Enhanced paclitaxel productivity and release capacity of Taxus chinensis cell suspension cultures adapted to chitosan Chang He Zhang a,b,c, * , Pedro S. Fevereiro b,c,d , Guangyuan He a , Zhenjia Chen c a School of Life Science & Technology, Huazhong University of Science & Technology, Wuhan 430074, China b Instituto de Biologia Experimental e Tecnolo ´gica (IBET), Apartado 12, 2781-901 Oeiras, Portugal c Instituto de Tecnologia Quı ´mica e Biolo ´gica (ITQB), Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal d Departamento de Biologia Vegetal, Faculdade de Cie ˆncias da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal Received 20 April 2006; received in revised form 23 July 2006; accepted 2 August 2006 Available online 22 August 2006 Abstract This work characterized the growth and paclitaxel production and extracellular release of Taxus chinensis cell suspension cultures adapted to chitosan stress by comparing with the unadapted cells. Without elicitor treatment, the paclitaxel yield of the chitosan-adapted cells was two-fold that of the unadapted cells. When both cell cultures were elicited by Ag + and methyl jasmonate (MJ), the paclitaxel yield of the chitosan-adapted cells was 4.6- and 3.2-fold that of the unadapted cells, respectively; the paclitaxel release ratio of the chitosan-adapted cells was 2.8- and 3.2-fold that of the unadapted cells, respectively. Furthermore, upon elicitor treatment, the activation of phenylalanine ammonia-lyase (PAL) activity, a common and important response of plant cells to biotic and abiotic stresses, as well as the cell viability and permeability of the chitosan-adapted cells was significantly higher, compared with the unadapted cell cultures, which corresponded well to the superior paclitaxel yield and release. These results suggest that adaptation to chitosan is an effective strategy for improving paclitaxel yield and release of T. chinensis cells. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Taxus chinensis; Paclitaxel; Plant cell suspension culture; Adaptation to chitosan stress; Secondary metabolism; Cell permeability 1. Introduction Paclitaxel is a novel anticancer drug, which has been approved by the United States Food and Drug Administration to treat breast, ovarian and lung cancers as well as AIDS-related Kaposi’s sarcoma. Since the supply of paclitaxel from the Taxus tree is in great shortage, large-scale cell suspension cultures of Taxus spp. are viewed as a promising production process. Various strategies have been examined to enhance the productivity of paclitaxel in Taxus cell suspension cultures, mainly including precursor [1] and nutrient (sugar) feeding [2], in vitro extraction (two phase culture) [3], and treatment with elicitors, such as, chitosan [poly (1,4-b-D-glucopyranosa- mine)], silver ion (Ag + ), benzoic acid, fungal elicitors, methyl jasmonate (MJ) and arachidonic acid [4]. Among them, elicitation is the most effective strategy. However, elicitors also severely reduce cell viability and growth at the optimal concentration [3]. So we have interest in improving cell tolerance against elicitor stress in Taxus cell cultures. Chitosan is the deacetylated form of chitin, which is the main component of the cell walls of some fungal species and of the exoskeletons of insects and crustaceans, being the second most abundant natural polysaccharide on the earth just next to cellulose. As a natural, biocompatible, cationic biopolymer, chitosan mimics the effects of some pathogenic microorgan- isms to stimulate plants to biosynthesize secondary metabo- lites. Therefore, chitosan has been widely applied as a potent elicitor in plant cell suspension cultures to enhance secondary metabolite production [5,6]. In addition, chitosan promotes plant secondary metabolite release from the cells as well [7] and has been shown to trigger paclitaxel production alone or in combination with MJ and Ag + in T. chinensis cell cultures [4]. Also it is shown that chitosan stimulates the immunity of plants, protects plants against microorganisms [8,9] and promotes plant growth and development [10]. Sub-lethal biotic or abiotic stress enhances the tolerance or resistance of plants against the same or similar stresses, although the molecular mechanism remains largely unclear www.elsevier.com/locate/plantsci Plant Science 172 (2007) 158–163 * Corresponding author. Tel.: +351 21 4469461; fax: +351 21 4421161. E-mail address: zhang@itqb.unl.pt (C.H. Zhang). 0168-9452/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2006.08.002