Published in Crop Sci. 44:76-80 (2004).
© 2004 Crop Science Society of America
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CROP BREEDING, GENETICS & CYTOLOGY
Isolation of a Natural Mutant in Castor with High Oleic/Low Ricinoleic Acid Content in the Oil
Pilar Rojas-Barros,
Antonio de Haro,
Juan Muñoz and
José María Fernández-Martínez*
Instituto de Agricultura Sostenible (CSIC), Apartado 4084, E-14080 Córdoba, Spain
* Corresponding author (cs9femaj{at}uco.es).
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ABSTRACT
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Castor (Ricinus communis L.) oil is characterized by high levels of ricinoleic acid content (about 900 g kg–1) and low levels of oleic acid (about 30 g kg–1). A total of 191 accessions of a germplasm collection of castor were evaluated for oil content and fatty acid composition of the seed oil with an attempt to widen the variability for these traits in this species. As a result of this evaluation, the natural mutant line OLE-1 with approximately 780 g kg–1 of oleic acid, compared with 40 g kg–1 of the standard castor oil, was identified. The dramatic increase in oleic acid was accompanied by a decrease in the level of ricinoleic acid to 140 g kg–1, compared with 870 g kg–1 in normal plants, and only very small changes in the proportions of other fatty acids. These proportions of oleic acid and ricinoleic acid are respectively the highest and the lowest yet reported in stable genotypes of castor. OLE-1 is a promising source of high oleic acid levels with potential industrial and food applications requiring very high oxidative stability and may be also useful for studying the biosynthesis and genetics of ricinoleic acid content in castor.
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INTRODUCTION
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THE INDUSTRIAL PROPERTIES and potential food applications of fats and oils stored in the seeds or fruits of oil crop plants are largely determined by their fatty acid (FA) composition. The industry demands oils with a maximum concentration of the desired fatty acid because the use of such oils contributes to a reduction in the amount of waste and represents considerable savings in processing costs (Lühs and Friedt, 1995; Murphy, 1999). Many efforts are now underway to manipulate the seed oil quality through production of new cultivars with relatively homogeneous oil composition targeted toward a specific end use (Murphy, 1999). Modifications in FA composition are the results of blockages in the steps of FA biosynthesis that have been accomplished in different ways (for review see Röbbelen, 1990). Natural variability for these traits has been observed in several oil crops such as safflower (Carthamus tinctorius L.; Knowles, 1989), peanut (Arachis hypogea L.; Norden et al., 1987), and rapeseed (Brassica napus L.; Stefansson and Hougen, 1964). However, no natural variability has been found in most cultivated oil seed species (Rattray, 1991), and additional variability has been induced only by mutagenesis or genetic engineering in many other crops (Ohlrogge, 1994; Velasco et al., 1999). Castor oil is characterized by high levels of ricinoleic acid (about 900 g kg–1) and low levels of other fatty acids (about 30 g kg–1 oleic, 40 g kg–1 linoleic, and 30 g kg–1 saturated fatty acids; Brigham, 1993). Ricinoleic acid (D-12-hydroxyoctadec-cis-9-enoic acid) is a hydroxylate fatty acid which has many industrial uses (Bonjean, 1991; Brigham, 1993). It has been identified as a constituent of the seed storage oil in at least 12 genera of higher plants (van de Loo et al., 1993), such as Linum (Linaceae) (Green, 1984), and Lesquerella (Brassicaceae) (Broun et al., 1998). Current knowledge on fatty acid biosynthesis in castor indicates that ricinoleic acid in maturing castor endosperm is synthesized by hydroxylation of an oleic acid precursor (Lin et al., 1996, 1998). The natural variability for ricinoleic acid contents of castor seed oil has been observed to range from 585 to 923 g kg–1 compared with 20 to 56 g kg–1 for oleic acid (Binder et al., 1962; Lakshminarayana et al., 1984; Da Silva Ramos et al., 1984; Bhardwaj et al., 1996).
Though the main use of castor oil is based on its high content of ricinoleic acid, which distinguishes it from other seed oils, the identification of variants with low levels or free from this fatty acid, and increased levels of oleic acid could provide wider markets for this crop. Oils with high oleic levels are optimal for food and industrial applications requiring high oxidative stability (Friedt, 1988) and have been developed in safflower (Knowles, 1989), canola (Auld et al., 1992), and sunflower (Soldatov, 1976). Moreover, the identification of the genes involved in the late steps of fatty acid desaturation and hydroxylation in castor would be of interest to study the genetic and biochemical mechanisms that regulate the stepwise desaturation from oleic to linolenic acid, and hydroxylation from oleic to ricinoleic acid. Toward achieving these objectives, we have identified a natural castor mutant with a very high oleic acid and low ricinoleic acid content through extensive screening of a world germplasm collection. This paper describes the screening strategy, isolation, and characterization of this mutant.
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MATERIALS AND METHODS
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In 1998, 191 accessions of castor seed, obtained from the Southern Regional Plant Introduction Station, USDA-ARS-NRGEEC, Griffin, GA, USA, were planted in the field at the experimental farm of the Institute of Sustainable Agriculture at Cordoba, Spain, in a nonreplicated, one-row plot 3 m long. At flowering, several plants of each entry were self-pollinated by bagging the first racemes with paper bags. Bulk samples of seeds from each S0 plant (S1 seeds) were screened for oil content, seed weight, and fatty acid composition. The oil content was measured by nuclear magnetic resonance (NMR). The fatty acid composition of the seed oil was determined by gas liquid chromatography (GLC).
On the basis of the results of this screening, S1 seed from one plant with contrasting high levels of oleic acid was further analyzed by the half seed method described in other oilseed crops (Knowles, 1989). A distal portion of the seed was removed with a scalpel and used to determine the fatty acid composition of seed lipids by GLC. The remaining portion of the seed containing the embryo, with a known fatty acid profile was transplanted into soil in pots and grown under greenhouse conditions [35/15°C (day/night) with 16-h daylength] in the winter of 1998 to obtain S1 plants. At flowering S1 plants were self pollinated and at maturity the S2 seeds of each plant were harvested separately and analyzed for fatty acid composition. The S1 seeds with high oleic acid/low ricinoleic acid content failed to germinate but three S1 plants derived from S1 seeds with standard fatty acid profile segregated for high oleic acid/low ricinoleic acid content. Half S2 seeds from these plants were transplanted into pots and grown outdoors in spring–summer 1999. As S2 half seeds with high oleic acid/low ricinoleic acid content, again failed to germinate, mature embryos of these seeds were rescued by in vitro culture with Knudson C Modified Orchid Medium (Knudson, 1946) and the plantlets obtained were then transplanted to pots. The progenies of S2 plants were analyzed by GLC. To confirm the fixation of the high oleic acid/low ricinoleic acid character and to identify heterozygous S2 plants, the half seed technique was applied to S3 seeds. Selected S3 half seeds were grown in a greenhouse in autumn–winter 1999. Half seeds with high oleic acid/low ricinoleic acid content were rescued as described above. To determine any possible alteration in the normal seed development, fatty acid composition, oil content, and seed volume were determined individually for all S3 seeds collected from two heterozygous S2 plants.
For fatty acid composition of bulk samples, 15 seeds or individual seeds were used. Lipids were simultaneously extracted and methylated following a modification of the procedure proposed by Garces and Mancha (1993). The seed samples, consisting of pieces of 15 seeds for bulk samples or half seeds for individual seed analyses, were placed into a 22-mL tube in a solution of methanol:toluene:H2SO4 (88/10/2, v/v/v) in a proportion of 50 mg of seed sample:5 mL of solution and heated at 80°C for 1 h. After cooling, 1 mL of heptane was added and mixed with each sample. The mixture was shaken for 30 s and a solution of NaCl was then added. After 30 min the fatty acid methyl esters were recovered from the upper phase and analyzed by GLC. The fatty acid composition of the seed oil was determined on a Perkin-Elmer Autosystem gas-liquid chromatograph (Perkin-Elmer Corporation, Norwalk, CT, USA) equipped with a flame ionization detector (FID) and a 2-m-long column packed with 3% SP-2310/2% SP-2300 on Chromosorb WAW (Supelco Inc., Bellefonte, PA, USA). The injector and flame ionization detector were held at 275, and 250°C, respectively. The gas chromatograph was programmed for an initial oven temperature of 190°C maintained for 10 min, followed by an increase of 5°C m–1 up to 225°C, holding for 7 min.
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RESULTS
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The analysis of the germplasm collection of castor showed rather sparse variability for total oil content (448–565 g kg–1) and fatty acid composition (Table 1). However, an individual plant of the accession PI 179729 from India showed a marked deviation from the range of other plants of the same accession and from the rest of accessions in oleic acid content (189 g kg–1 compared with 36–56 g kg–1) and in ricinoleic acid content (714 compared with 844–880 g kg–1) (Table 1). Individual S1 seeds belonging to this variant plant were analyzed by GLC using the half seed technique. The seed to seed variation showed a tremendous range for oleic (17–832 g kg–1) and ricinoleic acid content (99–886 g kg–1) (Table 2). S1 seeds from the variant plant were sown. All the seeds with high oleic acid/low ricinoleic acid content failed to germinate because of a breakdown of the endosperm and death of the embryo and no S1 plants were obtained. In contrast, S1 plants from S1 seeds from the variant plant with standard oleic and ricinoleic values were established and S2 seeds from these plants were analyzed by GLC using the half seed technique. Three of these S1 plants, Or80-3, Or80-6, and Or80-13, were heterozygous and segregated for high oleic acid/low ricinoleic acid content (Fig. 1). S2 seeds obtained from these three plants showed a bimodal distribution for oleic acid content (Fig. 1) with a low-intermediate class ranging from 16 to 230 g kg–1 and a high oleic class with values higher than 700 g kg–1 with approximately four times more individuals in the low-intermediate than in the high oleic acid class. Variation in oleic acid and ricinoleic acid in these seeds maintained a strong negative correlation (0.99, P < 0.001). Most of the S2 seeds from these plants, with a high oleic acid/low ricinoleic acid concentration, again failed to germinate, but six S2 plants from seeds with this phenotype could be established after in vitro culture of mature embryos. Taking advantage of the perennial habit of castor, these plants were established in big pots in the greenhouse to produce seeds in following years.
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Table 1. Fatty acid composition of the seed oil of 191 accessions from a castor germplasm collection, and normal and variant plants of accession PI 179729.
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Table 2. Fatty acid composition of individual S1 castor seeds derived from the variant S0 plant of accession PI 179729.
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Fig. 1. Pedigree and oleic acid concentration in S0, S1, S2, and S3 generations of a variant castor plant of PI 179729: a) fixation of ‘OLE-1’ mutant through the S1 plant Or80-3; b) fixation of ‘OLE-1’ mutant through the S1 plant Or80-6; c) fixation of ‘OLE-1’ mutant through the S1 plant Or80-13. In S0 generation, bulk seed samples were analyzed. In S1, S2, and S3, half-seeds were analyzed.
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In the next generation, all the S3 seeds of the six S2 plants obtained from high oleic acid/low ricinoleic acid S2 seeds expressed the character uniformly with oleic acid values ranging from 734 to 832 g kg–1 (Table 3, Fig. 1) and ricinoleic acid values ranging from 101 to 188 g kg–1 (Table 3). These results indicated that the high oleic acid/low ricinoleic acid levels were effectively fixed in the S3 generation (Fig. 1). OLE-1 was formed by bulking seeds from these six S2 plants. The oleic acid content of the seed oil of the natural mutant OLE-1 had an average of 784 g kg–1, which represented a greater than 20 fold increase in oleic acid compared with the accession PI 179729 with standard composition (Table 3). Conversely, the average value of ricinoleic acid content was 140 g kg–1 compared with 869 g kg–1 of control plants. The proportion of stearic acid and linoleic acid was lower than that of the control while the content of palmitic acid was higher (Table 3). Similar levels of oleic acid and ricinoleic acid were observed in seeds of OLE-1 in subsequent generations. S3 plants breeding true for the mutant character as well as heterozygous plants were established in large pots and the field for further genetic and biochemical studies. The oil content, seed weight, seed volume, and weight/volume of individual seeds developed under the same environmental conditions having standard (low) and high oleic acid content were determined. The seeds of the mutant OLE-1 showed a significant reduction compared with standard seeds in oil content (368 vs. 530 g kg–1), seed weight (0.39 vs. 0.54 g), and weight/volume (0.42 vs. 0.57 g cm–3).
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Table 3. Fatty acid content of S3 castor seeds from S2 plants of natural mutant OLE-1 derived from S2 high oleic/low ricinoleic seeds selected from S1 plants Or80-3, Or80-6, and Or80-13 and of seeds of normal plants of accession PI 179729 (control line) .
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DISCUSSION
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The variability of 191 accessions for oil content and for oleic acid and ricinoleic acid content, with the exception of the variant plant that produced the mutant line OLE-1, was lower than previously reported for this species (Binder et al., 1962; Lakshminarayana et al., 1984; Da Silva Ramos et al., 1984; Bhardwaj et al., 1996). However, the average concentration of oleic acid (about 780 g kg–1) and ricinoleic acid (about 140 g kg–1) of the line OLE-1 largely exceeded the ranges reported by these authors for castor oil (20–56 g kg–1 for oleic and 590–920 g kg–1 for ricinoleic acid). The high oleic acid content of OLE-1 was almost exclusively at the expense of ricinoleic acid with no significant changes in the concentration of other fatty acids, with the exception of a slight decrease in linoleic acid and increase in palmitic acid concentration with respect to the control line. The strong negative correlation between oleic acid and ricinoleic acid, not previously reported, indicated that the relative ratios of oleic acid and ricinoleic acid content may be under the control of one genetic system. The biosynthetic pathway leading to the production of ricinoleic acid in developing seeds of castor is generally considered to involve the insertion of a hydroxyl (OH) group in the twelfth carbon of an oleic acid precursor by the oleoyl-12-hydroxylase enzyme located in the endoplasmic reticulum (Lin et al. 1996, 1998). Because oleic acid is the precursor for the synthesis of ricinoleic acid, it seems that the concomitant increase in oleic acid and decline in ricinoleic acid observed in OLE-1 is the result of a natural mutation or mutations in the gene or genes responsible for the hydroxylation of oleic acid. The high oleic acid/low ricinoleic acid mutant OLE-1 was identified in S1 seeds, with similar concentrations of these fatty acids being found in the following generations, indicating that the gene(s) controlling this character was homozygous in some seeds of the original (S0) plant analyzed. Moreover, the seed to seed segregation observed in the heterozygous S0 and S1 plants, obtained from seeds with standard, low oleic acid/high ricinoleic acid, content indicated that the control of the character high oleic acid/low ricinoleic acid depends on the genotype of the embryo and is recessive as was the case of the high oleic acid concentration in safflower (Knowles, 1989) and low erucic acid concentration in rapeseed (Stefansson and Hougen, 1964). The phenotype with high oleic/low ricinoleic acid content was associated with very poor germination and lower seed weight and oil content compared with seeds of the same plant with normal phenotype. Apparently, the homozygous state of the high oleic acid/low ricinoleic acid trait brought gross changes in the physiological as well as biochemical pathway for fatty acid acid synthesis during the seed development stage.
The interest in genetically engineering ricinoleic acid accumulation into oilseeds crops more agronomically productive than the castor plant has stimulated extensive research on the enzymes involved in the hydroxy FA synthesis. This has led to the cloning of the gene encoding castor oleate 12-hydroxilase enzyme (van de Loo et al., 1995). However, the poor accumulation of ricinoleic acid (200 vs. 90 g kg–1 ricinoleic acid in castor) in transgenic seeds with castor oleate 12 hydroxylase cDNA clone (van de Loo et al., 1995; Broun and Somerville, 1997) indicates that there are other enzymes with specificity for transfer of hydroxy fatty acid into triacylglycerols (TAG). The low ricinoleic mutant OLE-1 identified in this study could be useful for characterizing those enzymes and developing transgenic plants that produce higher levels of ricinoleic acid. Moreover, the high oleic acid levels (>750 g kg–1) of this line are optimal for food and/or industrial applications requiring high oxidative stability. Crosses between the high oleic acid genotype and different low oleic acid lines are in progress to carry out genetic studies and to search for a further increase in oleic acid content.
In conclusion, the present research has identified the natural mutant OLE-1 in castor that has the highest oleic acid and the lowest ricinoleic acid content known to date in this crop. Ricinoleic acid has many industrial uses but it is undesirable in a vegetable oil for human consumption. Oleic acid is associated with oxidative stability and heart-healthy properties. Therefore, the high oleic acid/low ricinoleic acid mutant OLE-1 could have industrial uses requiring high oxidative stability, such a biofuel, or pharmaceuticals applications requiring lower ricinoleic levels than the standard castor oil. Moreover, OLE-1 signifies an important advance toward the development of ricinoleic acid free/very high oleic acid castor oil lines, which would be of interest for the edible oil market. Finally, it is an excellent trait for further studies in the characterization of the genes involved in the biosynthesis of ricinoleic acid.
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ACKNOWLEDGMENTS
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The technical assistance of Angel Benito and Antonia Escobar is gratefully acknowledged. The authors thank the USDA for providing seed of the castor germplasm collection. We thank Dr. L. Velasco for his critical review of the manuscript. This work includes a portion of a Ph.D. thesis by the first author and was supported by European Community Research Project AIR3 CT 94-2324.
Received for publication February 2, 2003.
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ABSTRACT
INTRODUCTION
