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Patricia E. Hung, Vincent A. Fritz, and Luther Waters Jr.

Sweet corn (Zea mays L. var. rugosa Bonaf.) seed carrying the mutant endosperm gene shrunken-2 (sh2) are very susceptible to seed rot and pre- and post-emergence damping off. Experiments were conducted to determine if selected organic solvents were suitable carriers for fungicide infusion of sh2 sweet corn seed for improved germination and stand establishment. Seed of `Florida Staysweet' and `Crisp-n-Sweet 710' were immersed in acetone, cyclohexane, decahydronaphthalene (Decalin), dimethylsulfoxide (DMSO), ethanol, or xylene for 5 seconds, 0.25, 0.50, 1.0, 2.0, 4.0, or 8.0 hours, air-dried, and subjected to a cold-stress test. Total germination and percentage of normal seedlings in both cultivars were significantly decreased after 8 hours of immersion in acetone. Average seedling dry weight, however, did not decrease. DMSO was highly toxic to both cultivars. Ethanol increased seed mortality with increasing immersion times. Cyclohexane, Decalin, and xylene caused erratic responses in all measured variables as immersion time increased. In a second experiment, the effects of immersion time up to 4 hours in acetone on germination and vigor of 11 sh2 cultivars were compared. There was no correlation between cultivar germination or vigor and immersion in acetone. Results indicate acetone could be used to infuse fungicides into the seed of some sh2 cultivars without compromising seed germination or vigor. However, each sh2 cultivar must be screened individually to determine if it is a suitable candidate for organic solvent infusion of fungicides.

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Patricia E. Hung, Vincent A. Fritz, Thor Kommedahl, Albert H. Markhart III, and Luther Waters Jr.

Experiments were conducted to determine if the fungicide imazalil infused into shrunken-2 sweet corn (Zea mays L. var. rugosa Bonaf.) seed via acetone could protect against soil- and seedborne fungi enough to improve germination and vigor. `Florida Staysweet' and `Crisp-n-Sweet 710' seeds were infused for 0.25 hours with 1% or 2% (w/w) imazalil-acetone (LA) solutions, air-dried, and subjected to a modified laboratory seedling growth cold test using sterile soil or soil inoculated with Fusarium moniliforme Sheldon. Both IA concentrations significantly reduced the incidence of diseased seedlings in soil inoculated with F. moniliforme when compared to nontreated controls. Neither treatment significantly reduced the incidence of seedborne fungi. Chemical name used: 1-[2-(2,4-dichlorophenyl)-2-(2-propenyloxy)ethyl-H-imidazole (imazalil).

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Kenneth G. McCabe, James A. Schrader, Samy Madbouly, David Grewell, and William R. Graves

). The use of chloroform as the organic solvent for PA, PLA, and TO made the coating process more difficult and hazardous when compared with coating with PU and required that the coating process be performed in a fume hood. Cost of coating with PA and PLA

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Niels O. Maness, Michael W. Smith, C. Zhang, and Gerald H. Brusewitz

Techniques to reduce the oil content of shelled pecans using supercritical CO2 have been developed, and the effect of partial oil extraction on kernel quality is being investigated. Extraction conditions induce little kernel damage and allow for up to 30% oil reduction. Extraction temperature, at 40 or 80C, influenced kernel color. Regardless of temperature, extracted nut meat was lighter in color. Testa color increased in redness for kernels extracted at 80C compared to kernels extracted at 40C. Extracted oil was amber. Fatty acid composition of oil obtained with supercritical CO2 was essentially the same as oil obtained by organic solvent extraction and by cold press. Investigations to determine the effect of oil reduction on pecan shelf life are described. This research was supported by U.S. Department of Agriculture grant 92-34150-7190, Oklahoma Center for Advancement of Science and Technology grant AR4-044, and the Oklahoma Agricultural Experiment Station.

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Jorge F.S. Ferreira, James E. Simon, and Jules Janick

Artemisinin (qinghaosu), an endoperoxide sesquiterpene lactone with antimalarial activity and little human toxicity, is produced in A. annua L., a short-day plant with the critical photoperiod of 13.5 h. Artemisinin peaks at full flowering when it is 11 times higher in inflorescences than in leaves. Supplementation of MS medium with BA, kinetin, CCC, or daminozide decreased content in shoot cultures as compared to the control, with the exception of CCC at 6.3 μM. Artemisinin content (percent dry weight) in this experiment did not correlate with shoot number (r = –0.198) but was highly correlated (r = 0.775**) with root number, indicating that although roots do not produce artemisinin, they may be involved in its synthesis. Maximum artemisinin was produced in hormone-free medium. Friable calli were obtained with a combination of BA (4.4 μM) and 2,4-D (4.5 μM), but artemisinin was absent from calli, cells, and cell culture media. Biseriate glandular trichomes are present in leaves and flowers from the earliest developmental stages. Artemisinin was extracted by a 1-min dip in organic solvents from flowers and leaves, indicating that the biseriate glandular trichomes of A. annua are the probable site of accumulation or sequestration of artemisinin.

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D.W. Lickfeldt, N.E. Hofmann, J.D. Jones, A.M. Hamblin, and T.B. Voigt

An efficient deoxyribonucleic acid (DNA) extraction procedure that yields large quantities of DNA would provide adequate DNA for a large number of different analytical procedures. This study was conducted to compare three DNA extraction procedures for cost, time efficiency, and DNA content while extracting DNA from Kentucky bluegrass (Poa pratensis L.). Three students at the Univ. of Illinois with varying levels of DNA extraction experience conducted DNA extractions using Plant DNeasy™ Mini Kits, Plant DNAzol® Reagent, and a PEX/CTAB buffer. Costs varied significantly with cost (US$) per DNA sample of $3.04 for the DNeasy™ method, $0.99 for the DNAzol® method, and $0.39 for the PEX/CTAB extraction. The DNAzol® method was the fastest; although extracting 2.8 ng less DNA than the DNeasy™ method, it did not require the use of hazardous organic solvents, and random amplified polymorphic DNA (RAPD) markers were satisfactory for DNA fingerprinting of Kentucky bluegrass cultivars. The PEX/CTAB method, which did not include a tissue homogenization step, did not have reproducible banding patterns due to miniscule and inconsistent quantities of DNA extracted, or possibly due to inadequate purification. The investigator with the least DNA extraction experience was the slowest, while extracting 75% more DNA. All three methods are easily adapted to laboratories having personnel with different levels of experience. The DNAzol® Reagent method should save time and money, with reproducible results when many individual plant samples need to be identified. Chemical names used: potassium ethyl xanthogenate (PEX); cetyltrimethyl ammonium bromide (CTAB)

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Stanley Ries, Rebecca Baughan, Muraleedharan G. Nair, and Robert Schutzki

Several plant species that are not consumed by animals were collected, extracted with organic solvents, and tested at different venues for their effectiveness as animal feeding repellents. Species with the most repellent activity were daffodil (Narcissus pseudo narcissus), bearded iris (Iris sp.), hot pepper (Capsicum frutescens), catnip (Nepeta cataria) and peppermint (Mentha piperita). Considerable effort was expended to isolate and identify compounds from these species responsible for repellent activity. Eight chemicals have been isolated and purified, and four of them have been identified. Both daffodil and catnip contain more than one repellent, but none of the four compounds identified were common to both species. Combinations of extracts from more than one plant species proved to have more repellent activity than extracts from individual species used alone. In several tests these plant extracts proved to be as effective or better than available commercial repellents. A plethora of additives and surfactants were tested to increase repellent activity by enhancing the spreading, penetration or persistence of the extracts.

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Deepak Dandekar, G. K. Jayaprakasha, and Bhimanagouda Patil

Citrus consumption has been shown to promote human health due to presence of several bioactive compounds. In the process of understanding the health benefits of citrus, we need to isolate and characterize these compounds. Limonoids are one of such prominent, but lesser-known phytonutrients that have been shown to prevent cancers of the mouth, skin, lung, breast, and colon. With the growing interest in the health-promoting properties of citrus limonoids, the demand for these bioactives has significantly increased. It has been critical to explore environment-friendly extraction methods rather than using hazardous organic solvents. A water-based hydrotropic extraction of limonoid aglycones from sour orange (Citrus aurantium L.) seeds was developed. Two hydrotropes, sodium salicylate (Na-Sal) and sodium cumene sulfonate (Na-CuS), were studied for extraction efficiency using the Box Behnken experiment design method. The extraction efficiency of prominent aglycone limonin was observed depending on hydrotrope concentration, extraction temperature, and percentage of raw material loading. Response Surface Analysis (RSA) of data predicted the optimum conditions for maximum yield. Recovery of aglycones from filtered extract is also easily achieved by mere dilution using water at pH 3 or 7 or by partitioning the extract with dichloromethane. At optimum conditions, limonin yield of 0.46 mg/g seeds in the case of Na-Sal extraction and 0.65 mg/g seeds in the case of Na-CuS extraction was achieved. The results demonstrated that the hydrotropic extraction process of limonoid aglycones has practical commercial importance. This project is based upon work supported by the USDA–CSREES IFAFS #2001 52102 02294 and USDA–CSREES #2005-34402-14401 “Designing Foods for Health” through the Vegetable and Fruit Improvement Center.

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Roseann Leiner, Abraham Smyth, Rudy Candler, and Patricia S. Holloway*

Berries and vegetables can be sources of beneficial phytochemicals that may have antioxidant activity in the human diet. Information on type and quantity of phytochemicals may open new crop opportunities for berries and vegetables harvested in Alaska. A method was developed for detecting ascorbic acid and eight phenolic acids on an HPLC instrument using a reverse phase Merck Chromolith C18 column. The method used UV absorbance detection at 280nm to separate a standard solution of the following nine phytochemicals: ascorbic acid, gallic acid, protocatechuic acid, p-hydroxybenzoic acid, p-hydroxyphenylacetic acid, caffeic acid, syringic acid, p-coumaric acid and ferulic acid. The mobile phase was a mixture (3.5% to 14% gradient) of organic solvent (5 parts acetonitrile: 2 parts methanol) and aqueous solvent (2 mmol aqueous trifluoroacetic acid - TFA) at a flow rate of 2 mL/min. In 2003, over 60 samples of berries and 60 samples of baby greens were extracted and analyzed. Plant samples were extracted by blending 10-20g of frozen plant tissue with 5 parts TFA. The extracts were centrifuged, diluted 4:1 and filtered (0.2 μm). Chromatograms from HPLC analysis for all samples were complex in peak size and number. Chromatograms for six extracts of high bush cranberries, Viburnum edule, exhibited intense peaks that indicate the presence of caffeic acid, based on retention times. Chromatograms for seven extracts of rose hips, Rosa acicularis, exhibited peaks that indicate the presence of ascorbic acid, based on retention times. Gallic acid and p-hydroxybenzoic acid are apparent minor components in the leaves of some baby greens. This research will continue in 2004 with more plant samples and further method development for detection of other phytochemicals.

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L.S. Boiteux, M.E.N. Fonseca, and P.W. Simon

Seven plant genomic DNA purification protocols were evaluated for genetic fingerprinting analysis using six tissues obtained from inbred carrot (Daucus carota L.) lines. Evaluations included 1) DNA yield, 2) DNA purity, 3) DNA cleavage with HindIII, 4) DNA integrity, and 5) DNA suitability for amplification in a random amplified polymorphic DNA (RAPD) system. Significant differences were observed among tissues and purification methods for the total amount of DNA. An extraction method using CTAB buffer + organic solvents gave the best results in DNA yield, purity, and HindIII cleavage when compared with the other six nonorganic extraction methods. Of the tissues examined, flowers yielded the most DNA (average value = 115 ng of DNA/mg of fresh tissue); followed by seeds (54 ng·mg-1), fresh leaves (48 ng·mg-1), lyophilized leaves (40 ng·mg-1), calli (22 ng·mg-1), and tap roots (4 ng·mg-1). For most of the preparations, the DNA showed no traces of degradation. However, DNA preparations were not consistently accessible to HindIII cleavage in all tissue-extraction method combinations. Uncut DNA was observed chiefly in extractions from flowers and fresh leaves suggesting a tissue-specific adverse effect on restriction endonuclease activity. Differences in RAPD band (amplicon) intensity and number were observed across tissues and DNA extraction methods using identical PCR conditions for RAPD. Callus was the best type of tissue for RAPD-based fingerprinting yielding a consistently higher number of more intense amplicons when compared to the other tissues. In flowers and seeds, only DNA obtained with the CTAB extraction method could be amplified. Polymorphisms deviating from genetic expectations were mainly observed in root and fresh leaf DNA, indicating that some RAPD markers may not present satisfactory levels of reproducibility. Judicious and uniform selection of DNA purification method as well as tissue source for DNA extraction are, therefore, important considerations for reliable RAPD-based DNA fingerprinting analysis in carrot. In addition, our studies allowed the identification of a better combination of procedures for use in routine manipulations of carrot DNA such as RFLP-RAPD-based cultivar fingerprinting, molecular mapping, screening of transgenic plants, construction of genomic libraries, and gene cloning.