The uptake and metabolism of exogenous tritium-labelled benzyl adenine was studied during the shoot induction period of petunia leaf explants in tissue culture. Transfer experiments with Petunia `MD1' leaf explants (1 cm2) on MS media with 2.2 uM BA show that 27% and 100% of the leaf explants are committed to shoot induction on days 6 and 10, respectively. To study BA uptake and metabolism, leaf explants were placed on media containing tritium-labled BA for 1, 3, 6 and 10 days. BA was taken up from the media on days 1-6. BA metabolizes were analyzed using HPLC, a UV absorbancc detector and enzymatic techniques. Metabolizes produced include: BA, BAdo, BA 7G, BA 9G, BAdoMP, BAdoDP, BAdoTP and 3 unidentified compounds. BA and BAdo were detected on days 1 and 3 but not during day 6-10, the time of shoot induction. The pool of ribotide metabolites decreased from days 1 to 10, from 26.5% of all metabolites to 1.6%. Glucosylated compounds, BA 7G and BA 9G, increased continuously from 24.9% to 69.8% between days 1 and 10. An unidentified compound C increased from 13% on day 3 to 24.8% on day 10. In separate experiments, BA uptake and metabolism were compared in two Petunia hybrida lines, St40 and TLV1, with different shoot organogenic responses in tissue culture. These data show interesting patterns of BA metabolism in relationship to shoot induction and organogenesis.
Carol Auer, Jerry D. Cohen, and Todd Cooke
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.
James F. Harbage, Dennis P. Stimart, and Carol Auer
The influence of root initiation medium pH on root formation was investigated in relation to uptake and metabolism of applied IBA in microcuttings of Malus ×domestica Borkh. `Gala' and `Triple Red Delicious'. Root formation and uptake of H 3-IBA were related inversely to root initiation medium pH. Maximum root count (10.3 roots) and IBA uptake were observed at pH 4.0. Regardless of pH, overall root count of `Gala' was higher (13.5 roots) than `Triple Red Delicious' (4 roots). Uptake of IBA was highest at pH 4.0 for `Gala' (1.7% uptake) and at pH 4 and 5 for `Triple Red Delicious' (0.75% uptake). Metabolism of IBA was the same regardless of root initiation medium pH or cultivar examined. One-half of the IBA taken up was converted to a compound that coeluted with IBAsp during high-performance liquid chromatography. Apparently, pH regulates root formation by affecting IBA uptake but not metabolism. The level of auxin in tissue appeared unrelated to root formation between genotypes. Chemical names used: 1H-indole-3-butyric acid (IBA); 5-H 3-indole-3-butyric acid (H 3-IBA); indole-3-butrylaspartic acid (IBAsp).