Photosynthetic Characteristics of Photoautotrophically Grown Tobacco Callus Cells

Abstract

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...

Effects of CO2 and O2 on Photosynthesis and Growth of Autotrophic Tobacco Callus

“…Previous demonstrations of inhibition of "'CO2 uptake by 100% 02, light-dependent 02 stimulation of "'CO2 release into C02-free air, and glycolate accumulation after treatment with a a-hydroxy-2-pyridinemethanesulfonate (1) provided evidence for the existence ofphotorespiration in autotrophic callus. In addition, it was shown that 60% 02 completely inhibited autotrophic growth in tobacco callus, but had no effect on heterotrophic growth.…”

Conditions for Strict Autotrophic Culture of Tobacco Callus

“…Although there is strong evidence that growth on solid media is predominantly autotrophic (1,8), there has been no demonstration that growth of illuminated colonies is strictly dependent on CO2 assimilation. This is of particular importance when the culture medium is solidified with an organic gelling agent.…”

“…Early reports of autotrophic growth of chlorophyllous plant cells in suspension (2,3) raised the possibility of obtaining photosynthetic mutants with these procedures. Autotrophic growth on solid media (1,8) established another important prerequisite, since most selection procedures hinge on the separation and immobilization of individual colonies.…”

Relationship between Net CO2 Assimilation and Dry Weight Accumulation in Field-Grown Tobacco

“…A estimation of a carbon budget; and (c) whether one could determine from a carbon budget why one variety produced more dry weight than the other. This investigation has demonstrated the usefulness of the C02-depletion method of assaying net CO2 exchange in field studies, and clearly established the close relationship between net CO2 exchange and dry weight accumulation.We have been involved in a continuing effort to modify genetically the biochemistry of C3 plants to produce tobacco mutants with decreased photorespiration and greater than normal rates of net CO2 assimilation per unit of leaf area (1,2,10,16). To confirm that varieties with superior rates of net photosynthesis under controlled conditions of the laboratory or greenhouse perform similarly in the field, an accurate yet convenient method of measuring net CO2 uptake under field conditions was sought.…”

“…We have been involved in a continuing effort to modify genetically the biochemistry of C3 plants to produce tobacco mutants with decreased photorespiration and greater than normal rates of net CO2 assimilation per unit of leaf area (1,2,10,16). To confirm that varieties with superior rates of net photosynthesis under controlled conditions of the laboratory or greenhouse perform similarly in the field, an accurate yet convenient method of measuring net CO2 uptake under field conditions was sought.…”