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Fatemeh Haddadi, Maheran Abd Aziz, Hossein Kamaladini, and Seyed Ali Ravanfar

this study was to investigate the optimal conditions for in vitro plant regeneration via organogenesis from leaf explants of strawberry using combinations of TDZ and BAP. The use of zeatin as an adenine-type cytokinin to induce adventitious shoot

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Samir C. Debnath and Danny L. Barney

growth and morphogenesis ( Miller, 1961 ). They induce cell division and shoot differentiation ( Skoog and Miller, 1957 ). Zeatin and its derivatives are the most abundant cytokinins in plants ( Letham and Palni, 1983 ). Zeatin was found effective for

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Ruth C. Martin, Machteld C. Mok, and David W.S. Mok

Cytokinins are widely used in tissue culture and transformation systems; however, little is known of their mode of action or the mechanisms regulating their levels in plant tissues. We are studying enzymes responsible for the metabolism of zeatin in immature seeds of Phaseolus. Selective expression of genes encoding such enzymes may regulate the level of active cytokinins during seed development as well as in in vitro systems. A zeatin O-xylosyltransferase, which mediates the formation of O-xylosylzeatin from trans-zeatin and UDP-xylose, has been isolated and monoclonal antibodies specific to the enzyme have been produced. Tissue print analyses demonstrated that the enzyme is primarily localized in the endosperm. ln situ localization and EM studies indicated that the enzyme is present in the cytoplasm and the nucleus. cDNA libraries were constructed from immature seed mRNA and immunopositive clones were selected. The products of these clones are being analyzed in E. coli and baculovirus expression systems.

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Tracy S. Hawkins, Nathan M. Schiff, Emile S. Gardiner, Theodor Leininger, Margaret S. Devall, Dan Wilson, Paul Hamel, Deborah D. McCown, and Kristina Connor

twice in sterile distilled water. Sterilized explants were placed on woody plant medium (WPM) ( Lloyd and McCown, 1980 ) supplemented with 1 μ m zeatin, and with a mixture of agar (4 g·L −1 ) and Gelright (1.4 g·L −1 ) added as gelling agents. Explants

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Dongliang Qiu, Xiangying Wei, Shufang Fan, Dawei Jian, and Jianjun Chen

that are equal to 1 cm. IBA = indole-3-butyric acid; WPM = woody plant medium; ZT = zeatin. Table 1. The number of adventitious shoots regenerated from leaf, stem, and callus explants of ‘Sunshine Blue’ cultured on woody plant medium supplemented with

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Barbara M. Reed and Ana Abdelnour-Esquivel

Explants of mature pot-grown Vaccinium corybosum L. cultivars were tested for initiation of new shoots using two growing conditions and four cytokinin treatments. Initiation tests with 12 genotypes showed significantly higher rates of new shoot growth on modified woody plant (MWPM) medium with 4 mg zeatin/liter at 25C under low light intensity than on any other treatment. Explants at 25C in light with 10 or 15 mg 2iP/liter initiated at a moderate rate, but significantly lower rates were found for all controls and at 4C in darkness. To determine the utility of zeatin for initiation of diverse genotypes, 96 Vaccinium accessions from the National Clonal Germplasm Repository, representing 22 species and 44 cultivars, were screened using 25C and low light intensity. Initiation rates higher than 60% were achieved for 89 of 96 accessions tested. Chemical name used: N6-[2-isopentenyl] adenine (2iP), 6-[4-hydroxy-3-methylbut-2-enylamino]purine (zeatin).

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James W. Rushing

Broccoli (Brassica oleracea L. var. italica `Citation') florets were treated postharvest with either benzyladenine or trans-zeatin at either 10 or 50 ppm before packaging in perforated polyethylene bags and storage at 16C. The most pronounced effects were observed with benzyladenine at 50 ppm. Compared to controls, respiration rate was reduced 50% and ethylene production increased 40% throughout the first 4 days of storage. Total chlorophyll content had dropped 60% in controls, but was unchanged in cytokinin-treated florets, which had a 90% longer shelf life than controls. These effects depended on the amount of cytokinin applied and were of greater magnitude with benzyladenine than with zeatin.

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Grzegorz Bartoszewski, Cesar V. Mujer, Katarzyna Niemirowicz-Szczytt, and Ann C. Smigocki

A Lycopersicon esculentum Mill. (tomato) cDNA clone with high similarity to a Nicotiana plumbaginifolia Viv. (tobacco) cytochrome P450 gene was isolated using 5' and 3' rapid amplification of cDNA ends (RACE). The isolated cDNA (GenBank Accession No. AF249329) has an open reading frame of 1494 base pairs (bp) and encodes a protein of 498 amino acids with 75% identity to the N. plumbaginifolia cytochrome P450 (CYP72A2) and 45% to a Catharanthus roseus G. Don (Madagaskar periwinkle) CYP72A1 protein sequence. By Southern-blot analysis, one or two highly homologous genes were detected in the L. esculentum genome. Expression of the cloned P450 gene was regulated by circadian rhythm and enhanced by wounding. Leaf transcripts were detected in the light but not dark. Highest transcript levels were observed 3 hours after mechanical wounding. No increase in expression was seen in response to applications of zeatin as with the N. plumbaginifolia gene. Of the tissues analyzed, shoot tips and young leaves and fruit had the highest detectable transcript levels. Attempts to transform more than 1400 cotyledon explants of L. esculentum with sense or antisense CYP72A2 gene constructs produced no transgenic plants.

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Samir C. Debnath

In an attempt to improve the micropropagation protocol for lingonberry (Vaccinium vitis-idaea L.) developed at the Centre, two lingonberry clones were compared for in vitro shoot proliferation on two different media supplemented with varying levels of thidiazuron (TDZ). TDZ supported proliferation at low concentrations (0.1 to 1 μm) but inhibited shoot elongation. However, usable shoots were obtained within 4 weeks by transferring shoot cluster to medium containing 1 μm zeatin. Genotypes differed significantly with respect to multiplication rate with `EL1' producing the most shoots per explant. In both genotypes, shoot proliferation was greatly influenced by explant orientation. Changing the orientation of explants from vertically upright to horizontal increased axillary shoot number, but decreased shoot height and leaf number per shoot. Proliferated shoots were rooted on a 2 peat: 1 perlite (v/v) medium, and the plantlets were acclimatized and eventually established in the greenhouse.

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Samir C. Debnath

The effects of TDZ (0, 0.1, 1, 5 and 10 μm) and explant orientation on adventitious shoot regeneration of `Erntedank' lingonberry were studied. Moderate concentration (1 to 5 μm) of TDZ supported bud and shoot regeneration, but strongly inhibited shoot elongation. TDZ initiated cultures were transferred to medium containing 1-2 μm zeatin and produced usable shoots after one additional subculture. Adventitious bud and shoot regeneration was greatly influenced by explant orientation. Elongated shoots were rooted on a 2 peat: 1 perlite (v/v) medium, and the plantlets were acclimatized and eventually established in the greenhouse with 80% to 90% survival rate.