Digitalis Cardenolides by Plant Tissue Culture II. EFFECT OF LIGHT AND PLANT GROWTH SUBSTANCES ON DIGITOXIN FORMATION BY UNDIFFERENTIATED CELLS AND SHOOT-FORMING CULTURES OF DIGITALIS PURPUREA L. GROWN IN LIQUID MEDIA

Undifferentiated, highly chlorophyllous cell cultures; undifferentiated white cell cultures; green, shoot-forming cultures; and white, shoot-forming cultures of Digitalis parpurea L. were established and subcultured every 3 weeks in liquid media in the light or in the dark. The digitoxin content, the chlorophyll content, and the ribulose bisphosphate carboxylase activity of these cultures were assayed. The light-grown, green, shoot-forming cultures accumulated considerable amounts of digitoxin (about 20 to 40 micrograms per gram dry weight), and the white, shoot-forming cultures without chloroplasts accumulated about one-third that amount of digitoxin. The chlorophyli content and the ribulose bisphosphate carboxylase activity of the undifferentiated green cells were about the same as they were in the green, shoot-forming cultures, but the digitoxin content of the former was extremely low (about 0.05 to 0.2 microgram per gram dry weight), which is about the same as that in undifferentiated white cells without chloroplasts. Thus, it was concluded that the chloroplasts are not essential for the synthesis of digitoxin in Digitalis cells. The optimum concentrations of the tested compounds for accumulation of digitoxin were: benzyladenine, 0.01 to 1 milligram per liter, indoleacetic acid, 0.1 to 1 milligram per liter, anaphthaleneacetic acid; 0.1 milligram per liter, and 2,4-dichlorophenoxyacetic acid, 0.01 milligram per liter. Digitalis cardenolides, especially digitoxin and digoxin, are important in medicine, and cardenolide production of cultured cells of Digitalis species has been investigated. Most workers have reported that undifferentiated cultured cells either did not produce cardenolides (2, 4, 6) or contained only trace amounts of cardenolides (8, 11). However, organ-redifferentiating cultures have been reported to accumulate considerable amounts of cardenolides (3, 6, 8). In an earlier report (5), we showed that even the first passage calli of six Digitalis species, including root-forming cafli, lacked the ability to accumulate cardenolides, but shoot-forming calli accumulated considerable amounts of cardenolides. In the present study, we have established four liquid-cultured cell lines of Digitalis purpurea L., ie. undifferentiated green cells; undifferentiated white cells; green, shoot-forming cultures; and white, shoot-forming cultures. These cell lines were used to study the effect of light on the expression of cardenolide-production. Furthermore, we have investigated the effect of several plant growth substances on growth and digitoxin formation ofthe green, shoot-forming cultures, which accumulated the highest amounts of digitoxin in the four cell lines. MATERIALS AND METHODS Undifferentiated Cell Cultures. Callus was induced from seedlings of Digitalis purpurea L. on the basal medium, supplemented with 3 mg/L IAA and 0.8% (w/v) agar. After 30 d, the callus was transferred into the liquid basal medium, supplemented with 1 mg/L IAA, and cultured in the light. Thus, undifferentiated green cell cultures were established and sub-cultured every 3 weeks. Undifferentiated white cell cultures were obtained by sub-culturing the green cells in the dark. Shoot-Forming Cultures. The shoot-forming cafli ofD. purpurea L., which had been established in the previous study (5), were transferred into the liquid basal medium, supplemented with 1 mg/L BA and 1 mg/L IAA, and cultured in the light. Thus, green, shoot-forming cultures without root were established and subcultured every 3 weeks. White, shoot-forming cultures were obtained by sub-culturing a portion of the green, shoot-forming cultures in the dark. Culture Condition. Murashige and Skoog medium (9)-with 1 mg/L thiamin-HCl and without agar, edamin, IAA, and kinetinwas used as the basal medium. Approximately 1.5 g fresh weight of cells were inoculated into a 500-ml Erlenmeyer flask containing 100 ml of liquid medium and cultured in continuous light (fluorescent lamp: about 4 x 105 erg/s.cm2) or in the dark at 28°C on a reciprocal shaker (100 strokes/min; 2.0 cm in length). Measurement of Growth. Fresh weight was measured after removing culture medium by suction filtration. The harvested fresh culture was lyophilized, and its dry weight was determined. Assay for Digitoxin. Lyophilized cells (0.1-2 g) were homogenized with 50 ml ethanol in a glass homogenizer. The homogenate was heated at 74°C for 4 h and filtered. The filtrate was dried in vacuo, and the residue was taken up in 2 ml ethanol and diluted with 18 ml H20. When necessary, the extract was further diluted with H20 to a desirable level. Determination of digitoxin concentration of the extract was done by radioimmunoassays, as described (5). Assay for Chi. Chl content was determined spectrophotometrically in an 80%o (v/v) acetone extract. Chl was extracted by the method of Sunderland (12), and its concentration was calculated using the equation derived by Arnon (1). Assay for RuBPCase' Activity. Five g of cooled fresh cells were homogenized in 10 ml of ice-cold buffer solution containing 25 mm Tris-HCl (pH 7.4), 1 mm EDTA, 2 mM MgCl2, 100 mM NaCl, and 80 mm 2-mercaptoethanol for 1 min in a Waring 1 Abbreviations: RuBPCase, ribulose bisphosphate carboxylase; NAA, a-naphthaleneacetic acid. 653 www.plantphysiol.org on August 15, 2017 Published by Downloaded from Copyright © 1982 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 69, 1982 Blendor. The homogenate was filtered through four layers of gauze, and the filtrate was centrifuged at 10,000g for 30 min. The supernatant was used as the enzyme solution. RuBPCase (EC 4.1.1.39) was assayed at 30°C by measuring the incorporation of 14CO2 into acid-stable compounds in reaction mixtures (pH 7.8) containing the following compounds: D-ribulose 1,5-bisphosphate, 0.25 ,LlM; NaH['4C]03 (0.36 ,uCi), 10 ,UM; MgCl2, 5 ,pM; EDTA, 0.03 ,UM; GSH, 3 ,AM; and 0.1 ml of enzyme solution; total volume, 0.42 ml (10). The reaction was started by adding D-ribulose 1,5-bisphosphate and was stopped by adding 0.2 ml HCOOH; then the reaction mixture was evaporated to dryness. The acid-stable '4C-product was dissolved in 0.5 ml of water and added to 10 ml scintillator (Instagel, Packard Instruments Company, Downers Grove, IL), and the radioactivity was counted with a liquid scintillation counter. The protein content in the enzyme preparation was determined by the method of Lowry et al. (7).