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Hae-Jeen Bang, Soo-Jung Hwang, Hee Chung, and Jung-Myung Lee

Soybean sprouts are one of the most-favored traditional vegetables around the world. The sprouts are usually consumed 7 to 10 days after sowing depending upon the growing conditions. High-quality sprouts should have less secondary roots, short and well-swollen hypocotyls in pure white color, and small cotyledons in hooked position. Cytokinins were reported to be effective in producing such sprouts by promoting sprout growth while inhibiting the excessive hypocotyl elongation and secondary root growth. Seeds of four soybean cultivars with different characteristics were soaked in water for 4 h and, 2 to 3 h after the imbibition, the seeds were soaked again in solutions of different cytokinins such as benzyladenine (BA), BA-riboside (BAR), BPA, 2iP, 2iP-riboside, 4-CPPU, and kinetin-riboside (KR) for 10 min. After the treatment, the sprouts were grown in a plastic tube (25 cm height × 10.5 cm diameter) a dark culture room with ample watering every 4 h. After 7 days of growth, uniform samples were taken from each treatment and the sprout characteristics were examined. Some cytokinins such as BA, BAR, 4-CPPU were highly effective in promoting the sprout growth (fresh weight) even though the hypocotyl length was markedly reduced. Other cytokinins such as 2iP, 2iPR, and KR had no effect on sprout growth. Hypocotyl diameter was markedly increased by BA and 4-CPPU treatment, thus resulting in short, strong and good quality sprouts. Cultivars responded differently to cytokinin treatment by showing different growth promotion depending upon the sprout parts. Injury-like symptoms, abnormal and twisted heads or cotyledons, appeared in cytokinin-treated sprouts at high concentrations and the symptoms were severe when the sprouts were grown at high temperatures. In all the cultivars tested, BAR appeared to be better than others in terms of sprout quality and growth promoting characteristics.

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Young Hee Joung, Mark S. Roh, Kathryn Kamo, and Jeoung Seob Song

The main goal of this research was to develop Campanula glaumerata `Acaulis' plants transformed with the isopentenyl transferase (ipt) gene for increased growth of the axillary buds and en-hanced insect resistance. Isopentenyl transferase is a first enzyme in the cytokinin biosynthetic pathway. For Campanula transformation, leaf discs were co-cultivated with Agrobacterium tumefacience LBA4404, which harbored the binary vector pBC34 (A. Smigocki, Beltsville, Md.) that codes for the nos-nptII gene and the ipt gene controlled by the CaMV35S promoter. The transformation frequency was about three times higher when leaf blade explants were infected with LBA4404 containing pBC34 as compared to infection with pGUSINT, which contains the gusint gene instead of the ipt gene. This difference in transformation frequency was attributed to expression of cytokinin from the ipt gene. Transgenic plant lines containing the ipt gene were verified by southern hybridization and divided into three groups by phenotype following culture in vitro on MS medium: 1) yellow/large leaves, no rooting; 2) green/large leaves, no rooting; 3) green/normal leaf size, rooting. These different phenotypes could be due to different levels of cytokinin expression in the transgenic plants.

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Robin A. Cohen and Duane W. Greene

Two experiments were conducted to examine the effects of growth regulator application on cytokinin levels in developing apple leaves. In experiment 1, gibberellin GA4/7 (100 mg·liter-1) and benzyladenine (BA, 100 mg·liter-1) were applied alone or in combination as a foliar spray to `Golden Delicious' 7 days after full bloom. In experiment 2, BA was applied at 50 or 100 mg·liter-1 as a foliar spray to `Morespur McIntosh' 18 days after full bloom. Trans-zeatin ribosidelike (t-ZR) levels were determined by immunoassay. In experiment 1, GA1/7 had no effect and BA increased t-ZR-like levels. In experiment 2, BA increased t-ZR levels quadratically with application rate. The increase in endogenous cytokinin levels with BA treatment occured within 2 hours. Initially, t-ZR-like levels were 47 times higher in the BA (100 mg·liter-1) treated leaves as compared to check leaves. Over the next 8 days, t-ZR-like content in treated leaves decreased in a cubic manner.

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William R. Woodson and Amanda S. Brandt

Treatment of cut carnation (Dianthus caryophyllus L. `White Sim') flowers with the synthetic cytokinin benzyladenine (BA) at concentrations >1.0 μm induced premature petal senescence. Flowers treated with 100 μm BA exhibited elevated ethylene production in styles and petals before untreated flowers. The gynoecia of BA-treated flowers accumulated 1-aminocyclopropane-l-carboxyllc acid (ACC) and enlarged before untreated flowers. Removal of the gynoecium (ovary and styles) or styles prevented BA-induced petal senescence and resulted in a substantial delay in petal senescence. In contrast, removal of the gynoecium had no effect on timing of petal senescence in flowers held in water. These results indicate BA stimulates petal senescence by inducing premature ACC accumulation and ethylene production in the gynoecium.

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Emily B. Merewitz, Thomas Gianfagna, and Bingru Huang

desiccation ( Fry and Huang, 2004 ). Plant adaptation to drought stress has been associated with the hormonal regulation of these processes. Changes in the level and proportion of endogenous phytohormones, such as cytokinins (CK) and abscisic acid (ABA

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Ann C. Smigocki

Cytokinins were first recognized as a class of phytohormones for their ability to promote cytokinesis in cultured plant cells and have since been shown to be involved in a wide range of physiological processes. Most recently, the availability of phytohorm one-specifying genes from Agrobacterium tumefaciens has allowed for direct in planta manipulation of cytokinin levels. Overexpression of the isopentenyl transferase (ipt) gene by constitutive promoters led to enhanced ability of plant cells to undergo shoot organogenesis but the high endogenous cytokinin levels almost completely suppressed root development. Transient overproduction of cytokinins using promoters regulated by environmental and/or developmental factors did not inhibit regeneration of rooted plants. Transgenic plants in which cytokinin levels can be modulated are being used to characterize the participation of cytokinins in fundamental regulatory mechanisms of morphogenesis, delayed senescence, disease resistance and directed nutrient transport. The potential for using reconstructed cytokinin biosynthesis genes in economically important crops is of tremendous agronomic significance.

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A.G. Reynolds, D.A. Wardle, C. Zurowski, and N.E. Looney

One of three levels (O, 1, 10 mg·liter-1) of the cytokinin-active substituted phenylurea compound CPPU was applied with or without 100 mg GA/liter to developing clusters of `Sovereign Coronation' and Summerland Selection 495 grapes (Vitis spp.). In a similar experiment, one of three levels (0, 1, 10 mg·liter-) of either CPPU or the related compound thidiazuron was applied to `Simone' and Summerland Selection 535. Both phenylurea chemicals tended to linearly increase cluster weight and berry weight while reducing degrees Brix, pH, and anthocyanins and increasing titratable acidity. A subsequent trial with O, 4, and 8 mg thidiazuron/liter on all four varieties yielded similar results. GA had no individual or synergistic effects. Due to the very low concentrations required, CPPU and thidiazuron show great promise as chemical tools for the increase of berry weight in seedless table grapes. Chemical names used: N-(2-chloro-4-pyridyl) -N'-phenylurea (CPPU); N1-phenyl-N'-l,2,3-thiadiazol-5-yl urea (thidiazuron);

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Lisa J. Rowland and Elizabeth L. Ogden

Conditions for improving the efficiency of shoot regeneration from leaf sections of highbush blueberry (Vaccinium corymbosum L.) were investigated. Effectiveness of tissue culture medium supplemented with the cytokinin conjugate zeatin riboside or the cytokinin zeatin at 10, 20, or 30 μm was compared with medium supplemented with the optimum 2iP concentration of 15 μm. Use of 20 μm zeatin riboside resulted in the most shoots per leaf section, » 6-fold higher than the number of shoots produced on 2iP medium. The number of shoots produced on medium supplemented with zeatin was not significantly higher than the number of shoots produced on 2iP medium. Consequently, we concluded that the cytokinin conjugate zeatin riboside was more effective than either of the free cytokinins, 2iP or zeatin, in promoting shoot regeneration from leaf sections of highbush blueberry. Chemical names used: 6-(y,y-dimethylallylamino)-purine (2iP); 6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin).

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Eric W. Mercure, Carol A. Auer, and Mark H. Brand

Tissue proliferation (TP) is characterized primarily by the formation of galls or tumors at the crown of container-grown rhododendrons propagated in vitro. However, TP of Rhododendron `Montego' is observed initially in in vitro shoot cultures and it is characterized by the formation of multiple shoots with small leaves and nodal tumors. The formation of shoots in `Montego' TP (TP+) shoot cultures occurs without the presence of exogenous cytokinin in the medium, unlike normal `Montego' (TP–) shoot cultures, which require cytokinin for shoot growth. Structural studies have shown that tumors are composed of many adventitious buds and parenchyma cells, suggesting that TP is a result of abnormal cytokinin regulation that is controlling tumor and shoot formation. Two approaches are being used to determine if differences in cytokinin concentration and/or metabolism exist between TP+ and TP– shoot cultures. In the first approach, shoot cultures are grown in vitro for 1 week in the presence of tritiated isopentenyladenine (iP). Cytokinin uptake and metabolism are analyzed using HPLC and other analytical methods. Experiments suggest that extensive degradation and N-glucoside conjugation occur in TP+ and TP– shoots, resulting in the removal of most of the exogenous iP. In the second approach, the levels of endogenous cytokinins such as iP, isopentenyladenosine, zeatin, and zeatin riboside, are being measured in TP+ tumors and shoots and in TP– shoots by an ELISA method.

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Sandra B. Wilson and Dennis R. Decoteau

Similarities exist between the effects of phytochrome and cytokinins on plant growth and development (e.g., chloroplast development, amaranthin synthesis, seed germination). It is unclear, however, if and how these two systems interact. The coaction between phytochrome and cytokinins was investigated by using Nicotiana plumbaginifolia plants transformed with the isopentenyl transferase (ipt) cytokinin gene and treated with end-of-day (EOD) red (R) and far-red (FR) light. The ipt gene was under control of either a constitutive cauliflower mosaic virus promoter (35S-plants) or an inducible, heat shock promoter (HS-plants). When treated with EOD FR light, whole plants were characterized by decreased chlorophyll concentrations and increased fresh weights. When treated with EOD R light, 35S-plants contained high concentrations of zeatin riboside (ZR) compared to plants treated with EOD FR light. When treated with EOD FR light, HS-plants contained high concentrations of ZR compared to plants treated with EOD R light. Both cytokinin responses were photoreversible. The reasons for the differences between the 35S- and HS-plant responses are not known. Results appear to implicate interactions between phytochrome and cytokinins in plant growth and development.