BIOTECHNOLOGY LETTERS Volume 14 No.lO (October 1992) pp.959-964 Received 22nd September EFFECTS OF INITIAL MEDIUM pH ON GROWTH AND METABOLISM OF CATHARANTHUS ROSEUS HAIRY ROOT CULTURES -A STUDY WITH 31p AND 13C NMR SPECTROSCOPY Chih-Huang Ho and Jacqueline V. Shanks* Department of Chemical Engineering Institute of B iosciences and Bioengineering Rice University, Houston, TX 77251-1892 SUMMARY Hairy root cultures of Catharanthus roseus were grown for 26 days in half-strength Gamborg's B5 liquid medium at different initial pH values of 4.2, 5.7, 6.5, and 7.3. Maximum growth was obtained for cultures with an initial medium pH 6.5. The lowest growth rate was found in cultures at initial pH values of 4.2 and 7.3. Roots in cultures at initial pH of 4.2 had a thickened and stunted morphology in contrast to the other cultures. Also, cultures at initial medium pH of 4.2 exhibited an increase in medium pH in the first few days instead of the characteristic acidification. All cultures maintained a cytoplasmic pH of 7.4 throughout the growth cycle. However, vacuolar pH was 5.1-5.2 in cultures of initial pH 4.2, as opposed to 5.4-5.5 for other cultures. Sucrose was hydrolyzed completely to glucose and fructose by day 26 except for cultures at initial pH of 7.3. Glucose was the preferred substrate throughout the growth cycle for cultures with initial pH values of 7.3 and 6.5, after day 20 for an initial pH of 5.7, and after day 26 for pH 4.2. INTRODUCTION Hairy root cultures, by virtue of their genetic stability, fast growth in phytohormone-free media, and level of morphological differentiation, are one of the most recent and promising tissue culture options for enhanced production of valuable secondary metabolites. We have established fast-growing hairy root lines of Catharanthus roseus (Bhadra et aL, 1992), the plant species from which the antineoplastic drug vinblastine and its two precursors vindoline and catharanthine are obtained. Cell suspension cultures of (7. roseus do not produce vindoline (Lapinjoki et al., 1987; Naaranlahti et al., 1989), while two of our clones have shown stable vindoline production (Bhadra et al., 1992). Since many indole alkaloids in our (7. roseus hairy roots are growth-associated, optimization of growth conditions should increase metabolite production. Growth and nutrient assimilation may be sensitive to medium pH (Kurkdjian and Guem, 1989). In this work, extracellular pH, cytoplasmic pH, vacuolar pH, uptake of inorganic phosphate, carbon utilization and growth were studied as a function of the initial medium pH of the cultures. In vivo 31p nuclear magnetic resonance (NMR) spectroscopy is employed to monitor subcellular compartmental pH and inorganic phosphate levels within hairy roots and 13C NMR is used to monitor carbon utilization. 959