Studies on the Isolation of Green Pigmented Callus Tissue of Tobacco and its Continued Maintenance in Suspension Cultures.

From stock cultures of tobacco callus tissue grown in a salt-sucrose medium supplemented with 1 × 10^-6 M indoleacetic acid (IAA) and 1 × 10^-6 M 2,4-dichlorophenoxyacetic acid (2,4-D), visibly-green callus cultures have been isolated both in solid and in liquid media. This green color can be maintained in continued subcultures by growing the cultures under very diffuse light conditions. Spectrophotometric determinations of the pigment contents of the green culture showed no difference in the nature of the pigment components or chlorophyll a : b ratio but contained only low concentrations (17-30%) of total pigments of that of the intact leaf in culture. The liquid culture was highly friable and green and maintained a very active growth rate in two different media, viz., IAA + 2,4-D and IAA + kinetin. Cultures growing in IAA + 2,4-D medium were extremely friable, soupy and light green whereas cultures growing in IAA + kinetin medium were less friable, much greener in appearance and slightly higher in pigment content. Pigment analysis of the liquid cultures in both media indicated only low concentrations of chlorophylls and carotenoids per g fresh weight. The round spherical green calli growing in IAA + kinetin cultures showed a higher pigment content than the mixture of cells growing in the supernatant medium. Plating of the liquid suspension containing single cells and cell aggregates on an agar plate produced visibly-green colonies within 2-3 weeks. The smallest of such a colony weighed as little as 4-6 mg fresh weight (2-3 mm diameter). These small colonies when extracted with 80% acetone showed delectable pigment separation into various components on thin-layer and paper chromatography. The results of the study are discussed suggesting that the development of chlorophyll and differentiation of chloroplasts in tissues cultured in vitro will depend on a number of chemical and physical factors employed under specific culture conditions, viz., growth factors, age of culture, light conditions, etc. [ABSTRACT FROM AUTHOR]