Anatomical and chemical studies of vitrified shoots of chestnut regenerated in vitro

The growth of crown-gall cells cultured in vitro ( Nicotiana tabacum L. cv. White Burley and Parthenocissus tricuspidata cv. Veitchii) is inhibited by alstonine (BG-8), a plant alkaloid, the anti-cancer effect of which has previously been demonstrated on animals and plants. The growth of normal cells is only slightly affected. The inhibitory effect of BG-8 on crown-gall cells is antagonized by indole-3-acetic acid (IAA) added to the culture medium. Kinetin associated with IAA does not prevent this inhibitory effect. BG-8 present in the culture medium containing the two types of hormones seems to modify the later hormonal requirement of Parthenocissus crown-gall tissues.
BG-8 exhibits high binding affinity for crown-gall DNA and, therefore, strongly inhibits its in vitro synthesis. The alkaloid has practically no effect on DNA from healthy cells. The inhibition by BG-8 is dependent on the level of DNA strand separation and on the origin and nature of the tissues (crown-gall DNA is more destabilized than healthy DNA; DNA from habituated tissues is intermediate). IAA and kinetin have opposite effects on the in vitro strand separation of the DNAs from crown-gall cells and, consequently, antagonistic effects on DNA replication (IAA stimulates and kinetin inhibits). It is possible to establish a close relationship between in situ development of crown-gall tissues of the two species studied (in the presence or absence of BG-8 or cell-growth factors), in vitro DNA synthesis and DNA strand separation.