BREEDING FOROF OLEIC ACID

HIGH CONTENTIN SI.JNFLOWER

(Helianthus annuus L) OIL

J. FERNANDEZ-IYTARTI NEZ, J. DOMINGUEZ-GIMENEZ, A. JIMENEZ.RAMIREZ

Department of Breeding and Agronomy, SIA,D.G.I.E.A. Junta de Andalucia, C6rdoba (Spain)

INTRODUCTION

Sunflower oil is widely used in Spain andaround the world. In Spain, where the area de-dicated to this crop has surpassed one millionhectares, the consumption oi sunflower oil is si-milar to that of olive oil (Oleo, 1986). A greatdeal of this expansion has been due to the impactof the breeding work through the developmentof hybrids with increased seed yield and seed oilcontent. Less emphasis has been given to aspectsof oil quality susceptible to be changed by geneticmeans as the case of fatty acid composition, mainindex to define the quality of vegetable oils. Thereare two main fatty acids, in sunflower oil, oleic(18:l) and linoleic (18:2), which together ac-count for about 90 % of the total fattyacids, corresponding the remaining l0 /s to pal-mitic ( l6:0) and stearic ( l8:0). In the commercialhybrids, oleic acid represents between l0 and50 % under field conditions depending on en-vironment, especially temperature during seeddevelopment, existing a strong inverse relations-ship with linoleic acid (Ki nm a n and E a r l e,1974; Fe r n â n d e z-M a r t i n e z et al., 1986).

As in other vegetable oils, the quality of sun-flower oil is a relative concept which dependson the use of that oil (Fernândez-Mar-t i n e z and Alba, 1984). For example, forfrying purposes, oils with a high degree of oxi-dative stability i.e. those with htgh content ofoleic acid, such as olive oil, or hydrogenatedvegetable oils, are prefered (F u I I e r et al.,1967). For soft margarine industry oils withhigher level of unsaturation, higher levels oflinoleic are prefered. Under the nutritional pointof view researchers do not completely agreewhich type is more advisable although recentworks reported that a diet rich in monounsatu-rated lattv acids reduced.cholesterol in blood-

plasmâ which is a risk f actor for coronaryheart disease (G reen dy, 1986).

The high oleic acid sunflower variety Perve-nets was developed in U.S.S.R. following a treat-ment of seed with dimethyl sulfate (S o I d a-tov, 1976). Oleic acid levels in this varietyaveraged 75 /s allhough individual plants ran-ged from 5O /s Io 80 % (Mi I I e r and Zim-m e r m a n, 1983) and, seed to seed variationwas even higher ranging from 19 to 94 %(U r i e, 1985). Selected'breeding true high oleicprogenies lrom this variety were very stablewhen grown under different temperature regi-mes with oil content of oleic higher than 83 /o(Urie, 1985; Fern â n de z-Martinez etal., 1988).

Several studies have been carried out to elu-cidate the inheritance of the high oleic contentirr germplasm derive<l [rorn Pervenets. U r i't'(1985) concluded that the high oleic characterwas controlled by one single dominant gene OLwith embryo genotype control. Miller et al.(1987) found a second gene modilying the oleiccontent of the OL gene. Fernândez-Mar-t i ne z et al. (1988) found segregations thatindicated the presence of one, two and even threemajor complementary dominant genes contro-lling the character, depending on the genotypeof the low oleic parent.

The variety Pervenets was introduced in ourDepartment in 1980. We started a breeding pro-gramme with the aim of studying the inheritanceof the high oleic trait and also incorporating itto known restorers an.d male sterile inbred linesin order to develop high oleic commercial hy-brids.

The present paper describes the breeding pro-gramme used and the high oleic material obtai-ned.

't'l

BREEDING MATERIAL

The,original sunflower material used as higholeic source was derived from the open pollinatédvariety Pervenets. This material was grown in1980 in the field at the experimental farm <if theAgricultural Research Center of C6rdoba(Spain). Some plants were selfed and other crosspollinated among them. Ten of these plants wereanalysed for fatty acid composition using bulksamples of l0 seeds. In plants with oleic acidcontent higher than 60 /s, analyses of individualseeds were made using the half seed technique(Fernândez-Martinez and Know-I e s, 1982) which allows growing the otherhalf. Pollen of plants grown from seeds with le-vels of oleic acid higher than 85 /s were used

OLEIC RECURRENT PARENT.RHA- 271 qnd tlA-89.ooruon't8ln?h'rlîo-r-,

@------_,

Fl

BC1 'F1

BC2 F2

BCg Ft

BC6 F2

Bc6F3 W-,

Selection schem'e usedand RHA-271 lines

HIGH OLEIC ttNES: R-OL-t ond ,HA-OL-9

for incorporating the higft oleic character into the HA-89

to hand pollinate emasculated capitula ofRHA-274, a low oleic selfcompatible reslorer line.Ft seeds were analysed using again the half seedtechnique and those plants with oleic acid contenthigher than 85 /e were selected and growrr. Afterthree generations of selfing and selecliori for higholeic, breeding true lines for high content of ttiisacid were identified. One of these lines, quite selfcompatible and with oleic acid levels higher thang0 %, was identified as AOP-l and used-as donor'parent of this character in a backcross program-me to incorporate it into commercial material.The low oleic rnaterials used as recurrent parentswere the inbred lines HA-89 and cms HA-8g andRHA-271, maintainer, and its cytoplasmic malesterile and restorer lines respectively released bythe USDA and widely used in breeding pro-grammes in Spain.

SELFTNG OF SELECTED PLANTS..

DISCARDING SEGREGANTTO SELFED AND TEST

CAPITULA ON BASECROSSED ANALYSED SEED.o

o12

Fig. I. -

*

BCl Fl

BC2 F1

LOW OLEIC MALESTEBILË LINE,cms. HA-89

F1

THE STXTH GENERATION OF

AS RECURRENT PARENT.

BC6 Fl

DISCARDING

MALE STERILE LINE.(qms l'lA'OL-9)

Fig. 2. - Selection scheme used in obtaining the high oleic male sterile line cms HA-OL-9

HIGH OLETC BCa Fl PLANTS.( Bockcross of HA-89 to A0-P-l )See Fig. 1 '

HIGH OLETC Bq Fl PLANTS.

HIGH OLEIC BC5 F1 PLANTS.

HIGH OLEIC BC6, F1 PLANTS.

OLEIC PLANTS FROM

BACKROSSI NG To HA - eg ( BCe [,BCa Fr,etc. )

TWIN PLANTS OBTENTIONSEEDLING EXCISSION.

HIGH OLEIC LINE,

/d"rrrr( HA-oL-s )

OIL ANALYSES

Fatty acid analyses were carried out on oilextracted from bulk seeds of l0 seeds or halfseed analyses. Bulk seeds or the half seed werecrushed ahd ground in a glass mortar and ex-tracted using redistilled petroleum ether. Methylesters of the fatty acids were used for gas chrô-matographic analyses. The half seeds were ob-tained by previously soaking the seeds for 5 mi-nutes in sodium hypochlorite for surface steriliza-tion followed by rinsing in water and rt moval <-r[

| /3 to | /2 o1 the stilar part for analysis. Methylesters were Dr('t)ared bv the standard proceduresand analysed uling a Hewlett Packard gas chro-

matograph carrying a flame detector. The columnwas packed with 5 /o diethylene glicolasuccinateon 80/100 W/An ch-rommosorb.

Oil content analyses were made by NuclearMagnetic Resonancé (NMR)

BREEDING METHODS

The backcross programme was started in1982 using the low oleic lineé HA-89 andRHA-271 as recurrent parents and the high oleicone AOP- I as donor for high oleic. HA-89 andRHA-271 were emasculated and hand pollinatedwith pollen of the AOP-I line.'F, seed was ana-lysed using the half seed technique to obtain

13

ooo ES ON

F' plants with high oleic.'Pollen of these plantswas used to pollinate the recurrent low qleic linesto obtain BCrFr seeds (^Fig. 1). Further backcros-ses were always made using the low oleic re-current parents as female which were emascula-ted prior pollinations. As the high oleic characterwas dominant and controlled by embryo genoty-pe, heterozygous high oleic BC.Fr seeds wereidentified using half seed technique to obtainBC,Fr plants. Only BCnFr plants f rom-seeds with levels of oleic higher than 85 /swere used as pollinators in the backcross pro-gramme. BCuFr plants frorr both backcrossedparents (HA-89 and RHA-271), identified ashigh oleic carriers were selfed. BGF, plantswere selected for high levels of oleic and sel-fed again and homozygous plants breeding truefor the high oleic character were identified sel-fing and testcrossing to the low oleic pargnt.

The maintainer line HA-89 was converted intomale sterile (petiolaris cytoplasm) by backcros-sing the high oleic material to the male sterilelow oleic line cms HA-89 (Fig. 2). This conversionto male sterility was made para-llel to the incorpo-ration of the high oleic:to the maintainef HA-89after the third backcross of the oleic character.In each backcrossing generation high oleicplants were used as male parents to pollinatelow oleic male sterile female parent, at thesame time that pollinated the HA-89 line in thebackcrossing programme for introducing the'high oleic character. Male sterile,: high oleicsegregants were selected in each generationusing the half seed technique. The final selec-tion of male sterile plants, breeding true forhi'gh oleic genes, was made-'ogr'the basis ofteJt crosseslAs the plants werêjmâle sterile ana@uate number of twins BQF, plants were ob-tained using the method described by P a w-I o w.s k i (l9ti9). One of the twin plants wastesf-crossed to l'ow oleic HA-89 and the otherbackcrossed to HA-89 breeding true for higholeic acid content' (Fig. 2\.

RESULTS AND DISCUSSION

The original material derived from Pervenetswas quite self incompatible and did not breedtrue for high levels of oleic acid under the en-vironmental conditions of C6rdoba (SouthernSpain). The high level of variation for cileic acidcontent observed among seeds within the samecapitulum, 30 to 94 /s, conlirmed the influenceof the genotype of the embryo in the control ofoleic acid content observed in other crosses withthe same material (Fernânde z-Marti-nez et al., 1988 and U r ie, lg85). The linesobtained using the method described above aremorphologically similar to the recurrent lowoleic inbred lines, RHA-271 and HA-89 andcms HA-89. The high oleic restorer line hasbeen registered in t-he Spanish Catalogue asR-OL-I and the maintainer and male-sterileas HA-OL-9 and cms HA-OL-9. The mean oleiccontent of the high oleic lines varies from88 to 94 % in comparison to 25 to a0 /o o1the isogenic low oleic lines (Table /). The oleicvalue of R-OL-I was slishtlv hisher than thatof HA-OL-9 and cms HA--OL-9 ùnder the fieldconditions of C6rdoba, the oil content being alsohigher. The oil content of the new lines was alsohigher than of its isogenic low oleic lines.

The breeding method described above, back-crossing with selection at the seed level, couldbe applied because of the dominant nature of thehigh oleic character and the strong influence ofthe genotype of the embryo which allowed theuse of the half seed technique. Due to the highstability of the high oleic character, this methodallows the obtention of several generations peryear using greenhouses-or growth chamber fa-cilities. The final evaluation of the lines obtained.has to be done under normal field conditions forevaluation of other plants characters. Given thedominant nature of this trait it could be incorpo-rated to only one of the parental lines of onehybrid. However due to the strong gametophytic

Table IPlant and seed characteristlcs of high oleic parental lines R-OL-I

and HA-OL9 in comparison with their isogenic ltnl-ZZt and HA-g9 lines

Paretrtalline

Type of line Days tof lowe-

rino

Recessivebranching

Resistanceto downymil dew

oil Fatty acid composition % l)Palmitic Stearic ulelc Ltnotelc

R-OL-l

HA-OL-9 andcms HA-OL-9

RHA-27r

HA-89 andcms HA-89

restorer

maintainerand

male sterile

restorer

maintainerand

male sterile

87

79

86

79

Yès

No

Yes

No

Yes

No

Yes

No

50.0

45.2

48. I

44.4

3.7-4.9

4.1-5.6

5.0-6.5

5.4-6.7

2.2-4.1

1.9-3.4

3.5-4.4

3. l -4.3

90.7-93.7

87.6-91.5

29.8-37.6

31.5-40.3

0.5-2.9

Ll-3.8

50.9-61.3

47.5-58.6

l) Data from seed obtained under lield conditions at Côrdoba

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embryo control and ihe genes involved (F e r-n â nd ez-Martîn ez et al., 1988) these hy-brids would have lower oleic content becausethe seed on F, plants would be F, seed andwould segregate for high, intermediate and lowtypes. The mean oleic content would be around70 /s or even lower iÎ these hybrids are grownwithout isolation. However combining onlyhigh oleic lines, homozygous for the high oleicgenes, 90 Yo oï oleic could be obtained. Thismaterial would be stable and isolation wouldnot be necessary.

Soldatov K. I., 1976. Chemical mutagenesis in sun-f loaer breeding. In : Proc. 7th Int. Sunflower ConÎ.,Krasnodar, U.S.S.R., 27 June - 3 July. Interna-tional Sunflower Association Vlaardingen, p. 352-357, The Netherlands.

U r i e L., 1985. Inheritance of high oleic acid in sunfloaer.Crop Sci. 25 : 986-989

RËFERENCES

Fernândez MarIinez, J. and Knowles P.F.,1982. Materia,l and embrgo effects on the oleic andlinoleic acid contents ol sunfloaer oil. Proc. l0'nInt. Sunil. Conf., Surfers Paradise, Australia,24t-243.

Fernândez Martinez, J. and Alba E., 1984.Breeding for oil and meal qualitg in sunf loaer. Proc.Int. Symposium on science and biotechnology for

. an integral sunflower utilizations, 75-102.

Fernândez Martinez J., Jimenez 4", Rami-rez A., Dominguez-Gimenez J. and Alc â n t a r a 4., 1986. Temperature effect on the oleicand linoleic acid ol three genotgtpes in sunflouer.Grass y Aceites, 37 :326:33 (In S.panish).

Feràândez Martinez J., Jimenez 4., Do-. minguez J., Garcia J.M.,Garces R.,and. Mancha M., 1988. Genetic analgsis of the high

oleic qcid cantent in cultiaated sunfloaer. Euphg-tica (ln press).

Fuller M., Diamond. I. and Appteuthite T.,1967. High oleic sunfloaer oil. Stability and chemïcal modification. J. Amer. Oil Chem. Soc. 44:264-267.

Grundy S.M., 1986. Comparison ol monounsaturatedfattg acids and carbohgdrates for louering plasmacholesterol. N. Engl. J. Med. 314 : 745-748.

Kinman M.L. and Earle F.R., 1964. Agronomicperformance anQ chemical composition ol the seed ofsunfloaer hgbrids and introduced oarieties. CropSci., 4 : 417-420.

Miller J.F. and. Zimmerman D.C., 1983. Inheri-tance ol high oleic fattg acid content in sunflouer.In: Proc. Sunflower Research Workshop, Fargo,N. D. 26 Januarv. National Sunflower Association.Bismark, N. D. -

Milf er J.F.. Zimmerman D.C. and Vick B.4..1987. Genetic control of high oleic acid content insunfloaer. Crop. Sci. 27 : 923-926.

O I e o, 1986. Edit. Tecnipulicaciones, S. A. . ladrid. ArioXXV, Octobre 1986 (In Spanish).

Pawlowski S.H., 1963. A method for obtaining gene-ticallg identical sunfloaer plants.. Can. J. of Bot.4l : 743-745.

AMÊLIORATION POUR HÂUTES TENEURS EN ACIDEoLÉIQUE CHEZ LE TOURNESOL (Helianthus annuus L.l

Résumë

L'obtention d'hybrides de tournesol avec des hautes te-neurs d'acide oléique dans I'huile pourraient ouvrir de nou-veaux marchés pour cette huile. Dans ce travail on décritla méthode d'amélioration utilisée pour I'incorporation du ca-ractère haut oléique dans dç-s lignées pures restauratriceset mâles stériles cytoplasmiques avec I'objectif iinal de dé-velopper des variétés hybrides avec ces caractéristiques, Laméthode utilisée a été Ie back-croSs et sélection de semencesindividuelles par le moyen de la technique de la demi-semence.La méthode décrite permet la réalisation de plusieurs gé-nérations chaque année et I'obtention des lignées avec dehautes teneurs en oléique dans une période relativement cour-te. On a obtenu une lignée restauratrice, R-OL-1, partant dela lignée RHA-271 et les lignees mainteneuses de cms et mâlestérile, HA-OL-9 et cms HA-OL-9, partant des ligneesHA-89 et cms HA-89. Les nouvelles lignées sont similairesaux lignées originales mais avec une teneur en oléique d'en-viron 90 /o et avec de plus grandes teneurs en huile. Ondiscute, en fonction du contrôle génètique du caractère hautoléique, la çonvenance de l'*tilisation de deux lignées pa-rentales avec le caractère incorporé, dans le développementdes variétés hybrides.

MEJORA DEL GIRASOL (HELIANIHUS ANNUUS L.'PARA ALTOS CONTENIDOS DE ACIDO OLEICO

EN SU ACEITE

Resrimen

La obtenciôn de hibridos de girasol con alto contenidode âcido oleico en su aceite puede abrir nuevos mercadosa este aceite. Se describe en este trabajo el método de mejoraseguido para la introducci6n del carâcter alto oleico a lineaspuras restauradoras y androestériles con el objectivo finaldel desarrollo de hibridos con estas caraçteristicas. El mé-todo utilizado ha sido el de retroôruzamiento y selecciôn de,semillas individuales mediante la técnica de media sernilla.El método descrito permito la realizaciôn de varias genera-ciones por afro y la obtenci6n de lineas alto olèico en unperiodo relatiVamente corto. Se ha obtenido una linearestauradora, R-OL-I, a partir de la linea RHA-271 y laslineas mantenedoras v androesteril HA-OL-9 y cmsHA-OL-9 a partir de las lineas HA-89 y cms HA-{i9. Lasnuevas lineas son similares a las originales pero con uncontenido de âcido oleico de alrededor de fi /s y mayorcontenido de aceite. Se discute en funciôn del controlgenético del carâcter alto oleico la conveniencia de la uti-lizaciôn de ambas lineas parentales con el carâcter incor-porado en el desarrollo de hibridos.

15