Tom XXVI ROŚLINY OLEISTE OILSEED CROPS 2005 Młgorzt Nogl-Kłuck, Czesłw Muśnicki*, Bogumił Kupczyk, Aldon Jsińsk-Stępnik, Ew Brtkowik-Fludr, Aleksnder Siger Akdemi Rolnicz im. A. Cieszkowskiego w Poznniu, Ktedr Biochemii i Anlizy Żywności * Ktedr Uprwy Roli i Roślin Preliminry studies of tocochromnol content in seeds of open pollinted nd hybrid vrieties of winter rpe Wstępne bdni zwrtości tokochromnoli w nsionch odmin populcyjnych i mieszńcowych rzepku ozimego Key words: rpeseed, open pollinted nd hybrid vrieties, homologous tocopherols, HPLC The objective of this study ws determintion of nturl ntioxidnts, such s tocochromnols, in seeds of fifteen vrieties of winter rpe registered by COORU in the yers 2002 2003. There were open pollinted vrieties: Polish Btory nd Bzyl nd foreign Rsmus, Digger, Cpio, Czek, Clifornium nd Spencer NL s well s Polish composite hybrids Kszub nd Mzur nd foreign restored hybrids Kronos, Bldur, Extrem nd Titn. Mterils from the first reproduction K 1 were supplied by seed producing compny OBROL. All studied rpeseeds cme from the crop of 2004. Tocochromnol composition of new vrieties ws compred to tht of vrieties lredy cultivted Kn, Lirjet, Lisek nd Bufflo, which hd lredy been studied erlier nd results were published in 2003. The mteril under investigtion included lso the seeds of winter turnip rpe (B. rp) Lufowy vriety nd winter flse flx Cmelin stiv Przybrodzk II vriety hrvested in 2003. The qulittive identifiction nd quntittive determintion of homologous tocopherols were crried out by HPLC fter sponifiction of smples nd extrction of unsponifible substnces. Tocopherol seprtion ws performed with the use of Wters HPLC system equipment with LiChrosorb Si 60 column. The mobile phse ws n-hexne nd 1,4 dioxne. Tocopherols were monitored by fluorimetric detector (295, 330 nm). Contents of individul tocopherol homologues were clculted on the bsis of clibrtion curves for pure forms of these compounds. Results obtined in this study indicted tht totl tocochromnol content in the mjority of rpeseed vrieties ws bove 300 mg/kg d.m of seeds. The gretest tocopherols content ws determined in two rpeseed vrieties restored hybrids Kronos nd Titn (bove 400 mg/kg d.m). Ech of four homologous tocopherols ws identified in ll investigted winter rpeseed vrieties. α- nd γ-tocopherols occurred in gretest quntities. The clculted rtio of α- to γ-tocopherol content rnged from 0.8 to 1.10 for open pollinted vrieties of rpeseed, nd it ws higher for the restored hybrids (1.32). Among the open pollinted vrieties of winter rpeseed the seeds of Lisek vriety were richest in tocopherols (383 mg/kg d.m.). Tocopherol content ws lso higher when compred to the 2000 2003 crops of this vriety. Słow kluczowe: nsion rzepku, odminy populcyjne i mieszńcowe, homologiczne tokoferole, HPLC W prcy bdno zwrtość ntywnych przeciwutleniczy, jkimi są tokochromnole, w nsionch 15 odmin rzepku ozimego, zrejestrownych przez COBORU w ltch 2000 2003. Bdno polskie
562 Młgorzt Nogl-Kłuck... odminy populcyjne Btory i Bzyl orz zgrniczne Rsmus, Digger, Cpio, Czek, Clifornium i Spencer NL, polskie mieszńce złożone Kszub i Mzur orz zgrniczne mieszńce zrestorowne Kronos, Bldur, Extrem i Titn. Mterił siewny pochodził z pierwszego rozmnożeni K 1 i zostł zkupiony w przedsiębiorstwie nsiennym OBROL. Do bdń włączono tkże nsion rzepku strszych odmin (Kn, Lirjet, Lisek i Bufflo). Wszystkie bdne nsion pochodziły ze zbiorów w 2004 r. Anlizowno również nsion rzepiku ozimego odminy Ludowy i lninki ozimej odminy Przybrodzk II (nmnożone przez ns w 2003 roku). Zwrtość homologicznych tokoferoli oznczno techniką HPLC, po uprzednim zmydleniu nsion i ekstrcji eterem etylowym. Do rozdziłu stosowno kolumnę LiChrosorb Si 60 (250 4,6 mm; 5 µm). Fzę ruchomą stnowił n-heksn z 1,4 dioksnem (97 : 3 v/v) o szybkości przepływu 1,5 cm 3 /min. Stosowno detektor fluorymetryczny (295, 330 nm). Zwrtość poszczególnych homologów tokoferoli obliczono n podstwie krzywych klibrcji wykonnych z pomocą próbek wzorcowych tokoferoli. We wszystkich nsionch rzepku, rzepiku i lninki zidentyfikowno cztery homologiczne tokoferole α-, β-, γ- i δ-t. Cłkowit ilość tokoferoli w nowych odminch populcyjnych rzepku ksztłtowł się od 295 mg/kg s.m. (Btory 2001 r.) do pond 397 mg/kg s.m. (Digger 2003 r.). We wszystkich bdnych odminch rzepku dominowły homologi α- i γ tokoferolu. W nsionch był identyfikowny w niewielkich ilościch β- i δ-tokoferol. Obliczony stosunek zwrtości α- do γ-tokoferolu ksztłtowł się dl odmin populcyjnych rzepku ozimego n poziomie 0,8 do 1,10. W odminch mieszńcowych zrestorownych był zncznie wyższy i sięgł wrtości 1,32. W bdnych nsionch odmin populcyjnych rzepku ozimego njwiększą zwrtość oznczono dl odminy Lisek (383,0 mg/kg s.m.), zncznie większą w porównniu do ilości tokochromnoli oznczonych w nsionch tej smej odminy ze zbiorów wcześniejszych (2000 2003 r.). Introduction The seeds of double low qulity (00) rpeseed cultivted in Polnd re vluble mteril for production of plnt oils nd fts. Oils obtined from rpeseed re chrcterized by high content of tricyloglycerols source of polyunsturted ftty cids (PUFAs). Rpeseed oil contins bout 20% of linoleic cid (C 18:2 n-6) nd bout 10% of linolenic cid (C 18:3, n-3) necessry in humn diet (Mikołjczyk et l. 1999). On the other hnd, high level of unsturted ftty cids (90%) cn influence decrese of the oxidtive stbility of oil nd other products contining this oil. Free rdicls nd lso peroxides nd other products of their conversion re formed during oxidtion, which cn cuse mny civiliztion diseses (Korczk et l. 1999, Cdens nd Dvies 2000, Wng nd Quinn 1999). By combining modern biotechnology methods with clssicl plnt breeding novel vrieties of rpeseed were generted hving specific properties nd chemicl composition (Leckbnd et l. 2002). The new geneticly modified vrieties of rpeseed re chrcterized by reduced levels of e.g. linolenic cid (Murphy nd Mithen 1995, Jourdren t l. 1996, Mikołjczyk et l. 1999, Spsibionek et l. 2000, Hom 2004). New methods of genetic modifiction cn give new rpeseed vrieties hving higher level of ntive oxidtion inhibitors such s tocopherols (Goffmn nd Becker 1998, 2001, 2002; Mrwede et l. 2004, 2005). Tocopherols re nturl ntioxidnts in plnt oils. All tocopherols re mphipthic molecules
Preliminry studies of tocochromnol content... 563 hving polr chromn rings nd hydrophobic prenyl chin (Hofius nd Sonnewld 2003). The min biochemicl function of tocopherols is protection of polyunsturted ftty cids ginst peroxidtion (Kml-Eldin nd Appelqvist 1996). The strongest biologicl ctivity is displyed by α-tocopherol wheres γ-tocopherol hs the strongest ntioxidnt ctivity (Goffmn nd Becker 2001, Nogl-Kłuck et l. 2003). Homologous tocopherols re synthesised by plnts nd photosynthetic orgnisms (Choon nd Coughln 2004, Sdre et l. 2001). Formtion of four tocopherols results from the rections of cycliztion nd methyltion of their romtic precursor homogentisic cid (Jniszowsk 1986). Four homologous tocopherols occur in rpeseed nd other photosynthetic plnts (Nogl-Kluck et. l. 2002). Among the tocopherols, α- (α-t) nd γ- (γ-t) re the predominnt forms. β- (β-t) nd δ-tocopherols (δ-t) re identified in smll (δ-t) or trce mounts (β-t) (Nogl-Kluck et l. 2002). Introducing new winter rpeseed vrieties with modified seeds qulity contributed to continution of erlier studies. The im of these studies is determintion of the qulity nd quntity of tocopherol contents in new winter rpeseed vrieties (open pollinted vrieties nd composite hybrids) in comprison to the vrieties lredy exmined. Mteril nd methods Seed smples of fifteen vrieties of winter rpeseed registered by COBORU in the yers 2002 2003 were investigted. There were open pollinted vrieties: Polish Btory nd Bzyl nd foreign Rsmus, Digger, Cpio, Czek, Clifornium nd Spencer NL s well s Polish composite hybrids (F 1z ) Kszub nd Mzur nd foreign restored hybrids (F 1 ) Kronos, Bldur, Extrem nd Titn. Mterils from first reproduction K 1 ws supplied by the seed producing compny OBROL. Four older vrieties: Kn, Lirjet, Lisek nd Bufflo were lso investigted. Seeds of ll studied vrieties cme from the crop of 2004, nd were collected during growing period from the experimentl fields of Poznń Agriculturl University (Przybrod). The mteril under investigtion included lso the seeds of winter turnip rpe Ludowy vriety nd winter flse flx (Cmelin stiv) Przybrodzk II vriety reproduced in 2003 by the uthors t the experimentl sttion Przybrod. Tocopherols determintion In order to determine the tocopherol content, smples of rpeseeds were sponified using 60% KOH. After sponifiction the unsponifible substnces were extrcted three times using the peroxide free diethyl ether. Then, the ether ws
564 Młgorzt Nogl-Kłuck... distilled off nd the residue ws dissolved in n-hexne. The qulittive identifiction nd quntittive determintion of homologous tocopherols were crried out by HPLC (Gogolewski et l. 2000, Nogl-Kłuck et l. 2002). The tocopherols were nlysed on the Wters HPLC system equipment with LiChrosorb Si 60 column (250 4.6 mm; 5 µm) nd precolumn LiChrospher Si 60. The mobile phse ws n-hexne nd 1,4 dioxne. The tocopherols were monitored by fluorescence detector (295, 330 nm). Contents of individul tocopherol homologoues were clculted on the bsis of clibrtion curves mde for pure forms of these compounds. Stndrds of α-, β-, γ- nd δ-tocopherol (α-t, γ-t, β-t, δ-t) (99%) were purchsed from Merck (Drmstdt, Germny). Two seed smples (3 g) were drwn from ech vriety nd ll nlyses were repeted three times. Sttisticl nlysis The results obtined were subjected to sttisticl nlysis. One-fctor nlysis of vrince nd post-hoc Tukey s tests were crried out with the sttisticl pckge progrm Sttistic (version 6.0) for the significnce level α = 0.05. Results nd discussion The new hybrid vrieties of winter rpeseed hve been of interest since 1995. Their cultivtion constntly increses, becuse the yields re high nd giving rpe seeds of good qulity. Breeding of new vrieties results in obtining lrger mount of pods with well developed seeds. The seeds re chrcterised by improved technologicl qulity higher level of ft (bove 38%) nd protein (bove 19%) (Wójtowicz et l. 2003). The level of ntive ntioxidnt in seeds of new vrieties of rpeseed hs not been tested untill now. Therefore, following current reserch trends in genetic modifiction, in breeding nd in cultivtion of new vrieties of rpeseed, determintion of tocochromnol content in selected vrieties of rpeseed took into ccount the studies which hd lredy been published (Nogl-Kłuck et l. 2002, 2003). In this study lkline hydrolysis nd chromtogrphic method (norml-phse high-performnce liquid chromtogrphy with fluorescence detection NP-HPLC- FLD) for determintion of ll forms of tocopherols were used. They re lso commonly used by other reserchers (Gogolewski et l. 2000, Nogl-Kłuck et l. 2002, Ryynänen et l. 2004). There re three mjor smple preprtion pproches for food smples: simple dilution of oil smples in orgnic solvent, direct extrction of the vitmins with orgnic solvents, nd extrction of the vitmins fter sponifiction (Eitenmiller nd Lnden 1999, Ryynänen et l. 2004). Cerels, seeds nd nuts re crushed to fine prticles before smple preprtion. Sponifiction is generl term referring to lkline hydrolysis. Usully, KOH
Preliminry studies of tocochromnol content... 565 is used, lthough NOH is specified by some procedures. Hydrolysis results in clevge of the ester bounds of tricyloglycerols, phospholipids nd sterols; it destroys pigments nd disrupts the smple mtrix which fcilittes vitmin extrction (Eitenmiller nd Lnden 1999, Wrolstd 2002). Tocopherols re reltively unstble in lkline conditions, nd cre must be tken to void their destruction. They re protected by using ntioxidnts (pyrogllol or scorbic cid), flushing the sponifiction vessel with nitrogen nd working under subdued light (Eitenmiller nd Lnden 1999, Eitenmiller nd Lee 2004). After sponifiction, the digest is diluted with wter or 1% NCl to inhibit emulsion formtion nd is extrcted with diethylether, petroleum ether, hexne or ethyl cette in hexne, or other solvent mixtures. The solvent is used to retrieve unsponifible components into the orgnic phse. Solvent nd queous phses re seprted fter centrifugtion or setting period. The unsponifible components, including tocopherols nd tocotrienols in smple solution, re extrcted into the solvent lyer. Most wtersoluble interfering substnces, including proteins, sugrs, nd fibers, remin in the wste (queous lyer) (Shin nd Godber 1994). Tocopherols were seprted by reversed-phse thin-lyer chromtogrphy (C 18 RP-HPTLC), norml-phse high-performnce liquid chromtogrphy (NP- HPLC), reversed-phse high-performnce liquid chromtogrphy (C 18 RP-HPLC), nd gs chromtogrphy (GC). Seprtion using C 18 RP-HPLC does not seprte β- nd γ-tocopherols. (Pyk nd Sliwiok 2001). GC seprtion of tocopherols is difficult to chieve. To decrese their boiling points nd to void decomposition of tocopherols t the high temperture of nlysis tht must be pplied in the GC method, derivtiztion rection is used to trnsform the hydroxyl groups of tocopherol to trimethylsilyl (TMS) derivtives, which results in lower boiling points. However, the conditions of derivtiztion re very criticl for obtining high yields of derivtives, nd high vribility my occur between duplictes (Lercker nd Rodriguez-Estrd 2000, Wrolstd 2002). Vitmin E nlysis is most commonly conducted with NP-HPLC-FLD. A fluorescence detector is preferred to n ultrviolet detector in nlysing tocopherols nd tocotrienols in complex food mtrices becuse of its specificity nd sensitivity (Eitenmiller nd Lnden 1999, Abidi 2000, Ryynänen et l. 2004). Two Polish composite hybrid vrieties Kszub nd Mzur (2001) were mong the investigted fifteen seed smples of rpeseed vrieties registered by COBORU in the yers 2000 2003. The remining were Polish nd foreign open pollinted vrieties. The vribility in tocopherol contents in the investigted rpeseed smples is presented in Tble 1. In ll smples of rpe seeds, turnip rpe nd flse flx seeds four homologous tocopherols α-, γ-, β- nd δ- were identified. The totl tocopherols content in new open pollinted vrieties rnged from 295 mg/kg dry mtter (Btory 2001) to bove 397 mg/kg seed d.m. (Digger 2003).
566 Młgorzt Nogl-Kłuck... Tble 1 Tocopherol content in investigted winter rpe seeds open pollinted, composite hybrids nd restored hybrids, lso in winter turnip nd winter cmelin Zwrtość homologicznych tokoferoli w nsionch rzepku ozimego odmin populcyjnych, mieszńców złożonych i zrestorownych orz rzepiku i lninki ozimej Vrieties Odminy Open pollinted Odminy populcyjne Kn 154,6 ± 4,9 c,d Tocopherol content Zwrtość tokoferoli [mg/kg d.m.] α-t β-t γ-t δ-t sum sum 2,2 ± 0,2 163,1 ± 4,6 f,g 6,8 ± 0,2,b,c 326,6 ± 9,9 d,e,f Rtio Współczynnik α-t / γ-t 0,95 e,f Btory 146,8 ± 3,4 c 5,3 ± 0,1 e,f 134,3 ± 2,7,b 8,8 ± 0,1 295,1 ± 6,3 b,c 1,09 i,j,k Bzyl 173,8 ± 3,5 f,g,h 6,1 ± 0,2 g 170,9 ± 13,2 g,h 9,0 ± 0,7 f,g 359,8 ± 17,7 h,i 1,02 g,h Lirjet 175,2 ± 3,6 g,h 2,2 ± 0,2 162.0 ± 5,2 6,4 ± 0,1,b 345,8 ± 9,1 f,g,h 1,08 i,j Lisek 180,4 ± 0,2 h 2,1 ± 0,1 194,5 ± 0,7 j,k,l 5,9 ± 0,1 382,9 ± 1,1 i,j 0,93 d,e Czek 157,4 ± 1,9 4,4 ± 0,2 b,c 147,3 ± 0,7 6,1 ± 0,1 315,3 ± 2,8 1,07 h,i Digger 206,7 ± 7,7 i,j 4,4 ± 0,2 b,c 179,9 ± 6,6 h,i 6,4 ± 0,6,b 397,4 ± 15,2 j,k 1,15 k Cpio 164,1 ± 1,7 d, 6,9 ± 0,3 h 164,7 ± 1,7 f,g 8,7 ± 0,5 344,5 ± 4,7 f,g,h 1,00 f,g Contct 160,1 ± 2,7 d,e 4,4 ± 0,1 b,c 186,6 ± 1,9 i,j,k,l 8,1 ± 0,7 d,e,f 359,3 ± 5,5 g,h,i 0,86 c Clifornium 167,8 ± 4,5 ± 0,1 9,3,h 152,4 ± 7,5,f 7,7 ± 0,2 332,5 ± 17,1 1,10 i,j,k Rsmus 159,8 ± 1,3 d,e 4,6 ± 0,1 150,8 ± 5,1,f 9,4 ± 0,1 g 324,7 ± 6,5 d,e,f 1,06 h,i Spencer NL 160,9 ± 1,5 d,e 6,1 ± 0,2 g 184,0 ± 0,4 h,i,j,k 12,3 ± 0,1 h 363,3 ± 2,2 h,i 0,87 c,d Composite (F 1z ) Odminy mieszńcowe złożone Kszub 120,3 ± 3,1 b 4,2 ± 0,1 b 146,5 ± 1,4 b,c 8,8 ± 0,1 279,8 ± 4,7,b 0,82 c Mzur 159,0 ± 1,9 4,8 ± 0,1 161,8 ± 1,2 d, 9,1 ± 0,2 f,g 341,0 ± 3,6,h 0,98
Preliminry studies of tocochromnol content... 567 Restored (F 1 ) Odminy mieszńcowe zrestorowne Bufflo 195,5 ± 0,6 i 4,5 ± 0,1 148,4 ± 5,8 b, 7,7 ± 0,6 356,1 ± 6,5 g,h,i 1,32 m Kronos 202,3 ± 8,7 i,j Bldur 177,4 ± 0,3 h 4,7 ± 0,4 5,0 ± 199,9 ± 7,6 0,3 d,e,f l 157,0 ± 1,4 c,d, 8,7 ± 0,6 8,7 ± 0,5 415,9 ± 17,2 k 347,8 ± 2,6 f,g,h 1,01 g,h 1,13 j,k Extrem 161,6 ± 4,2 ± 0,2 5,7 d,e,f b 128,7 ± 1,6 7,5 ± 0,2 302,0 ± 7,7 1,25 l Titn 210,1 ± 1,4 j 5,4 ± 0,2 f 198,5 ± 0,3 k,l 9,1 ± 0,0 f,g 423,1 ± 2,0 k 1,06 h,i Others Inne Rzepik ozimy Ludowy Lnink ozim Przybrodzk II 127,1 ± 0,3 b 11,6 ± 0,1 2,1 ± 0,1 2,4 ± 0,0 180,0 ± 6,8 ± 0,1 0,2 h,i,j,b,c 244,7 ± 0,1 m 6,5 ± 0,1,b 316,1 ± 0,7 265,3 ± 0,3 0,71 b 0,05 * vlues followed by different letters re sttisticlly significnt t α = 0.05 wrtości oznczone różnymi litermi różnią się sttystycznie istotnie przy α = 0,05 In ll nlysed smples of rpe seeds α- nd γ-tocopherols occurred in gretest nd usully equivlent quntities. The mount of α tocopherol ws between 120 mg/kg d.m in the composite hybrid Kszub 2001 nd 200 mg/kg d.m. in the restored hybrid Titn (Tble 1). These vrieties contined similr mounts of γ-tocopherol. The restored hybrids vrieties Kronos nd Titn were chrcterized by the highest totl content of tocohromnols bove 415 mg/kg d.m. Among the investigted seed smples of open pollinted rpeseed vrieties two were richest in vitmin E-ctive compounds. Lisek vriety (registered in 1998) contined 380 mg/kg d.m. of these compounds. In 2004 the tocopherol content in this vriety nd lso in Lirjet (registered in 1994) nd Bufflo (registered in 1999) ws higher in comprison to the tocopherol content determined in evluted seed smples of these vrieties from cultivtions in 2001 nd 2002 (Nogl-Kłuck et l. 2003). The rtio of the α- / γ-tocopherols content ws clculted for ll tested rpe seed smples (Tbles 1). This rtio is prmeter pointing to the increse of the ntioxidnt content s the effect of genetic modifiction of rpe in mny ppers (Goffmn nd Becker 2001, 2002; Mrwede et l. 2004, 2005). The clculted rtio ws greter thn 1.0 in eight mong twelve investigted open pollinted vrieties of winter rpeseed nd in four vrieties it ws little lower (Tble 1). In ll seed smples of foreign restored hybrids the clculted rtio ws bove 1.0 nd for Bufflo nd Extrem it ws 1,3. For Polish composite hybrids this prmeter ws below 1.0 (Mzur 0.98 nd Kszub 0.82). These vrieties contined smller mounts of α- thn γ-tocopherol.
568 Młgorzt Nogl-Kłuck... In the investigted smples of turnip rpe seeds the rtio of the α- to γ-tocopherols content ws smller (0.71). The seeds of winter flse flx Przybrodzk vriety contined smll mount of α-tocopherol s compred to higher mount of γ-tocopherol, so the rtio ws only 0.05. The clculted rtio of the α- to γ-tocopherols content in seed for ll tested rpeseed vrieties ws similr to the vlues published by Goffmn nd Becker (2001, 2002). Conclusions Four homologous tocopherols were identified in seed smples of ll investigted winter rpeseed vrieties nd lso in turnip rpe nd flse flx seeds. α- nd γ-tocopherols occurred in seeds in gretest nd similr quntities. The totl tocochromnol content in the mjority of rpeseed vrieties ws bove 300 mg/kg d.m. The gretest tocopherol content ws found in two rpeseed vrieties restored hybrids Kronos nd Titn (bove 400 mg/kg d.m). The clculted rtio of the α- / γ-tocopherol content rnged from 0.8 to 1.10 for exmined open pollinted vrieties of rpeseed. For the restored hybrid vrieties the rtio of the α- / γ-tocopherol content ws higher nd rnged from 1.01 to 1.32. References Abidi S.L. 2000. Chromtogrphic nlysis of tocol-derived lipid ntioxidnts. Review. J. Chrom. A., 881: 197-216. Cdens E., Dvies K.J.A. 2000. Mitochondril free rdicl genertion, oxidtive stress nd ging. Free Rd. Biol. Med., 29: 222-230. Choon E.B., Coughln S.J. 2004. Metbolic redesign of vitmin E biosynthesis in plnts for tocotrienol production nd inoresed lipid solube ntioxidnt content. 16th Plnt Lipid Symposium, Budpest, 1-4 June 2004. Progrmme nd bstrcts of presenttions, 7. Eitenmiller R.R., Lnden W.O. 1999. Vitmin E: tocopherols nd tocotrienols. W: Eitenmiller R.R., Lnden W.O. (Eds.). Vitmin nlysis for the helth nd food science. CRC Press, Boc Rton, FA, 109-148. Eitenmiller R.R., Lee J. 2004. Anlysis of tocopherols nd tocotrienols in food. W: Eitenmiller R.R., Lee J. (Eds.). Vitmin E: food chemistry, composition nd nlysis. Mrcel Dekker Inc., 323-424. Goffmn F.D., Becker H.C. 1998. Phenotypic vribility for tocopherol content nd composition in seeds of winter rpeseed (Brssic npus L.) (In Germn). Vortr. Pflnzenzüchtung, 42: 105-106. Goffmn F.D., Becker H.C. 2001. Dillel nlysis for tocopherol contents in seeds of rpeseed. Crop Sci., 41: 1072-1079.
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