Crop Science 41:577-579 (2001)
© 2001 Crop Science Society of America
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Vitamin e concentration in upland cotton seeds
C.Wayne Smitha and
Robert A. Creelmanb
a Dep. of Soil and Crop Sciences, Texas A&M Univ., College Station, TX 77843-2474
b Mendel Biotechnology, Inc., 21375 Cabot Blvd, Hayward, CA 94545
Corresponding author (cwsmith{at}tamu.edu)
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ABSTRACT
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Vitamin E, tocopherol, is a naturally occurring antioxidant that has been implicated in human health issues such as decreased risk of cardiovascular disease and some forms of cancer, improved immune functions, and in slowing the progress of degenerative diseases. Tocopherol affects plant health much as it does human health, i.e., by scavenging free radicals, thus protecting plant membrane integrity. Tocopherol is a strong antioxidant and increases oil oxidative stability in cottonseed oil. Little is known about the tocopherol content of cotton (Gossypium hirsutum L.) seed. ‘Acala 1517-88’ and ‘Acala 1517-SR2’ are reportedly higher in 
-tocopherol than ‘Deltapine 50’ or ‘Stoneville 825.’ Objectives of this research were to determine the variability in vitamin E content among several current upland cotton genotypes grown during 1997 and 1998 at College Station, TX, and when grown at College Station and Chillicothe, TX, in 1997. Tocopherol was extracted from cotton seeds with hexane and levels were determined by HPLC. Years were significant for - and 
-tocopherol and location was significant for ß/
-tocopherol. However, no differences were detected among 18 genotypes grown during 1997 and 1998 at College Station nor among 13 genotypes grown at College Station and Chillicothe during 1997.
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INTRODUCTION
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VITAMIN E is a naturally occurring antioxidant that has been implicated in decreased risk of cardiovascular disease and some forms of cancer, improved immune functions, and in slowing the progress of some degenerative diseases in humans (Fryer, 1992; Traber and Sies, 1996; Brigelius-Flohe and Traber, 1999). Vitamin E is a lipid soluble antioxidant that occurs most frequently in vegetable oils. There are four naturally occurring forms, (5, 7, 9-trimethyltocol), ß (5,8-dimethyltocol), (7,8-dimethyltocol), and (8-methyltocol). Alpha is the most important form from a human health perspective because it has the highest vitamin E activity, and is recommended for human consumption at 10 to 13 IU per day. Demurin et al. (1996) reported that tocopherol was a strong antioxidant and increased oil oxidative stability of linoleic and oleic types (unsaturated fatty acids) of oil by 1.2 to 3 times. Cotton, Gossypium spp, seeds are approximately 73% unsaturated fatty acids and 26% saturated (Gupta, 1995).
Dupont et al. (1990) reported that seeds of cotton contain one of highest levels of vitamin E, reported as alpha and gamma forms only, of 13 plant and animal sources of fats and oils. Soybean [Glycine max (L.) Merr.], corn (Zea mays L.), sunflower (Helianthus annuus L.), sesame (Sesamum indicum L.), safflower (Carthamus tinctorius L.), rapeseed (Brassica napus L.), and palm (family Palmae) oils also were high in total tocopherol (Shintani and DellaPenna, 1998). Tocopherol affects plant health much as it does human health, i.e., by scavenging free radicals, thus protecting plant membrane integrity (Fryer, 1992). Several authors have reported that plants exhibiting high levels of antioxidants have greater resistance to oxidative damage (Harper and Harvey, 1978; Dhindsa and Matowe, 1981; Wise and Naylor, 1987; Monk and Davies, 1989; Spychalla and Desborough, 1990).
Limited information is known about the tocopherol content of cotton seed. Abdel-Nabey et al. (1991) reported that -tocopherol ranged from 237 to 674 mg kg-1 crude oil while -tocopherol ranged from 284 to 534. They reported levels of ß-tocopherol from "non-detectable" to 21 mg kg-1 crude oil and -tocopherol ranging from a "trace" to 55 mg kg-1 crude oil across 14 cotton genotypes. Their study included both upland, G. hirsutum, and Egyptian, G. barbadense L., cultivars. Four cultivars were from the USA, ‘Acala SJ-1’, ‘Acala SJ-2’, ‘Acala SJ-5’, and an unidentified U.S. cultivar. These values indicate that -tocopherol represents approximately 50% of total tocopherol while -tocopherol makes up 35% on average. Gossett et al. (1994) reported that Acala 1517-88 and Acala 1517-SR2 were salt tolerant and contained 312 to 420% higher levels of -tocopherol than Deltapine 50 or Stoneville 825, both defined as salt sensitive.
The objective of this research was to survey the variability in vitamin E content among currently available upland cotton genotypes across years at one location and across locations in a single year.
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Materials and Methods
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Genotype x Year
Eighteen cultivars were evaluated for yield and quality parameters at College Station, TX, during 1997 and 1998. These cultivars were grown under irrigated conditions in 12- by 1-m plots replicated four times. Cultural practices were common for central Texas. Aliquots of spindle-harvested seedcotton were taken from two replications and ginned on a 10-saw laboratory gin. Tocopherol content was determined in November 1998. Thus, seeds obtained in 1997 were held in paper bags under general warehouse conditions for approximately 12 mo while those obtained in 1998 were stored for less than 3 mo. The effects of storage and conditions of storage were not considered.
Genotype x Location
Thirteen cultivars and strains were grown under irrigated culture at College Station, TX, approximately 31°N latitude, during 1997 and at Chillicothe, TX, approximately 34°N latitude. College Station is located in the humid southeastern part of Texas with cultural practices similar to those used throughout eastern Texas, the Lower Rio Grande Valley of Texas, and the Midsouth and southeastern regions of the U.S. Cotton Belt. Chillicothe is located in the semiarid, Rolling Plains region of Texas and production practices are similar to those utilized on the Texas High Plains and in Oklahoma. These two sites were chosen because of the potential differences in weather as well as differences in production practices other than irrigation.
Genotypes were grown in 12- by 1-m plots and replicated four times. Cultural practices were common for both locations. Aliquots of spindle-harvested seedcotton were taken at harvest and ginned on a 10-saw laboratory gin. Seeds thus obtained were held in paper bags for about 9 mo in a seed room at approximately 9°C.
Tocopherol Extraction and Determination
Tocopherol content was measured by modifications of various methods (Weber, 1984; Slack, 1987). Dry cotton seeds were cracked coarsely by using a common, household coffee grinder. Seed coats were removed by hand and the remaining cotton seed meats were ground to a fine powder with a coffee grinder, weighed, and transferred to a cellulose thimble. To estimate tocopherol recovery, 12.5 ng tocol (Matreya, Inc., Pleasant Gap, PA) were added to the ground cotton seed powder before the samples were exhaustively extracted in a Sohxlet apparatus for 90 min with hexane containing 1 g kg-1 BHT. The hexane was removed by rotary evaporation and the resulting crude cotton oil dissolved in a small amount of hexane. The tocopherol composition was analyzed from 10 µL of this crude oil solution by reverse phase chromatography (Waters C18 column, 250 mm by 4.5 mm; Waters Corp., Milford, MA) with a solvent composition of 0.3 g kg-1 isopropanol in hexane at a flow rate of 1 mL min-1. The HPLC system consisted of two Model 6000 pumps (Waters), a Model 630 gradient controller (Waters), WISP Model 710 autosampler (Waters), and a data integrator (Milton Roy Co., Ivyland, PA). Tocopherols were detected with a Perkin Elmer (Norwalk, CT) LS4 fluorescent detector (290 nm excitation and 330 nm emission). Peak areas for -, ß/-, and -tocopherols were compared with the standard curve generated from different amounts of tocopherols (Matreya, Inc.) to calculate the relative amounts of tocopherols.
-Tocopherol, ß/-tocopherol, and -tocopherol content across genotypes and years and across genotypes and locations were analyzed as a split plot arrangement of a randomized complete block design. Years and locations were main effects split to genotypes with all effects considered fixed. Analyses of variance were developed with SAS GLM procedure with means separated by the Waller-Duncan LSD.
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Results and Discussion
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Genotype x Year
The analysis of variance indicated a significant difference between years for -tocopherol and -tocopherol content when cultivars were averaged within 1997 and 1998 (Table 1). Across these 18 cultivars and strains, -tocopherol averaged 125 µg g-1 fresh weight in 1997 and 244 µg g-1 fresh weight in 1998. Both tests were irrigated and managed similarly, but 1998 was an exceptionally hot year at College Station with 30 consecutive days of 38°C or higher and a total of 45 d of 38°C or higher. Additional research is necessary to determine if the unusually prolonged high temperatures such as those encountered during the 1998 growing season will result in an increase in -tocopherol. If -tocopherol is affected by such environmental differences, then it will affect a breeder's ability to select genotypes with higher -tocopherol content. On the positive side from a breeding program perspective, however, is the fact that the cultivars reacted similarly to the 2 yr as the analysis of variance indicated no cultivar x year interaction.
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Table 1. Analysis of variance components for seed tocopherol content of 18 cultivars or strains evaluated at College Station, TX, in 1997 and 1998
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The average -tocopherol content of the 18 genotypes grown at College Station during 1997 and 1998 ranged from 150 µg g-1 fresh weight for the ‘Stoneville 474’ to 224 µg g-1 fresh weight for ‘Paymaster 1330 B’ (Table 2). A CV of 22.7 suggest that improved sampling techniques or improved statistical precision could detect differences and thus the opportunity to identify varying levels of -tocopherol.
The same comments can be made relative to -tocopherol (Tables 1 and 2). Yearly means of -tocopherol, however, were opposite those for -tocopherol. Year had no impact on ß/-tocopherol content. There was not a significant interaction of cultivar and year, but again the environmental component of the phenotypic variation exceeded the genetic component.
Genotypes x Location
Eleven cultivars and two experimental strains were grown at College Station and Chillicothe during 1997. The analysis of variance indicated no effect of location on - or -tocopherol content (Table 3). These genotypes averaged 121 µg g-1 fresh weight -tocopherol at College Station and 135 µg g-1 fresh weight at Chillicothe (Table 4). The location means for -tocopherol were near identical, 1.85 and 1.82 for College Station and Chillicothe, respectively. A difference (P = 0.01) was noted for location relative to ß/-tocopherol, where the 13 genotypes averaged 79 µg g-1 fresh weight at Chillicothe but only 35 µg g-1 fresh weight at College Station. As with the evaluation across years, statistical analysis indicated no variation due to genotype for or -tocopherol content. However, there were differences (P = 0.06) in ß/-tocopherol content of these genotypes. TAM 91D-92, a breeding line of the Texas A&M, contained more ß/-tocopherol than AgriPro AP 6101, AgriPro AP 4103, and Deltapine 33B. There were no significant interactions of location and genotype indicating that the genotypes performed similarly at both locations for all forms of tocopherol.
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Table 3. Analysis of variance components for seed tocopherol content of 13 cultivars or strains grown at College Station and Chillicothe, TX, during 1997
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Table 4. Tocopherol content of cotton seed of cultivars grown at Chillicothe and College Station, TX, during 1997
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These data suggest that breeders will have difficulty selecting for increased tocopherol in nonacala upland cotton. However, while no statistical differences among genotypes for -tocopherol across years or across locations were noted, there were two encouraging results. First, no interactions of genotype x year or genotype x location for -tocopherol content were detected. This suggests that selections will be stable across environments. Second, relatively large coefficients of variation suggests that better sampling or greater statistical precision could improve the probability of detecting differences among cotton genotypes for tocopherol content. Conversely, the large environmental component of variance across years, 180 times greater than the genotypic component (Table 1), suggests that it will be difficult for breeders to use simple, single plant selection, the most preferred selection option, to select for increased -tocopherol content in cotton seed.
Biotechnology offers an alternative for the improvement of vitamin E (-tocopherol) concentration in plants. Shintani and DellaPenna (1998) obtained over-expression of a -tocopherol methyltransferase (-TMT) gene in Arabidopsis seeds which resulted in an almost quantitative conversion of -tocopherol to -tocopherol. Since cotton seeds contain a significant amount of -tocopherol, targeted overexpression of -TMT in cotton should increase -tocopherol levels by approximately 50%.
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NOTES
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Research conducted by the Texas Agric. Exp. Stn., the Texas A&M University System. Supported by the National Cottonseed Products Association.
Received for publication April 25, 2000.
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REFERENCES
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ABSTRACT
INTRODUCTION
