1054 the following conditions:gradient elution-acetonitrile Table 1 Lithospermum canescens hairy roots clone Lcl biomass (40-0 ml)/0.04 M orthophosphoric acid (60-100 ml):flow increase based on fresh weight estimated after 6 weeks of culture rate 1.5 ml min;column:EC 250/4.6 Nucleosil 120- Line of Medium 127 mm Cis (Macherey-Nagel),and monitoring eluent at hairy roots LS LS+GA3 SH SH+GA3 215,278,514,and 320 nm.The extract of natural roots of A 11±3.46a 9±2.31b 12±4.95b 11±4.00b L.canescens collected in June 2001 in Togo,Canada was B 9±3.51a 8±1.53b 11±5.77b 11±4.03b used as a control material.Shikonin (Wako)and its two derivatives acetylshikonin (ACS)and isobutyrylshikonin C 11±7.00a8±3.21b 15±4.11b 9±3.61b D 8±2.56a 6±151a (IBS)isolated previously from natural roots of L.canescens 6±3.51a6±3.05a (Pietrosiuk and Wiedenfeld 2005)were used as the stan- E 13±1.73b10±4.04b12±5.02b 11±2.08b F 16±4.36c 6±1.00a 21±4.15c 19±5.65c dards and analyzed using the same conditions.Peaks were assigned by spiking the samples with the standards and G 8±1.41a 9±1.00b 7±3.21a 7±1.15a comparison of the retention times and UV spectra. H 16±7.73c10±3.25b14±6.61b 12±3.06b To obtain PA fractions,the same samples of transformed Values are means+SE of three experiments with two replicates per roots were sonicated with methanol (4 x 50 ml)for 30 min treatment at 40C.The combined methanolic extracts were evapo- Values within a single column followed by the same letter were not rated under reduced pressure to dryness.The residue was significantly different according to the LSD at the 5%level purified using dichloromethane and ethylic ether,sepa- rately,at pH 2.The total PA fraction was isolated from with strain LBA 9402,respectively.ATCC 15834 was the second most efficient bacterial strain used to transform the extract by shaking two times with dichloromethane after clones Lel,Lc2 and Le7 with 10,3,and 8 roots,respec- adjustment to pH 9 with 25%ammonia and with salting out the water phase after the first stage.TLC,TLC preparative tively.No transformed roots were obtained after treatment (silica gel F254,CH2Cl2-CH3OH-NH4OH 25%,85:14:1). of the plantlet clone Lc9 and only two roots appeared when and then GC-MS analysis,according to Wiedenfeld et al. plantlets of clone Lc8 were infected with strains LBA 9402 and ATCC 15834.A visual estimation of roots obtained af- (2003)were used for the detection of PA in the root extracts ter transformation with LBA 9402 strain revealed that they with the conditions GC-MS:GC:150C(5 min)-250C, 10/min;HP-1,25m×0.32;nj.:250°C,det.:280°C; did not produce shikonin derivatives.Roots were white in comparison to hairy roots obtained after transformation MS:220°C:interface:250°C:2000emV. with ATCC 15834 strain (after the same time of culture). A voucher specimen of L.canescens (Michx.)Lehm. from South of Togo,Saskatchewan,found in sandy soils,is HPLC-UV studies demonstrated only traces of ACS and IBS in these white roots (data not shown).Among all deposited at The W.P.Fraser Herbarium (SASK),Univer- sity of Saskatchewan,Saskatoon,Saskatchewan,Canada, induced roots,eight lines (A-H)developed as a result of infection with A.rhizogenes ATCC 15834 of the plantlet accession number 94815. clone Lc1,demonstrated a sufficient and stable biomass increase (Table 1)and they were submitted to further investigations.These lines of hairy roots were marked Results and discussion with letters from A to H.PCR analyses confirmed their transformation status (data not shown).Roots of eight lines The present study was concerned with the establishment of transformed hairy root cultures of L.canescens and evalu- growing on solid LS medium with GA3 0.25 mg I-1 were ation of their ability to produce shikonin derivatives.The long with good lateral branching(Fig.1).The young roots phytochemical studies were focused on the detection of the (1-2-week-old)were white with red apexes while older dyes acetylshikonin (ACS)and isobutyrylshikonin (IBS) ones (4-6-week-old)became deeply red (Fig.2).These which could be of biological importance as indicated in our earlier studies(Pietrosiuk et al.2003a,b,2004a,b). The variations in the growth and pigment content between different hairy root lines were investigated to evaluate the importance of the clonal selection in shikonin derivative accumulation.The infection of L.canescens stems of 6-week-old plantlets with the A.rhizogenes strains(ATCC 15834,LBA 9402 and NCIB 8196)resulted in hairy root formation.The first root from the infected point emerged 11 days after treatment of the plantlet clone Lc7 with A.rhizogenes LBA 9402.Six weeks later we observed 55 roots after treatment with LBA 9402,and only 22 and three roots when ATCC 15834 or NCIB 8196 were used,respectively.The transformation rate was strongly dependent on the clone and the bacterial strain used.The Fig.1 Hairy roots (1-2-week-old)of Lithospermum canescens highest numbers of developing roots 19,15,and 20 were grown on solid LS medium supplemented with GA3 (line Lc1D). observed after transformation of clones Lc1,Lc2 and Lc7 red apexes are seen (5:1)1054 the following conditions: gradient elution—acetonitrile (40–0 ml)/0.04 M orthophosphoric acid (60–100 ml); flow rate 1.5 ml min−1; column: EC 250/4.6 Nucleosil
r 120– 127 mm C18 (Macherey-Nagel), and monitoring eluent at 215, 278, 514, and 320 nm. The extract of natural roots of L. canescens collected in June 2001 in Togo, Canada was used as a control material. Shikonin (Wako) and its two derivatives acetylshikonin (ACS) and isobutyrylshikonin (IBS) isolated previously from natural roots of L. canescens (Pietrosiuk and Wiedenfeld 2005) were used as the stan￾dards and analyzed using the same conditions. Peaks were assigned by spiking the samples with the standards and comparison of the retention times and UV spectra. To obtain PA fractions, the same samples of transformed roots were sonicated with methanol (4 × 50 ml) for 30 min at 40◦C. The combined methanolic extracts were evapo￾rated under reduced pressure to dryness. The residue was purified using dichloromethane and ethylic ether, sepa￾rately, at pH 2. The total PA fraction was isolated from the extract by shaking two times with dichloromethane after adjustment to pH 9 with 25% ammonia and with salting out the water phase after the first stage. TLC, TLC preparative (silica gel F254, CH2Cl2–CH3OH–NH4OH 25%, 85:14:1), and then GC–MS analysis, according to Wiedenfeld et al. (2003) were used for the detection of PA in the root extracts with the conditions GC–MS: GC: 150◦C (5 min)–250◦C, 10◦/min; HP-1, 25 m × 0.32; Inj.: 250◦C, det.: 280◦C; MS: 220◦C; interface: 250◦C; 2000 emV. A voucher specimen of L. canescens (Michx.) Lehm. from South of Togo, Saskatchewan, found in sandy soils, is deposited at The W.P. Fraser Herbarium (SASK), Univer￾sity of Saskatchewan, Saskatoon, Saskatchewan, Canada, accession number 94815. Results and discussion The present study was concerned with the establishment of transformed hairy root cultures of L. canescens and evalu￾ation of their ability to produce shikonin derivatives. The phytochemical studies were focused on the detection of the dyes acetylshikonin (ACS) and isobutyrylshikonin (IBS), which could be of biological importance as indicated in our earlier studies (Pietrosiuk et al. 2003a, b, 2004a, b). The variations in the growth and pigment content between different hairy root lines were investigated to evaluate the importance of the clonal selection in shikonin derivative accumulation. The infection of L. canescens stems of 6-week-old plantlets with the A. rhizogenes strains (ATCC 15834, LBA 9402 and NCIB 8196) resulted in hairy root formation. The first root from the infected point emerged 11 days after treatment of the plantlet clone Lc7 with A. rhizogenes LBA 9402. Six weeks later we observed 55 roots after treatment with LBA 9402, and only 22 and three roots when ATCC 15834 or NCIB 8196 were used, respectively. The transformation rate was strongly dependent on the clone and the bacterial strain used. The highest numbers of developing roots 19, 15, and 20 were observed after transformation of clones Lc1, Lc2 and Lc7 Table 1 Lithospermum canescens hairy roots clone Lc1 biomass increase based on fresh weight estimated after 6 weeks of culture Line of Medium hairy roots LS LS + GA3 SH SH + GA3 A 11 ± 3.46a 9 ± 2.31b 12 ± 4.95b 11 ± 4.00b B 9 ± 3.51a 8 ± 1.53b 11 ± 5.77b 11 ± 4.03b C 11 ± 7.00a 8 ± 3.21b 15 ± 4.11b 9 ± 3.61b D 8 ± 2.56a 6 ± 3.51a 6 ± 3.05a 6 ± 1.51a E 13 ± 1.73b 10 ± 4.04b 12 ± 5.02b 11 ± 2.08b F 16 ± 4.36c 6 ± 1.00a 21 ± 4.15c 19 ± 5.65c G 8 ± 1.41a 9 ± 1.00b 7 ± 3.21a 7 ± 1.15a H 16 ± 7.73c 10 ± 3.25b 14 ± 6.61b 12 ± 3.06b Values are means ± SE of three experiments with two replicates per treatment Values within a single column followed by the same letter were not significantly different according to the LSD at the 5% level with strain LBA 9402, respectively. ATCC 15834 was the second most efficient bacterial strain used to transform clones Lc1, Lc2 and Lc7 with 10, 3, and 8 roots, respec￾tively. No transformed roots were obtained after treatment of the plantlet clone Lc9 and only two roots appeared when plantlets of clone Lc8 were infected with strains LBA 9402 and ATCC 15834. A visual estimation of roots obtained af￾ter transformation with LBA 9402 strain revealed that they did not produce shikonin derivatives. Roots were white in comparison to hairy roots obtained after transformation with ATCC 15834 strain (after the same time of culture). HPLC-UV studies demonstrated only traces of ACS and IBS in these white roots (data not shown). Among all induced roots, eight lines (A–H) developed as a result of infection with A. rhizogenes ATCC 15834 of the plantlet clone Lc1, demonstrated a sufficient and stable biomass increase (Table 1) and they were submitted to further investigations. These lines of hairy roots were marked with letters from A to H. PCR analyses confirmed their transformation status (data not shown). Roots of eight lines growing on solid LS medium with GA3 0.25 mg l−1 were long with good lateral branching (Fig. 1). The young roots (1–2-week-old) were white with red apexes while older ones (4–6-week-old) became deeply red (Fig. 2). These Fig. 1 Hairy roots (1–2-week-old) of Lithospermum canescens grown on solid LS medium supplemented with GA3 (line Lc1D), red apexes are seen (5:1)