2,4-D-Sustained Photomixotrophic Growth of a Chlorophyllous Cell Suspension Culture of Nicotiana tabacum

A chlorophyllous, photomixotrophic cell suspension culture of tobacco (Nicotiana tabacum L.) was established using medium containing 30 g/liter of sucrose and 1.5 μM 2,4-D. The 2,4-D-sustained photomixotrophic line was able to show rapid regreening in the light after bleaching in the dark and characterized with a much slower and longer growth cycle than a heterotrophic line derived from the same original callus (cell doubling time of 100 h vs. 40 h and duration of logarithmic phase of 17 days vs. 7 days). The photomixotrophic line took up sucrose more rapidly than the heterotrophic line and accumulated starch during the early logarithmic phase when it showed a maximum photosynthetic capacity on a chlorophyll basis (6.3μmol O2/min/mg Chl). Chlorophyll content and photosynthetic capacity on a per cell basis and on a cell fresh weight basis, on the other hand, decreased during this phase and reincreased later to reach maximum levels (310 μg Chl/g fr wt; 1.4 μmol O2/min/g fr wt) when the line exhibited the highest activities of dark respiration (1.0 μmol; O2/min/g fr wt) and cell division (mitotic index of 3.0%). These characteristics of the photomixotrophic line were lost if it was grown in the dark to become non-chlorophyllous. Although net O2 evolution could not be detected in the photomixotrophic line throughout the growth cycle when assayed under suboptimum light intensity, reaccumulation of starch and a marked increase in cell fresh weight upon addition of minerals, vitamins and 2,4-D without sucrose at the late logarithmic phase indicated the development of photosynthetic activity under the culture conditions.