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revised 6 April 2004

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Inheritance of High Oleic/Low Ricinoleic Acid Contentin the Seed Oil of Castor Mutant OLE-1

Pilar Rojas-Barros, Antonio de Haro, and Jose Marıa Fernandez-Martınez*

ABSTRACT low ricinoleic mutant OLE-1 could have industrial usesrequiring high oxidative stability such as for biofuel, orA mutant line of castor (Ricinus communis L.), OLE-1, was re-pharmaceutical applications requiring lower ricinoleiccently identified. It has a 20-fold increase in oleic acid (C18:1, about

780 g kg�1) and a six-fold decrease in ricinoleic acid content (C18:1-OH, levels than the standard castor oil. Moreover, this mu-about 140 g kg�1) compared with standard castor oil (C18:1, about tant signifies an important advance toward the develop-40 g kg�1; C18:1-OH, about 870 g kg�1). The objective of this research ment of ricinoleic acid free/high oleic acid castor oilwas to determine the inheritance of the high oleic/low ricinoleic trait germplasm with potential for the edible oil market.in this mutant. Reciprocal crosses were made between the mutant One requisite for the commercial use of the new oil isOLE-1 and castor line A74/18/10 with standard composition. Although the incorporation of the modified biosynthetic pathwaya slight maternal effect for oleic and ricinoleic content was observed

into commercial cultivars with good agronomic perfor-in the analysis of F1 seeds, the genetic control was mainly embryonic.mance, which requires a knowledge of the genetic be-The standard oleic/ricinoleic content was dominant over high oleic/lowhavior of the trait. The inheritance of high oleic/lowricinoleic content. Oleic acid content of F2 seeds segregated in bimodalricinoleic content in castor remains unexplained to date.patterns, each consistent with a ratio of 13 to 3 for low-intermediate oleic

content (�110 g kg�1) to high oleic content (�650 g kg�1), respectively. However, the genetic control of high oleic content hasThis segregation was consistent with the action of two independent been found to be simply inherited in different mutantsmajor genes (ol, Ml) with epistatic interaction. The high oleic/low ricino- of several oilseed crops. In soybean [Glycine max (L.)leic phenotype was homozygous for the genotypes with the recessive Merr.] and safflower (Carthamus tinctorius L.), it is con-allele ol, and heterozygous or homozygous for the dominant allele trolled by multiple recessive alleles at a single locus (Ta-Ml. The dominant allele Ml would release the action of the recessive kagi and Rahman, 1996; Knowles and Hill, 1964). Theallele ol, controlling the oleic and ricinoleic content. This mo_el was

content of oleic acid in the corn oil (Zea mays L.) wasconfirmed in the BC1F1 to OLE-1, which segregated following a 1:1shown to be controlled by single major gene (Widstrom(low-intermediate:high) ratio, and F3 segregations. The informationand Jellum, 1984) or by two independent genes (De laprovided by this genetic study will facilitate the transfer of the highRoche et al., 1971). In peanut (Arachis hypogaea L.),oleic/low ricinoleic trait to castor cultivars.Moore and Knauft (1989) found two loci, designed Ol1

and Ol2, controlling the high oleic/low linoleic ratio inseed oil. In sunflower (Helianthus annuus L.), the highThe quality of seed oils both for food and nonfoodoleic acid content was found to be controlled by a singleapplications is largely determined by their fatty acidpartially dominant gene designated Ol (Fick, 1984) orcomposition. The seed oil of castor normally containsa dominant gene (Urie, 1984). However, later studiesabout 900 g kg�1 of ricinoleic acid (D-12-hydroxyocta-demonstrated that the genetic control of the high oleicdec-cis-9-enoic acid) (Brigham, 1993) and is too highacid trait in sunflower was more complex. Urie (1985)for use as an edible oil but gives the oil its traditionalreported the existence of reversal in dominance andindustrial usage in manufacture of polymers, lubricants,modifying genes. Additionally, Miller et al. (1987) de-polyurethane coatings, cosmetics, plastics, and otherscribed a second locus, Ml, whose recessive alleles mlmlthings (Bonjean, 1991; Brigham, 1993). However, a nat-were necessary for the expression of the high oleic acidural mutant of castor, OLE-1, with significantly lesstrait, and Fernandez et al. (1999) also postulated a two-ricinoleic acid (C18:1-OH, about 140 g kg�1 comparedgene (Ol and Ml) model, in which the high oleic acidwith about 900 g kg�1 in commonly grown cultivars) andphenotypes are the result of the expression of the geno-increased oleic acid (C18:1, about 780 g kg�1 comparedtype ololMlMl. Finally, Fernandez-Martınez et al. (1989)with about 40 g kg�1 in standard castor bean oil) hasidentified three complementary dominant genes (Ol1,been developed by selection from a germplasm acces-Ol2, and Ol3) controlling the high oleic acid trait in sun-sion with high oleic content (Rojas-Barros et al., 2004).flower seed oil.This mutant probably has altered gene(s) encoding for

The objective of this study was to determine the inher-the oleoil-12-hydroxylase enzyme which catalyses theitance of the high oleic/low ricinoleic acid content ofhydroxylation of oleic to ricinoleic acid (Lin et al., 1996,the castor mutant line OLE-1.1998). Since high oleic acid levels are associated with

oxidative stability (Friedt, 1988), the oil of the high oleic/MATERIALS AND METHODS

P. Rojas-Barros, A. de Haro, and J.M. Fernandez-Martınez, Instituto Plant Materialsde Agricultura Sostenible (CSIC), Apartado 4084, E-14080 Cordoba,Spain. Received 16 Apr. 2004. *Corresponding author (cs9femaj@ The castor germplasm used in this study were (i) OLE-1,uco.es). a high oleic/low rinoleic mutant line developed by selection

from a germplasm accession with high oleic/low ricinoleic con-Published in Crop Sci. 45:�–� (2005).© Crop Science Society of America677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: GLC, Gas-liquid Chromatography.

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2 CROP SCIENCE, VOL. 45, JANUARY–FEBRUARY 2005

Table 1. Mean fatty acid content and their standard deviations of the oil for the castor line A74/18/10, mutant OLE-1, and their reciprocalF1 seeds, based on analysis of half-seeds from plants grown in Cordoba (Spain) during 1999.

Fatty acid (g kg�1) †

Germplasm n (p ) ‡ C16:0 C18:0 C18:1 C18:2 C18:3 C18:1-OH

A74/18/10 109 (5) 12 � 2 b§ 15 � 2 b 33 � 6 c 57 � 6 a 5 � 1 b 870 � 10 aF1 (A74/18/10 � OLE-1) 98 (2) 12 � 1 b 18 � 4 a 36 � 7 c 49 � 3 b 6 � 1 a 870 � 9 aF1 (OLE-1 � A74/18/10) 49 (1) 10 � 3 c 14 � 1 b 68 � 28 b 55 � 7 a 4 � 1 bc 840 � 25 bOLE-1 206 (2) 17 � 3 a 10 � 1 c 742 � 23 a 32 � 5 c 4 � 2 c 183 � 21 c

† C16:0 � palmitic acid, C18:0 � stearic acid, C18:1 � oleic acid, C18:2 � linoleic acid, C18:3 � linolenic acid, C18:1-OH � ricinoleic acid.‡ Number of half-seed (number of single-plants) analysed.§ Mean values of parents and reciprocal crosses for each fatty acid that have the same letter are not significantly different (LSD, p � 0.05).

tent (Rojas-Barros et al., 2004) and (ii) the line A74/18/10, with was assigned to phenotypic classes on the basis of the appear-a standard seed oil fatty acid profile (low oleic/high ricinoleic) ance of discontinuities in the frequency distribution and theselected from breeding material of PROTOSEMENCES Tou- values found in the parentals grown under the same environ-louse (France). The fatty acid composition of these materials mental conditions. The proportion of seeds observed in eachis shown in Table 1. phenotype class was compared with those expected on the

basis of appropriate genetic hypotheses. The goodness of fitto tested ratios was measured by the �2 statistic. HeterogeneityGenetic Study�2 for families within a cross was nonsignificant so that data

Seeds of OLE-1 and A74/18/10 were individually analyzed for families for the same cross were pooled for analysis.for fatty acid composition by the half seed method, describedfor castor by Rojas-Barros et al. (2004), to ensure that the plants

Fatty Acid Analysesused in the genetic study bred true for seed oil fatty acid composi-tion. A distal portion of the seed was removed with a scalpel The fatty acid composition of the seed oil was determined byand used to determine the fatty acid composition of seed lipids simultaneous oil extraction and methyl esterification followingby gas–liquid chromatography (GLC). The remaining portion the procedure described by Rojas-Barros et al. (2004) thenof the seed containing the embryo, with a known fatty acid analyzed by GLC on a PerkinElmer Autosystem gas–liquidprofile was used for planting. As the trait high oleic/low ri- chromatograph (PerkinElmer Corporation, Norwalk, CT)cinoleic is associated with very poor germination (Rojas-Barros equipped with a flame ionization detector and with a 2-m-longet al., 2004), mature embryos of half seeds with this trait, were column packed with 3% SP-2310/2% SP-2300 on Chromosorbrescued by in vitro culture with Knudson C Modified Orchid WAW (Supelco Inc., Bellefonte, PA). The injector, and flameMedium (Knudson, 1946). Plants of OLE-1 were reciprocally ionization detector were held at 275 and 250�C, respectively.crossed with plants of A74/18/10 in the greenhouse in 1999. The gas chromatograph was programmed for an initial ovenIn all cases, paper bags were placed over racemes to prevent temperature of 190�C maintained for 10 min, followed by ancross-pollination with external pollen. Crossing was done by increase of 5�C m�1 up to 225�C, holding for 7 min.emasculating immature flower buds in the raceme of the fe-male parent followed by immediate pollination of their stigmaswith fresh pollen from open flowers of the male parent. RESULTS AND DISCUSSION

F1 half seeds from reciprocal crosses as well as seeds fromthe parents were analyzed for fatty acid composition from The average oleic and ricinoleic acid content of theplants grown in the greenhouse in 2000. F1 plants from recipro- mutant line OLE-1 were 20-fold higher and five-foldcal crosses were self-pollinated to obtain F2 seeds and also lower, respectively, than those of the standard low oleic/backcrossed to both parents. Reciprocal crosses were repeated high ricinoleic line A74/18/10 (Table 1). The oleic andagain to obtain F1 seeds grown under the same environment ricinoleic acid content in reciprocal F1 seeds differedas the F2 and BC1F1 seeds. An evaluation of the fatty acid

significantly indicating the presence of partial maternalcomposition of F1 plants was made by averaging the GLCeffects for these traits (Table 1). However, these differ-analyses of the F2 seeds from each individual F1 plant. Fattyences were much smaller than those between the F1 andacid composition was determined on a total of 999 individual

F2 seeds, 272 BC1F1 to A74/18/10 seeds, and 204 BC1F1 to each of the parents indicating that the genetic controlOLE-1 seeds. of the contents of these fatty acids was mainly embryonic

A total of 21 F2 half-seeds representing all the classes for with a minor maternal effect. The differences betweenoleic acid concentration detected in this generation were se- reciprocal F1 seeds were not observed between F1 plantslected, germinated, and grown in a field screenhouse in 2001 (F2 seeds averaged) (Table 2) revealing an absence ofto obtain the F3 generation. The study of this generation was

cytoplasmic effects for both fatty acids. Since no signifi-performed through the analysis of 100 to 200 F3 seeds fromcant cytoplasmic effects could be detected the data fromeach segregating F2 plant and about 20 to 100 seeds for eachreciprocal F2 seeds in Fig. 1 and F1, F2, and BC1F1 seeds innonsegregating F2 plant.Fig. 2 were combined. Similar results, embryonic controlwith a partial maternal effects of low magnitude in someStatistical Analysescrosses and the absence of cytoplasmic effects, have been

Mean oleic and ricinoleic acid content was calculated for reported for oleic acid content in safflower (Knowlesthe parental lines, the F1, and F2 generations and comparedand Hill, 1964), sunflower (Urie, 1984, 1985; Fick, 1984;by the LSD test. Since the results did not reveal importantMiller et al., 1987; Fernandez-Martınez et al., 1989),maternal effects for oleic and ricinoleic acid content, the fattyrapeseed (Schierholt et al., 2001), and soybean (Takagiacid composition of segregating generations was analyzed on

single seeds. The oleic acid content of BC1F1, F2, and F3 seeds and Rahman, 1996; Rahman et al., 1994). There are no

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ROJAS-BARROS ET AL.: INHERITANCE OF HIGH OLEIC CONTENT IN CASTOR 3

Table 2. Oleic acid content of F1 half-seeds and mean and range of oleic and ricinoleic acid content of the oil of F2 seeds from individualF1 plants of reciprocal crosses between castor lines OLE-1 and A74/18/10 grown in Cordoba (Spain) during 2000.

C18:1 content in F2 seeds C18:1-OH content in F2 seedsF1 plant (F2 averaged seeds) C18:1 content inor parent seed F1 half-seeds n† Mean Range Mean Range

g kg�1

A74/18/10 63 31 19–46 872 843–893OLE-1 100 751 685–795 176 151–239F1–1 (A74/18/10�OLE-1) 45 150 199 a‡ 21–798 710 a 117–936F1–2 (A74/18/10�OLE-1) 25 149 210 a 19–835 697 a 86–887F1–3 (A74/18/10�OLE-1) 29 100 197 a 24–787 709 a 132–88.8F1–4 (OLE-1�A74/18/10) 109 150 146 a 19–802 769 a 115–902F1–5 (OLE-1�A74/18/10) 110 150 174 a 17–833 741 a 103–899F1–6 (OLE-1�A74/18/10) 19 150 186 a 20–792 729 a 141–892F1–7 (OLE-1�A74/18/10) 25 150 173 a 31–812 734 ab 117–879

† Number of analysed half-seeds; C18:1 � oleic acid; C18:1-OH � ricinoleic acid.‡ Means values for each fatty acid of the reciprocal in F2 families that have the same letter are not significantly different (LSD test, p � 0.05).

Fig. 1. Scatter plot of oleic and ricinoleic acids content in the oil of F2 seeds of reciprocal crosses between castor A74/18/10 and OLE-1.

previous studies on maternal and cytoplasmic effects on cinoleic acids are under the control of one genetic system.The oleic acid values of the F2 seeds from OLE-1 �the content of ricinoleic concentration.

The average oleic-acid content of F1 seeds from recip- A74/18/10 and A74/18/10 � OLE-1 crosses revealed aclear bimodal distribution (Fig. 1 and 2). The first classrocal crosses (52 g kg�1) was very close to that of the

standard low line A74/18/10 (Table 1) and much lower with seeds having �110 g kg�1of oleic acid was assignedto the combined category “low-intermediate” with anthan the midparent value (387 g kg�1) indicating almost

complete dominance of the standard low over the in- oleic acid range between 19 and 110 g kg�1 and the secondclass to the “high” category (oleic acid � 650 g kg�1).creased oleic acid content. This result is in agreement

with those obtained in safflower (Knowles and Hill, 1964). The observed data satisfactorily fit a phenotypic ratio13 low-intermediate: 3 high for these classes (Table 3).However, in sunflower several authors reported domi-

nance of high oleic over low oleic acid content (Miller et This segregation suggests that the high oleic/low ri-cinoleic content is determined by an interaction betweenal., 1987; Fernandez-Martınez et al., 1989). The average

ricinoleic-acid content in F1 generation (855 g kg�1) was a recessive allele at one locus and a dominant allele ata second locus (dominant and recessive epistasis). Thesesimilar to that of the standard low oleic/high ricinoleic

line A74/18/10 (Table 1) and higher than the mid-parent alleles were designated ol and Ml respectively, followingthe symbols previously assigned to genes controllingvalue (526 g kg�1) suggesting dominance of standard

over reduced ricinoleic acid concentration. oleic acid content in sunflower (Miller et al., 1987). Theproposed genotypes for the high oleic-low ricinoleic acidThe analysis of fatty acid composition of individual

F2 seeds from reciprocal crosses between OLE-1 and mutant line OLE-1 was ololMlMl and for the low oleic-high ricinoleic acid line A74/18/10, OlOlmlml. With thisA74/18/10, showed a considerable variation for oleic

and ricinoleic acids (Fig. 1), which were strongly and genetic model the genotypes with high levels of oleicacid would be homozygous for the recessive allele ol.negatively correlated (r � –0.99, P � 0.001). There was

no substantial variation for the other fatty acids. This The olol is recessively epistatic to Ml locus and geno-types with these alleles (ololMl_) would produce highindicated that the relative proportions of oleic and ri-

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4 CROP SCIENCE, VOL. 45, JANUARY–FEBRUARY 2005

expected to be in the low-intermediate class in the back-cross to the low oleic parent A74/18/10. The data ob-served (Fig. 2) fitted satisfactorily the theoretical ratios(Table 3) supporting the proposed model.

As a further confirmation of the genetic model pro-posed a progeny test was conducted on crosses by ana-lyzing the F3 from each of 21 F2 plants. These plantswere selected on the basis of the oleic acid content of thecorresponding F2 half-seeds, which covered the wholerange of oleic levels observed in the F2 population. TheF3 seeds derived from F2 half-seed plants with low andintermediate oleic values (23-128 g kg�1) showed threedifferent patterns (Table 4). Four of them bred true forvalues of this fatty acid below 50 g kg�1. The genotypesof these plants were identified as homozygous either forthe ml or the Ol allele (genotypes _ _mlml or OlOl_ _).Five segregated for high (�700 g kg�1) oleic acid valueswith a 3:1 (low-intermediate:high) ratio and nine segre-gated with a 13:3 (low-intermediate:high) ratio. The seg-regation 3:1 (one locus) would correspond to a genotypeOlolMlMl and the segregation 13:3 (two loci) would beexpected for the progeny of the F2 genotype OlolMlml.In contrast, all the F3 progenies derived from F2 half-seeds plants with oleic acid content higher than 700 g kg�1

showed no segregation for this fatty acid, the oleic acidcontent of all the F3 seeds being higher than 700 g kg�1.The genotypes of these plants were identified as homo-

Fig. 2. Frequency of distribution of oleic acid (C18:1) in oil from zygous for ol and Ml allele (genotype ololMlMl). How-individual seeds of castor A74/18/10, OLE-1 and their F1, F2 andever, according to the genetic model proposed, F2 half-BC1F1 populations of combined data of reciprocal crosses.seeds plants with high oleic acid values (�700 g kg�1)

Table 3. Number of seeds having different oleic acid (C18:1) content and Chi-square analyses in the F2 and BC1F1 seeds from crossesbetween the standard castor line A74/18/10 and the mutant line OLE-1.

No. of seeds with C18:1 content

Low-intermediate HighGeneration (�110 g kg�1) (�650 g kg�1) �2 (p )†

F2 (A74/18/10�OLE-1) 118 32 0.60 (0.44)F2 (A74/18/10�OLE-1) 115 34 1.41 (0.24)F2 (A74/18/10�OLE-1) 78 22 0.61 (0.43)F2 (OLE-1�A74/18/10) 128 22 1.99 (0.16)F2 (OLE-1�A74/18/10) 122 28 0.00 (0.98)F2 (OLE-1�A74/18/10) 121 29 0.03 (0.86)F2 (OLE-1�A74/18/10) 125 25 0.46 (0.50)Pooled 807 192 0.14 (0.70)Heterogeneity 4.96 (0.55)BC1F1 ((A74/18/10�OLE-1)�A74/18/10) 74BC1F1 ((A74/18/10�OLE-1)�A74/18/10) 82BC1F1 ((OLE-1�A74/18/10)�A74/18/10) 66BC1F1 ((OLE-1�A74/18/10)�A74/18/10) 50BC1F1 ((A74/18/10�OLE-1)�OLE-1) 25 23 0.08 (0.77)BC1F1 ((OLE-1�A74/18/10)�OLE-1) 60 43 2.88 (0.09)BC1F1 ((OLE-1�A74/18/10)�OLE-1) 28 25 0.17 (0.68)Pooled 113 91 2.40 (0.12)Heterogeneity 0.73 (0.69)

† Ratios tested: F2 generation � 13:3 and BC1F1 to OLE-1 generation � 1:1, and p is the probability level for significance.

oleic/low ricinoleic whereas the ololmlml genotypes heterozygous for the Ml gene (genotype ololMlml) wouldbe expected to segregate 1 low-intermediate (genotypewould produce low oleic/high ricinoleic. The F2 pheno-

typic expression of these genotypes would be in the ololmlml): 3 high (genotypes ololMlMl and ololMlml).The absence of this segregation in the progeny of highratio 13 low-intermediate (including genotypes Ol_Ml_,

Ol_mlml and ololmlml): 3 high (genotype ololMl_). oleic/low ricinoleic F2 half-seeds plants may be attributedto the fact that only three F3 families from high oleicAccording to the proposed genetic model, a ratio of

1 low-intermediate (including genotypes OlolMlMl and F2 half seeds were evaluated due to the poor germinationof F2 half seeds with this phenotype (Rojas-Barros et al.,OlolMlml): 1 high (including genotypes ololMlMl and

ololMlml) was to be expected in the backcross to the 2004) and apparently none of them had the ololMlmlgenotype.high oleic plant OLE-1, whereas all the individuals were

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ROJAS-BARROS ET AL.: INHERITANCE OF HIGH OLEIC CONTENT IN CASTOR 5

Table 4. Number of F3 castor seeds having a different oleic acid (C18:1) content in the analysis of 21 F3 families from the cross A74/18/10 � OLE-1 and Chi-square (�2) analyses.

No. F3 seeds in C18:1 classes�2 (p ) �2 (p )

F3 family C18:1 in F2 half-seed C18:1 of F3 family † Low-intermediate High 1-gene 3:1 2-genes 13:3

g kg�1

F3–36 23 196 122 33 0.60 (0.44)F3–73 33 39 20F3–48 37 178 121 29 0.03 (0.86)F3–678 39 43 20F3–669 39 210 112 36 0.04 (0.84)F3–675 39 269 78 35 1.89 (0.17)F3–83 44 43 20F3–63 48 147 119 23 0.67 (0.41)F3–72 51 40 20F3–664 57 233 176 61 0.07 (0.79)F3–707 78 223 125 42 0.00 (0.96)F3–690 87 171 128 28 0.07 (0.80)F3–698 89 200 117 32 0.66 (0.41)F3–674 104 192 118 32 0.60 (0.44)F3–75 104 217 113 37 0.01 (0.92)F3–706 105 181 168 43 0.35 (0.55)F3–686 109 191 120 32 0.48 (0.49)F3–114 128 190 118 30 0.21 (0.65)F3–684 788 789 148F3–99 791 832 116F3–702 815 751 100

† Mean value of analysed F3 seeds.

No previous studies have been reported on inheri- ACKNOWLEDGMENTStance of altered oleic acid content in castor. However, This work was supported by the European Commission un-similar genetic systems with two genes and epistatic der the project No AIR3-CT93-2324.interaction have been proposed for the control of oleicacid content in sunflower (Miller et al., 1987; Fernandez REFERENCESet al., 1999). These studies concluded that the desatura-

Bonjean, A. 1991. Le Ricin. Une culture pour la chimie fine. Castortion of oleic to linoleic acid was controlled by a major cultivation for chemical applications. Galileo/ONIDOL, Les Lilas,gene which action was modified by a second gene. Marker France.

Brigham, R.D. 1993. Castor: Return of an old crop. p. 380–383. Inanalyses supported that the oleoyl-PC-desaturase locusJ. Janick and J.E. Simon (ed.) New crops. John Wiley & Sons,(OLD7) was altered in the high oleic sunflower mutantNew York.(Perez-Vich et al., 2002) and other molecular studies De la Roche, I.A., D.E. Alexander, and E.J. Weber. 1971. Inheritance

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high oleic acid content in sunflower oil. J. Genet. Breed. 53:99–103.OLD7 locus on the high oleic trait. In the present study,Fernandez-Martınez, J.M., A. Jimenez, J. Domınguez, J.M. Garcıa,the recessive gene ol present in the high oleic/low ri- R. Garces, and M. Mancha. 1989. Genetic analysis of the high oleic

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Fick, G.N. 1984. Inheritance of high oleic in the seed oil of sunflower.droxylation of oleic acid to synthesize ricinoleic acid.p. 9. In Proc. Sunflower Research Workshop, Bismark, ND. 1 Feb.Recessive alleles at the Ml locus would suppress the National Sunflower Association, Bismark, ND.

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