Flower Formation in Excised Tobacco Stem Segments

Abstract

A method has been developed that extracts DNA from stem tissue of flowering tobacco plants, Nicotiana By in vitro cultivation of excised stem segments, flowering tobacco plants have been shown to have a pronounced apex to base gradient in the capacity to form floral buds (1, 2), which truly reflects a difference in the capacity to flower and not merely a decrease in the over-all capacity to form buds (32). Appropriate exogenous supplies of IAA effected a transition from floral to vegetative bud formation in such segments (31).

Rapid Initiation of Thymidine Incorporation into Deoxyribonucleic Acid in Vegetative Tobacco Stem Segments Treated with Indole-3-acetic Acid

“…Recently, a correlation between in vitro floral expression and DNA content in stem segments from a flowering tobacco plant has been reported (32). The DNA content in stem segments above the gradient was 1 0-fold greater (on a fresh weight basis) than in stem segments below it, and the DNA content decreased roughly in parallel with the gradient itself.…”

“…For each treatment (most experiments consisted of about 10 treatments), the stem bottoms of two shoots were placed in contact with 0.25 ml of labeling solution (0.1 mCi (methyl-3H)thymidine, 25 ,ug/ml chloramphenicol) contained in a 5-ml beaker. Then the shoots were illuminated as previously described (32). They absorbed the labeling solution in 30 min.…”

“…Of the remaining stem tissue (7.5 cm), a fraction containing the DNA was extracted from a homogenate as previously described (32) but with the following changes. One mg of unlabeled spooly fraction (1.25 mg/ml), extracted from stem tissue homogenates of flowering tobacco plants (32), was added as carrier to 5 ml of grinding medium contained in a mortar. (Wardell and Skoog have reported that DNA cannot be isolated from vegetative stem tissue homogenates alone.)…”

“…The remaining portions (1.9 ml/treatment) of the separate fractions per experiment were pooled, and the concentration and the radioactivity of the pooled fraction were determined (duplicate 0.05-ml samples). The rest of the pooled fraction was mixed with solid sodium perchlorate, and the DNA was extracted (32) without any further changes. Then, the absorbancy of the DNA and the radioactivity in the DNA were measured.…”

“…Next, the radioactivities in the samples were measured as described previously (32) except that a Nuclear-Chicago Mark I liquid scintillation system was used for counting. The remaining portions (1.9 ml/treatment) of the separate fractions per experiment were pooled, and the concentration and the radioactivity of the pooled fraction were determined (duplicate 0.05-ml samples).…”