JOURNAL OF FERMENTATION AND BIOENGINEERING Voi. 77, No. 2, 178-182. 1994 Stimulation of Emergence of Root Apical Meristems in Horseradish Hairy Root by Auxin Supplementation and Its Kinetic Model YUTAKA NAKASHIMADA, NOBUYUKI UOZUMI, AND TAKESHI KOBAYASHI* Department of Biotechnology, Faculty of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan Received 25 March 1993/Accepted 3 October 1993 1-Naphthaleneacetic acid (NAA) in the medium strongly inhibited horseradish hairy root elongation. In batch culture, NAA in the medium was absorbed by the root at the beginning of the culture (0-4 d). After the depletion of NAA, root apical meristems emerged, and then the growth rate of the root increased dramatically. The batch culture with 1 x 10-3 kg/m 3 of NAA reached the highest biomass, 10.9 kg-dry weight/m 3 at 21 d. Modificaton of a kinetic model previously developed resulted in a favorable fit with the growth curve of horseradish hairy root treated with NAA. Furthermore, in repeated batch culture, the NAA-treated root cul- ture exhibited a 1.7-fold increase in dry weight, 57 kg/m 3 at 40 d, compared with that of no NAA-treated root. The modified kinetic model also correlated well with the experimental results in repeated batch culture. Plant hairy roots, which are generated by integration of the T-DNA on the Ri plasmid of Agrobacterium rhizogenes into plant genomic DNA, are seen as the differentiated organs which have the potential to produce valuable materials such as secondary metabolites (1--4). To obtain a large amount of hairy roots efficiently, a suitable bioreactor system for hairy root culture has been developed (5). Furthermore, high biomass yields of 30.1 kg-dry weight/m 3 at 38 d (carrot hairy root) and 27.2 kg-dry weight/m 3 at 39d (Ajuga hairy root) with fed-batch culture using monosaccharide as a carbon source in a turbine blade reactor were obtained (6, 7). However, a dramatic improvement in growth characteris- tics would be difficult using a technological approach only because the growth rate is regulated by the number of root apical meristems of the hairy roots. In a previous paper, we reported that the emergence of root apical meristems in horseradish hairy root was stimulated by auxin supplementation, although hairy roots have usually been cultured without growth regula- tors (4). Since the growth properties of hairy root de- pend on both the elongation rate and the number of root apical meristems, an increase in the number of root apical meristems of hairy root by auxin supplementation should significantly increase the growth rate. In this paper, the effects of auxin supplementation on the growth behavior of horseradish hairy root were eval- uated and the kinetic model for the growth studied. MATERIALS AND METHODS Plant materials and culture Horseradish hairy root (Armoracia rusticana) induced by the leaf disk method with A. rhizogenes A4 as described previously (5) was used as the model hairy root in all experiments. The hairy root was maintained by regular subculture in the dark for 3 weeks at 25°C on growth regulator-free Mura- shige and Skoog (MS) medium (8) supplemented with 2% (w/v) sucrose. For batch or repeated batch cultures, about 1 × 10 -4 kg of hairy root (fresh weight) was inocu- lated into 4× 10-Sm 3 MS medium containing 2% su- * Corresponding author. crose and various concentrations of NAA in a 1 × 10 -4 m a Erlenmeyer flask. The culture was maintained on a rotary shaker at 100rpm in the dark at 25°C. The dry weight of the culture was estimated from the decrease in conductivity of the medium as described previously (5). For measurement of the root elongation rate under a constant NAA concentration, one root fragment (5 x 10-3m in length) with a root apical meristem excised using a razor was inoculated on 5 x 10 -5 m 3 solid MS me- dium containing 1% (w/v) agar. The length of the frag- ment was manually measured photomicrographically every day until 2 d after the inoculation in order to evalu- ate the daily root elongation rate. The number of root apical meristems in liquid MS medium containing various NAA concentrations was measured at 7 d, and the dry weight of the root was gravimetrically measured after drying the root at 60°C for 24 h. The number of root apical meristems per meter was estimated from overall root length calculated from the dry weight using Eq. 16 (described below). Analytical methods Sucrose, glucose and fructose concentrations in the medium were assayed using a high performance liquid chromatograph (HPLC) (Tri Rotor- V, JASCO, Tokyo) equipped with an Aminex HPX-87C column (BIO-RAD Laboratories, Richmond, CA, USA) as described previously (5). The NAA concentration was also determined by HPLC with a Finepak Sil C-18 column (JASCO) and UV/VIS detector (870-UV; JASCO) at a wavelength of 280 nm. The solvent system used was 0.1% aqueous phosphoric acid: acetonitrile (40:60, v/v %) with a flow rate of 1.0 × 10 -6 m3/min. Scanning electron microscopy For scanning elec- tron microscopy, the root was fixed with 2% glutaralde- hyde in 10mM phosphate buffer (pH 6.0) for 2 h. After drying with a critical point dryer (HCP-2; Hitachi, Tokyo) and sputtering with ion sputter (El01; Hitachi), micrographs were taken using a Hitachi S-570 scanning electron microscope. RESULTS AND DISCUSSION Selection of growth regulator for biomass increase At first, the effects of growth regulators on growth be- 178