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Qinglu Ying, Yun Kong, and Youbin Zheng

. (Oakville, ON, Canada). To measure the total chlorophyll and carotenoid contents, ≈20 mg of each frozen sample was used. The samples were re-suspended with 1 mL ice-cold 100% methanol in 1.7 mL pre-chilled Eppendorf tubes. The samples were stored on ice

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Brian J. Just* and Philipp W. Simon

While the carotenoid biosynthetic pathway has been studied several horticultural and agronomic crops, very little information exists for this conserved pathway in carrot, a primary source of dietary carotenoids. Though orange carrots are the most familiar color to Western consumers, yellow, red, and white carrots also exist and have been historically important. Modern carrot breeders are showing renewed interest in these unusual color phenotypes. Beta- and alpha-carotene are the primary pigments in orange carrot roots. Yellow carrots accumulate xanthophylls (oxygenated carotenes), red carrots accumulate lycopene (the precursor to alpha- and beta-carotene), and white carrots accumulate no detectable pigments. Differences between these phenotypes are usually monogenic or oligogenic. Our research has focused on identifying putative genes for carotenoid biosynthetic enzymes in the carrot genome, mapping them, and examining expression patterns in various tissues and carrot root pigment phenotypes. We are using this information to create a carrot pigment biosynthesis function map incorporating biosynthetic enzymes, major carrot color genes, and gene expression information.

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José A. Narváez, Patricia Flores-Pérez, Virginia Herrera-Valencia, Fernando Castillo, Roberto Ku-Cauich, Blondy B. Canto-Canché, Nancy Santana Buzzy, and Renata Rivera-Madrid

The tropical plant Bixa orellana L. (annatto) is the sole source of bixin, the most frequently employed natural pigment in the food industry. Little is known about the physiology, biochemistry, and molecular genetics of this crop. Our purpose was to establish a set of analytical tools that could be applied in the genetic improvement of B. orellana, particularly for the screening of characteristics such as bixin content and resistance to diseases or pests. Some preliminary results on the study of carotenoid synthesis are presented. In vitro cultures from several B. orellana tissues were established and DNA, RNA, and proteins were extracted from them and analyzed. Similarly, bixin and total carotenoids were quantified.

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Haejeen Bang*, Daniel I. Leskovar, and Kilsun Yoo

This experiment was conducted to determine the effects of deficit irrigation and growing season on fruit quality, carotenoid content and yield of red-, orange-, and yellow-fleshed diploid and triploid watermelon. Irrigation rates were 1.0 evapotranspiration (ET) and 0.5 ET. Diploid cultivars were Summer Flavor 710 (red), Tendersweet (orange), and Summer Gold (yellow). Triploid cultivars were Summer Sweet 5244 (red), Sunshine (orange), and Amarillo (yellow). Four-week old containerized transplants were planted in the field at TAES-Uvalde on 27 Mar. and 21 May 2003. Deficit irrigation imposed after plants were fully established reduced the individual fruit weight and size in the early planting. Soluble solids content (SSC) and firmness was not affected by irrigation rate in both plantings. SSC varied across cultivars and increased with maturity, particularly for the triploid cultivar Amarillo. In general, triploids were firmer than diploid cultivars. Total carotenoid content was not affected by irrigation during early planting. Diploid and triploid red-fleshed watermelon cultivars had significantly higher carotenoid content than orange- and yellow-fleshed cultivars. The major carotenoid was lycopene (more than 65%), followed by prolycopene (20%) and B-carotene (7%).

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Haejeen Bang*, Sungil Kim, Daniel Leskovar, and Stephen King

Carotenoids are plant compounds that serve a variety of essential functions in the plant and have also been found to have several health-promoting activities in humans. Carotenoids found in watermelon (Citrullus lanatus) flesh are responsible for the various colors such as red, yellow and orange. Previous inheritance studies of flesh color revealed that six genes were involved in color determination. The relationship and interaction of these genes suggests that some color-determining genes may be the result of mutations on the structural genes encoding enzymes in the carotenoid biosynthesis pathway. In this study we were able to isolate and sequence six genes encoding enzymes involved in the carotenoid biosynthetic pathway, and determine their expression in different colored watermelon fruit. The cDNA was synthesized from total RNA using RACE (Rapid Amplification of cDNA ends) kit (SMART RACE cDNA Amplification Kit; Clontech, Palo Alto, Calif.). Degenerate primers were designed based on published homologous genes from other species and were used to isolate gene fragments and full-length cDNAs of phytoene synthase, phytoene desaturase, _-carotene desaturase, β-cyclase, β-carotene hydroxylase and zeaxanthin expoxidase. RT-PCR was carried out to examine any differential expression of cloned genes in white, yellow, orange and red-fleshed watermelon. All cloned enzyme-encoding genes were expressed regardless of flesh colors. These results indicate that carotenoid biosynthesis may be regulated at the post-transcriptional level. One interesting feature supports this hypothesis. In case of β-cyclase, a 229-bp leader intron was identified, and an unspliced mRNA with this leader intron existed dominantly in cDNA pool of all samples.

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Ingrid M. Fordham, Beverly A. Clevidence, Eugene R. Wiley, and Richard H. Zimmerman

Autumn olive (Elaeagnus umbellata Thunb.) has edible fruit with brilliant red or yellow pigmentation. An analysis of the pigment in fruit of five cultivars and six naturalized plants showed that the berries contain lycopene, α-cryptoxanthin, β-cryptoxanthin, β-carotene, lutein, phytoene, and phytofluene. The lycopene content per 100 g ranged from 15 to 54 mg in fresh fruit from the naturalized plants and from 17 to 48 mg in the four cultivars with red-pigmented fruit. A cultivar with yellow fruit had only 0.47 mg/100 g fresh fruit. In contrast, fresh tomato fruit, the major dietary source of lycopene, has a lycopene content per 100 g of ≈3 mg. This newly identified source of lycopene may provide an alternative to tomato as a dietary source of lycopene and related carotenoids.

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Khalid Ibrahim and John Juvik

Vegetables are a rich source of dietary carotenoids and tocopherols, powerful antioxidants that have the capacity to protect cells against oxidative damage caused by free radical reactions. There is evidence for a negative correlation between the incidence of certain types of cancer, age-related macular degeneration, cataract development, and cardiovascular disease with increased carotenoid and tocopherol intake. Development of elite vegetable germplasm with enhanced levels of these phytochemicals will potentially promote health among the consuming public. To assess the feasibility for genetic improvement in phytochemical content, it is necessary to partition the phenotypic variability into its component sources (genotype, environment, and genotype by environment interaction). To provide data for comparison and partition of phenotypic variation, 41 sweet corn and 13 broccoli genotypes were grown and harvested in one location for 3 years and analyzed for phytochemical content by HPLC. The most abundant form of carotenoids and tocopherols were lutein and gamma-tocopherol in sweet corn and beta-carotene and alpha-tocopherol in broccoli. Analysis of variance showed that, in sweet corn, the differences among genotypes described most of the phenotypic variation (76% for lutein, and 78% for gamma-tocopherol). Genotype by year interaction was a second significant factor, while variation affiliated with the year was found to be a minor component. In contrast, in broccoli, the three sources of variability contributed equally to describe the total phenotypic variation for beta-carotene and alpha-tocopherol. These results suggest that elite sweet corn and broccoli germplasm with improved carotenoid and tocopherol levels can be developed using conventional breeding protocols.

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Mark G. Lefsrud and Dean A. Kopsell

Plant growing systems have consistently utilized the standard Earth day as the radiation cycle for plant growth. However, the radiation cycle can easily be controlled by using automated systems to regulate the exact amount of time plants are exposed to irradiation (and darkness). This experiment investigated the influence of different radiation cycles on plant growth, chlorophyll and carotenoid pigment accumulation in kale (Brassica oleracea L. var. acephala D.C). Kale plants were grown in growth chambers in nutrient solution culture under radiation cycle treatments of 2, 12, 24, and 48 h, with 50% irradiance and 50% darkness during each time period. Total irradiation throughout the experiment was the same for each treatment. Radiation cycle treatments significantly affected kale fresh mass, dry mass, chlorophyll a and b, lutein, and beta-carotene. Maximum fresh mass occurred under the 2-h radiation cycle treatment. The maximum dry mass occurred under the 12-h radiation cycle treatment, which coincided with the maximum accumulation of lutein, beta-carotene, and chlorophyll a, expressed on a fresh mass basis. The minimum fresh mass occurred during the 24 h radiation cycle treatment, which coincided with the largest chlorophyll b accumulation. Increased levels of chlorophyll, lutein and beta-carotene were not required to achieve maximum fresh mass production. Environmental manipulation of carotenoid production in kale is possible. Increases in carotenoid concentrations would be expected to increase their nutritional contribution to the diet.

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Khalid E. Ibrahim, Kanta Kobira, and John A. Juvik

Genotype-by-environment interaction (G×E) is a fundamental concern in plant breeding since it hinders developing genotypes with wide geographical usefulness. Analysis of variance (ANOVA) has been widely used to interpret G×E, but it does not elucidate the nature and causes of the interaction. Stability analysis provides a summary of the response patterns of genotypes to different growing environments. Two classes of phytochemicals with putative health promoting activity are carotenoids and tocopherols that are relatively abundant in broccoli. Growing clinical and epidemiological evidence suggests that vegetables with enhanced levels of these phytochemicals can reduce the risk of cancer, cardiovascular, and eye diseases. The objective of this study is to have better understanding of the genetic, environmental and G×E interaction effects of these phytochemicals in broccoli to determine the feasibility of the genetic enhancement. The ANOVA and Shukla's stability test were applied to a set of data generated by the HPLC analysis of different carotenoid and tocopherol forms for six broccoli accessions grown over three environments. The ANOVA results show a significant G×E for both phytochemicals that ranged from 22.6% of the total phenotypic variation for beta-carotene to 54.0% for delta-tocopherol while the environmental effects were nonsignificant. The genotypic effects ranged from as low as 1% for alpha-tocopherol to 31.5% and 36.0% for beta-carotene and gamma-tocopherol, respectively. Stability analysis illustrated that the most stable genotype for all phytochemicals is Brigadier. The results suggest that feasibility of the genetic enhancement for major carotenoids and tocopherols. A second experiment that includes a larger set of genotypes and environments was conducted to confirm the results of this study.

Free access

Kanta Kobira, Khalid Ibrahim, Elizabeth Jefferey, and John Juvik

Considerable epidemiological evidence exists on the association between consumption of antioxidant-rich vegetables and incidence of chronic diseases, including cancer and cardiovascular disease. Broccoli (Brassica oleracea L. sp. italica) florets are relatively abundant sources of antioxidants, and potentially amenable to genetic manipulation to enhance this vegetable's health-promoting properties. This investigation focuses on the identification of chromosomal segments in the nuclear genome of broccoli associated with antioxidant carotenoid and tocopherol variability. A broccoli F2:3 population consisting of 163 families derived from a cross between two parents (VI-158 and BNC) and previously mapped with 62 polymorphic SSR and SRAP marker loci was evaluated for carotenoid and tocopherol concentration in floret tissue over two growing seasons. Significant differences were observed among F2:3 family means for concentrations of lutein (10-fold difference between the lowest and highest family), beta-carotene 17-fold), alpha-tocopherol (8-fold) and gamma-tocopherol (6-fold). On a concentration basis, beta-carotene, lutein, alpha-tocopherol, and gamma-tocopherol were the most abundant antioxidant forms in broccoli. Heritability estimates of primary phytochemicals ranged from 0.35 to 0.38, 0.40, and 0.44 for beta-carotene, alpha-tocopherol, gamma-tocopherol, and lutein, respectively. Composite interval mapping (CIM) identified two quantitative trait loci (QTL) associated with carotenoid variability on two linkage groups and five QTL associated with tocopherol variability on four linkage groups. The QTL identified in this study have potential for use in marker-assisted crop improvement programs to develop elite germplasm designed to promote health among the consuming public.