1057 on LS with GA3 medium (Tables 2 and 3).However,the References addition of GA3 to SH medium resulted in lower levels of ACS and IBS.In the presence of GA3,the growth Birgham LA,Michaels PJ,Flores HE (1999)Cell-specific produc- rate of all root lines diminished slightly (Table 1),but tion and antimicrobial activity of naphthoquinones in roots of shikonin derivative production was not affected (Tables 2 Lithospermum erythrorhizon.Plant Physiol 119:417-428 and 3).Different results were obtained by Yoshikawa et al. Boom R.Sol C.Beld M.Weel J.Goudsmit J.Wertheim-van Dillen P (1999)Improved silica-guanidiniumthiocyanate DNA isolation (1986)who observed that GA3 strongly inhibited shikonin procedure based on selective binding of bovine alpha-casein to formation in L.erythrorhizon callus culture but had no silica particles.J Clin Microbiol 37:615-619. effect on cell growth.The line Lc1-D appeared the most Chang HN,Sim SJ(1996)Genetic transformation of Lithospermum suitable for ACS and IBS production on all media tested. erythrorhizon for increased production of shikonin.In:Bajaj This root line yielded ACS and IBS levels ranging from YPS (ed)Biotechnology in agriculture and forestry,vol 38. Plant protoplasts and genetic engineering VII.Springer-Verlag. 0.712 to 2.720 mg g-of dry weight and from 0.0402 to Berlin Heldelberg.pp 233-242 0.307 mg g-of dry weight,respectively.The content was Fujita Y,Hara Y,Suga C,Morimoto T(1981)Production of shikonin not accompanied by abundant biomass increase (Table derivatives by cell suspension cultures of Lithospermum 1).which was the lowest among all investigated hairy erythrorhizon.A new medium for the production of shikonin derivatives.Plant Cell Rep 1:61-63 root lines.This result was consistent with those reported Hiroshi K.Hitoshi S (1988)Production of naphthoquinone by Payne et al.(1991)who found that many secondary compound.JP-J63230093 metabolites were produced by cultures.which were either Hosokawa K.Matsuki R.Oikawa Y.Yamamura S (1997)Genetic not growing at all or growing very slowly.The presence of transformation of gentian using wild-type Agrobacterium rhizogenes.Plant Cell Tissue Organ Cult 51:137-140 PA was also examined in our lines.We detected traces of PA Kropczyriska D,Kawka B,Pietrosiuk A,Furmanowa M,Wiedenfeld in hairy roots of L.canescens (data not shown).The most H(2004)The effect of pyrrolizidine alkaloids on the Colorado sensitive methods for detecting PA on TLC using Ehrlich potato beetle (Leptinotarsa decemlineata Say).Acta Biol reagent (4-dimethylaminobenzaldehyde:Mattocks.1967) Cracoviensia Ser Zool 46:83-86 showed the presence of PA in each examined line of L Linsmaier EM,Skoog F(1965)Organic growth factor requirement in relationship to cytokinin in"normal"and "mutant"strains of canescens hairy roots.This result was confirmed by gas tobacco callus.Planta 72:146-154 chromatography combined with mass spectrometry (GC- Mattocks AR (1967)Detection of pyrrolizidine alkaloids on MS).a valuable and highly sensitive means for the detection thin-layer chromatograms.J Chromatogr 27:505-508 and identification of pyrrolizidine alkaloids.The GC-MS Papageorgiou VP.,Assimopoulou AN.Couladouros EA.Hepworth D,Nicolaou KC,(1999)The chemistry and biology of alkannin, spectrum of investigated compounds shows the [M]-peak shikonin,and related naphtazarin natural products.Angew at 399 indicating the molecular formula C2oH33NO7.The Chem Int Ed 38:270-300 further fragmentations are the same for 7-0-(3-hydroxy- Payne GF,Bringi V,Prince C,Shuler ML(1991)Quantifying growth 3-methyl-butanoyl)-9-0-(+)-trachelanthoyl-heliotridine and product synthesis:kinetics and stoichiometry.In:Payne GF.Bringi V.Prince C.Shuler ML (eds)Plant cell and tissue (canescine)and 7-0-(3-hydroxy-3-methyl-butanoyl)-9-0- culture in liquid systems.Hanser Publishers.Munich Vienna (-)-viridifloryl-heliotridine (canescenine),as described New York Barcelona,pp 48-70 earlier (Wiedenfeld et al.2003). Pietrosiuk A,Furmanowa M,Kropczynska D.Kawka B,Wieden- To summarize briefly,we can state that L.canescens is feld H (2003a)Life history of the two-spotted spider mite susceptible to genetic transformation with A.rhizogenes. (Tetranychus urticae Koch)feeding on bean leaves treated with pyrrolizidine alkaloids.J Appl Toxicol 23:187-190 ACS and IBS are the main compounds among the other Pietrosiuk A.Kedzia B,Holderna-Kedzia E.Wiedenfeld H. unidentified shikonin derivatives in transformed hairy roots Malinowski M,Furmanowa M(2003b)Antimicrobial activity of L.canescens.ACS and IBS contents in hairy roots con- of naphthoquinones from Lithospermum canescens Lehm. stitute ca.10%of those observed in natural roots of L. Herba Polonica 49(3/4):209-215 Pietrosiuk A.Skopiniska-Rozewska E.Furmanowa M.Wiedenfeld canescens(24.35 and 14.48 mg g-DW,respectively).The H,Sommer E,Sokolnicka I,Bany J,Malinowski M(2004a) HPLC-UV results show that shikonin itself could not be Immunomodulatory effect of shikonin derivatives isolated from detected in the transformed hairy roots but more shikonin Lithospermum canescens on cellular and humoral immunity in derivatives were found in transformed hairy roots of L. Balb/c mice.Die Pharmazie 59:640-642 Pietrosiuk A,Furmanowa M,Skopiniska-Rozewska E.Sommer E. canescens than in control roots of plants growing in their Skurzak H,Bany J(2004b)The effect of acetylshikonin isolated natural habitat (Fig.3).Line Lc1-D proved to be a good from Lithospermum canescens roots on tumor-induced cuta- source of shikonin derivatives and will be used to scale up neous angiogenesis.Acta Polon Pharm Drug Res 61(5):379- ACS and IBS production. 382 Pietrosiuk A.Wiedenfeld H(2005)Shikonin derivatives from Lithos- Acknowledgements This investigation was supported by a research permum canescens (Michx.)Lehm.Pharm Biol 43(2):189-191 grant PBZ-KBN-092/P05/2003 from the State 'Committee of Sci- Schenk RU.Hildebrandt AC (1972)Medium and techniques for entific Research.We are thankful to Dr.Branka Barl,chief scien- induction and growth of monocotyledonous and dicotyledonous plant cell cultures.Can J Bot 50:199-204 tist.New Era Nutrition.Inc..Edmonton.Alberta.for plants and Shimomura K.Sudo H.Saga H.Kamada H (1991)Shikonin seeds of L.canescens.We are also grateful to Professor Mondher Jaziri from the Laboratory of Biotechnology and Plant Morphol- production and secretion by hairy root cultures Lithospermum ogy,Free University of Brussels for the bacterial strain A.rhizo- erythrorhizon.Plant Cell Rep 10:282-285 Syklowska-Baranek K,Pietrosiuk A,Dluska H,Furmanowa M genes NCIB 8196.We thank Mr Ireneusz Rudnicki,Photodepart- (2004)Clonal multiplication of Lithospermum canescens ment of Medical University of Warsaw,Poland for photographic (Michx.)Lehm.and Onosma paniculatum (Bur.and Franch). documentation. Herba Polonica 51(2):38-471057 on LS with GA3 medium (Tables 2 and 3). However, the addition of GA3 to SH medium resulted in lower levels of ACS and IBS. In the presence of GA3, the growth rate of all root lines diminished slightly (Table 1), but shikonin derivative production was not affected (Tables 2 and 3). Different results were obtained by Yoshikawa et al. (1986) who observed that GA3 strongly inhibited shikonin formation in L. erythrorhizon callus culture but had no effect on cell growth. The line Lc1-D appeared the most suitable for ACS and IBS production on all media tested. This root line yielded ACS and IBS levels ranging from 0.712 to 2.720 mg g−1 of dry weight and from 0.0402 to 0.307 mg g−1 of dry weight, respectively. The content was not accompanied by abundant biomass increase (Table 1), which was the lowest among all investigated hairy root lines. This result was consistent with those reported by Payne et al. (1991) who found that many secondary metabolites were produced by cultures, which were either not growing at all or growing very slowly. The presence of PA was also examined in our lines. We detected traces of PA in hairy roots of L. canescens (data not shown). The most sensitive methods for detecting PA on TLC using Ehrlich reagent (4-dimethylaminobenzaldehyde; Mattocks, 1967) showed the presence of PA in each examined line of L. canescens hairy roots. This result was confirmed by gas chromatography combined with mass spectrometry (GC– MS), a valuable and highly sensitive means for the detection and identification of pyrrolizidine alkaloids. The GC–MS spectrum of investigated compounds shows the [M]+-peak at 399 indicating the molecular formula C20H33NO7. The further fragmentations are the same for 7-O-(3-hydroxy- 3-methyl-butanoyl)-9-O-( + )-trachelanthoyl-heliotridine (canescine) and 7-O-(3-hydroxy-3-methyl-butanoyl)-9-O- ( − )-viridifloryl-heliotridine (canescenine), as described earlier (Wiedenfeld et al. 2003). To summarize briefly, we can state that L. canescens is susceptible to genetic transformation with A. rhizogenes. ACS and IBS are the main compounds among the other unidentified shikonin derivatives in transformed hairy roots of L. canescens. ACS and IBS contents in hairy roots con￾stitute ca. 10% of those observed in natural roots of L. canescens(24.35 and 14.48 mg g−1 DW, respectively). The HPLC-UV results show that shikonin itself could not be detected in the transformed hairy roots but more shikonin derivatives were found in transformed hairy roots of L. canescens than in control roots of plants growing in their natural habitat (Fig. 3). Line Lc1-D proved to be a good source of shikonin derivatives and will be used to scale up ACS and IBS production. Acknowledgements This investigation was supported by a research grant PBZ-KBN-092/P05/2003 from the State Committee of Sci￾entific Research. We are thankful to Dr. Branka Barl, chief scien￾tist, New Era Nutrition, Inc., Edmonton, Alberta, for plants and seeds of L. canescens. We are also grateful to Professor Mondher Jaziri from the Laboratory of Biotechnology and Plant Morphol￾ogy, Free University of Brussels for the bacterial strain A. rhizo￾genes NCIB 8196. We thank Mr Ireneusz Rudnicki, Photodepart￾ment of Medical University of Warsaw, Poland for photographic documentation. References Birgham LA, Michaels PJ, Flores HE (1999) Cell-specific produc￾tion and antimicrobial activity of naphthoquinones in roots of Lithospermum erythrorhizon. Plant Physiol 119:417–428 Boom R, Sol C, Beld M, Weel J, Goudsmit J, Wertheim-van Dillen P (1999) Improved silica-guanidiniumthiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles. J Clin Microbiol 37:615–619. Chang HN, Sim SJ (1996) Genetic transformation of Lithospermum erythrorhizon for increased production of shikonin. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 38. Plant protoplasts and genetic engineering VII. Springer-Verlag, Berlin Heldelberg, pp 233–242 Fujita Y, Hara Y, Suga C, Morimoto T (1981) Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. A new medium for the production of shikonin derivatives. Plant Cell Rep 1:61–63 Hiroshi K, Hitoshi S (1988) Production of naphthoquinone compound. JP–J63230093 Hosokawa K, Matsuki R, Oikawa Y, Yamamura S (1997) Genetic transformation of gentian using wild-type Agrobacterium rhizogenes. Plant Cell Tissue Organ Cult 51:137–140 Kropczynska D, Kawka B, Pietrosiuk A, Furmanowa M, Wiedenfeld ´ H (2004) The effect of pyrrolizidine alkaloids on the Colorado potato beetle (Leptinotarsa decemlineata Say). Acta Biol Cracoviensia Ser Zool 46:83–86 Linsmaier EM, Skoog F (1965) Organic growth factor requirement in relationship to cytokinin in “normal” and “mutant” strains of tobacco callus. Planta 72:146–154 Mattocks AR (1967) Detection of pyrrolizidine alkaloids on thin-layer chromatograms. J Chromatogr 27:505–508 Papageorgiou VP, Assimopoulou AN, Couladouros EA, Hepworth D, Nicolaou KC, (1999) The chemistry and biology of alkannin, shikonin, and related naphtazarin natural products. Angew Chem Int Ed 38:270–300 Payne GF, Bringi V, Prince C, Shuler ML (1991) Quantifying growth and product synthesis: kinetics and stoichiometry. In: Payne GF, Bringi V, Prince C, Shuler ML (eds) Plant cell and tissue culture in liquid systems. Hanser Publishers, Munich Vienna New York Barcelona, pp 48–70 Pietrosiuk A, Furmanowa M, Kropczynska D, Kawka B, Wieden- ´ feld H (2003a) Life history of the two-spotted spider mite (Tetranychus urticae Koch) feeding on bean leaves treated with pyrrolizidine alkaloids. J Appl Toxicol 23:187–190 Pietrosiuk A, K˛edzia B, Hołderna-K˛edzia E, Wiedenfeld H, Malinowski M, Furmanowa M (2003b) Antimicrobial activity of naphthoquinones from Lithospermum canescens Lehm. Herba Polonica 49(3/4):209–215 Pietrosiuk A, Skopinska-R ´ o´zewska E, Furmanowa M, Wiedenfeld ˙ H, Sommer E, Sokolnicka I, Bany J, Malinowski M (2004a) Immunomodulatory effect of shikonin derivatives isolated from Lithospermum canescens on cellular and humoral immunity in Balb/c mice. Die Pharmazie 59:640–642 Pietrosiuk A, Furmanowa M, Skopinska-R ´ o´zewska E, Sommer E, ˙ Skurzak H, Bany J (2004b) The effect of acetylshikonin isolated from Lithospermum canescens roots on tumor-induced cuta￾neous angiogenesis. Acta Polon Pharm Drug Res 61(5):379– 382 Pietrosiuk A, Wiedenfeld H (2005) Shikonin derivatives from Lithos￾permum canescens (Michx.) Lehm. Pharm Biol 43(2):189–191 Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204 Shimomura K, Sudo H, Saga H, Kamada H (1991) Shikonin production and secretion by hairy root cultures Lithospermum erythrorhizon. Plant Cell Rep 10:282–285 Sykłowska-Baranek K, Pietrosiuk A, Dłuska H, Furmanowa M (2004) Clonal multiplication of Lithospermum canescens (Michx.) Lehm. and Onosma paniculatum (Bur. and Franch). Herba Polonica 51(2):38–47