An analysis of the stages of organogenesis in leaf explants of Nicotiana tabacum L. CV. xanthi NC. cultured in vitro

The culture conditions of tobacco leaf explants were perfected to produce a large number of either roots or shoots in vitro with the least possible callus production, in the shortest possible time. Experimental variation due to the source of the explant was also reduced. The origins of the roots and shoots were examined under the light and scanning electron microscope. Roots were produced directly from the bundle-sheath and vein parenchyma cells. Shoots were produced indirectly from nodules of dividing cells originating mainly from the division of bundle-sheath and palisade mesophyll cells. Both organs were produced initially at the explant edge, with root production spreading along the veins towards the centre of the explant. Various aspects of the uptake of tritiated 3-indolebutyric acid (IBA-5-8H) from the culture medium by the explants were studied to find their relationship to the pattern of root formation.Through transfer experiments between different media the leaf explants were found to be competent for root production immediately upon excision with the first roots becoming determined after only a few hours on root inducing medium. But the explants required two days on either basal medium (BM) or shoot inducing medium (SIM) to become competent for shoot production, followed by a four day period during which SIM was required for determination. The period necessary on SIM for determination could be reduced to one day by extended culture on BM.The maintenance of competence in explants on BM for organ production varies according to the type of organ induced. Once competent for shoot production the explants remain competent for at least 30 days. The competence for root production, however, declines sharply within a few days of culture. To determine whether the decreased competence for root production was related to a decrease in uptake of auxin from the medium, the uptake of IBA-5-*H was examined. This revealed an overall increase in uptake of IBA with increased length of culture on BM, while uptake per mg fresh weight remained steady. The point at which inhibitory levels of growth regulators act upon explants was also examined through transfer experiments and only contact with the explant at the time of determination and in the two days following this was organ production inhibited.