Maataloustieteellinen Aikakauskirj a Journal of Agricultural Science in Finland Vol. 58: 103—141, 1986 AN ANALYTICAL AND BREEDING STUDY ON FATTY ACIDS IN SUMMER TURNIP RAPE (Brassica campestris L. var. annua) Selostus: Tutkimus kevätrypsin (Brassica campestris L. var. annua) rasvahappojen analytiikasta ja jalostuksesta INTO LAAKSO Division ofPharmacognosy, School of Pharmacy, University of Helsinki, SF-00170 HELSINKI, Finland ACADEMIC DISSER TA TION To be presented, with the permission of the Faculty of Science of the University of Helsinki, for public criticism in Auditorium XU on December 3rd, 1986, at 12 o’clock. SUOMEN MAATALOUSTIETEELLINEN SEURA, HELSINKI https://www.c-info.fi/en/info/?token=GDUTZELXwtey6yVm.DsaU0ra2aLyGWKgYk_XhWg.ClPc6YCvfNwlgKX22w65GFbe3QY_U_Dfqe5BEyNLewMB0yv9t2r5yvSFvLdAYs7F4KrgDZLwyJyQENGpfSQLu6pIZ5AN2wS9zNT0aUSbuJTbPtjVXTJdekeNVM23Smx-CdnU8jnP0IaX8a025WHuTEuCeDxl98gUVQ Preface The present study was carried out at the Division of Pharmacognosy, School of Pharmacy, University of Helsinki, during the years 1978—86. I owe my deepest gratitude to Professor Max von Schantz, Head of the Division, for suggesting the subject of this study, his interest and encouragement in my work and for perusing the manuscript. I am especially grateful to Associate Professor RaimoHiltunen, for his innumer- able advice and the support he has given me over a long period of time and his re- peated encouragement to complete this study. I am also grateful to Hankkija Plant Breeding Institute, Hyrylä, for fruitful co- operation during these years. I wish to thank Professor Erkki Kivi, Head of the Institute, for advice concerning the manuscript. I extend special thanks to Simo Ho- vinen, Lic.Agr. & For., not only for several discussions on the area of plant breeding but also for keeping the large material included in this study under control. I would like to thank Professor J. Johan Lindberg, Department of Wood and Polymer Chemistry, and Seppo Räisänen, Head of the Instrument Centre of Chemis- try, for providing advice and facilities in capillary column technology. I wish to thank Professor Aarre Huhtikangas, University of Kuopio, for sev- eral fruitful discussions. I express my appreciation to Tuulikki Seppänen, M.Sc., Jorma Kajaste, M.Sc. and Pertti Koiranen, M.Sc., for their excellent assistance in our laboratory. The work done by Ms Outi Kovanen and Ms Hannele Uusitalo at Hankkija labora- tory is gratefully acknowledged. I wish also thank my colleagues and personnel at our division. My thanks are also due to John Derome, M.Sc., who translated this thesis into English and has checked the language of the separate papers. This study was supported by grants from the Academy of Finland, Farmasian Opettajien ja Tieteenharjoittajien Seura r.y. and Suomen Apteekkariyhdistys r.y. I am grateful to the Scientific Agricultural Society ofFinland for including this study in their series of publications. Helsinki, October 1986 Into Laakso JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND Maataloustieteellinen A ikakauskirja Vol. 58: 107—141, 1986 An analytical and breeding study on fatty acids in summer turnip rape (Brassica campestris L. var. annua ) Abstract. The fatty acid composition of the seed oil of summer turnip rape (Brassica cam- pestris L. var. annua) was investigated by gas liquid chromatography (GLC). The reliability of conventional sampling methods in capillary GC was compared with that of the new on- column and PTV (programmed temperature vaporizer) techniques, with particular reference to the determination of fatty acid variation. In order to develop new, well-adapted turnip rape strains with improved oil quality, a breeding programme for a higher linoleic acid content, based on individual plant selection, was performed in 1978—85. The results showed that the conventional sampling techniques involving sample transfer to a hot injector were very unreliable as regards precision and accuracy. This was especially the case in the determination of trace fatly acid levels. The PTV methods with splitless and solvent split mode were as precise as cold on-column injection. The PTV sampling modifica- tions, which are all superior toclassical techniques, were even more suitable for routine analy- ses than on-column injection, where several restrictions are met. The analytical error with PTV for most of the compounds represented less than 1 % of the variation found for fatty acids within a turnip rape variety. The breeding experiments indicated that the level of linoleic acid can be increased under open-pollinated conditions in the field without affecting the a-linolenic acid content. The green- house conditions, on the other hand, were found to have a considerable environmental in- fluence on the variation of these compounds, resulting in no response to linoleic-acid selecion. In field trials, several strains with yields comparable to the varieties commonly cultivated in Finland were selected with a higher linoleic acid content (up to 25 %). Most of them also con- tained no erucic acid. The new evidence concerning its beneficial physiological effects indicate that rapeseed oil should be considered as a serious alternative among sources of essential fatty acids. Such aspects should also be taken into account in future breeding of rapeseed fatty acids. Index words: Turnip rape, fatty acids, breeding, GLC, PTV sampling technique List of publications I Hiltunen, R., Laakso, L, Hovinen, S. and Derome, J. 1982. Sampling techniques in the glass capillary gas chromatography of fatty acids of rape-seed, J. Chro- matogr. 237, 41—48. II Laakso, L, Hiltunen, R., Hovinen, S., v. Schantz, M. and Huhtikangas, A. 1982. Selection of high linoleic acid content in summer turnip rape (Brassica campest- ris L.). I. Variationof fatty acids in an irradiated crossing material, Acta Agrig. Scand. 32, 397—404. 11l Laakso, L, Hiltunen, R., Seppänen, T. and v. Schantz, M. 1983. Relationships between some fatty acid isomers in rapeseed oil, Acta Pharm. Fenn. 92, 127—135. IV Laakso, L, Hiltunen, R. and Hovinen, S. 1983. Selection of high linoleic acid content in summer turnip rape (Brassica campestrisL.). 11. Variation in linoleic acid content in successive generations, Proc. 6th Int. Rapeseed Conf., Paris, France, C: 607—612. V Laakso, I. 1985. Selection of high linoleic acid content in summer turnip rape (Brassica campestris L.). 111. Effects of selection on fatty acid composition, Acta Pharm. Fenn. 94, 51—57. VI Laakso, L, Hiltunen, R., Kajaste, J. and v. Schantz, M. 1985. Single seed fatty acid analysis of rapeseed, Acta Pharm. Fenn. 94, 59—65. VII Laakso, 1., Hovinen, S. and Hiltunen, R. 1986. Selection of high linoleic acid content in summer turnip rape (Brassica campestris L. ssp. oleifera var. annua). IV. Selection of improved oil yield, Acta Agric. Scand. 36, 347—351. In the text the papers are referred to by their Roman numerals. 109 Contents ABSTRACT 107 LIST OF PUBLICATIONS 108 INTRODUCTION 11l THE AIMS OF THE STUDY 113 REVIEW OF THE LITERATURE 114 A. GAS LIQUID CHROMATOGRAPHY OF FATTY ACIDS 114 1. Sample preparation 114 2. Stationary phases 115 3. Conventional sampling methods 115 4. Cold sampling methods 116 5. Precision of the analyses 116 B. BREEDING FOR FATTY ACID COMPOSITION IN RAPESEED OIL 116 1. Biogenetic dependances 117 2. Erucic acid-free rapeseed oil 119 3. Breeding for polyunsaturated fatty acids 120 C. THE ROLE OF POLYUNSATURATED FATTY ACIDS IN THE DIET 121 EXPERIMENTAL 123 A. MATERIALS 123 B. METHODS 123 1. Analytical methods 123 2. Statistical analysis 125 RESULTS 126 A. GAS CHROMATOGRAPHY 126 1. Conventional vs. on-column technique 126 2. Programmed temperature vaporization (PTV) techniques 127 B. BREEDING EXPERIMENTS 128 1. Variation in fatty acids 128 2. The long-term effects of selection 128 3. Interrelationships between fatty acids 130 DISCUSSION 131 A. COMPARISON OF SAMPLING TECHNIQUES IN FATTY ACID ANALYSIS 131 110 B. SELECTION FOR HIGHER LINOLEIC ACID CONTENT IN RAPE- SEED OIL 132 REFERENCES 135 APPENDIX 140 SELOSTUS 141 Introduction The cultivation of oil plants as the most economic way of producing fats has consid- erably increased during the last decades along with the development of cultivation tech- niques and industrial processing know-how. Soybean holds first place among oilseeds, ac- counting for one third of the world’s produc- tion of vegetable oil. It is followed in impor- tance by oil palm, sunflower and rapeseed (Brassica sp.). Due to its excellent adaptabil- ity to different climatic conditions, rapeseed has enabled vegetable oil to be produced in areas ever further to the north. This expan- sion has been speeded up by compositional improvements achieved through intensive breeding such as elimination of erucic acid (22: lw9) from the oil, and reduction of the glucosinolate content in the meal (Downey 1983, Pigden 1983, Fochem 1985). In Finland, turnip rape (Brassica campes- tris L.) is cultivated in the southern and cen- tral parts of the country, while rape (B. napus L.) only produces a high-quality crop in a narrow zone running along the southwestern and southern coasts. At present, summer va- rieties (var. annua) only are cultivated (Hovi- nen 1985). Following the introduction (in 1976) of practically erucic acid-free varieties, cultivation has come under special direction of the government in order to increase self- sufficiency in domestic vegetable oil (Anon 1978, 1982). Finnish rapeseed oil is of very high quality and the norms as regards erucic acid are especially tight, since the maximum permitted level in sown seed is 0.5 % (Hovi- nen 1985). The oil produced has an erucic acid content clearly below the EEC’s recom- mended maximum level of 5 °7o (Anon 1980). Elimination of erucic acid associated with a simultaneous increase in the polyunsaturated linoleic (18: 20j6) and a-linolenic (18:3w3) acid contents, has put rapeseed in a new position among sources of edible oils. These two es- sential components account for one third of the fatty acid content, thus making the new rapeseed oil rather competitive with many other vegetable oils. However, the content of linoleic acid is still very low compared to that of soybean or sunflower oil, for instance. Achieving a further increase in the amount of linoleic acid, which is nutritionally the most important constituent of the oil, is therefore the primary goal of breeding work (Thies 1968, Downey and McGregor 1975, Röbbe- len 1976, Jönsson 1977 b). Modification of the proportions of linoleic and a-linolenic acids is, however, a rather laborious process due to their limited range of variation. Their inheritance is far more complex than that of erucic acid and, in ad- dition, the variation is influenced by the en- vironment to a considerable degree (Kondra and Thomas 1975, Bartkowiak-Broda 1983, Stefansson 1983). In such cases a reproduc- ible analytical technique is of decisive impor- tance in separating the effects of genetical properties and the environment. Gas liquid chromatography (GLC) has been the main analytical method used for studying fatty acids ever since the time it was first de- veloped. It has not only shown that the group of naturally occurring fats is much more di- verse than was first suspected, but has also 111 played an important role in breeding work on rapeseed fatty acids. The capillary technique has increasingly superceded the use of packed columns, and new stationary phases have made it possible to analyse ever more compli- cated mixtures of isomers (Lie Ken Jie 1980). The present-day technique is called with good reason high resolution gas chromatography. It is usually coupled with the latest applica- tions of sampling methods. On-column and programmed temperature vaporizer (PTV) in- jection systems, which have enabled consid- erable strides to be made in quantification, have been developed alongside the traditional GC techniques (Schomburg et al. 1977, Grob and Grob 1978, Poy et al. 1981). The so-called half-seed technique is con- sidered to be the best tool in breeding for higher linoleic acid content in rapeseed oil. Crosses can be made on plants with known chemotypes, and the greenhouse offers better controlled conditions (Jönsson 1977b). How- ever, no studies have been reported on the longterm effects of linoleic acid selection based on individual plants grown under open- pollinated field conditions. When carrying out breeding trials over a number of years, opti- mization of the analytical techniques is there- fore a basic prerequisite in elucidating the ef- fects of selection. 112 The aims of the study The aims of this study were: 1. To study the reliability of the traditional gas chromatographic methods in fatty- acid analysis, and to optimize, using the latest injection technique, the GC method best suited for the breeding work in ques- tion. 2. To study the variation of fatty acids in the seed oil of summer turnip rape and to in- crease the linoleic acid content through in- dividual plant selection. 3. To study the effects of selection on the fatty-acid composition and the yield of the breeding lines. 113 Review of the literature A. Gas liquid chromatography of fatty acids Gas chromatography is the most suitable of the methods applied in the quantitative and qualitative analysis of fatty acids. Owing to its speed, sensitivity and accuracy it has large- ly replaced traditional techniques such as pa- per and column chromatography (Thiele 1979). A decisive improvement in gas chroma- tographic separation has been achieved by employing glass and silica capillary columns, whose thermal stability has been further im- proved by the development of deactivation and phase techniques (e.g. chemical bonding). These topics have been dealt with extensively in the review articles of e.g. Lee and Wright (1980) and Haken (1984). The column prepa- ration methods and modifications in fatty- acid analysis have been described in a large number of studies (Schomburg and Husmann 1975, Grob and Grob 1976, Sisfontes et al. 1981, Arrendale et al. 1983, Lercker 1983, Bohov et al. 1984, Golovnya et al. 1984). Detection of fatty acids in GLC is usually done by a flame ionization detector (FID), as well as mass spectrometrically using a mass- selective detector (MS). The differences in the FID responses of e.g. palmitic, oleic, linoleic and a-linolenic acids, are insignificant with respect to stearic acid (F = 1.00—1.01) (Ba- dings and de Jong 1983), although with longer carbon chain compounds and higher degrees of unsaturation the differences may become considerable (22:10j9, F = 1.23; 22:60)3, F = 1.59) (Slover and Lanza 1979). However, the values are specific for each in- strument and are affected to some extent by e.g. the »dead volumes» of the detector and the flow ratios of the gases (Yang and Cram 1979). Despite this, the main sources of error are the sampling technique and a large num- ber of GC process-phenomena associated with sampling. 1. Sample preparation Most of the naturally occurring reserve fats have the structure of triacylglycerols. These neutral fats are best extracted using non-polar organic solvents such as petroleum ether or chloroform (Thiele 1979). The fat is saponi- fied and the fatty acids are converted into more volatile derivatives such as methyl esters using e.g. methanolic bortrifluoride (Ackman et al. 1971, Slover and Lanza 1979) or methanolic sulphuric acid (Sebedio and Ack- man 1978). Transesterification, which is done in water-free conditions using sodium meth- oxide as catalyst, is a fast method which is widely used. A numberof modifications of the method have been presented in the literature (Thies 1971, Ackman et al. 1977, Johansson and Uppström 1978, Hiltunen et al. 1979, Badings and de Jong (1983). Metcalf and Wang (1981) and Badings and de Jong (1983) have, for instance, used derivatization of the free fatty acids in connection with trans- esterification in their work. In the analysis of complicated mixtures of isomers produced in the hydrogenation of fatty oils, the cis and trans forms are co-eluted on a number of columns. Therefore, a prepa- rative separation before quantification has been found necessary. In addition to gas chro- 114 matographic fractionation, other possible methods include column and thin-layer chro- matography and, more recently, high per- formance liquid chromatography (HPLC). The last-mentioned method has been used by e.g. Sebedio et al. (1982) and Svensson et al. (1982). Separation of positional and geomet- rical isomers of mono-unsaturated fatty acids in GC analysis is such a difficult task that it is often taken as a measure of the resolution when comparing stationary phases. 2. Stationary phases The best separation is achieved in fatty-acid analysis using polar stationary phases. Jae- ger et al. (1975) analysed a number of iso- mers using a 50-m glass capillary column con- taining an FFAP polyether phase, but found that the separation of elaidic (trans- 18: lco9) and vaccenic acid (cis- 18: lw7) was insufficient for automatic integration. According to Sis- fontes et al. (1981), exact quantification of isomers is not possible in the analysis of hy- drogenated oils using a Silar lOC column (50 m). An extremely polar cyanopropyl siloxane phase (SP 2340) has been used by e.g. Hec- kers et al. (1977) and Slover and Lanza (1979) and Lanza and Slover (1981). The last-mentioned authors achieved good preci- sion with very long columns (60—100 m) in analysing the trans fatty-acid contents of foodstuffs. Quite recently, Bohov et al. (1984) separated the four isomers of linoleic and oleic acid using this phase on a 78-m-long capillary column. The authors noted, in ad- dition, that the separation number (TZ = 0.26/m) was considerably smaller than that obtained by Jaeger et al. (1975) using an FFAP column (TZ = 0.93/m). Simultaneous analysis of esterified and free fatty acids on an OV-351 silica capillary column (15 m) has been utilized in clinical studies carried out by Penttilä et al. (1984). Since unprocessed fat- ty oils do not in practice contain any trans fat- ty acids, the resolution of even short columns (Carbowax 20M, 15 m; Silar 10C, 25 m) is suf- ficient for screening tests where the time taken to carry out the analysis is of decisive impor- tance (Lercker 1983, Arrendale 1983). 3. Conventional sampling methods Sampling techniques have received special attention in recent years. One of the greatest drawbacks of such methods is considered to be the use of high temperatures for vaporizing the sample. The traditional split and splitless injection techniques have proved to be un- reliable both as regards precision and accu- racy. The main reasons for this are decom- position of the components and selective vola- tilization of different-sized molecules from the injector needle, which in turn results in an unequal distribution between the split and the column. The split ratio can also vary as a result of pressure effects in the injector caused by different-sized sample volumes (Schom- burg et al. 1977, Grob and Neukom 1979, Schomburg 1979, Galli and Trestianu 1981). Comparison of different injection methods in fatty-acid analysis has shown that the split ratio and sample size have a decisive effect on the quantitative results (Hiltunen et al. 1982). In addition to these discriminating factors, adsorption of the components on the needle, septum and injector, and a reduction in resolution caused by the presence of non- volatile contaminants, are all possible sources of error (Grob and Neukom 1979, Grob and Grob 1979). According to Schomburg et al. (1977), achieving optimal quantitative and qualitative results presupposes: sufficient resolution high reproducibility of retention high precision and accuracy in quantifica- tion, i.e. there is no discrimination of the components with respect to volatilization, polarity or concentration, and there is minimal thermal and catalytic de- gradation of labile components. In addition to the instrumental errors, fac- tors attributable to the sample preparation such as incomplete esterification, side reac- 115 tions, evaporation, adsorption or inaccuracies split method with a cold or hot injector, and in the isolation process should also be taken into account (Badings and de Jong 1983). 4. Cold sampling methods The direct, cold on-column injection meth- od involves transferring the sample onto the column without a vaporizing injector (Schomburg et al. 1977, Grob and Grob 1978). The on-column technique has been found to be indisputably superior to the split and splitless methods in comparative studies and, as a result of eliminating the discrimina- tion phenomena, it has been possible to achieve considerably better analytical preci- sion and accuracy (Schomburg et al. 1981, Munari and Trestianu 1981). Volatilization of the sample before reaching the column can be prevented using a secondary cooling sys- tem. This ensures that the total sample is transferred onto the column (Galli and Tres- tianu 1981). Deterioration will occur in the separation efficiency of the column unless relatively pure samples are used in the on-column technique, i.e. no non-volatile contaminants should be introduced into the column (Grob 1978). Fast injection of large sample volumes should also be avoided in order to prevent back-flushing of excess volatilized sample from the column. Thus the temperature of the column should not be greater than the boiling point of the solvent (Grob and Neukom 1980). Despite the excellent precision and accuracy, the resolu- tion can sometimes be considerably inferior to that obtained with split injection. The most problematic factors causing band-broadening in the on-column technique are large sample volumes and, in particular, the injection of polar solvents into non-polar columns (Grob 1981, Sandra et al. 1983). A programmed temperature vaporizer (PTV), in which the sample is vaporized fol- lowing injection at a low temperature by raising the temperature quickly to the final level, is the latest type of injection technique. This system permits cold splitless injection, the a special solvent elimination technique. The last-mentioned method can be used if the dif- ference between the boiling points of the sol- vent and the components to be analyzed is suf- ficiently large. Opening and closing the split valve can be regulated automatically using a programming unit (Poy et al. 1981, Poy 1982). Schomburg et al. (1983 a) have since developed a temperature-programmed (TP) injector. Caplan and Cronin (1983) have presented a special version of their »solvent- free» injection system, in which the solvent is removed in the tube prior to the sample being transferred into the injector, and have applied the technique in fatty-acid analysis. 5. Precision of the analyses The precision of a method is usually ex- pressed using the standard deviation (S.D.) or the relative standard deviation (S rel , ®/o), i.e. the coefficient of variation (C.V. %). The pre- cision of different injection methods is pre- sented in Table 1. The data published by the authors are not reported here in full in all cases, and in order to obtain a uniform com- parison the C.V. values for samples C and D are derived from the mean and S.D. values of the original fatty acid data. A satisfactory precision level in the high- resolution capillary technique is considered to be less than 1 % (C.V.) when determined from the normalized area of thepeaks (Yang et al. 1978). B. Breeding for fatty acid composition in rapeseed oil Evidence indicating the use of Brassica seed oil for cooking, illumination and medicinal purposes already in ancient times indicates that these plants have been among the earliest ones domesticated by man. Almost every plant part, such as the roots, stems; leaves and seeds, have been utilized and different forms of Brassica species have been developed 116 Table 1. Precision of fatty-acid analyses (C.V. %) calculated from the normalized data. Fatty Sampling technique a °' C* Conventional split On-column »Solvent-free- injection» A B C A D E F Mean C.V. Mean C.V. Mean C.V.* Mean C.V. Mean C.V.* Mean C.V. Mean C.V. 16:0 3.0 2.1 15.2 1.7 26.5 3.9 3.0 2.7 23.4 0.9 24.7 0.2 6.3 1.6 18:0 1.6 2.4 11.8 1.1 9.3 2.7 1.4 0.5 11.6 0.5 33.5 0.2 4.8 0.6 18: lo>9 58.6 0.3 22.9 1.3 30.1 1.0 57.6 0.1 27.9 0.7 32.9 0.2 19.0 0.3 18: lw7 1.9 1.2 18:2o>6 20.2 0.5 17.8 1.3 6.8 4.3 20.7 0.5 1.5 1.5 3.2 0.3 14.4 0.4 18: 30j3 12.7 0.5 1.1 2.3 1.1 6.5 13.0 0.3 2.5 2.3 1.1 0.7 55.4 0.1 20:0 0.5 8.1 0.4 4.8 0.4 1.8 0.6 4.4 1.1 0.9 20:1«9 1.7 6.7 1.8 2.2 20: 2u6 0.2 21.6 0.2 5.2 22:0 0.3 14.1 0.2 4.7 0.5 3.7 22: lu9 1.3 7.8 1.7 3.5 Samples: A Rapeseed oil (Hiltunen et ai. 1982) (I) D Milk fat (Badings and de Jong 1983) B Shortening (Slover and Lanza 1979) E Cocoa butter (Geeraert et al. 1983) C Human milk lipids (Haug et al. 1983) F Linseed oil (Caplan and Cronin 1983) * includes extraction, methylation and GLC through natural selection and breeding (Dow- ney 1983). Turnip rape (B. campestris L. ssp. oleife- ra) is one of the basic oilseed species in the Cruciferae family, which by an interspecific cross with cabbage (B. oleracea L.) produces an amphidiploid rape (B. napus L. ssp. olei- fera). Turnip rape is also a parental species for Indian mustard (B. juncea (L.) Czern.) (Bengtsson et ai. 1972, Downey 1983). In the recent literature new systematic names for the family (Brassicaceae), turnip rape (B. rapa ssp. oleifera or B. rapa var. silvestrjs) and rape (B. napus ssp. napus or B. napus L. var. napus) have been given (Ehrendorfer 1983, Frohne and Jensen 1985). 1. Biogenetic dependances The formation of oleic acid plays a key role in the biosynthesis of fatty acids in plants. The chloroplasts of the leaf tissue and the pro- plastids of the embryo are most probably the only site of de novo synthesis involving the formation of palmitoyl-, stearoyl- and oleyl- ACP (acyl carrier protein) complexes. Oleyl- ACP (18:1 ACP, Scheme 1) is hydrolyzed rapidly by an enzyme and the product, oleic acid, is transported from the organelles to the cytoplasm where it is subsequently modified in a number of reactions (Scheme 1) (Stumpf and Pollard 1983). It has been proposed that the fatty acids in rapeseed are formed via the following bio- genetic pathways (Scheme 2). The scheme is based on literature presented by Downey and Craig (1964), Appelqvist (1968), Thies (1968), Brar and Thies (1978) and Downey (1983). The development of new rapeseed varieties during the last twenty years has provided the foodstuff, animal-feed and chemical indus- tries with an ever more versatile source of raw- materials. This success is primarily due to the changes brought about in the fatty acid com- position, which is considered to be one of the greatest efforts made in the area of plant breeding. Direct biosynthetic studies carried out with I4C-labeled precursors have shown that eico- senoic (20:1 oo9) and erucic acids (22: 1oj9) are 117 formed from oleic acid (18: lw9) as a result of chain elongation (pathway C, Scheme 2) (Downey and Craig 1964). Multiple alleles located at a single locus in diploid turnip rape control the synthesis of these C20—C 22 com- ponents (Jönsson 1977 a). Downey and Craig (1964) found, furthermore, that the formation of saturated fatty acids (pathway A) is relatively independent of changes in monoenoic components (pathway C). Since genetical blocking of the formation of eico- senoic and crude acids also results in the in- hibition of the formation of the corresponding lco7 isomers (pathway B), parallel elongation of lu9 and lw7 components is assumed to be under the control of a single genetic system (Appelqvist 1968). The main pathway of polyunsaturated fatty acids starts with the de- saturation of oleic acid into linoleic acid (18:2co6) and subsequently a-linolenic acid (18:3ou3) (pathway D). Biosynthetic studies carried out on rape embryos have shown that Scheme I. The role of oleic acid in the synthesis of fatty acids in different plant tissues. Scheme 2. Biogenesis of rapeseed fatty acids. 118 2 hexadecatrienoic acid (16:3c03) is also a pos- sibleprecursor of a-linolenic acid (pathway F) Brar and Thies 1978). Rakow (1973) has earlier suggested that two independent enzyme systems are involved in the production of a- linolenic acid. A number of genes have been found to control the linoleic and a-linolenicacid levels in rape (Kondra and Thomas 1975). If a- linolenic acid is formed through two pathways (D and F, Scheme 2), then they are controlled at two locii in turnip rape and at four locii in amphidiploid rape. Thus one allele would de- termine only 1/8 of the total a-linolenic acid content in rape (Stefansson 1983). The a- linolenic and oleic acid contents are deter- mined genetically by the genotype of the mother plant alone, and not that of the em- bryo. The linoleic acid level is mainly regu- lated by the mother plant, the effect of the genotype of the embryo being four times smaller. In addition, environmentalconditions have also been found to modify considerably the contents of these C lB fatty acids (Bartkowiak-Broda 1983). As far as breeding work is concerned, the frequently rather high correlations which exist between the fatty acids in rapeseed oil have provided useful, although indirect, evidence for the biogenetic interrelationships between these components. The unusually high nega- tive correlation (r = —0.975) found in bio- logical materialbetween oleic and erucic acids by Craig (1961), has since been shown to be a biosynthetic relationship (Downey ,and Craig 1964). The situation between eico- senoic and erucic acid is, however, more complicated since the correlation is positive up to an erucic acid content of 25 %, and be- comes negative at higher levels (Jönsson 1977 a). Oleic acid is, furthermore, a precur- sor of linoleic acid (Stearns 1970). Accord- ing to Kondra and Thomas (1975), the simi- lar behaviour of these fatty acids in crossings, as well the very high negative correlation, in- dicates that the formation of linoleic acid is controlled by a single gene system. On the other hand, the correlation between linoleic and a-linolenic acids is considerably smaller than the above and, in addition to ordinary positive correlation, negative correlations have also been occasionally found (Kondra and Wilson 1976, Jönsson 1975 a). 2. Erucic acid-free rapeseed oil The variation in the erucic acid content was found, already many years ago, to be impor- tant when comparing different varieties of rape in breeding programmes (Craig and Wetter, 1959). The half-seed technique proved to be a valuable tool in breeding work, especially after the erucic acid content was found to be determined on the basis of the genotype of the embryo (Harvey and Dow- ney 1964). In this method, one of the cotyle- dons of the embryo is analysed and the other one allowed to develop into a normal plant (Downey and Harvey 1963, Thies 1971). Erucic acid-free seed material was found in rape varieties (Stefansson et al. 1961, Ste- fansson and Hougen 1964) and turnip rape varieties (Downey 1964) already at the begin- ning of the 1960’5. However, these varieties did not fully meet the requirements when grown under European conditions, and hence they had to be crossed with European varie- ties (Röbbelen 1976). Varieties which have al- ready become adapted have been used by, e.g. Jönsson (1973), in breeding erucic acid-free turnip rape. The typical composition of high and low erucic-acid rapeseed oil is presented in Table 2. In addition to oleic acid, the most marked change has taken place in the amounts of the polyunsaturated acids, linoleic and a- linolenic, which have approximately doubled in comparison to the levels in traditional rape- seed oil (Table 2). Analytical studies on the isomers have, furthermore, shown that the vaccenic acid content (18: la/7) rises to rather high levels, even to over 3 %, in low erucic- acid material (Hougen and Wasowicz 1978). 119 Table 2. Fatty acid composition of a traditional and a new rapeseed variety. Compound Traditional New rapeseed oil 1 rapeseed oil 2 Mean (%) Mean (%) 16:0 4.0 3.8 18:0 1.3 1.2 18: la)9 16.4 53.5 , 18:1«7 1.0 } 18:2u6 12.7 23.5 18:3w3 5.3 14.0 20:0 0.9 0.3 20: lv)9 9.0 1.4 . 20; loil 1.4 J 20:2c06 0.3 0.1 22:0 0.6 0.2 22: lw9 44.4 1.0 > 22: loi? U 2 }_ 1 Ackman 1966, 2 Ackman & Sebedio 1978 Only very small amounts ( 0.01 %) of trans isomers, which are usually C l 5 fatty acids, have been found in unprocessed rapeseed oil (Sebedio and Ackman 1979). The oil content of rapeseed is usually about 40—50 °7o, most of it (c. 95 %) in the form of triacylglycerols (Appelqvist 1972). Re- placement of erucic acid by a fatty acid with a smaller molecule (/. e. oleic acid) has thus, to some extent, resulted in a reduction in the total amount of oil. Despite the low variation, it has been possible to increase the oil content by applying continuous selection (Krzymans- ki 1984), and by favouring yellow-seeded ma- terial, which also has a lower fibre content, over the brown-seeded form (Jönsson 1975 b). The above-mentioned changes in the composition have naturally increased the com- mercial possibilities of utilizing erucic acid- free rapeseed oil in the foodstuff industry. A new term (»Canola») has been adopted in e.g. Canada, to differentiate new rapeseed produc- tion from that of traditional rapeseed varie- ties (Paszkowski 1983). 3. Breeding forpolyunsaturatedfatty acids After the erucic acid problem had been solved, the primary task in the breeding of new varieties has been to bring about a con- siderable increase in the amount of essential linoleic acid as opposed to the normal (20 —22 %) level. Furthermore, the a-lino- lenic acid content should be decreased from the present level of 10—12 °7o, down to as low a level as possible (c. 3—4 °/o). Being an easily oxidized component, a-linolenic acid is par- ticularly problematic for the margarine indus- try. Other aims of breeding are considered to be a relatively high content of (-10 %) pal- mitic acid (16:0) in order to improve the physi- cal properties of the fat (Thies 1968, Downey and McGregor 1975, Röbbelen 1983, Jöns- son and Persson 1983). Since the variation in the amounts of poly- unsaturated fatty acids is relatively small, it has been suggested that the genetic variabili- ty could be increased by treating the seed ma- terial with mutagens (Thies 1968). Rakow (1973) has observed considerable differences, independent of the linoleic acid content, in the a-linolenic acid level (4 —20 %) in material induced in this way. According to Röbbelen and Nitsch (1975), it is not promising to select for linoleic and a-linolenic acid contents simultaneously in order to obtain the desired combination of these polyenoic fatty acids. Relatively high heritability values (h 2 ) of 0.56 (Bartkowiak-Broda 1978), 0.26—0.59 (Kondra and Thomas 1975) for rape and 0.44—0.76 for turnip rape (Jönsson 1975 a) have been obtained for linoleic acid in con- trolled crossings. Jönsson (1975 a) has noted that the effect of the environment is consid- erably lower than would be expected and that increasing the linoleic acid level of summer turnip rape up to 40 % is a realistic target. Such results could be obtained by using the half-seed technique and carrying out the breeding experiments in the greenhouse where constant conditions can be maintained, e.g. with respect to day length and temperature. The first marked changes with respect to polyunsaturated fatty acids were found in rape material treated with a mutagen. The low a- linolenic acid content (4 %) was combined with a high linoleic acid level (40 %) (Röb- belen and Nitsch 1975). Jönsson and Pers- 120 son (1983) have since used this material in bolized into arachidonic acid (Holman 1970, their breeding experiments and achieved even higher linoleic acid contents. Since, in addi- tion, the amount of palmitic acid increased at the same time up to 10 %, the fatty acid pro- file was rather close to the composition of soybean oil. Achieving such a composition for summer turnip rape too, supports the 50 % linoleic acid content found in its seed. Cross pollination restricts the breeding of turnip rape, and selfpollination cannot be uti- lized in the same way as with rape. The com- bination and retention of improvements in the quality of cultivable material, however, form the most problematic stage since a number of factors have to be taken into account. The ma- terial should primarily be resistant to the weather, insect pests and plant pathogens, and to fulfill the quantity and quality criteria set on the yield before it can be considered as a variety suitable for commercial use (Lööf and Appelqvist 1972). C. The role of polyunsaturated fatty acids in the diet Linoleic (18:2w6) and a-linolenic acid (18:3o>3) are both essential constituents for hu- man physiology because the body is not cap- able of synthesising them or interconverting these two fatty acid series (w 6 and w3) (Hol- man 1970). These fatty acids have, as is usually the case with naturally occurring un- saturated fatty acids, a cis configuration (Thiele 1979). Retention of this configura- tion in the structure of linoleic and a-linolenic acids is further a basic prerequisite for the for- mation of the prostaglandin precursors such as homogammalinolenic (20:30j6), arachi- donic (20:4u6) and eicosapentaenoic acids (20:5w3, EPA) (Vane and Moncada 1979). The enzyme, A 6 desaturase, which can be in- hibited by a number of factors such as satu- rated and trans fatty acids, plays a central role in the formation of these precursors (Horro- bin 1982). a-Linolenic acid also has an inhibi- tory effect when linoleic acid is being meta- Seher et al. 1983). When the desaturation stage of linoleic acid is passed, its following metabolite, gammalinolenic acid (18:3co6), has a clearly more pronounced effect than its pre- cursor (Horrobin 1982). The w 3 fatty acids have been looked at in a new light during the last few years following the finding that a fish diet, and especially the EPA to be found in fish, have a beneficial effect on the function- ing of the heart and circulatory system (Dyerberg et al. 1978, Hamilton et al. 1980, Hay et al. 1982). The large amount of fat and high propor- tion of saturated fatty acids in the diet of west- ern peoples is considered to be a significant factor contributing towards the high incidence of cardiac and circulatory diseases (Gander 1984, Öster and Schlierf 1982). However, this is not necessarily a result of the increase in the consumption of fats proper (butter, margarine, vegetable oils), but rather the con- siderable rise in the proportion of so-called hidden fats in the diet (Fondu 1981, Masson 1981). In the Finnish diet, these hidden fats can constitute as much as over 50 % of the total intake of fat (Anon 1981). Monitoring studies carried out on sections of the Finnish population have shown that there is a connection between a high level of saturated and low level of polyunsaturated fatty acids in the serum phospholipids and the incidence of ischaemic heart disease (Mietti- nen et ai. 1982). In addition, the serum sele- nium level has been found to be lower in high- risk groups (Miettinen et ai. 1983). A high cholesterol level is also considered to be a result of too high a fat consumption and an imbalance between the intake of saturated and polyunsaturated fatty acids (Vartiainen et ai. 1984). Fish offers excellent possibilities for achiev- ing a balanced intake of fats since the propor- tion of polyunsaturated fatty acids in the lipids of the flesh and roe of the fish commonly eaten can be as high as 50 °/o even (Kaitaran- ta 1981). The cj6/w3 fatty acid ratio in fish oil is extremely low (0.1 —0.3). In contrast, the 121 most balanced ratio in vegetable oils along 1984). with increasing a-linolenic acid content is in soybean (7.0), rapeseed (1.9) and linseed oils (0.3) (Laakso et ai. 1984). Some vegetable oils, such as sunflower and safflower oils, have a ratio of as high as 150—220/1 even. In England, the preference for vegetable oils of this type is considered to have had a detri- mental effect, along with the decrease in the consumption of fish, on the intake of o 3 fat- ty acids. One proposed solution to this prob- lem is the addition of a-linolenic acid to edible oils such as olive or soybean oils (Hamilton et al. 1980). We have long been uncertain about the effects of a-linolenic acid, and it has only recently been shown to act as a precur- sor of EPA in humans (Sanders and Younger 1981, BuDOWSKiet al. 1984). A more balanced ratio of linoleic and a-linolenic acids in the diet is being emphasized more and more, and it has even been suggested that linoleic acid has been favoured too much in the west at the expense of a-linolenic acid (Budowski et al. As far as rapeseed oil is concerned, early studies with laboratory animals and especial- ly with the rat indicated that that the myocar- dial lesions which developed were due to the high erucic acid concentration. However, those results cannot be applied in humans as such. It is now apparent that low erucic-acid rapeseed oil is like other vegetable oils, a safe substance for human consumption (Grice and Heggtveit 1983). A considerable decrease in the serum cho- lesterol level has been described after a fat diet containing rapeseed oil in a number of studies as reviewed by McDonald (1983). Rapeseed oil has been found to be effective in decreasing the total cholesterol and increasing the HDL cholesterol levels also in the treatment of familial hypercholesterolemia (Savoie et al. 1983). One of the most important findings is that rapeseed oil is capable of increasing the eicosapentaenoic acid (EPA) content in the serum (Lassere and Jacotot 1983). 122 Experimental A. Materials The practical breeding work and yield trials included in the present study have been done at Hankkija Plant Breeding Institute at Hy- rylä in 1978—85. The starting material was two summer turnip rape populations of Ca- nadian origin which were erucic acid-free, had a low glucosinolate content and about 70 °7o yellow seed. The two populations are referred to in the text as numbers 7622 and 7629. In addition to the breeding tests, these popula- tions were also used as the control material. Material grown in the greenhouse or in the field are marked with the symbols G (green- house) and F (field). The breeding program- me, which is described in detail in papers 11, IV and V, is summarized in Scheme 3. The individuals with the best agronomic properties were always taken for further breeding via phenotypic selection, and the final selection of the linoleic acid lines was done on the basis of the yields. The yield trials have been carried out parallely on summer turnip rape varieties (e.g. Emma, Ante and Span) commonly cultivated in Finland (VII). B. Methods 1. Analytical methods Usually c. 10—15 seeds (30—50 mg) were taken the from the yield of each individualand the fatty acids derivatized using the trans- esterification method described by Hiltunen et ai. (1979). The gas chromatographic analy- ses were done using glass capillary columns on a number of different instruments. The rou- tine analyses were carried out on a Carlo Erba Fractovap 2300 and a Dani 3200 GC fitted with a split-splitless injector system. FFAP (free fatty acid phase) was used as the phase on the columns of different length, and the runs were usually carried out at 200°C using hydrogen (H 2) as the carrier gas. All the in- struments were fitted with a flame ionization detector (FID). The split ratio was set at 15:1, and the amount of sample injected was 1 /d (I, HI). A 55-m-long FFAP column, pretreated with an aqueous solution of Ba(OH) 2 and carbon dioxide in order to form a layer of barium car- bonate, was prepared for the isomer studies (III) (Grob and Grob 1976). The phase dis- solved in dichlormethane was run through the column using the dynamic, so-called mercury drop method according to Schomburg and Husman (1975). A Dani 3200 gas chromatograph fitted with an on-column injector and a secondary cool- ing system was used in comparing different in- jection techniques. The injection temperature in the on-column analysis was 35°C, and the oven programmed to 210°C at a rate of 10°C/min (I). The fatty acid analyses were further run on a Dani HR 3800 PTV instrument fitted with a PTV (programmed temperature vaporizer) injector and control unit (PTV 382). The column was a FFAP (15 m, i.d. 0.33 mm) and the carrier gas hydrogen (H 2, flow rate 2.5 ml/min). In the solvent split method an ini- tial injector temperature of 70°C was used. 123 Scheme 3. 124 After introducing the sample, the split was kept open for 8 s. The split was then closed, the injector temperature increased to 250°C, and the split then opened again after 70 s. The oven was programmed from 70°C to 205°C at a rate of 10°C/min (VI). The area of the peaks was determined on Infotronics CSR-208, Hewlett-Packard 3390A or Shimadzu C-RIB integrators, and nor- malized to 100 % before carrying out statis- tical analysis (I, 111, IV). The components were identified by com- paring them with the retention times (III) for pure compounds of the fatty acid methyl esters (Applied Science Labs.; Nu Chek Prep. Inc.), and mass spectrometrically using a Hewlett- Packard 5890 GC fitted with an HP 5970 mass selective detector (VI). The precision and ac- curacy of the analytical conditions were opti- mized on the basis of the results obtained with the on-column method and a mixture of pure compounds (I). The raw fat content of the breeding mate- rial was determined using the NIR (near infra- red reflectance) technique (VII). 2. Statistical analysis The Student’s t-test was used in comparing the mean values. The equality of variances was studied using the F test, and when necessary a modification of the t-test was applied ac- cording to equations by Snedecor and Coch- ran (1973) (I, 11, IV—VI). The heritability of linoleic acid was determined in two separate generations using the offspring-midparent equation (h2 = bOP), and with the realized heritability (h2 = R/S) for the whole material throughout the course of the breeding period (Simmonds 1979, Falconer 1981) (IV, V). Analyses of correlation and variance, as well as comparison between two correlation co- efficients, were carried out using the equations presented in the literature (Snedecor and Cochran 1973) (11, 111, V). 125 Results A. Gas chromatography The variation in the fatty acid isomers in the heterogeneous seed material was studied using an FFAP column (55 mm) especially prepared for this purpose. A gas chromato- gram of the fatty acids in traditional rapeseed oil is presented in Fig. 1 (III). A total of 13 different components were identified. Iw7 isomers were represented by vaccenic, 13-eicosenoic and 15-docosenoic acids (peaks 4, 9 and 13 in Fig. 1). The re- suits obtained following the esterification of triolein, which is a compound where the acyl groups are formed only from oleic acid (18: lw9), showed that neither the esterifica- tion method nor gas chromatography resulted in the conversion of oleic acid to vaccenic acid (HI). 1. Conventional vs. on-column technique The significance of sample injection in the fatty acid analysis of rapeseed oil was studied Fig. I. The fatty acid composition of a high erucic acid rapeseed variety. 126 in detail using a number of different instru- ments (I, 111, VI). The conventional split tech- nique was compared with the on-column method, and the variation and differences between the means were tested statistically (I). The mean precision of the two methods for 13 fatty acid compounds is presented in Ta- ble 3. Table 3. Estimates of the precision of the conventional split and on-column techniques. Method Split Split On-column (I) (HI) (I) Mean precision (C.V. %) 6.4 5.7 2.3 The results show that the precision of the on-column method is clearly superior to that of the split technique. Compared to the on- column analyses, the variation in the C20 — C 22 fatty acids with the split method was greater (P<0.01), and also a large number of highly significant differences were obtained between the mean values (I). The effect of the sample size and the split ratio on the quantitative results of the split technique was studied using low erucic acid samples, and the results then compared to those obtained with the splitless and on- column methods (I) (Table 4). The use of a large sample size (2.4 /d, Ta- ble 4) or small split ratio (3:1) resulted in a considerable analytical error when deter- mining low erucic acid levels, the amount being in some cases less than half (0.7 %) the value obtained with the on-column method (1.7 %). With the split method the same quan- titative level was obtained with a very small sample size (0.1 /d) as with the splitless tech- nique (I). 2. Programmed temperature vaporization (PTV) techniques Preliminary tests withPTV showed that the splitless method has a similar precision to that of the on-column technique. The variation error in the split and solvent split runs (injec- tions at 45°C and 70°C respectively) was found to account for about 10 and 40 % re- spectively of the variation associated with con- ventional hot injection (250°C). PTV split in- jection also gave the most accurate result com- pared to the on-column method (Laakso et ai. 1983). The precision of the PTV solvent split tech- nique was determined using samples with dif- ferent erucic acid contents. The derivatization method and the GC process were repeated by carrying out the runs on six samples taken from the same extractant (VI). The estimates of the mean precision (C.V. %) in PTV analy- sis are presented in Table 5. Table 5. Estimates of the mean precision of the instru- ment and the whole process in PTV analysis. Sample Mean precision (C.V. %) Table 4. Effect of sample size and split ratio in fatty acid analysis. Intra-assay Inter-assay Zero High Zero erucic erucic erucic 2.0 1.9 2.2 Method Sample Split Peak area {%) Peak area ratio size 0*1) ratio (16:0/22:1) Split 0.1 15:1 2.9 1.4 2.1 2.4 15:1 4.3 0.7 6.1 1.0 3:1 4.2 0.7 6.0 Splitless 0.3 2.9 1.4 2.1 On-column 1.0 3.0 1.7 1.8 127 It can be concluded from Table 5 that at least the same degree of precision can be ob- tained using the PTV solvent split technique as with the on-column method (Table 3). The combined mean variation (mean C.V. = 2.2 °7o) of the esterificationprocedure and the GC analysis indicate that the proportion of the variation attributable to derivatization is in practice negligible (VI). The PTV solvent split analyses were carried out on a 15-m-longFFAP column, which sep- arated vaccenic acid (18: lu7) from oleic acid with a precision of 4 % even (VI). The fatty acid variation in the high erucic- acid (c. 7 %) seed material was determined using the conventional method (III) and the PTV technique (VI). The proportion of the analytical error (variance) was then subtracted from the total variation (N = 30). The most marked differences between the methods are presented in Table 6. Table 6. Proportion of biological variation as deter- mined by two different injection techniques. Sampling method Variation of biological origin (%) Compound Mean (%) Conven- PTV tional solvent split splitsplit (Ill) (VI) 16:0 20:0 3.6 78.3 99.4 0.4 66.7 92.6 20:2u6 22:0 0.5 83.6 93.7 0.2 68.4 99.3 The proportion of the analytical error caused by the conventional split method out of the total variation is approximately one quarter in the case of palmitic (16:0), arachi- dic (20:0), eicosadienoic (20:2c06) and behe- nic (22:0) acids (Table 6), while for the three main components it is below 3 % (III). With the PTV method, on the other hand, the proportion of biological variation was found to be over 90 °Io for all the fatty acids, and in most cases the analytical error re- mainedbelow 1 % of the total variation (VI). B. Breeding experiments I. Variation in fatty acids When the mode of inheritance of the fatty acids was being studied, the frequency distri- butions ofpalmitic, stearic, oleic, linoleic, a- linolenic, arachidic and behenic acids were found to be continuous and to conform to some extent to the normal distribution. The distributionof eicosadienoic acid, on the other hand, was skewed strongly to the right and levels many times greater than the normal one (0.1 —0.2 %) were found in both the bred ma- terial and in the controls. Similarly, increased concentrations of eicosenoic acid were found occasionally (II). The effect of environmental factors on the fatty acid composition was studied by growing the control material in four different blocks in the field. The results of replicate block trials showed that the replicates did not differ sig- nificantly from each other as regards selection for linoleic acid (II). However, the variation in the linoleic acid content of this material was approximately double when grown in the greenhouse (IV, V). Single seed analyses done on these individuals showed that there were rather large differences between different pods in the variance (s 2) of both linoleic (0.5 —7.7) and a-linolenic acids (0.6—4.3) (VI). The variance of the main components oleic, linoleic and a-linolenic acids in the control material never exceeded, to a signifi- cant degree, the corresponding values for the bred material, and the relative standard de- viation in both materials was almost without exception below 10 % (C.V.). The range for linoleic acid in different generations was about 10 %-units, the lowest individual content being 18.1 °/o (M 4) and the highest 34.1 % (M 8) (11, V). 2. The long-term effects of selection The linoleic acid content of every one of the generations grown in the field was significant- ly greater (P <0.001) than that of the controls, 128 and the oleic acid content correspondingly lower. No statistically significant differences were found, on the other hand, in the green- house (MrBOG). The smallest increase in the amount of linoleic acid achieved under field conditions was 1.7 ®/o-units (M 2, M s), and the largest 3.8 %-units (M 8). The amount of eicosadienoic acid, which belongs to the same series of u 6 fatty acids, was at least signifi- cantly higher (P<0.05) than that in the con- trols (11, IV, V). The heritability (h 2) of linoleic acid was de- termined using the offspring-midparent re- gression for the M 2 and M 5 populations. The values of the regression coefficients were 0.14 and 0.12 respectively (IV). The value of the realized heritability (R/S), in which the total amount of the selection performed is included, was 0.11 (V). The variance of linoleic acid was found to decrease in comparison to that for the con- trols during the course of the selection process. The changes in the coefficient of variation (C.V.) indicated a similar trend (V). The a-linolenic acid content had increased significantly with respect to that for the con- trols within two generations (M 4, M 7) (V). The increase in the linoleic acid content brought about through selection caused an opposite effect on the oleic acid level. On the other hand, the trend in the case of the a-lino- lenic acid level, starting from the M 5 prog- eny, was opposite to that for linoleic acid. Ex- ceptions in the case of saturated fatty acids were also found, the palmitic acid content showing an increasing trend as the levels for other components fell (V). Erucic acid occurred on the average at a concentration of 0.1 % per generation throughout the duration of the breeding pro- gramme (V). Finally, the yield was determined on the breeding lines showing the highest linoleic acid content. The fatty acid composition, crude fat content and oil yield of the material obtained from replicate block experiments were com- pared to the corresponding values for controls and summer turnip rape varieties commonly cultivated in Finland (VII). The lines with the best linoleic acid contents and oil yields are presented in Table 7. The lines with the highest linoleic acid con- tents (97703 and 97712) proved to have an oil yield comparable to that of the ’Emma’ va- riety in yield trials carried out in two succes- sive years. The crude fat content of the bred material was c. 2 %-units higher than that of the standard varieties. Line 97711 promises to give a very high oil yield. Most of the linoleic acid lines were erucic acid-free and, in addi- tion, the a-linolenic acid level did not signif- icantly differ from that of the controls (VII). Table 7. Quality characteristics of summer turnip rape varieties and the control and selected lines in Trial 1984 (N = 3). Material Fatty acids (%) Seed yield Crude fat Oil yield 18:2o>6 18:3u3 22: lu9 (reL) (%) (rd) Varieties Ante 21.2 10.8 0.6 86 43.8 82 Emma 22.4 11.4 0.5 117 44.6 113 Controls 22.4 12.1 100 46.1 100 Selection Hja 97815 25.5 11.8 103 46.6 104 97712 25.2 12.1 115 46.4 116 97703 25.2 12.2 110 45.7 109 97832 24.3 12.3 114 47.0 116 97824 24.3 12.6 124 46.2 125 97711 23.6 11.8 0.9 133 46.5 139 97828 23.5 12.3 124 46.6 126 129 3. Interrelationships between fatty acids The saturated fatty acids stearic (18:0) and arachidic acids (20:0) were positively correlated (P< 0.001) with each other, as was also the case with arachidic and behenic acids (22:0). Oleic acid was positively correlated (P< 0.001) with stearic acid, and negatively correlated (P< 0.001) with linoleic acid (VI). The correlation between linoleic and a-lino- lenic acids remained positive (r = 0.07—0.44) in all the generations, and in most cases was statistically significant. Significant negative correlations were also found between these compounds both in some individual yields (VI) and within some families in the material cultivated in the field (V). The correlation between linoleic and a- linolenic acids was also calculated on indi- viduals selected as parents and on their prog- eny. In the material cultivated in the field, in particular, the correlations were found to be of the same order of magnitude (V). The interrelationships between the lco9 fatty acids and their corresponding la/7 isomers were studied in material containing different levels of erucic acid (0.3—37 %). The lw7 iso- mers (18: lco7, 20: lco7 and 22: lu7) were found to be intercorrelated (P< 0.001), similarly as was the case between the lw9 isomers. The correlation between the two fatty acid series (lw9 and lco7) was positive and highly signifi- cant (III). 130 Discussion A. Comparison of sampling techniques in fatty acid analysis The use of capillary columns in analytical gas chromatography has rapidly become wide- spread during the past decade. Considerably higher resolution can be obtained in compari- son to packed columns, and it has been pos- sible to shorten the analysis time without any loss in resolution. The detection of com- pounds in ever smaller amounts has also be- come possible in trace-component analysis. However, the small diameter of the column means that the capacity of the column is re- duced and special injection systems are needed. The traditional split and splitless in- jection techniques have been the object of con- siderable criticism owing to their inadequate precision in quantitative work (Schomburg et al. 1977, Grob and Grob 1979, Grob and Neukom 1979, Schomburg 1979, Munari and Trestianu 1981). Traditional sample-injec- tion techniques were compared with new in- jection methods in the study in hand. In ad- dition, the magnitude of the analytical error in the GC process and the suitability of the various methods from the point of view of fatty-acid breeding work were examined (I, 111, V). The split technique, which is the method most commonly used for routine work, proved to be rather unreliable in the analysis of fatty acids. The effects of sample size and the split ratio on the quantitative results were especial- ly pronounced when low levels of erucic acid were being determined. It was not possible to analyse, even with carefully optimized split and splitless injection, the erucic acid levels attainable with the on-column method (I). Taking the precision level of below 1 % (C.V.) proposed by Yang et al. (1978) as the crite- rion, the split method has given a degree of precision of this magnitude for only 3 of the main components (>lO %) (I, III). The mean precision of the on-column tech- nique (C.V. = 2.3 %) was clearly better than that of the conventional split method (C.V. = 5.7 °7o and 6.4 %) (Table 3). The CV values for compounds present at levels of be- low 1 % have decreased considerably (Table 1,1), and are in good agreement with the pre- cision level obtained by Badings and de Jong (1983) and Geeraert et al. (1983) in their fatty acid analyses (Table 1). The superiority of this technique in comparison to traditional sample injection methods has been demon- strated in a large number of studies during the past few years (e.g. Grob and Neukom 1980, Schomburg et al. 1981, Munari and Tres- tianu 1981). Despite the good precision and accuracy of the on-column method, its applicability is re- stricted by the unvolatilized impurities that ac- cumulate at the column inlet and subsequent- ly bring about a reduction in the resolution (Schomburg et al. 1977, Bayer and Liu 1983). The solvent in on-column injection has also been found to sometimes produce peak broadening and thus reduce the resolution (Grob 1982, Sandra et al. 1983). Introducing the sample with an extremely thin needle is further considered to be a drawback (Poy et al. 1981). The on-column technique is pre- ferred when there is no need for automation 131 of the analyses (Venema 1983). The precision (mean C.V. = 2.0 % and 1.9 %) of the PTV solvent split technique was found to be of the same order of magnitude as that for the on-column technique (mean C.V. = 2.3 %) (Tables 3 and 5). The results also show that the variation caused by the derivatization method is insignificant com- pared to the gas chromatographic error (Table 5). The precision (C.V. = 0.1—0.2 °/o) of the main components (10 %) is in good agreement with values obtained with on-column or »sol- vent-free» injection (Geeraert et al. 1983, Caplan and Cronin 1983) (Table 1). Earlier analyses carried out using PTV showed that the splitless technique has the best precision, and that the split method gives the most ac- curate results compared to on-column injec- tion (Laakso et ai. 1983). Schomburg et ai. (1983 a, 1983 b) have also found that tem- perature programmed (TP) injection carried out using the splitless or split method does not cause any marked discrimination. Using the PTV method in place of the on-column meth- od has proved to be very suitable for, e.g. chemotaxonomic studies on volatile oils (Ho- lopainen et ai. 1983). Detailed comparison of the sampling methods showed that a high in- jection temperature resulted in a many times greater analytical error, especially in the case of volatile monoterpenes (Hiltunen et ai. 1983 a). The headspace technique also gives better precision than manual hot injection for these components (Hiltunen et ai. 1983 b). When the proportion of the analysis error was compared to the fatty acid variation in turnip rape, values of over 99 °7o were ob- tained for the variation of a number of com- ponents with the PTV technique (VI). The good separation of the components is illus- trated by the determination of vaccenic acid with a precision of 4 % (C.V.) even, using a column only 15 m long (VI). Separation is pre- sumably improved by the fact that there is a greater difference between the volatilization instant of the solvent and that of the fatty acids in temperature-programmed injection than in hot injection. The advantages of the temperature-pro- grammed injection technique in gas chromato- graphic analysis are considered to be so clear that it will presumably replace the classical split and splitless hot injection methods (Grob 1984). In this study, the PTV tech- nique was found to be even more suitable than the on-column method as far as the studied fatty acids were concerned. Impurities in the sample are not as critical a factor as in on- column injection, analysis on short columns even gave good separation and, in addition, it is possible to automate the method. B. Selection for higher linoleic acid content in rapeseed oil As was the case in the breeding of erucic acid-free varieties, the half-seed technique is considered to be the best method for further modifying the proportions of different fatty acids. Only a relatively small amount of ma- terial is required, crossings can be done between known chemotypes, and cultivation in the greenhouse makes the breeding work in- dependent of the season. Even a 5 to 6 %-unit increase in the linoleic acid content has been achieved through a single crossing (Jönsson 1977 a). However, the linoleic acid level of the common summer turnip rape varieties is still only about 22 % (Canadian »Canola» type oil) (Daun 1983). A phenotypic selection for single plants best suited for cultivation under field conditions was carried out in this study on summer tur- nip rape. The individuals with the highest linoleic acid content were selected for further breeding on the basis of the fatty acid com- position of their yields, and the lines finally obtained were culled on the basis of their yield (11, IV, V, VII). The fatty acid variation of the main com- ponents under field conditions was rather constant during successive years, the relative standard deviation (C.V.) usually being under 10 % (V). On the other hand, the environment had a considerable effect under greenhouse conditions. The linoleic, and especially the a- 132 linolenic acid content, tended to increase and almost double values were found in the vari- ances. Neither was it possible to show the effect of selection on the linoleic acid content (IV, V, VI). Although it is impossible to car- ry out exact comparison between the green- house conditions, the result goes against the belief concerning the better suitability of greenhouse conditions in breeding work (Jönsson 1975 a, Thies 1971). The clearer effect of the environment on a-linolenic than linoleic acid agrees with the results obtained by Jönsson (1975 a) in breeding experiments with turnip rape. As the individualplant selec- tion on linoleic acid content was effective only in the material grown in the field (V), it would indicate that a more reliable selection can also be made on single seeds from such material. The only increase which systematically fol- lowed that of the linoleic acid content under field conditions was in the level of eicosadi- enoic acid (20:2w6) (11, V). This compound is an expected elongation product of linoleic acid (Stearns 1970). However, the analytical error was rather large when determining the variation of this component (III). Higher con- tents (0.3 —0.6 %) have usually been obtained with high erucic-acid varieties (Ackman 1966) and elevated values occasionally occurred also in the present material when the distributions of eicosenoic and eicosadienoic acid were studied (II). This may mean that eicosenoic acid (20: Icj9) is also a precursor for eico- sadienoic acid. The close dependance between loj7 isomers of C 18—C 22 fatty acids, indicated by highly significant correlations, as was also the case between corresponding lu9»isomers (III), would support the hypothesis of Ap- pelqvist (1968) that elongation of these iso- mer series takes place in parallel under the control of a single gene system. The breeding trials carried out in the field demonstrated that the linoleicacid content can be increased, using individual plant selection, without affecting the a-linolenic acid content (V). It was possible, following the field trials, to select erucic acid-free lines with elevated linoleic acid contents that had yields which were fully comparable to the varieties present- ly cultivated in Finland (VII). The effect of selection on the linoleic acid level during eight generations was very small (22 —25 °7o). This corresponds well to the realized heritability of 0.11 (h 2 = b = R/S) obtained in the study (V). However, it is a spe- cific estimate for the varieties and is highly dependent on the method of breeding (Sim- monds 1979). A low value is to be expected when breeding polyunsaturated fatty acids of turnip rape under uncontrolled crossing con- ditions (Jönsson 1975 a). Individual selection with respect to the oil content has also given corresponding heritability values (Krzymans- ki 1983). The response to selection for linoleic acid varied considerably during the course of the breeding work (V). When the analytical pre- cision of fatty acids and environmental con- ditions are the same for the selection lines and controls, as can be assumed to be the case here, the selection intensity is one of the most important factors affecting the response (Falconer 1981). The proportion of selected individuals out of the basic population varied between 9 to 32 %, the selection of the M 7 generation (9 %), for instance, having the greatest intensity and also giving the greatest difference in linoleic acid content with respect to the controls (3.7 %) (V). Increasing the re- sponse by means of high selection intensity is, however, complicated because the variance is expected to decrease as a result of directional selection, and hence the genetic base will also narrow (Falconer 1981). Repeated irradia- tion may be one possible means of again in- creasing the genetic variability of linoleic acid in the breeding material after its variation has already fallen to the level of the controls (V). One interesting feature was the change in the response ofa-linolenic acid as opposed to that of linoleic acid (V). The fact that the par- tial independence of these two components, which is supported by studies on both their composition and biosynthesis (Rakow 1973, Brar and Thies 1978), does not appear until in field conditions, would indicate that the 133 individual selection made on greenhouse ma- trol of the erucic acid levels, as well as im- terial is also ineffective when a high linoleic/ a-linolenic acid ratio is being aimed for. Jönsson (1975 a) has found that the corre- sponding independence between these com- pounds in turnip rape is variety dependent. The correlations between linoleic and a- linolenic acids in the parents and their prog- enies corresponded rather well with each other during the breeding period (V). Therefore, taking the a-linolenic acid content also into account may probably be advantageous in modifying the fatty acid ratio, despite the fact that no rapid results can be expected by simul- taneous selection (Röbbelen and Nitsch 1975). However, due to the new evidence for the role of a-linolenic acid the balance be- tween linoleic and a-linolenic acids probably should not be too radically altered (Ackman 1983). The results of this study show that selection for higher linoleic acid content can form part of breeding work designed to produce good- yielding summer turnip rape varieties adapted to Finnish field conditions. The improved ana- lytical techniques permit more accurate con- proving the reliability of selection in modi- fying the polyunsaturated fatty acid compo- sition in rapeseed oil. Furthermore, it may be possible to achieve a greater elevation in the linoleic/a-linolenic acid ratio by more effec- tive exploitation of extreme chemotypes. Despite the low content of polyunsaturated fatty acids, rapeseed oil has been shown to have an appreciable effect on serum lipid pat- terns, lowering the cholesterol level, in par- ticular. (McDonald 1983, Savoie et al. 1983). Furthermore, a-linolenic acid is considered to be responsible for increasing the content of eicosapentaenoic acid (EPA) (Lassere and Jacotot 1983). As these constituents have been one of the foremost questions in studying the risk factors of coronary heart disease in Finnish populations (Miettinen et ai. 1982, Vartiainen et ai. 1984), determining the role of rapeseed oil in our diet is of prime impor- tance. Rapeseed oil is, in respect to linoleic and a-linolenic acid, an exeptionally balanced vegetable oil, which must be considered as a serious alternative among sources of essential fatty acids. 134 3 References Ackman, R.G. 1966. Analysis of the monoethylenic fatty acids of rapeseed by open tubular gas chromato- graphy. J. Am. Oil Chem. Soc. 43: 483—486. —, Hooper, S.N. & Hingley, J. 1971. Monoethylenic fatty acids of a partially hydrogenated herring oil. J. Am. Oil Chem. Soc. 48: 804—806. , Barlow, S.M. & Duthie, I.F. 1977. Erucic acid in edible fats and oils: a collaborative study on deter- mination by open-tubular(capillary) gas-liquid chro- matography. J. Chromatogr. Sci. 21: 87—93. &Sebedio, J.-L. 1978. The minor fatty acids of rape- seed oil. Proc. slh5 lh Int. Rapeseed Conf., Malmö, Sweden, F: 9—12. 1983. »Chemical Composition of Rapeseed Oil» in Kramer, Sauer & Pigden, High and Low Erucic Ra- peseed Oils. Academic Press, 582 p. Toronto—New York —London. Anon. 1978. Laki kotimaisen öljykasvituotannonedistä- misestä. Suomen Asetusk. n:o 262. , 1980. Commission directive of 25 July, 1980relating to the Community method of analysis for determining the erucic acid content in oils and fats intended to be used as such for human consumption and foodstuffs containing added oils or fats, (80/89/EEC). Offical Journal of the European Communities L 254: 35—41. , 1981, Kasviöljyt. Margariiniteollisuuden tiedotuskes- kus. 16 p. , 1982. Laki kotimaisesta öljykasvituotannosta. Suo- men Säädösk. n:o 965. AppELQVtsT, L-A. 1968. Lipid patterns in Cruciferae. Acta Univ. Lund. Sectio 11, No. 7: 1—25. —, 1972. »Chemical Constituents of Rapeseed» in Ap- pelqvist & Ohlson, Rapeseed: cultivation, composi- tion, processing and utilization. Elsevier, 391 p. Amsterdam—London—New York. Arrendale, R.F., Chapman, G.W. & Chortyk, O.T. 1983. Gas chromatographicanalyses of fatty acids on laboratory-prepared fused silica Silar 10C capillary columns. J. Agric. Food Chem. 31: 1338—1342. Badings, H.T. & De Jong, C. 1983. Glass capillary gas chromatography of fatty acid methyl esters. A study of conditions for the quantitative analysis of short- and long-chain fatty acids in lipids. J. Chromatogr. 279: 493—506. Bartkowiak-Broda, I. 1978. Inheritance of fat content and fatty acid composition in seeds of zero-erucic winter rape (B. napus). Proc. sth5 th Int. Rapeseed Conf., Malmö, Sweden, B: 119—123. & Krzymanski, J. 1983. Inheritance of C-18 fatty acids composition in seed oil zeroerucic winter rape Brassica napus L. Proc. 6,h Int. Rapeseed Conf., Pa- ris, France, G: 477—482. Bayer, E. & Liu, G.H. 1983. New split injection tech- nique in capillary column gas chromatography. J. Chromatogr. 256: 201—212. Bengtsson, L., v.Hofsten, A. & Lööf, B. 1972. »Botany of Rapeseed» in Appelqvist & Ohlson, Rapeseed: cul- tivation, composition, processing and utilization. Else- vier, 391 p. Amsterdam—London—New York. Bohov, P., Balä2, V. & HrivSäk, J. 1984. Analysis of fatty acid methyl esters on an SP 2340 glass capillary column. J. Chromatogr. 286: 247—252. Brar, G.S. & Thies, W. 1978. Biosynthesis of a-linolenic acid in leaves and seeds of rape (Brassica napus L.) Proc. slh5 lh Int. Rapeseed Conf., Malmö, Sweden, F: 27—30. Budowski, P., Trostler, N., Lupo, M., Vaisman, N. & Eldor, A. 1984. Effect of linseed oil ingestion on plasma lipid fatty acid composition and platelet ag- gregability in healthy volunteers. Nutr. Res. 4: 343—346. Caplan, P.J. & Cronin, D.A. 1983. Simple device for solvent-free injection of high-boiling compounds in capillary gas chromatography. J. Chromatogr. 267: , 19—28. Craig, B.M. & Wetter, L.R. 1959. Varietal and en- vironmental effects on rapeseed. 11. Fatty acid com- position of the oil. Can. J. Plant Sci. 39: 437—442. —, 1961. Varietal and environmental effects on rapeseed. HI. Fatty acid composition of 1958 varietal tests. Can. J. Plant Sci. 41: 204—210. Dalin, J.K. 1983. »The Introduction of Low Erucic Acid Rapeseed Varieties into Canadian Production» in Kra- mer, Sauer & Pigden, High and Low Erucic Rapeseed Oils. Academic Press, 582 p. Toronto —New York — London. Downey, R.K. & Harvey, B.L. 1963. Methods of breed- ing for oil quality in rape. Can. J. Plant Sci. 43: 271—275. 135 —, 1964. A selection of Brassica campestris L. contain- ing no erucic acid in its seed oil. Can. J. Plant Sci. 44: 295. & Craig, B.M. 1964. Genetic control of fatty acid biosynthesis in rapeseed (Brassica napus L.) J. Am. Oil Chem. Soc. 41: 475—478. & McGregor, D.I. 1975. Breeding for modified fat- ty acid compostion. Adv. Plant Sci. 12: 151—167. —, 1978. Breeding for quality. Proc. sth5 th Int. Rapeseed Conf., Malmö, Sweden, B: 106—112. —, 1983. »The Origin and Description of the Brassica Oil- seed Crops» in Kramer, Sauer & Pigden, High and Low Erucic Rapeseed Oils. Academic Press, 582 p. Toronto —New York —London. Dyerbero, J., Bang, H.0., Stoffersen, E., Moncada, S. & Vane, J.R. 1978. Eicosapentaenoic acid and pre- vention of thrombosis and atherosclerosis? Lancet, ii: 117—119. Ehrendorfer, 1983. »Evolution und Systematik» in Strasburgers Lehrbuch der Botanik (v. Denffer, Zieg- ler, Ehrendorfer & Bresinsky). Gustav Fischer Verlag. 32. Aufl. 1161 p. Stuttgart—New York. Falconer, D.S. 1981. Introduction to Quantitative Gene- tics, 2nd ed. Longman Group Ltd. 340 p. London— New York. Fochem, H. 1985. Der Weltmarkt der Pflanzenöle, ihre Produktion, Verwendung und Vermarktung. Fette— Seifen—Anstrichmittel 87(2): 47—52. Fondu, M. 1979. Chemistry of dietary oils and fats. Acta Cardiolog. Suppl. XXIII: 74—94. Frohne, D. & Jensen, U. 1985. Systematik des Pflanzen- reichs. 3. Aufl. Gustav Fischer Verlag. 355 p. Stuttgart—New York. Galli, M. & Trestianu, S. 1981. Benefits of a special cooling system to improve precision and accuracy in non-vaporizing on-column injection procedures. J. Chromatogr. 203: 193—205. Gander, K-F. 1984. Die Fettversorgung des Menschen aus wirtschaftlicher und technologischer Sicht. Fette — Seifen—Anstrichmittel 86(1): I—B. Geeraert, E., Sandra, P. & De Schepper, R. 1983. On- column injection in the capillary gas chromatographic analysis of fats and oils. J. Chromatogr. 279; 287—295. Golovnya, R.V., Kuzmenko, T.E. & Vasilev, A.V. 1984. Stable and reproducible selective glass capillary columns with polysiloxane stationary phases for the analysis of fatty acid methyl esters. J. Chromatogr. 292: 49—55. Grice, H.C. & Hegotveit, H.A. 1983. »The Relevance to Humans of Myocardial Lesions Induced in Rats by Marine and Rapeseed Oils» in Kramer, Sauer & Pig- den, High and Low Erucic Rapeseed Oils. Academic Press, 582 P. Toronto —New York —London. Grob, K. & Grob, G. 1976. A new, generally applicable procedure for the preparation of glass capillary columns. J. Chromatogr. 125: 471—485. —, 1978. On-column injection onto capillary columns. Part 2: Study of sampling conditions; practical recom- mendations. J. High Resolut. Chromatogr. & Chro- matogr. Comm. 1: 263 —267. & Grob, K. Jr. 1978. On-column injection on to capillary columns. J. Chromatogr. 151: 311—320. & Grob, G. 1979. Practical capillary gas chromatog- raphy a systematic approach. J. High Resolut, Chromatogr. & Chromatogr. Comm. 2: 109—117. Grob, K, Jr.& Neukom, H.P. 1979. The influence of the syringe needle on the precision and accuracy of va- porizing GC injections. J. High Resolut. Chromatogr. & Chromatogr. Comm. 2: 15—21. & Neukom, H.P. 1980. Factors affecting the accuracy and precision of cold on-column injections in capil- lary gas chromatography. J. Chromatogr. 189: 109—107. —, 1981.Peak broadening or splitting caused by solvent flooding after splitless or cold on-column injection in capillary gas chromatography. J. Chromatogr. 213: 3—14. —, 1984. Movements of the sample plug in the column inlet after on-column injection in capillary gas chro- matography. J. Chromatogr. 283; 21 —35. Haken, J.K. 1984. Developments in polysiloxane station- ary phases in gas chromatography. J. Chromatogr. Chromatogr, Rev. 300: 1—77. Hamilton, D.V., Lea, E.J.A. & Jones, S.P. 1980. Die- tary fatty acids and ischaemic heart disease. Acta Med. Scand. 208: 337—340. Harvey, B.L. & Downey, R.K. 1964, The inheritance of erucic acid content in rapeseed (Brassica napus). Can J. Plant Sci. 44: 104—111. Hauo, M., Dieterich, L, Laubach, C., Reinhardt, D. & Harzer, G. 1983. Capillary gas chromatography of fatty acid methyl esters from human milk lipid sub- classes. J. Chromatogr. 279: 549—553. Hay, C.R.M., Durber, A.P. & Saynor, R. 1982. Effect of fish oil on plateletkinetics in patients with ischaemic heart disease. Lancet i: 1269—1272. Heckers, H., Melcher, F.W. & Schloeder, U. 1977. SP 2340 in the glass capillary chromatography of fatty acid methyl esters. J. Chromatogr. 136: 311 —317. Hiltunen, R., Huhtikangas, A. & Hovinen, S. 1979. Breeding of a zero erucic spring turnip-rape cultivar, Brassica campeslhs L., adapted to Finnish climatic conditions. I. The use of glass capillary column gas chromatography in fatty acid analysis. Acta Pharm. Fenn. 88; 31—34. —, Laakso, L, Hovinen, S. & Derome, J. 1982. Sam- pling techniques in the glass capillary gas chromato- graphy of fatty acids of rape-seed. J. Chromatogr. 237; 41—48. —, Holopainen, M., Laakso, 1. & v.Schantz, M. 1983 a. Comparison between a programmed tempera- ture vaporizer (PTV) and other sampling techniques in the GLC-analysis of volatile oils. Acta Pharm. 136 137 Fenn. 92: 197—201. , Laakso, I, & Scheffer, J.J.C. 1983 b. Application ofheadspace gas chromatography in essential oil anal- ysis. I. Precision. Acta Pharm. Fenn. 92: 209—21. Holopainen,M., Hiltunen, R., Forsen, K. & v. Schantz, M. 1983. Comparisons between steam-distillation and extraction in the analysis of tansy oil (Tanacelum vulgare L.) for chemotaxonomic studies. Acta Pharm. Fenn. 92: 203—208. Holman, R.T. 1970. Biological activities of and require- ments for polyunsaturated acids. Prog. Chem. Fats 9: 611—682. Horrobin, D.F. 1982. »Essential Fatty Acids: A Review» in Horrobin, Clinical Uses ofEssential Fatty Acids. Eden Press, 214 p. Montreal—London, Houoen, F.W. & Wasowicz, E.W. 1978. Isomeric mono- unsaturated fatty acids in edible fats and oils with reference to myocardial lesions in animals. Proc. sth Int. Rapeseed Conf., Malmö, Sweden, F: 13—15. Hovinen, S. 1985. »Öljykasvit». Siemenjulkaisu. Hank- kijan Kasvinjalostuslaitos. 188 p. Jaeger, H., Klör, H-U., Bros, G. & Ditschuneit, H. 1975. Reliable separation of cis and trans fatty acids by gas liquid chromatography on glass capillary columns. Chromatographia 8(9); 507—510. Johansson, S-Ä. & Uppstrom, B. 1978. Analys av olje- växtfrö: Bestämning av fettsyrasammansättning, Sver. Utsädesför. Tidskr. 88: 35—44. Jönsson, R. 1973. Breeding for low erucic acid content in summer turnip rape (Brassica campeslris L. var. annua L.). Z. Pflanzenziichtg. 69: 1 —lB. , 1975 a. Förädling för förbättrad fettsyrasammansätt- ning i oljeväxter. 111. Höjd linolsyrahalt i värrybs (Brassica campeslris L. var. annua L.). Sver. Ut- sädesför. Tidskr, 85: 19—29. , 1975 b. Gulfröighet i raps och rybs. IL Växtföräd- ling för förbättrad olje- och mjölkvalitet i gulfröigt material. Sver. Utsädesför. Tidskr. 85: 271—278. , 1977 a. Erucic-acid heredity in rapeseed (Brassica napus L. and Brassica campeslris L.). Hereditas 86: 159—170. , 1977 b. Breeding for improved oil and meal quality in rape (Brassica napus L.) and turnip rape (Brassica campeslris L.). Hereditas 87: 205—218. & Persson, C. 1983. Breeding for improved fatty acid composition in rapeseed. Proc. 6th Int. Rapeseed Conf., Paris, France, C: 311 —314. Kaitaranta, J. 1981. Fish roe lipids and lipid hydrolysis in processed roe of certain Salmonidae fish as studied by novel chromatographic techniques. Research Re- ports 14/1981, p. 31 +app. p, 49, Techn. Res. Cen- tre of Finland. Kondra, Z.P. & Thomas, P.M. 1975. Inheritance of oleic, linoleic and linolenic acids in seed oil of rape- seed (Brassica napus). Can. J. Plant Sci. 55: 205—210. &Wii,son, T.W. 1976. Selection for oleic, linoleic and linolenic acid content in F 2 populations of rape. Can. J. Plant Sci. 56: 961—966. KrzymAnski, J. 1984. Possibilities of breeding for higher oil and protein contents in winter rape seeds. Fette— Seifen—Anstrichmittel 86(12): 468—470. Laakso, L, Holopainen, M., Hiltunen, R. & v. Schantz, M. 1983. Application ofa programmed temperature vaporizer (PTV) in the gas chromatographicanalysis of volatile oils and fatty acids. Abstr. ler Gesamt- kongr. Pharm. Wissensch., Miinchen, GFR, 296— 297. —, Hiltunen, R., Koiranen, P. & Seppänen, T. & v. Schantz, m , 1984. Vegetable seed and fish oils as a source of polyunsaturated fatty acids (PUFA) of the «6 and o>3 series. Farm. Tijdschr. Belg. 61(3): 373—374. Lanza, E. & Slover, H.T. 1981. The use of SP 2340 glass capillary columns for the estimation of Irons fatty acid content of foods. Lipids 16(4): 260 —267. Lassere, M. & Jacotot, B. 1983. Effects ä long terme de plusieurs graisses alimentaires (dont I’huile de colza) sur les lipides seriques d’une population de religieu- ses benedictines. Proc. 6th Int. Rapeseed Conf., Paris, France, K: 1653—1659, Lee, M.L. & Wright, B.W. 1980. Preparation of glass capillary columns for gas chromatography. J. Chro- matogr. Rev. 184; 235—312. Lercker, G. 1983. Short capillary columns in the analy- sis of lipids. J. Chromatogr. 279: 543—548. Loop, B. & Appelqvist, L-Ä. 1972. »Plant Breeding for Improved Yield and Quality» in Appelqvist & Ohl- son, Rapeseed: cultivation, composition, processing and utilization. Elsevier, 391 p. Amsterdam—Lon- don—New York. Lie Ken Jie, M.S.F. 1980. The characterization of long- chain fatty acids and their derivatives by chromato- graphy. Adv. Chromatogr. 18: 1—57. Masson, L. 1981. Relative nutritional value of various dietary fats and oils. J. Am. Oil. Chem. Soc. 58(3); 249—255. McDonald, B.E. 1983. »Studies with High and Low Erucic Acid Rapeseed Oil in Man» in Kramer, Sauer & Pigden, High and Low Erucic Rapeseed Oils. Aca- demic Press, 582 p. Toronto—New York —London. Metcalfe, L.D. & Wang, C.N. 1981. Rapid preparation of fatty acid methyl esters using organic base-catalyzed transesterification. J, Chromatogr. Sci. 19: 530—535. Miettinen, T.A., Naukkarinen, V., Huttunen, J.K., Mattila, S. & Kumlin, T. 1982. Fatty-acid compo- sition of serum lipids predicts myocardial infarction. Br. Med. J. 285: 993—996. —, Alethan, G., Huttunen, J.K. Pikkarainen, J., Naukkarinen, V., Mattila, S. & Kumlin, T, 1983. Serum selenium concentration related to myocardial infarction and fatty acid content of serum lipids. Br. Med. J. 287: 517—519. Munari, F. & Trestianu, S. 1981. Comparison ofsome quantitativeresults obtained with non-vaporizing cold on-column and vaporizing split-splitless injection tech- niques. In Kaiser (ed.) Proc. 4th Int. Symp. Capil- lary Chromatography, Hindelang, 349—369. Lööf, B. 1972. »Cultivation of Rapeseed» in Appelqvist & Ohlson, Rapeseed: cultivation composition, proces- sing and utilization. Elsevier, 391 p. Amsterdam— London—New York. Öster, P. & Schlierf, G. 1982. Kardiovakuläre Risiko- faktoren 1982. Fette-Seifen—Anstrichmittel 84(2): 621—622. Paszkowski, W.A. 1983. The Canadian Canola industry. Proc. 6th Int. RapeseedConf., Paris, France, A: 3—B. Penttilä, L, Huhtikangas, A., Herranen, J., Eskelinen, S. & Moilanen, O. 1984. Simultaneous measurement of free and esterified fatty acids by gas chromato- graphy from normal and type IV hyperlipoprotein- aemic sera. Ann. Clin. Res. 16: 13—17. Pigden, W.J. 1983. »World Production and Trade of Rapeseed and Rapeseed Products» in Kramer, Sauer & Pigden, High and Low Erucic Rapeseed Oils, Aca- demic Press, 582 p. Toronto —New York—London. Poy , F., Visani, S. & Terrosi, F. 1981. Automatic injec- tion in high-resolution gas chromatography: A pro- grammed temperature vaporizer as a general purpose injection system. J. Chromatogr. 217; 81—90. —, 1982. A new temperature programmed injection tech- nique for capillary GC: Split mode with cold intro- duction and temperature programmed vaporization. Chromatographia 16: 345—348. Rakow, G. 1973. Selektion auf Linol- und Linolensäu- regehalt in Rapssamen nach mutagener Behandlung. Z. Pflanzenziichtg. 69: 62—82. Röbbelen, G. & Nitsch, A. 1975. Genetical and physi- ological investigations on mutants for polyenoic fat- ty acids in rapeseed, Brassica napus L. I. Selection and description of new mutants. Z. Pflanzenziichtg. 75(2): 93—105. —, 1976. Ziichtung und Erzeugung von Quaitätsraps in Europa. Fette—Seifen—Anstrichmittel 78(1); 10—17. —, 1983. Fortschritte in der Welterzeugung von Rapssaa- ten. Fette—Seifen—Anstrichmittel 85(10): 395—398. Sanders, T.A.B. & Younger, K.M. 1983. The effect of dietary supplements of w3-polyunsaturated fatty acids on the fatty acid composition ofplatelets and plasma choline phosphoglycerides. Br. J. Nutr. 45: 613—616. Sandra, P., v. Roelenbosch, M., Verzele, M. & Bicchi, C. 1983. Experiments with cold on-column injection. J. Chromatogr. 279: 279—286. Savoie, L., Navratil, 0., Moorjani, S. & Lupien, P.J. 1983. Effects comparatifs de I’huile de tournesol et de I’huile de colza ä faible teneur en acide erucicdans le traitement de I’hypercholesterolemie familiale heterozygote. Proc. 6th. Int. Rapeseed Conf., Paris, France, K: 1700—1705. Schomburo, G. & Husmann, H. 1975. Methods and tech- niques of gas chromatography with glass capillary columns. Chromatographia 8(9): 517—530. —, Behlau, H., Dielmann, R., Weeke, F. & Husmann H. 1977. Sampling techniques in capillary gas chro- matograhy. J. Chromatogr. 142: 87—102. —, 1979. Practical limitations of capillary gas chromato- graphy. J. High Resolut. Chromatogr. & Chromatogr. Comm. 2; 461 —474. —, Husmann, H. & Rittman, R. 1981. »Direct» (on- column) sampling into glass capillary columns. Com- parative investigations on split, splitless and on-column sampling. J. Chromatogr. 204: 85—96. —, Husmann, H., Behlau, H. & Schulz, F. 1983 a. Cold sample injection with either the split or splitless mode of temperature-programmed sample transfer. Design and testing of a new, electrically heated con- struction for universal application of different modes of sampling. J. Chromatogr. 279: 251—258. —, Husmann, H., Schulz, F., Teller, G. & Bender, M. 1983 b. Cold sample injection with eithei the split or splitless mode of temperature-programmed sample transfer. Comparison tocold on-column injection with a commercial device. J. Chromatogr. 279; 259—267. Sebedio, J-L. & Ackman, R.G. 1979. Some minor fatty acids of rapeseed oils. J. Am. Oil Chem. Soc. 56: 15—21. —, Farquharson, T.E. & Ackman, R.G. 1982. Improved methods for the isolation and study of the Clg , C,„ and C22 monoethylenic fatty acid isomers of biologi- cal samples: Hg adducts, HPLC, AgN03-TLC/FID, and ozonolysis. Lipids 17(6): 469—475. Seher, A., Werner, G., Krohn, M. & Petersen, U. 1979. Ernährungsphysiologische Wirkung erucasäu- rearmer Rapsöle auf Schweine. 3. Wirkung auf De- potfette und Leberlipide. Fette—Seifen—Anstrich- mittel 81(5): 187—192. Simmonds, N.W. 1979. Principles of Crop Improvement, Longman Group Ltd. 408 p. London —New York. Sisfontes, L., Nyborg, G., Svensson, L. & Blomstrand, R. 1981. Separation of complex long chain fatty acid mixtures by high-performance glass capillary gas chro- matography. J. Chromatogr. 216: 115—125. Slover, H.T. & Lanza, E. 1979. Quantitative analysis of food fatty acids by capillary gas chromatography. J. Am. Oil Chem. Soc. 56: 933—943. Snedecor, G.W. & Cochran, W.G. 1967. Statistical Methods, 6th ed. lowa State University Press, 593 p. Ames, lowa, USA. Stage, H. 1982. Physical refining and deodorization of food oils. Fette—Seifen—Anstrichmittel 84(10); 377— 395. Stearns, E.M. Jr. 1970. Biosynthesis of unsaturated fat- ty acids in higher plants. Prog. Chem. Fats 9(4): 453—516. Stefansson, 8.R., Hougen, F.W. & Downey, R.K. 1961. Note on the isolation of rape plants with seed oil free from erucic acid. Can. J. Plant Sci. 41: 218—219. & Hougen, F.W. 1964. Selection of rape plants (Bras- sica napus) with seed oil practically free from erucic 138 acid. Can. J. Plant Sci. 44: 359—364. —, 1983. »The Development of Improved Rapeseed Cul- tivars» in Kramer, Sauer & Pigden, High and Low Erucic Rapeseed Oils. Academic Press, 582 p. To- ronto—New York—London. Stumpf, P.K. & Pollard, M.R. 1983. Pathways of fat- ty acid biosynthesis in higher plants with particular reference to developing rapeseed. ibid. Svensson, L., Sisfontes, L., Nyborg, G. & Blomstrand, R. 1982. High performance liquid chromatography and glass capillary gas chromatography of geometric and positional isomers of long chain monounsaturated fatty acids. Lipids 17(1): 50—59. Thiele, O.W. 1979. Lipide, Isoprenoide mit Steroiden. Georg Thieme Verlag, 415 p. Stuttgart. Thies, W. 1968. Die Biogenese von Linol- und Lino- lensäure in den Samen höherer Pflanzen, insbesondere Raps und Riibsen, als Problem der Ölpflanzenziich- tung. Angew. Bot. XLII (3/4): 140—154. —, 1971. Schnelle und einfache Analysen der Fettsäure- zusammensetzung in einzelnen Raps-Kotyledonen. I. Gaschromatographische und papierchromatogra- phische Methoden. Z. Pflanzenziichtg. 65(3): 181—202. Vane, J.R. & Moncada, S. 1979. Polyunsaturated fatty acids as precursors of protaglandins. Acta Cardiol. Suppl. XXIII: 21—37. Vartiainen, E., Puska, P., Pietinen, P., Nissinen, A., Leino, U. & Uusitalo, U. 1984. Ruokavalion vaiku- tus suomalaisten lasten korkeaan seerumin kolestero- litasoon. Suom. Lääkäril. 39: 3054—3058. Venema, A. 1983. Potential of capillary gas chromato- graphy in industrial research laboratories. J. Chro- matogr. 279: 103—110. Yang, F.J. Brown, A.C. & Cram, S.P. 1978. Splitless sampling for capillary-column gas chromatography. J. Chromatogr. 158; 91—lO9. & Cram, S.P. 1979. Characteristics and performance of gas chromatographic detectors with glass capillary columns. J. High Resolut. Chromatogr. & Chroma- togr. Comm. 2: 487—496. 139 140 APPENDIX Trivial and systematic names for fatty acids treated in this study are listed below. Omega (o>) is used to denote the first double bond position in the chain counting from the terminal methyl group. Trivial name Systematic name Abbreviation palmitic acid hexadecanoic acid 16:0 stearic acid octadecanoic acid 18:0 oleic acid cw-9-octadecenoic acid 18: lu9 vaccenic acid c/s-l 1-octadecenoic acid 18: lu7 linoleic acid all-c/s-9, 12-octadecadienoic acid 18; 2u6 y-linolenic acid all-cis-6, 9, 12-octadecatrienoic acid 18:3co6 a-linolenic acid all-c/s-9, 12, 15-octadecatrienoic acid 18:3u3 arachidic acid eicosanoic acid 20:0 eicosenoic acid c/s-11-eicosenoic acid 20: lu9 cis- 13-eicosenoic acid 20: lu7 eicosadienoic acid all-cis-11, 14-eicosadienoic acid 20: 2u6 homo-y-linolenic acid all-c/s-8, 11, 14-eicosatrienoic acid 20:3c06 arachidonic acid all-c/s-5, 8, 11, 14-eicosatetraenoic acid 20:4<06 eicosapentaenoic acid all-cts-5, 8, 11, 14, 17-eicosapentaenoic acid 20;5u)3 behenic acid docosanoic acid 22:0 erucic acid c/s-13-docosenoic acid 22; lu9 c/s-15-docosenoic acid 22: lu7 SELOSTUS Tutkimus kevätrypsin (Brassica campestris L. var. annua) rasvahappojen analytiikasta ja jalostuksesta Into Laakso Helsingin yliopiston farmasian laitos 00170 Helsinki Kotimainen rypsiöljy on viimeisen kymmenen vuoden aikana saanut yhä tärkeämmän aseman elintarviketeol- lisuuden raaka-aineena. Nykyisistä rypsilajikkeista saa- tava öljy on varsin kilpailukykyistä, sillä erityisesti eru- kahappopitoisuuden suhteen laatuvaatimukset meillä ovat varsin tiukat. Viljelyä ei voida kuitenkaan suuressa määrin lisätä, sillä jalostavan teollisuuden mahdollisuudet kor- vata tuontiöljyjäkotimaisella raaka-aineella ovat suhteel- lisen rajoitetut johtuen ensisijaisesti rypsiöljyn alhaises- ta linolihappopitoisuudesta. Tämän tutkimuksen tarkoituksena oli yksilövalintaa apuna käyttäen kohottaa kevätrypsin siemenöljyn lino- lihappopitoisuutta sekä lutkia valinnan vaikutuksia ras- vahappokoostumukseen ja satoisuuteen. Tutkimuksessa verrattiin edelleen perinteisten ja uusimpien kaasukroma- tografisten menetelmien luotettavuutta rasvahappoana- lytiikassa ja pyrittiin optimoimaan jalostustyöhön par- haiten soveltuva analyysitekniikka. Jalostuskokeet tehtiin Hankkijan kasvinjalostuslaitok- sella Hyrylässä vuosina 1978—85 kanadalaista alkuperää olevasta kevätrypsiaineistosta. Linolihappolinjojen lopul- linen karsinta suoritettiin satoisuuden perusteella. Vapaapölytteisissäpelto-oloissa tehdyt jalostuskokeet osoittivat, että linolihappotasoa on mahdollista kohot- taa yksilövalinnalla vaikuttamatta kuitenkaan toisen mo- nityydyttämättömän komponentin, a-linoleenihapon, määrään. Parhaat valintalinjat sisälsivät linolihappoa n. 25 %, minkä lisäksi useat olivat erukahapottomia ja sa- toisuudeltaan nykyisten lajikkeiden veroisia. Kasvihuo- neolosuhteiden todettiin sitävastoin aiheuttavan huomat- tavaa lisävaihtelua, eikä valinnan vaikutusta linolihappo- tasoon voitu osoittaa. Pyrittäessä edelleen kohottamaan rypsiöljyn linolihappopitoisuutta saattaisi alhaisen a- linoleenihappotasonhuomioiminen yksilövalinnassa sa- manaikaisesti olla merkityksellistä linolihappovalinnan te- hostamiseksi. Alhaisesta monityydyttämättömien rasvahappojen määrästä huolimalta rypsiöljyllä on todettu olevan var- sin edulliset fysiologiset vaikutukset eritoten kolesteroli- tasoon. Tässä valossa on rypsiöljyä pidettävä vakavasti otettavana vaihtoehtona välttämättömien rasvahappojen lähteenä, mikä myös tulevaisuudessa on huomioitava ras- vahappokoostumusta jalostettaessa. 141