JOURNAL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA OF FERMENTATION AND zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA BIOBNGINJIERING Vol. 81, No. 5, 374-378. 1996 Efficient Regeneration from GUS-Transformed Ajuga Hairy Root NOBUYUKI UOZUMI,‘+ YASUKI OHTAKE, 1 YUTAKA NAKASHIMADA, ’ * YUTAKA MORIKAWA, l NOBUKAZU TANAKA,Z AND TAKESHI KOBAYASHI’* zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR Department of Biotechnology, Faculty of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-01’ and Center for Gene Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 724,2 Japan Received 7 December 1995/Accepted 5 February 1996 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ A procedure for the systematic regeneration from Ajuga hairy root was developed. Supplementation of an auxin, naphthaleneacetic acid (0.1 mg/& in the hairy root growth culture enhanced the cell growth rate. Mechanical fragmentation for 10 s in a commercial blender enabled a large number of root fragments to be produced efficiently. Supplementation of a cytokinin, benzyladenine (10 mg/l), exhibited the highest plantlet formation frequency in the plantlet formation stage. The plantlets longer than 4 mm in length were able to grow into whole plants on agar containing modified MS medium. Ajuga hairy root in which the ,9-glucuronidase (GUS) gene was introduced under control of the tomato ribulose-l&bisphosphate carboxylase/oxygenase small subunit (rbcS3B) promoter was constmcted by the Agrobacterium rhizogenes-mediated co-transforma- tion. The GUS-transformed hairy root could also be efficiently regenerated into plantlets through this proce- dure. GUS activity was detected in leaf tissue of the regenerated plants. [Key words: regeneration, hairy root, micropropagation, Ajuga, transgenic plant] Micropropagation through plant regeneration is seen as a means of allowing the clonal propagation of a large number of disease-free propagules. The technology has applications in a diverse range of plant species, particu- larly when a liquid-based system is used, as a means of low-cost and high-volume propagation (1). Infection of dicotyledonous plants by Agrobacterium rhizogenes causes the formation of hairy root as a differentiated organ (2). Hairy roots have the potential to produce useful materials such as enzymes (3, 4) and secondary metabolites (5). Applications of cultured hairy roots have chiefly focused on the large-scale production of useful products or secondary metabolites. However, plant regeneration from hairy roots raised the possibility of other potential uses, such as agronomic applications. To date, plant regeneration from hairy roots has been reported in several plant species (6-11) and characteris- tics such as the stable regeneration ability of hairy root have implications for the development of plant regenera- tion. In addition, the use of A. rhizogenes offers the opportunity of introducing foreign genes into plant genomes when a hairy root is induced, enabling alteration of plant properties by gene manipulation (12). Thus, the micropropagation of elite hairy roots offers attractive advantages in the agricultural and biochemical produc- tion of useful materials. To date, fragmented hairy root segments (13), and adventitious shoot primordia on hairy roots in horseradish (14) have been studied for their application as artificial seeds. Ajuga produces an insect-molting hormone, 20-hydro- xyecdyson, which can be used commercially as insectside and steroid compounds. Ajuga hairy roots had higher content of 20-hydroxyecdyson than the original plant (15). Systematic studies for efficient micropropagation * Corresponding author. + Present address: Bioscience Center, Nagoya University, Ch’kusa-ku, Nagoya 464-01, Japan. 1 Present address: Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Kagamiyama, Higashi-Hiroshima 724, Japan. from hairy roots have been done in only horseradish (13, 14), and it is not known whether the micropropaga- tion procedure developed in horseradish can be applied to other hairy roots or not. Here, we describe the regeneration from Ajuga hairy root to develop an efficient regeneration process of Ajuga hairy root and the hairy root possessing a GUS gene under control of the light-inducible promoter (16, 17) as an example of plant transformation. MATERIALS AND METHODS Plant materials and culture Ajuga reptans var. atropurpurea hairy root (18) was used. The cells were maintained by subculturing as described previously (18). For Erlenmeyer flask culture, roots were grown for about 25 d and then 0.1 g (fresh weight) portions were transferred aseptically into lOO-ml flasks containing a 40- ml aliquot of modified Murashige-Skoog (MS) medium with 2% glucose (18). Auxins or cytokinins supplement- ed as additives were aseptically mixed with the medium after filtration through a cellulose nitrate membrane (Advantec Co., Tokyo). The auxins used were j-indole- butyric acid (IBA), 1-naphthaleneacetic acid (NAA), 4- chloro-indoleacetic acid (4-ClIAA), 3-indoleacetic acid (IAA) and trifluoro-indolebutyric acid (TFIBA). Cul- tures were maintained on a gyratory shaker at 1OOrpm in the dark at 25°C. The final dry cell mass was mea- sured gravimetrically after drying the roots at 60°C for 3 d. On-line estimation of the cell concentration during culture was based on conductometry, as reported previ- ously (19). The conductometrical final cell mass was cor- rected by the gravimetrical final cell mass. Regeneration from hairy root fragments Root frag- ments were obtained using a blade-type blender (MX-30; Matsushita Electric Industrial Co., Osaka). The roots were fragmented in the blender (11,000 rpm) containing 200ml of modified MS medium. Root fragments less than 100 pm in length were removed by sieving through a stainless steel mesh and discarded. The root fragments (100 /*m or more) were then transferred aseptically to 374