IUNG ed., 1988, R(241): 19-28. buckvvheat]. IUNG Pulawy, 1991, R(279): 5-30. 40. Ruszkovvski MJ [Changes in yield components and 51. Ruszkovvski MJ The study on buckvvheat plant habitus сапору architecture as affected by husbandry factors]. and productivity in relation to assimilate leaf surface. IUNG ed., 1988, R(241): 41-66. Proc. 5th Intl.Symp.Buckwheat. China, Taiyuan, 1992: 41. Ruszkovvski MJ Structure of yield architecture and 357. buckvvheat productivity. Proc. of 4th 52. Ruszkovvski MJ [Productivity of buckvvheat in relation Intl.Symp.Buckwheat, Orel, USSR, 1989, Buckvvheat to changes in assimilation leaf area on lst order Research. Part 1: 7-34. branches]. ART Olsztyn, 1993.* 42. Ruszkovvski M, Holubovvicz-Kliza Gj [Integrated 53. Ruszkovvski MJ [Yield components and сапору buckvvheat production technology. Extension instruction architecture of buckvvheat at different plant spacings on no 24/89] Ed. Pulawy, 1989: 1-18. a unit area]. ART 01sztyn, 1993* 43. Ruszkovvski MJ [The phenomena of domination, 54. Ruszkovvski MJ [Yield components and сапору competition and compensation in the production of architecture of buckvvheat at different leaf assimilation buckvvheat]. Niektore Zagadnienia z zakresu produkcji areas on the main stalk and lst order branches]. ART zbož. IUNG Pulawy, 1990 R(279): 31-60. 01sztyn, 1993* 44. Ruszkovvski M.: The phenomenon of domination, 55. Ruszkovvski M.: [Effect of removing leavs on the main competition, compensation and overcompensation in stalk and the lst order branch at different grovvth buckvvheat productivity. 1990, Fagopyrum 10: 56. stages on the changes in habitus and productivity]. ART 01sztyn, 1993*
*items 52 through 55 are presentations delivered at the 8th National Buckwheat Symposium
Biography and bibliography by IUNG, Pulawy INSTRUCTIONS FOR AUTHORS
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