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John J. Frett and Richard McCardell

Several cytokinins at various concentrations were tested to determine which would stimulate the most synchronous shoot initiation. Kinetin was effective only at concentrations of 50 mg/L, while 2iP and zeatin where effective from 5 to 50 mg/L. BA at 10 mg/L produced the most synchronous and the greatest number of shoots. This treatment was used to determine at what point in development cytokinins stimulate shoots. Tissue was grown in the presence and absence of BA for various lengths of time. Application of BA for at least 10 days was required to initiate shoots. Explants were not effected by BA during the first 5 days of culture. Starving tissue for various periods caused a proportional lag in shoot production. Short pulses of BA at different developmental stages did not alter the cytokinin response. Vacuum infiltration of cytokinins prior to culture did not increase the BA response.

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John J. Frett and Richard McCardell

Several cytokinins at various concentrations were tested to determine which would stimulate the most synchronous shoot initiation. Kinetin was effective only at concentrations of 50 mg/L, while 2iP and zeatin where effective from 5 to 50 mg/L. BA at 10 mg/L produced the most synchronous and the greatest number of shoots. This treatment was used to determine at what point in development cytokinins stimulate shoots. Tissue was grown in the presence and absence of BA for various lengths of time. Application of BA for at least 10 days was required to initiate shoots. Explants were not effected by BA during the first 5 days of culture. Starving tissue for various periods caused a proportional lag in shoot production. Short pulses of BA at different developmental stages did not alter the cytokinin response. Vacuum infiltration of cytokinins prior to culture did not increase the BA response.

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Greg S. Rogers and John Frett

A problem in immunocytochemistry is obtaining acceptable fixation of tissue while retaining antigenicity. Two concentrations (1% and 2.5%) of glutaraldehyde, with and without secondary fixation in 1% Osmium Tetroxide (OsO4) and varying fixation times were used.

Fixation in 1% glutanddehyde for 3 h was adequate to preserve the tissue. Some loss of fine structure was visible under an electron microscope. A solution of 2.5% glutaraldehyde was more effective in preserving fine structure. At 2 h fixation the tissue was well preserved and only slight loss of tine detail was observed. A longer fixation results in better ultrastructural preservation, but can cause loss of antigenicity.

OsO4 fixes lipids and acts as an electron dense stain. OsO4 has a negative effect on antigenicity. The use of OsO4 had little effect upon the preservation of ultrastructural detail and-did not improve staining; therefore, it was omitted in later fixations. Based on this experimental evidence, initial localization experiments will utilize tissue fixed in 1% glutaraldehyde for 3 h.

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Stephen Redcay and John J. Frett

Seeds of Chionanthus virginicus L. have a double dormancy associated with germination. Seeds are inhibited by a hard bony endocarp. Inhibition may also be due to the endosperm and possibly embryo dormancy. Experiments were performed on excised embryos in sterile culture. Little growth was noted on excised embryos, which possibly indicates dormancy within the embryo. In another experiment, whole seeds, seeds with endocarp removed, and acid scarified seeds were germinated in moist peat moss to observe inhibition by the endocarp. Seeds with endocarps removed, germinated quicker and in higher percentages than whole seed or scarified seed. Scarified seeds showed no improvement over whole seeds and radicles which were produced tended to be less vigorous. Whole seeds were also soaked for 24 hours in 1000 ppm GA and germinated in moist peat moss. Treatment with GA did improve radicle emergence.

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Gregory S. Rogers and John J. Frett

Nicotiana transformed with the isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens was fixed for 1 h in 1% glutaraldehyde and 4% formaldehyde. Ultrathin sections were collected on nickel grids. Grids were treated with polyclonal anti-IPTase antibody raised in rabbits and visualized with 10 nm, protein-A–labeled colloidal gold. Gold label was found throughout the cell, including the cell wall, vacuole, rough ER, and organelles. Cell wall and vacuole labeling appears to be due to non-specific binding and is greatly reduced by a BSA block. Mitochondria and chloroplasts also showed gold label, but not greater than established background levels. Labeling above background levels on the rough ER, free polysomes, and further label in the free cytoplasm indicate a cytoplasmic role for IPTase.

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Kathryn R. Kleiner and John J. Frett

A greenhouse study was designed to determine the relative heat tolerance of 10 lima bean cultivars and to evaluate the effects of high temperature on lima bean yield. Cultivars were arranged in a randomized complete block with three blocks per treatment. The temperature treatments were 25C day/15C night and 35C day/25C night. Cultivars varied in their response to the higher temperature, allowing for classification into three heat response groups: intolerant, average, and tolerant. Heat-intolerant plants did not experience a significant reduction in number of pods, but number of beans and total bean weight were reduced at the higher temperature. Number of seeds per pod and average weight per bean also tended to decrease in intolerant plants at 35C. In future experiments, these data will be correlated with random amplified DNA (RAPD) markers. These markers will be evaluated for their potential for heat tolerance screening.

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Gregory S. Rogers and John J. Frett

Nicotiana transformed with the isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens was fixed in 1% gluterdehyde and 4% formaldehyde for 1 h. Grids were treated with polygonal anti-IpTase antibody raised in rabbits and visualized with 10 nm protein-A-labeled colloidal gold. Initial localization was performed on Nicotiana transformed with the ipt gene under the control of the 35S promoter from cauliflower mosaic virus. Colloidal gold was found throughout the cell, including the cell wall, vacuole, and rough ER. Cell wall and vacuole labeling appears to be due to nonspecific binding and is greatly reduced by a BSA block. Colloidal gold label on rough ER provides preliminary evidence that translation occurs here rather than on free polysomes. General reaction throughout the cell indicates cytoplasmic activity of the enzyme. Future research will attempt to localize IPTase in wild-type Nicotiana and in plants transformed with the ipt gene under the control of the hsp 70 heat shock promoter.

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John Frett, W. Edward Kee, and Stephen Redcay

Lima bean yields are lower in Delaware than in other lima-bean-producing states. One of the factors that contributes to the low production is the high temperatures that occur during production. Six commercial varieties of lima beans, both fordhook and baby lima bean types, were grown in a glass greenhouse at either 25C or 35C daytime temperatures to screen for heat tolerance. Plants grown at high temperature were typically shorter and more bushy than plants grown at 25C. Few, if any, buds, flowers, or early pods remained on plants at harvest if the plants were grown at 25C, while plants grown at 35C were still producing buds and flowers. Lima bean yields were generally reduced at 35C. The magnitude of the effect on yield ranged from `F1072', which had a 100-fold decrease in yield, to `Early Thorogreen', which demonstrated a slight increase in yield in response to increased temperatures.

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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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Wallace G. Pill, John J. Frett, and Ian H. Williams

Matrically priming seeds of common Kentucky bluegrass (Poa pratensis L.) and `SR8300' tall fescue (Festuca arundinacea Schreb.) in fine, exfoliated vermiculite (-1.5 MPa, 20 °C, 4 days) increased subsequent germination rate but did not increase germination percentage or synchrony. The lowest seed: vermiculite ratio (dry weight basis) to provide full priming benefit for seeds of Kentucky bluegrass and tall fescue was 1:10 and 1:20, respectively. Storing Kentucky bluegrass seeds primed in 1:10 (seed:vermiculite) in moist vermiculite for 10 days at either 5 or 20 °C did not reduce germination rate in comparison to primed seeds that were not stored. Primed tall fescue seeds could be stored in moist vermiculite (1:20, seed:vermiculite) for up to 10 days at 5 °C with no loss of priming benefit, but storage for only 2 days at 20 °C resulted in germination. Primed seeds of Kentucky bluegrass (stored for 0 or 10 days at 5 or 20 °C) or tall fescue (stored 0 or 10 days at 5 °C or 2 days at 20 °C) resulted in more rapid germination and seedling emergence, and greater seedling shoot fresh and dry masses than was the case for nonprimed seeds.