Nuclear DNA content and phytohormone requirements of normal, crown gall and habituated tissues of Nicotiana tabacum var. White Burley

Abstract

Determination of the nuclear DNA content of leaves and normal, habituated and Crown gall callus tissues of Nicotiana tabacum var. White Burley were performed using cytophotometry on Feulgen stained preparations. Several aspects concerning the reliability of the Feulgen technique for DNA determinations were investigated. Crown gall callus tissue used in this study had both a higher nuclear DNA content and chromosome number than normal callus (3.2C versus 2.5C). Both have a higher DNA content than the diploid tobacco leaf cells (2C). The normal callus tissue failed to grow on medium without indole acetic acid and kinetin when cultured in tubes. From this normal callus two habituated lines growing without both phytohormones were selected by culturing the normal callus first in the absence of either indole acetic acid or kinetin. Changing the culture conditions of the normal callus by using culture flasks instead of tubes resulted in a remarkably faster growth rate of the tissue. This was accompanied by an acquisition of the habituation characteristics since it was possible now to grow this tissue also directly on medium lacking both phytohormones. All habituated tissues showed a higher nuclear DNA content compared to the normal callus tissue from which they were derived. Interestingly, one of the tissues acquired a nuclear DNA content not different from that of Crown gall tissue. By changing the culture conditions of Crown gall callus tissue no concomitant change in nuclear DNA content occurred. The results suggest a correlation between the acquisition of a special chromosome complement and the loss of phytohormone requirement resulting in autonomous growth.