The Time Course of Xylem Differentiation and Its Relation to Deoxyribonucleic Acid Synthesis in Cultured Coleus Stem Segments

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Abstract
The relationship between DNA synthesis and wound xylem differentiation was investigated in cultured stem segments of Coleus blumei. The addition of 50 micrograms of indoleacetic acid per liter to the culture medium resulted in a 400 to 500% increase in the number of wound vessel members formed in 7 days. However, the time course of wound vessel member formation was similar in segments cultured in the presence and absence of auxin. In either case, no wound vessel members appeared before the 3rd day of culture, while the majority of wound vessel members appeared on the 4th and 5th days of culture. (3)H-Thymidine incorporation into DNA was used to measure changes in the DNA synthetic activity of the tissues during the culture period. Comparatively little (3)H-thymidine incorporation occurred during the 1st day of culture. Maximum (3)H-thymidine incorporation was observed on the 2nd day of culture, 2 days before the peak period of xylem differentiation. The rate of incorporation of (3)H-thymidine into DNA decreased with increasing time in culture after the 2nd day. Auxin at 50 micrograms per liter had no effect on the time course of (3)H-thymidine incorporation, although somewhat more (3)H-thymidine was incorporated into DNA throughout the culture period in the presence of auxin. The magnitude of this effect was small when compared to the effect of auxin on xylem differentiation. The antimetabolite 5-fluorodeoxyuridine was shown to block DNA synthesis in the cultured stem segments. When the tissues were isolated on media containing 10(-6)m 5-fluorodeoxyuridine, wound vessel member differentiation was inhibited by approximately 80%, in both the presence and absence of auxin. Thymidine at 10(-5)m completely overcame the 5-fluorodeoxyuridine inhibition of wound vessel member formation. 5-Fluorodeoxyuridine was effective in blocking xylogenesis only when this substance was supplied to the tissues during the early part of the culture period. 5-Fluorodeoxyuridine had no effect on xylem differentiation when it was applied after the 3rd day of culture.