Photoautotrophic Growth and Photosynthesis in Tobacco Callus Cells

Abstract

Haploid tobacco (Nicotiana tabacum L.) cell cultures derived from quite different cultivars have been grown photoautotrophically in medium lacking sucrose and with 1.6 pM naphtbaleneacetic acid and 1.5 AM isopentenylamino.purine. Cells were grown for 5 months on agar medium in Petri plates in air with dry weight increases of 1.5-to 3-fold per month. Callus cells were also grown photoautotrophically for at least three consecutive transfers 3 weeks apart in shallow liquid medium in horizontally placed gaswashing bottles where they were gassed continuously with air or air enriched with CO2. Raising the CO2 level in the air surrounding the cells increased the growth rate, and after about 3 weeks in 1% CO2 the dry weight was approximately 3-fold greater than the inoculum. Growth rates remained about the same after each consecutive transfer. Autotrophic growth with this regime is not restricted to specific clones or cultivars.Photosynthetic measurements in an atmosphere containing "4CO2 established that rates of CO2 assimilation in the callus cells at high CO2 levels were similar to those of leaves on a chlorophyll basis, but were much slower on a fresh weight basis. Photosynthetic light saturation was achieved at an irradiation of about 125 ,ueinsteins m-2 sec-1 (400-700 nm). The availability of photosynthetically dependent haploid cells provides an opportunity to select photosynthetic mutations which can be expressed in plants regenerated from these cells.Despite the widely held view that plant cell cultures are not photosynthetically self-sufficient a few cases of photoautotrophically grown plant cells have been reported (2,6,7,10 Leaves of 918.11 had a fast photorespiration in the "4C-assay (19) and a net photosynthesis of only 16.7 mg CO2 per dm2 -hr. Leaves of 918.23 had a slower photorespiration and a net photosynthesis of 23.6 mg CO2 per dm2 -hr. These cell cultures were isolated and generously provided by our colleagues P. R. Day and S. Anagnostakis.Culture Media. Cultures were maintained on Linsmaier and Skoog medium (14) with substitution of the Fe-EDTA solution of Kasperbauer and Collins (12) and with 16 jAM naphthaleneacetic acid (3 mg/l), 1.5 ;AM isopentenylaminopurine (0.3 mg/l), and 2% sucrose. The other organic constituents added to the medium were inositol (100 mg/l) and thiamin (1 mg/l). Photoautotrophic cultures were grown on this medium lacking sucrose and with 1.6 jAM naphthaleneacetic acid (0.3 mg/l). In some experiments, where indicated, photoautotrophic cultures were grown without any organic supplements.One per cent agar was used for solid media and Petri plates were sealed with 1-inch rubber bands. Experiments in which cultures were aerated with air or air-CO2 mixtures at flow rates of 6 to 10 ml/min were conducted in horizontally placed 500-ml gas washing bottles with small volumes of liquid media forming a shallow liquid phase (e.g., 1 g of tissue in 10 ml of medium). The incoming gas mixture passed through a cotton filter anda 0.20-,um membrane filter in an in-line filter holder. ...

Photosynthetic Characteristics of Photoautotrophically Grown Tobacco Callus Cells

“…The callus cells used were obtained from haploid Nicotiana tabacum plants of the variety John Wlliam Broadleaf (JWB su) as previously described (1). Cultures of the variety Wisconsin 38 were kindly supplied by J. C. Polacco.…”

“…Cultures of the variety Wisconsin 38 were kindly supplied by J. C. Polacco. Autotrophic medium (1,5) was solidified with 1% agar. Cultures were incubated at 28 C in 47-liter transparent plastic cabinets and illuminated continuously with fluorescent lights at an irradiance of approximately 200 yIE m-2 sec'1 of photosynthetically active irradiation for growth studies and at 200, or 120 to 150 IE m-2 sec'1 for metabolic studies.…”

“…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.…”

Development of Nitrogen Assimilation Enzymes during Photoautotrophic Growth of Chenopodium rubrum Suspension Cultures

“…Plant tissue cultures have proved very useful not only as models systems, but also as a means to manipulate genetic expression by regenerating whole plants from the cultured tissue (3). Although the tissue culture system might be ideal for studying and selecting photosynthetic characteristics, achieving photoautotrophic growth of plant cells in tissue culture has been difficult (1,3).…”

“…Although the tissue culture system might be ideal for studying and selecting photosynthetic characteristics, achieving photoautotrophic growth of plant cells in tissue culture has been difficult (1,3). A number of tissues, including carrot and tobacco, have been grown photoheterotrophically (7,17,29).…”