Rooting and the Metabolism of Nicotine in Tobacco Callus Cultures

The usefulness of exogenous nicotine as a factor in the induction of morphogenesis in a tobacco tissue culture medium has been demonstrated. Nicotiana rustica callus cell cultures were grown on a modified Murashige and Skoog medium with 2 mg/l indoleacetic acid (IAA) and 0.2 mg/l kinetin (MMS). Root morphogenesis was induced in roller tube callus cell cultures and solid callus cell cultures grown on MMS without kinetin supplemented with 10–100 mg/l nicotine. Optimal nicotine concentration for root induction was 50 mg/l. Other tests using varying combinations of IAA, kinetin and nicotine produced no obvious morphogenesis, although some changes in the amount of callus growth and endogenous protein concentration did correlate with nicotine concentration relative to the presence of IAA and/or kinetin. In liquid MMS medium, ¹⁴C-nicotine was primarily incorporated into the protein fraction of cultured cells while primarily incorporated into the cell wall and/or cell membrane fraction of cells cultured on MMS without kinetin in the medium. In MMS without IAA and MMS without both IAA and kinetin, there was incorporation, but to a lesser extent in both the protein and the cell wall and/or cell membrane fractions.     

Ultrastructural Localization of Ions: IV. LOCALIZATION OF CHLORIDE AND BROMIDE IN NITELLA TRANSLUCENS AND X-RAY ENERGY SPECTROSCOPY OF SILVER PRECIPITATION PRODUCTS

The cytoplasmic distribution of Cl^– in Nitella translucenswas assessed by means of two contrasting approaches. The firstinvolved the histochemical precipitation of Cl^– with Ag⁺followed by X-ray analytical verification of the silver precipitationproducts, and the second, quench-freezing of whole Nitella cellsfollowed by freeze-substitution in the presence of silver underanhydrous conditions. Both methods produced identical evidencefor Cl^– distribution, showing that a large proportionof the Cl^– is present in the stationary cortical gel layerwhich includes the chloroplasts. However, the chloroplasts appearedto be low in Cl^– content while the bulk of the Cl^–appeared to be situated between the chloroplasts and plasmalemma. Experiments were carried out in other to detect the pathwayof the ‘fast component’ of halide ion transfer tothe vacuole. Br^– was supplied for various time intervalsto low Cl^– Nitella cells, followed by attempts to differentiatebetween AgCl and AgBr deposits. Solutions of various strengthof NH₄OH or ammonium carbonate were used to remove AgCl (butnot AgBr) deposits by formation of a Ag(NH₃)₂⁺ complex. AlthoughX-ray analytical verification showed that the method had somepotential usefulness it could not be carried out successfullybecause of loss of structural detail caused by NH⁺₄. The distribution and density of deposits near the plasmalemmasuggested the occurrence of a process in which cytoplasmic loadingis achieved by a sequential rupture and repair of the plasmalemmamembrane. Vesicles and reticulate structures in the streamingcytoplasmic phase generally showed very little deposit, butthese structures, together with the tonoplast, became greatlyenriched with deposits when cells had been given a brief exposers(3 min) to a Cl^– or Br^– solution. These rapid changesmay possibly be related to the ‘fast component’of halide ion transfer to the vacuole.