1053 To the best of our knowledge,there are no data available fied with 8 g 1-Phytagar(GIBCO).The incubation was concerning the hairy root cultures of L.canescens and their performed at 25C and under 40 umol/m2 s-!fluorescent secondary metabolites,especially shikonin derivatives and lights for 18 h per day.The roots emerging from the in- pyrrolizidine alkaloids.Recently,an effective method of fected sites were transferred to the liquid hormone-free clonal multiplication of L.canescens has been elaborated LS medium supplemented with 0.05%Claforan(Roussel, (Syklowska-Baranek et al.2004). France)after autoclaving and cultured individually at 25C Of the fifty Lithospermum species,Lithospermum ery- in the dark on a gyratory shaker(New Brunswick Scien- throrhizon growing in Asia has been the most thoroughly tific)at 120 rpm.The established hairy root cultures were studied.Transformed roots of L.erythrorhizon have been maintained on hormone-free LS medium supplemented investigated as a promising source of shikonin (Shimo- with 0.25 mg I-1 GA3,starting from the 12th passage also mura et al.1991;Chang and Sim 1996;Yazaki et al.1998). on hormone-free media LS,SH(Schenk and Hildebrandt Shikonin was the first plant secondary metabolite produced 1972)and SH with addition of 0.25 mg 1-1 GA3.All the on industrial scale from plant cell cultures of this species media were solidified with 8 g 1-1 Phytagar. (Fujita et al.1981;Tabata 1985).Most research to date has In further investigations,the eight lines of clone Lcl se- focused on increasing the production of this compound be- lected from 81 lines were also cultivated in liquid hormone- cause of its value as a pharmaceutical agent but shikonin free LS medium.The cultures were carried out in the dark at derivatives could be also of interest from a biological point 25C with subculturing every 6 weeks.To estimate growth, of view.For instance,acetylshikonin is bioactive and shows about 0.3 g fresh weight of 6-week-old hairy roots was lower toxicity than shikonin (Papageorgiou et al.1999). placed on 30 ml of solid medium in 100 ml Erlenmeyer The acetylshikonin and B-hydroxyisovalerylshikonin iso- flasks.Biomass increase was evaluated on the basis of fresh lated from hairy roots of L.erythrorhizon proved to be the weight after 6 weeks of culture as a ratio of harvest weight most biologically active compounds against soil-borne bac- to initial inoculum weight.To determine the accumulation teria and fungi.However,quantitative analysis of shikonin of secondary metabolites,hairy roots from two flasks of derivatives of these roots was not performed(Birgham et al. each root line were harvested after 6 weeks of culture. 1999). Statistical analysis was performed using the StatSoft A method for obtaining transgenic roots of some STATISTICA software. Lithospermum species and recovering naphthoquinone secreted from the medium has been patented(Hiroshi and Hitoshi 1988). Confirmation of the transformed nature of the roots The objective of this study was to obtain hairy root cultures of L.canescens as a new alternative source of PCR reaction was performed for T-DNA detection in roots shikonin derivatives.The growth of hairy root cultures and of the analyzed plants.Total DNA was isolated from pow- acetylshikonin and isobutyrylshikonin contents were in- dered roots frozen in liquid nitrogen and extraction of vestigated.The selection of a transformed root line of L. chromosomal DNA using DNAzol Reagent(GIBCO-BRL) canescens should allow the production of higher amounts of with purification on silica particles(Boom et al.1999).For shikonin derivatives through large-scale cultures in biore- some samples,especially those containing shoots,addi- actors. tional phenol/chloroform extraction and precipitation were performed.The following oligomer primers were used as a primers:CTGTACCTCTACGTCGACT,TCAGTC- Materials and methods GAGTGGGCTCCTTG,according to the programme:ini- tial denaturisation at 94C for 1.5 min;30 cycles at 94C for Transformed hairy root culture 20 s.60C for 40 s.and then 72C for 1.5 min (Hosokawa et al.1997).For all samples,except control plants,the Transformed hairy root cultures of L.canescens were es- predicted product(1.1 kb)was obtained (data not shown). tablished using three strains of Agrobacterium rhizogenes: ATCC 15834.LBA 9402 and NCIB 8196.The bacteria were grown on YEB solid medium (Vervliet et al.1975) Analytical methods 24 h at 24C,in the dark.Next,single colonies were inoc- ulated into 50 ml YEB liquid medium and cultured 72 h Shikonin derivatives and pyrrolizidine alkaloids(PA)were at 24C in the dark,on a gyratory shaker(New Brunswick extracted from samples of eight lines of L.canescens Scientific)at 120 rpm.The bacterial cultures were diluted transformed roots.For obtaining dye fractions.the (1:4)with YEB liquid medium before transformation.The powdered sample (0.28-1.00 g)of lyophilized trans- leaves and stems of ten 6-week-old shoots of plantlet clones formed roots was sonicated with n-hexane (6 x 50 ml) (Lc1,Lc2,Lc7,Lc8 and Lc9)obtained from seedlings for 30 min at 40C.The extracts were filtered through of L.canescens micropropagated separately (Syklowska- Whatman No.2 paper and the solvent was evaporated Baranek et al.2004)were directly wounded with ster- from the extract solution under reduced pressure.Dry ile needles containing bacteria.The infected shoots were residue was dissolved in methanol and investigated in a placed on hormone-free LS medium (Linsmaier and Skoog DIONEX HPLC system,equipped with an automated 1965)with the addition of sucrose (30 g 1-)and solidi- sample injector (ASI-100),and UVD 340S detector using1053 To the best of our knowledge, there are no data available concerning the hairy root cultures of L. canescens and their secondary metabolites, especially shikonin derivatives and pyrrolizidine alkaloids. Recently, an effective method of clonal multiplication of L. canescens has been elaborated (Sykłowska-Baranek et al. 2004). Of the fifty Lithospermum species, Lithospermum ery￾throrhizon growing in Asia has been the most thoroughly studied. Transformed roots of L. erythrorhizon have been investigated as a promising source of shikonin (Shimo￾mura et al. 1991; Chang and Sim 1996; Yazaki et al. 1998). Shikonin was the first plant secondary metabolite produced on industrial scale from plant cell cultures of this species (Fujita et al. 1981; Tabata 1985). Most research to date has focused on increasing the production of this compound be￾cause of its value as a pharmaceutical agent but shikonin derivatives could be also of interest from a biological point of view. For instance, acetylshikonin is bioactive and shows lower toxicity than shikonin (Papageorgiou et al. 1999). The acetylshikonin and β-hydroxyisovalerylshikonin iso￾lated from hairy roots of L. erythrorhizon proved to be the most biologically active compounds against soil-borne bac￾teria and fungi. However, quantitative analysis of shikonin derivatives of these roots was not performed (Birgham et al. 1999). A method for obtaining transgenic roots of some Lithospermum species and recovering naphthoquinone secreted from the medium has been patented (Hiroshi and Hitoshi 1988). The objective of this study was to obtain hairy root cultures of L. canescens as a new alternative source of shikonin derivatives. The growth of hairy root cultures and acetylshikonin and isobutyrylshikonin contents were in￾vestigated. The selection of a transformed root line of L. canescensshould allow the production of higher amounts of shikonin derivatives through large-scale cultures in biore￾actors. Materials and methods Transformed hairy root culture Transformed hairy root cultures of L. canescens were es￾tablished using three strains of Agrobacterium rhizogenes: ATCC 15834, LBA 9402 and NCIB 8196. The bacteria were grown on YEB solid medium (Vervliet et al. 1975) 24 h at 24◦C, in the dark. Next, single colonies were inoc￾ulated into 50 ml YEB liquid medium and cultured 72 h at 24◦C in the dark, on a gyratory shaker (New Brunswick Scientific) at 120 rpm. The bacterial cultures were diluted (1:4) with YEB liquid medium before transformation. The leaves and stems of ten 6-week-old shoots of plantlet clones (Lc1, Lc2, Lc7, Lc8 and Lc9) obtained from seedlings of L. canescens micropropagated separately (Sykłowska￾Baranek et al. 2004) were directly wounded with ster￾ile needles containing bacteria. The infected shoots were placed on hormone-free LS medium (Linsmaier and Skoog 1965) with the addition of sucrose (30 g l−1) and solidi- fied with 8 g l−1 Phytagar (GIBCO). The incubation was performed at 25◦C and under 40 µmol/m2 s−1 fluorescent lights for 18 h per day. The roots emerging from the in￾fected sites were transferred to the liquid hormone-free LS medium supplemented with 0.05% Claforan (Roussel, France) after autoclaving and cultured individually at 25◦C in the dark on a gyratory shaker (New Brunswick Scien￾tific) at 120 rpm. The established hairy root cultures were maintained on hormone-free LS medium supplemented with 0.25 mg l−1 GA3, starting from the 12th passage also on hormone-free media LS, SH (Schenk and Hildebrandt 1972) and SH with addition of 0.25 mg l−1 GA3. All the media were solidified with 8 g l−1 Phytagar. In further investigations, the eight lines of clone Lc1 se￾lected from 81 lines were also cultivated in liquid hormone￾free LS medium. The cultures were carried out in the dark at 25◦C with subculturing every 6 weeks. To estimate growth, about 0.3 g fresh weight of 6-week-old hairy roots was placed on 30 ml of solid medium in 100 ml Erlenmeyer flasks. Biomass increase was evaluated on the basis of fresh weight after 6 weeks of culture as a ratio of harvest weight to initial inoculum weight. To determine the accumulation of secondary metabolites, hairy roots from two flasks of each root line were harvested after 6 weeks of culture. Statistical analysis was performed using the StatSoft
r STATISTICA software. Confirmation of the transformed nature of the roots PCR reaction was performed for T-DNA detection in roots of the analyzed plants. Total DNA was isolated from pow￾dered roots frozen in liquid nitrogen and extraction of chromosomal DNA using DNAzol Reagent (GIBCO-BRL) with purification on silica particles (Boom et al. 1999). For some samples, especially those containing shoots, addi￾tional phenol/chloroform extraction and precipitation were performed. The following oligomer primers were used as a primers: CTGTACCTCTACGTCGACT, TCAGTC￾GAGTGGGCTCCTTG, according to the programme: ini￾tial denaturisation at 94◦C for 1.5 min; 30 cycles at 94◦C for 20 s, 60◦C for 40 s, and then 72◦C for 1.5 min (Hosokawa et al. 1997). For all samples, except control plants, the predicted product (1.1 kb) was obtained (data not shown). Analytical methods Shikonin derivatives and pyrrolizidine alkaloids (PA) were extracted from samples of eight lines of L. canescens transformed roots. For obtaining dye fractions, the powdered sample (0.28–1.00 g) of lyophilized trans￾formed roots was sonicated with n-hexane (6 × 50 ml) for 30 min at 40◦C. The extracts were filtered through Whatman No. 2 paper and the solvent was evaporated from the extract solution under reduced pressure. Dry residue was dissolved in methanol and investigated in a DIONEX HPLC system, equipped with an automated sample injector (ASI-100), and UVD 340S detector using