In vitro culture of tobacco callus on medium containing peptone and phytate leads to growth improvement and higher genetic stability.

Growth and genetic stability of Nicotiana tabacum L. callus were strongly improved by replacing the inorganic nitrogen and phosphorus of the Murashige and Skoog's medium by a soybean peptone and phytate, respectively. Cell proliferation after subcultivation on the modified medium was highly stimulated as evidenced by a strong biomass increase; this improvement was mainly due to the organic N source. In addition, while calluses grown under standard conditions displayed various cell sizes and DNA contents, subcultivation on the modified medium led to homogeneous cell size distribution and stable 4C-8C DNA contents through several subcultures. This improved genetic stability was due to replacement of inorganic P by phytate, provided the presence of peptone. Such new media composition could be useful for slow-growing cell suspensions or calluses.

Local expression of the ipt gene in transgenic tobacco (Nicotiana tabacum L. cv. SR1) axillary buds establishes a role for cytokinins in tuberization and sink formation.

The developmental characteristics of a transgenic tobacco line (BIK62) expressing the ipt cytokinin-biosynthetic gene under the control of a tagged promoter were analysed. In situ hybridization and cytokinin immunocytochemistry revealed that the ipt gene was mainly expressed in the axillary buds after the floral transition. The ipt-expressing axillary buds presented morphological alterations such as short and narrow scale-leaflets, and swollen internodes filled with starch grains, giving rise to short and tuberized lateral branches. In addition, the modification of the endogenous cytokinin balance in the axillary meristems resulted in a fast rate of leaf initiation and cytokinins accumulated mostly in the lateral zones of the reactivated axillary meristems, suggesting a role in leaf organogenesis. Cell cycle analysis revealed that the reactivated axillary meristems were characterized by predominant S+G2 nuclei. Terminal internodes displayed low levels of hexose and sucrose concomitant with starch accumulation. Extracellular invertases (EC 3.1.26) were also present in higher amounts in the tuberizing internodes compared to the axillary buds of wild-type tobacco. These results underline the role of cytokinins in cell cycle regulation and in the creation of a sink--source effect. They also provide new information about cytokinin involvement in the process of tuberization and their overproduction in axillary buds giving rise to tuberized lateral branches in a naturally non-tuberizing species.