HORTSCIENCE 38(4):595–598. 2003. regions of Texas. In the spring and early sum- mer, tagging trips were taken to locate evening primrose species while they were in bloom Evaluation of Gamma-linolenic Acid so that identification could be made. Voucher specimens were collected for each species of Composition of Evening Primrose the study. Each collection site was recorded us- ing a hand-held GeoExplorer global positioning (Oenothera) Species Native to Texas unit. In the late summer or fall, plant popula- tions were relocated and ripened seed capsules S.A. Balch1, C.B. McKenney2, and D.L. Auld3 were collected. A threshing machine was used Department of Plant and Soil Science, Texas Tech University, Lubbock, to break open capsules and separate seed from TX 79409-2122 other plant debris. Seed was further cleaned with a seed blower (General Mfg.). Cleaned Additional index words. evening primrose oil, GLA seed was stored in air-tight plastic containers in a refrigeration unit held at 4 °C. Abstract. The oil of evening primrose (Oenothera sp.) is an important source of gamma- Seeds from each collection site were ana- linolenic acid (GLA). GLA [C18:3¨6,9,12] is an unsaturated fatty acid in demand for its lyzed for fatty acid composition (Speed, 1995). nutritional and pharmaceutical application. Oenothera biennis L. is the primary com- A300-mg sample of seed from each accession mercial source of evening primrose oil. A study was conducted to determine if species of was used. Oil was extracted from the seed by Oenothera, adapted to Texas, produce GLA levels comparable to O. biennis. This project grinding whole seeds in 20 mL of hexane us- identified and evaluated the fatty acid composition of eight species of evening primrose ing a Polytron grinder. The oil/hexane mixture native to Texas. GLA levels of 54 accessions evaluated from collected seed ranged from was poured through a Whatman filter into 20- 0.0% to 11.0%. Field experiments were then conducted to determine oil content, fatty mL scintillation vials. Vials were placed into acid composition, seed yield, and potential adaptation to commercial production of a heating block at 50 °C and the hexane was selected accessions. Mean GLA levels of cultivated seed from these accessions ranged evaporated with a continuous flow of nitrogen from 0.0% to 10.1%. Mean seed oil content ranged from 7.3% to 21.7%. Of the spe- gas. Methyl esters of the oil were made by cies examined, O. elata subsp. hirsutissima (A. Gray ex S. Watson) W. Dietrich and O. adding 1 mL of methylation mixture to each jamesii (Torrey & Gray) demonstrated GLA levels and seed yields adequate for com- sample and inverting the capped sample for mercial production. Based on these results, O. elata subsp. hirsutissima and O. jamesii mixing. The methylation mixture was com- demonstrated sufficiently high GLA levels, oil content, and seed yields to be considered posed of 29.1 mL of 14% borontrifluoride in for commercial production. methanol, 20 mL of toluene, and 50.9 mL of methanol. Samples were placed in a heating In recent years, there has been increased Evening primrose (Oenothera biennis L.) block at 100 °C for 20 min. Samples were then interest in plants that produce gamma-linolenic was the first plant used for the isolation of GLA removed from the heating block and allowed to acid (GLA). In humans and other mammals, (Gunstone, 1992), and it is the primary source cool for 10 min. Each sample was rinsed with 2 GLA is an important intermediate in the conver- of GLA used in clinical and pharmaceutical mL of deionized water and 1 mL of hexane and sion of linoleic acid to prostaglandins, throm- applications (Phillips and Huang, 1996). GLA shaken to mix well. The mixture was allowed boxanes, and other long-chain polyunsaturated was extracted from the oil in the seeds of this to separate into two layers and the hexane layer fatty acids (Horrobin, 1992). This compound plant. Hudson (1984) reported that evening was pipetted into vials for gas chromatograph is considered an essential fatty acid, since, like primrose seed contains 24% oil with gamma- analysis. Nitrogen gas was blown into each vitamins, it cannot be synthesized by animals linolenic acid comprising 7% to 14% of the sample vial before it was capped to prevent and must be consumed in the diet from plant fatty acid component. Evening primrose oil is oxidation of the sample. sources (Lehninger, 1982). Prostaglandins available as a nutritional supplement in over Samples were analyzed using a Hewlett and thromboxanes are hormone regulators 30 countries and Oenothera biennis is grown Packard 5890 Series II Gas Chromatograph which affect many cellular functions and commercially in at least 15 countries (Carter, equipped with a Supelco 30 m × 0.53 mm × regulate some blood platelet activities. The 1988). In 1988, Canada and the United States 0.5 µm fused silica capillary column and flame rate-determining step in the bioconversion of produced 300–400 tons of evening primrose ionization detector (Speed, 1995). The initial linoleic acid to arachidonic acid, the precursor seed. Most of the commercial production of oven temperature of 210 °C was increased at of prostaglandins and thromboxanes, is be- evening primrose oil in North America is the rate of 4 °C per minute until a final tem- lieved to be the formation of the double bond located in Canada and in the eastern United perature of 240 °C was reached. The injector at the C-6 carbon by the enzyme ¨6-desaturase States (Brandle et al., 1993; Hall et al., 1988). port was set at 200 °C and the flame ionization (Horrobin, 1992). This enzyme catalyzes the These are areas of high rainfall and moderate detector was set at 250 °C. Output of the flame conversion of linoleic acid to gamma-linolenic temperatures. Little work has been done to ionization detector was recorded on an HP 3396 acid. Although most humans consume suffi - evaluate the fatty acid composition of Oeno- Series II Integrator. Fatty acid composition was cient quantities of linoleic acid, it has been thera species native to semiarid regions of calculated by summing the total area of all shown that certain factors such as stress, aging, the United States. A native evening primrose fatty acids detected and dividing each fatty diabetes, and alcohol consumption can inhibit with commercial levels of GLA would have acid by the total area. Peaks were identified or reduce the activity of the ¨6-desaturase en- potential as a high-value oil crop in areas of by comparing retention times to reference zyme (Horrobin, 1992; Phillips and Huang, high temperatures and low annual rainfall. The standard retention peaks of known composi- 1996). In recent medical research, it has been objectives of this project were to: 1) identify tion (Sigma AOCS No. 3, Sigma AOCS No. found that dietary supplementation of GLA can and collect species of evening primrose native 5, Alltech Methyl Ester Standards). be effective in the treatment of these conditions to the western regions of Texas, 2) analyze the Seeds harvested from cultivated plots were (Carter, 1988). fatty acid composition of the seed oil of these analyzed for percent oil at the Univ. of Idaho, species, and 3) evaluate their adaptability to using a Newport MKIIIA nuclear magnetic commercial field production. Received for publication 13 Nov. 2001. Accepted for resonance instrument (Newport of North publication 23 Aug. 2002. Texas Tech Univ. Paper America, Villanova, Pa.). Analyses were made No. T-4-516. This research was funded in part by Methods and Materials on oven-dried seed, using a canola standard the Texas Dept. of Agriculture. of 36.2% oil. 1Former Graduate Student. The collection area included the Northern Species with high levels GLA were evalu- 2Assistant Professor. High Plains and Southern High Plains, the ated in field studies in 1996, 1997, 1998, and 3Professor. Texas Rolling Plains, and the Edwards Plateau 1999. Seeds were sown in a soilless mix in HORTSCIENCE, VOL. 38(4), JULY 2003 595 24-7116, p595-598 595 6/29/03, 3:56:43 PM BREEDING, CULTIVARS, ROOTSTOCKS, & GERMPLASM RESOURCES the greenhouse in January of each year and in 1996 and 0.0 % in 1997. All accessions of showed a modest correlation in concentration transplanted to the field in May. Plants were O. grandis, O. missouriensis, O. brachycarpa, of GLA levels (r = 0.73**, df = 12). Seeds from grown at the Erskine Research Farm in Lub- and O. laciniata had GLA levels of <0.5%. cultivated plants of O. jamesii had GLA levels bock, Tex., under drip irrigation. The soil at Statistical analyses were not conducted on fatty ranging from 8.3% to 10.5% (data not shown) this site was an Amarillo fine sandy loam. Each acid composition of native collected seed due in 1998 and 6.8% to 10.7% in 1999 compared species was planted in a completely random- to the low numbers of accessions collected to the 1997 native collection with 11% and the ized block design with four replicates. Seeds of some species. Accessions with high levels 1998 native collection with 6.8%. Oenothera were harvested at maturity and threshed and of GLA were included in subsequent field jamesii was collected in a mesic habitat that analyzed for fatty acid composition using the evaluations. may have greater water availability comparable above procedure. Each collected species was grown in to field conditions. In all three years of culti- Data were subjected to analysis of variance the field for a minimum of 2 years. Species vation, Oenothera elata demonstrated greater procedures (SAS Institute, 1998). Mean separa- exhibiting <2.0% GLA levels were not evalu- GLA levels (8.0% to 10.1%) than the original tions were performed using Fisher·s Protected ated in subsequent years.