T ABLE OF CONTENTS The Biological Activity of Some Mixed Sucrose and Glucose Esters from Exotic Tobacco Cultivars. Cutler, H. G., R F. Severson, P. D. Cole, V. A. Sisson, M. D. Jackson, M. G. Stephenson ................................... 1 Increasing Artemisinin Production Through Biotransformation of Precursors. Chen, P. K., C. Lukonis, L. Go, G. R Leather .......................... 2 Lysophosphatidylethanoleamine, A Natural Lipid, Enhances Ripening and Improves Keeping Quality of Tomato Fruits Without Damage to Leaves. Farag, K. M., J. P. Palta ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 9 Chitosan, Does It Have a Place in Agriculture? Freepons, D ................................................... 11 Enhancing Nutritional Status of Plants With Bioregulators. Stutte, C. A., T. H. Clark, C. Guo ................................... 20 Stimulation of Growth and the Monoterpene Production of Salvia officinalis by Benzyladenine in vitro. Tawfik, A. A., P. E. Read, S. Cuppett ................ 21 Uptake, Translocation and Metabolism of Dihydrozeatin Applied to the Surface of Leaves of Whole Plants. Sanderson, K. J., P. E. Jameson ........................ 23 The Effect of Release and Seeding Rate on Rice Production. Dunand, R T. . ............ 31 Plant Growth Regulator Type and Rate Influence Micropropagation of Chestnuts (Castanea spp.). Auko, C. 0., P. E. Read, S. D. Kachman, K. M. Eskridge .............. 33 Growth and Lipid Content of Osmo-Tolerant and Sensitive Algae. Orcutt, D. M., C. L. Goedhart ................................................. 34 Detection of Herbicidal Properties: Scope and Limitations of the Etiolated Wheat Coleoptile Bioassay. Jacyno, J. M., H. G. Cutler ......................... 36 Effect of Benzyladenine on the Oil Constituents of Rosemary Plants Regenerated from Callus. Tawfik, A. A., P. E. Read ................................... 37 Effect of Duration of Low Temperature Treatment on Flowering of Containerized 'Tommy Atkins'Mango. Nunez-Elisea, R, T. L. Davenport ........................ 39 Control of Anthesis in Coffee with Gibberellins. Crisosto, C. H., R. V. Osgood, J. L. Ingamells ...... ',' ......................................... 42 Shoot Emergence, Growth and Leaf Development of Citrus Seeds Treated with Uniconazol. Fucik, J. E., D. Davila ........................................... 45 Mechanical Stress and Drought Stress Effects on Height Control of Greenhouse Crops. Barrett, J. E., T. A. Nell .......................................... 50 111 Pac1obutrazol Reduces Insecticide Phytotoxicity Damage on Salvia. Latimer, J. G., R. D. Oetting .................................................. 57 The Effect of a Transpiration Minimizer and A Hydrophilic Water Absorbant on Establishment and Growth of White (Concolor) Fir Transplants. Carroll, R., A. R. Templeton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Hormonal Control of Sexual Differentiation in Lagenaria siceraria. Ying, Z., K. R. Narayanan ............................................... 62 Effects of Dikegulac-Sodium and BA Plus G~+7 to Enhance Lateral Branching of Golden Pothos. AI-Juboory, K. H., D. J. Williams ............................. 64 Cultivar Response of Marigold to Triazole Compounds. Gilbertz, D. A. . .............. 70 Beneficial Applications of Plant Growth Regulators to Economically Important Seaweeds. Bradley, P. M., D. P. Cheney ...................................... 72 H202-Scavenging Enzymes and Antioxidants in Echinochloafrwnentacea as Affected by Triazoles. Sankhla, N., A. Upadhyaya, T. D. Davis, D. Sankhla ............... 78 Triazole Plant Growth Regulators Influence Somatic Embryogenesis and Plantlet Regeneration in Tissue Cultures of Echinochloafrwnentacea. Sankhla, A., T. D. Davis, D. Sankhla, N. Sankhla, A. Upadhyaya, S. Joshi ................ 84 Partial Safening of Clomazone in Com With Two Phenoxy acetic Acid Compounds. Devlin, R. M., 1. 1. Zbiec .......................................... 90 Effe9ts of Several Plant Growth Regulators on Lignin Content in Wheat Straw. Buta, J. G., D. W. Spaulding ................................. 97 Comparison of Growth Retardants on Potted Impatiens. Holcomb, E. J., M. Rose, L. Gohn ..................................................... 99 Effects of Temperature Regimes on Growth and GA Levels in Genetic Dwarfs of Apple. Steffens, G. L., P. Hedden ....................................... 105 Thidiazuron: In vitro Shoot Proliferation and Leaf Modification of Prunus serotina. Eliasson, M. K., C. A. Beyl ....................................... 106 Aspirin and Sulfanilamide Improve Processed Potato Quality. Nickell, L. G ............ 115 Effect of Flavonoids on Soybean Nodulation and Yield. Clark, T. H., C. A. Stutte 121 Video graphic Analysis and Root Dynamics in Cotton and Soybeans. Ball, R. A., C. A. Stutte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Response of Maize and Rice to 9-B-L(+) Adenosine Applied under Different Environmental Conditions. Ries, S., V. Wert ..................................... 131 Lipid Antioxidants and Soybean Seed Storage. Aho, D. W., D. J. Parrish, D. M. Orcutt 133 Video graphic Classification Analysis of Bioregulator Treated Crops. Stutte, C. A., T. C. Clark, R. A. Ball .......................................... 134 iv Tmfgrass Response to Cimectacarb. Vitolo, D. B., P. J. Porpiglia, J. W. Peek, R. L. Brooks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Influence of Atrinal, Figaron and RSW 0411 Treatments on Calendula ojficinalis, L. Plants. AlBadawy, A. A., N. M. Abdalla, A. A. El-Sayed ........................ 140 Microbial Transformations of Bioactive Substrates. Hufford, C. D. . ................ 148 The Role of Polyamines in in vitro Cold Hardiness of White Clover (Trifpliwn repens L.). MacLeod, K. D. A., J. Nowak ..................................... 154 After Discovery: The Issue of Supply in the Development of Plant Drugs. McChesney, J. D ............................................... 160 Brushing Vegetable Transplants for Height Control: Survey of Cultivar Responses. Latimer, J. G., S. A. Baden ....................................... 166 Environmental Factors in Blossom Thinning Apple Trees With Monocarbamide Dihydrogensulfate (MCDS). Curry, E. A., M. W. Williams ................. 168 Injury, Regrowth and 14C-Sucrose Translocation in Canada Thistle Following Treatments of Mefluidide, Flurprimidol and Systemic Herbicides. Tworkoski, T. J., J. P. Sterrett ................................................. 169 Effect of a PGR Product and FE on Water-Stressed Kentucky Bluegrass. Nabati, D. A., R. E. Schmidt, D. J. Parrish ....................................... 170 Effects of Biozyme TF on Russeting in Golden Delicious Apple. Ramirez, H., A. Macias, A. Kamara . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Index of Authors .................................................... 175 v The Biological Activity of Some Mixed Sucrose and Glucose Esters From Exotic Tobacco Cultivars l l l Horace G. Cutler , Ray F. Severson Patsy D. Cole 2 2 3 Vernon A. Sisson , Michael D. Jackson , Michael G. Stephenson Sets of mixed sucrose and glucose esters, sucrose esters, and glucose esters were isolated from exotic tobacco species and evaluated for their biological activity in the etiolated wheat coleoptile bioassay and in impregnated disk assays against selected Gram-positive and Gram negative bacteria. Tops of selected field grown tobacco were harvested and dipped into methylene chloride; the resulting solution was evaporated in vacuo to remove the solvent and the extracts containing the sugar esters were separated by chromatography. These were subjected to capillary gas chromatography to obtain the mass, molecular formulae, and fragmentation patterns. Following segregation into groups of a) mixed sucrose and glucose esters; b) mixed sucrose esters; and c) mixed glucose esters, extracts of each tobacco species were tested for biological activity. Plant bioassays were conducted on etiolated wheat coleoptiles obtained from 4-day-old Triticum aestivum L. cv. Wakeland seedlings. Etiolated seedlings were harvested under a safelight at 540 nm and roots and caryopses were discarded. The apical 2 mm were cut away and discarded while the next 4 mm were cut and retained for bioassay. Compounds were dissolved in acetone and 5 ml of phosphate-citrate buffer at pH 5.6, supplemented with 2% sucrose to make a 10-3 M stock solution. From these, dilutions were made to 10-4, 10-5 and 10-6 M; ten 4 mm coleoptiles were added to each test tube and incubated for - 18 hours. Sections were then measured relative to controls. Bacterial bioassays were carried out on Bacillus subtilis, B. cereus, Mycobacterium thermosphactum (all Gram-positive) and Escherichia coli, E. cloacae, and Citrobacter freundii (all Gram-negative). Organisms were densely seeded onto nutrient agar plates and 4 trim disks were impregnated with each compound to be tested at 50, 250, and 500 ~g, then incubated at 37°C overnight. Those species predominantly containing both sucrose and glucose esters were Nicotiana cavicola, N. debneyi, N. noctijlora, N. maritima, and N. palmerii. Sucrose esters appeared in N. simulans, N. plumbaginifolia, and N. langsdoifii. Glucose esters were present in N. miersii. Acid chainlengths ranged from C4-C7 and contained CH branching. With the exception of N. 3 3 langsdoifii, all exhibited inhibitory activity against growth of etiolated wheat coleoptiles at 10- to 10-4 M. Activity against bacteria varied, depending upon whether they were Gram-positive or Gram-negative, and was species dependent. lUSDA, ARS, Richard B. Russell Center, Athens, GA 30613; 2USDA, ARS, Oxford, NC 27565; 3USDA, ARS, Tifton, GA 31793 1 INCREASING ARTEMISININ PRODUCTION THROUGH BIOTRANSFORMATION OF PRECURSORS l l l 2 P. K. Chen , C. Lukonis , Linda Go , and G. R. Leather ABSTRACT Artemisinin, a sesquiterpene lactone with an endoperoxide moiety, is an antimalarial agent isolated from sweet wormwood (Artemisia annua). Recent studies have shown that artemisinin has good potential as a natural herbicide. Five different monoterpenes (borneol, camphor, limonene, menthol, and pinene) and two sesquiterpenes (artemisic acid and arteannuin B) were used in biotransformation studies to increase !. artemisinin production in annua green callus tissue and leaf tissue homogenate. The results revealed that borneol, pinene,and a combination of artemisic acid and arteannuin B can increase artemisinin production significantly. Addition of glucose oxidase and tyrosinase to the tissue culture of leaf homogenate did not further increase the yield of artemisinin. lDepartment of Biology, Georgetown University, Washington, D.C. 20057. 2Foreign Disease-\'leed Science Research Unit, ARS, U. S. Department of Agriculture, Frederick, MD 21702 2 INTRODUCTION Artemisinin is an antimalarial agent that was isolated from sweet wormwood (Artemisia annua) by Chinese and American scientists (Klayman 1985, Luo and Shen 1987). The herbicidal potential of artemisinin has been reported by Chen and Leather (1990), Chen et al. (1991), and Duke et al. (1987). Chen and Leather have also demonstrated that artemisinin caused phytotoxicity in a number of weeds and vegetable plants except corn and flax. The mode of action of artemisinin in plants is different than that of two commonly used herbicides 2,4-D and glyphosate in mungbean and Lemna minor bioassay systems (Chen, et al. 1991). Akhila et al. (1987) reported that artemisic acid and arteannuin Bare immediate precursors of artemisinin. However, the content of artemisinin in !. annua is very low. On the other hand, the contents of its precursors such as artemisic acid (AA) and arteannuin B (AB) are two to ten times higher than artemisinin. Therefore, this study was to explore whether plant tissue culture and plant leaf tissue homogenates could use artemisic acid, arteannuin B, and other precursors to increase the yield of artemisinin. MATERIALS AND METHODS Fresh leaf tissue of A. annua was harvested from the garden grown plants and chopped into small pieces. About two grams of this chopped tissue were transferred to a flask containing 50 ml of 20% methanol in a sodium acetate and phosphate buffer (pH 7.0, 0.02 M) solution. After one day incubation, all the cultures were extracted with ethyl acetate to isolate artemisinin. 3 Green callus tissue cultures were established from lateral buds of selected greenhouse grown seedlings. This culture has been maintained in a modified Murashige and Skoog shoot multiplication medium A (half of the recommended amount and supplemented with naphthalene acetic acid at 2 mg/l), and transferred at monthly intervals for more than 30 times. For transformation experiments, about two grams of the green callus tissue were transferred to 50 ml of the same liquid medium for seven days, then the precursors were added. Five different monoterpenes borneol, camphor, limonene, menthol, and pinene, two sesquiterpenes namely, artemisic acid and arteannuin B (kindly provided by Drs. Klayman and N. Acton, Water Reed Army Institute of Research, Washington, D.C.) were incorporated into the medium. All the cultures were incubated in a growth chamber (25 C, continuous light) for 72 hours, then 100 ml of ethyl acetate was added. The artemisinin was extracted overnight in a hood at room temperature. The ethyl acetate fraction was obtained after filtration and partition, and then the volume was reduced in vacuum and dried under nitrogen in an N-vap. Qualitative and quantitative analyses were performed by TLC and HPLC and by the method described by Zhao and Zeng (1986). All experiments were prepared in triplicates and repeated once. Authentic artemisinin was used to prepare a standard curve. RESULTS AND DISCUSSION The data revealed that among the monoterpenes used in this study only borneol and pinene s110wed a stimulatory effect on artemisinin production (Table 1). 4 Table 1. The effect of monoterpenes on production of artemisinin (relative to control) Control Borneol Camphor Linonene Menthol Pinene 1.000 1.098 1.029 0.947 0.821 1.210 +0.141 +0.025 +0.025 +0.065 +0.050 +0.051 Our results also revealed that when artemisic acid and arteannuin B were incorporated into the medium separately, there was little stimulatory effect on artemisinin production. However, when these two precursors were incorporated together into the medium, the artemisinin production increased signficiantly. The data are shown in Table 2. Table 2. Effect of precursors on the production of artemisinin in callus culture Precursor (mg) Yield (mg/g) ReI. -% l. Control 0.61 100.00 2. Artemisic acid (10) 0.60 98.36 3. (25) 0.87 142.62 4. Arteannuin B (10) 0.71 116.39 5. (25) 0.73 119.67 6. (2) + (4) 3.07 503.28 7. (3) + (5) 3.02 495.08 Artemisinin has an endoperoxide moiety, thus two enzymes namely, glucose oxidase and tyrosinase, which are known to generate hydrogen peroxide in the presence of their respective substrate glucose and tyrosine, were selected to investigate whether their addition could affect the production of artemisinin. Although the addition of artemisic 5 acid and arteannuin B significantly increased the production of artemisinin, the addition of glucose oxidase decreased production of artemisinin (Table 3). The green tissue culture turned to be somewhat greenish yellow when it was terminated for extraction and analysis. This may be due to the bleaching effect of free peroxide generated by glucose oxidase. Addition of glucose and glucose oxidase showed similar effects in leaf tissue homogenate. Tyrosinase treated leaf tissue homogenate showed 4.99 times more artemisinin than i:he con(:col. In the tissue culture system, the production of artemisinin was 1.58 and 3.15 (with and without tyrosinase) times higher than the controls, respectively (Table 3). Table 3. The influence of glucose oxidase and tyrosinase on precursor transformation to artemisinin in green callus culture and leaf tissue homogenate. ti~sue Precursor Enzyme Substr. Art~misinin* in system with AA AB Glucose oxidase Tyrosinase Green Callus Tissue Culture + 3.70+ 3.57 3.72+3.72 + + + 20.llJ.+ 4.23 11.72+5.98 + + + 1.- 12.05+17.0 5.89+4.50 Leaf Tissue Homogenate + 3.37+ 1.57 2.22+2.22 + + + 20.88+ 3.28 5.42+5.33 + + + + 18.40+11. 3 11.07+1. 73 *yield of artemisinin = mg/g of tissue According to Klayman (1985) and Luo and Shen (19B?), artemislc acid 6