We previously reported continuous photoautotrophic growth of haploid tobacco cells from diverse genetic backgrounds with rates of C02 assimilation of 2.5 to 5 1tmol/g fresh wt * hr at high C02 levels (1). The cells were illuminated and grown in shallow liquid cultures in a medium without sucrose, with sterile C02-enriched air passing over the cells providing the carbon source for growth. The cells increased about 3-fold in dry wt in 21 days. Since then the continuous photoautotrophic growth of cell suspensions of Chenopodium rubrum in two-tiered culture vessels has been described with the highest growth rate reported as about a doubling in fresh wt in 18 days (3). A 2-to 4-fold increase in fresh wt/28-day passage in photoautotrophic cells of Ruta graveolens has also been obtained (8).We have modified our previous system to expedite the growing of large numbers of cultures necessary for selection of mutants. We now report comparable growth on solid medium in plates maintained in a transparent illuminated chamber through which C02-enriched air is passed. Properties related to photorespiration l This work was supported in part by a grant from the Rockefeller Foundation.have been examined in these cultures. Kennedy (4) has observed photorespiratory C02 evolution from heterotrophic callus cultures of Streptanthus tortuosus. We studied glycolate synthesis and metabolism in autotrophically and heterotrophically grown tobacco callus cells, the effect of increasing temperature on photosynthesis of such tissue, and the effects of 02 and C02 concentrations on photosynthesis and growth of autotrophic cultures. A mixture of 1% or 3% C02 in air was passed continuously through the cabinets at a flow rate of approximately 20 ml/min. Total fresh wt and sample dry wt determinations were made at the end of 3-week passages. MATERIALS AND METHODSSa_Bmp Procedure. Because of the heterogeneous nature of callus tissue grown on solid medium, it was necessary to use a systematic procedure to produce samples of uniform particle size, friability, and appearance. The total amount of tissue required for each assay was selected from the greenest tissue present on a Petri plate. Matched samples from autotrophic and heterotrophic plates for glycolate accumulation experiments were similar in appearance. This tissue was then broken into small pieces with forceps, and the pieces were randomly spread in a single layer in a Petri plate. The total was then divided into sectors equivalent to the number of samples required in the assay. The entire tissue in each sector was transferred to an assigned vessel and its fresh wt determined.Assay for Photosynthesis. In the standard assay 300 to 1,000 mg of callus tissue were weighed into 75-ml Warburg vessels covered on the bottom with Miracloth (Chicopee Mills) discs, and 0.35 ml of water was added. A measured quantity of NaH"4CO3 solution was added to the side arm of the vessel and the sidearm was covered with a rubber serum cap. The vessels were attached to manometers and, with the manometer taps open, pla...