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Sandra L. Barbour 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, photomorphogenesis). It is unclear. however, if and how these two systems interact.

To determine the effects of phytochrome activity on cytokinin synthesis and ultracellular plant development, we utilized tobacco transformed with the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene. This gene encodes for isopentenyl transferase (iptase) which is an enzyme active in cytokinin biosysthesis.

Ipt-transgenic tobacco cultures were treated with end-of-day red or far-red light for 15 minutes. After 15-30 days of treatment, the plant tissue was harvested and ipt expression was verified by SDS-PAGE and western blot analysis. Polyclonal antibodies specific to iptase were used as a primary antibody. Colloidal gold conjugated to goat. anti-rabbit antiserum served as an electron dense, secondary antibody and a probe to light-influenced iptase synthesis and distribution within the cell.

A Hitachi 600AB transmission electron microscope was used to determine the influence of phytochrome/light treatments on the ultrastructure of ipr-transgenic cells.

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Sandra L. Barbour and John J. Frett

Isopentenyl transferase, encoded by the ipt gene of Agrobacterium tumefaciens T-DNA, is an enzyme active in cytokinin biosynthesis. The ipt gene was cloned into the pMAL-c2 vector (New England Biolabs, Beverly, MA) and expressed as a fusion protein. The production of this fusion protein was induced by a 2 hour exposure to IPTG. The fusion protein was then purified by a mini-aggregate procedure and visualized by SDS-PAGE. To verify that the correct protein was purified, antibodies specific to the conserved region of the fusion protein were used to probe a western blot. Secondary antibodies were biotinylated. Rabbits were immunized to raise polyclonal antibodies against iptase. Using a slot format blotting apparatus, serum was titered. These antibodies will be used to probe western blots from transgenic plants transformed with various ipt constructs.

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Sandra L. Barbour, D.A. Schaff, and J.J. Frett

Cytokinins are involved in in vitro shoot initiation, although little is known about their mode of action. As the first step in localizing cytokinin synthesis, we present a cloning and expression strategy for the isopentenyl transferase (ipt) gene. The source of the Agrobacterium tumefaciens ipt gene was a 7.2 kb Eco RI fragment isolated from pBREF7 (Dr. Ann Smigocki, USDA, Beltsville, MD). The ipt gene was amplified by polymerase chain reaction (PCR) and cloned into pMal-cRI (New England Biolabs, Beverly, MA). Using the pMAL-cRI expression and purification system, the isopentenyl transferase protein will be purified for antibody production. These antibodies will be used in future work to localize the isopentenyl transferase enzyme.

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Sandra L. Barbour, Margaret J. McMahon, John J. Frett, 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, photomorphogenesis). It is unclear, however, if and how these two systems interact.

As a beginning step to determine cytokinin-phytochrome interactions, we developed a strategy utilizing ipt -transgenic tobacco in phytochrome/light treatment investigations. The sour-cc of the ipt gene was Agrobacterium tumefaciens Ti plasmid 15955. This gene encodes for isopentenyl transferase which is an enzyme active in cytokinin biosynthesis.

Ipt -transgenic tobacco cultures (grown on MS medium supplemented with kanamycin but no plant growth regulators) were treated with end-of-day red or far-red light for 15 minutes. After 30 days of treatment, the plant tissue was harvested and either homogenized for SDS-PAGE or fixed for transmission electron microscopic analysis.

Results from immuno-gold labelling using polyclonal antibodies specific to iptase will he used to Indicate the influence of phytochrome on cytokinin activity. Also, structural changes at the ultra-cellular level will be determined.

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Sandra L. Barbour, Kathy H. Brock, B.A. Fortnum, and Dennis R. Decoreau

Pest control-related problems jeopardize the advancement of our nation's vegetable industry. Because of the adverse effects of many fumigants. the grower is increasingly pressured to utilize sustainable. environmentally sound agricultural practices yet still maintain a marketable, blemish-free product.

The effects of wavelength selective mulches and three different fumigants on overall plant development and nematode control were studied in field grown, staked tomatoes. Plots were fumigated with methyl bromide. Telone II, or Telone C17. Within rows, mulch color was established by application of either white or red exterior enamel paint to the black plastic surface of polyethylene mulch. Reflective light from each mulch color was measured using a LiCor 1800 Spectroradiometer. Temperature below the mulch surface was monitored with a datalogger.

Prior to the first marketable harvest, plants grown on white mulch produced greater fruit weight and total dry weight than plants grown on black or red mulch. Total marketable yields, however. were not significantly different between the three mulches. Early and marketable yields from fumigated plots did not differ from control treatments. The lack of response due to fumigation may have been due to low initial nematode populations in the field.

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Sandra L. Barbour, D.A. Schaff, A.C. Smigocki, and J.J. Frett

The ipt gene of Agrobacterium tumefaciens T-DNA encodes for isopentenyl transferase, which is an enzyme active in cytokinin biosynthesis. While it is known that cytokinins are associated with in vitro promotion of cell division and stimulation of shoot production, little is known about their mode of action. As the first step in localizing cytokinin synthesis, we present a cloning and expression strategy for the ipt gene. The source of the ipt gene was Agrobacterium tumefaciens octopine Ti plasmid 15955. The ipt gene was amplified by the polymerase chain reaction (PCR) and cloned into pMal-c2 (New England Biolabs, Beverly, MA). This construct was transformed into E. coli and the ipt gene was expressed as a fusion protein. The protein was purified by affinity chromatography to serve as an antigen for polyclonal antibody production. These antibodies will be used to localize isopentenyl transferase in plant tissue.