ORIGINAL ARTICLE Anna Maria Pirttila¨ ® Hanna Laukkanen ® Anja Hohtola Chitinase production in pine callus (Pinus sylvestris L.): a defense reaction against endophytes? Received: 21 April 2001 / Accepted: 22 October 2001 / Published online: 18 January 2002 Ó Springer-Verlag 2002 Abstract In shoot tip-derived tissue cultures of Scots pine (Pinus sylvestris L.), browning and subsequent degeneration of the culture is accompanied by lipid peroxidation and lignification of cells, which are char- acteristic features of a plant defense reaction. Since chitinases are enzymes acting primarily in plant defense, their expression was studied in pine callus in order to elucidate the defense reaction. Chitinases were present diversely in tissue cultures originating from shoot tips and embryos of P. sylvestris, in contrast to Pinus nigra embryogenic callus, where production of chitinases or browning was not detected. Because endophytic microbes had earlier been detected in buds of Scots pine, their subsequent presence in the tissue cultures was considered a potential cause of the defense reaction. Therefore, the presence of endophytes in the tissue cul- tures was examined by in situ hybridization. Endophytes were found to colonize heavily in 45% of the tissue cultures of P. sylvestris and to form biofilms, while the P. nigra callus was not found to contain any microbes. The endophytes seemed to propagate uncontrollably once a tissue culture of P. sylvestris was initiated. Regardless of the high level of chitinase production in the callus, the control of the endophytes presumably becomes inadequate during the tissue culture of P. sylvestris. Keywords Chitinase ® Defense reaction ® Endophyte ® Pinus (defense) ® Tissue culture Introduction Plant defense systems are comprised of a number of different enzymes, of which probably only a fraction are known today. Chitinases are the largest group, having diverse roles in plant defense. The chitinases are espe- cially interesting because their substrates do not origi- nate from plants, but rather from fungal cell walls and the exoskeleton of arthropods (Collinge et al. 1993). Although the main role of chitinases is in defense, primarily acting on fungal pathogens, they also have a non-defensive role in plant development. The basic chitinases belonging to class I become activated in tobacco explants during flower formation, but not during the formation of vegetative shoots (Meeks- Wagner et al. 1989; Neale et al. 1990). In other plant species, such as cucumber, petunia, and Arabidopsis, different chitinases work in distinct organs of the flower, or are activated differentially during flower development (Lawton et al. 1994; Lotan et al. 1990; Trudel et al. 1989; Samac and Shah 1991). Roots, fruits, and seeds are other tissues in which high chitinase activities have been detected (Graham and Sticklen 1994; Salzman et al. 1998; Regalado et al. 2000). Some of the chitinases detected in seeds may only be present in seeds, and not in the mature plants (Graham and Sticklen 1994). Specific chitinases belonging to class II are produced during somatic embryogenesis (De Jong et al. 1992; Mo et al. 1996; Dong and Dunstan 1997; Domon et al. 2000), and chitinases are also found in cell-suspension cultures (Kragh et al. 1991; Masuta et al. 1991; Arie et al. 2000). Whereas several studies have concentrated on chitinases in elicited or induced suspension or tissue cultures (Masuta et al. 1991; Mason and Davis 1997; Popp et al. 1997; Wu et al. 1997; Jayasankar et al. 2000), the non- induced, non-embryogenic callus culture has, to our knowledge, been examined only rarely for chitinase production (Mayer et al. 1996; Domon et al. 2000). However, browning or blackening is a typical feature in tissue cultures of woody plants. This phenomenon is also continually detected in the tissue cultures derived from shoot tips of mature Scots pine (Pinus sylvestris L.; Laukkanen et al. 1997). The browning of a tissue culture is caused by oxidation of phenols, which occurs as a result of cellular degradation (Lee and Whitaker 1995). Planta (2002) 214: 848–852 DOI 10.1007/s00425-001-0709-x A.M. Pirttila¨ (&) ® H. Laukkanen ® A. Hohtola Department of Biology/Botany, University of Oulu, POB 3000, 90014 Oulu, Finland E-mail: am.pirttila@oulu.fi Fax: +358-8-5531500