Plant Cell Reports (1991)10:282-285 Plant Cell Reports C Springer-Verlag 1991 Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon Koichiro Shimomura1,Hiroshi Sudo2,Hitoshi Saga2,and Hiroshi Kamada2 1Tsukuba Medicinal Plant Research Station,National Institute of Hygienic Sciences,1 Hachimandai,Tsukuba,Ibaraki,305 Japan 2 Institute of Biological Sciences,University of Tsukuba,Tsukuba,Ibaraki,305 Japan Received March 1,1991/Revised version received June 17,1991-Communicated by F.Constabel Summary Hairy root cultures of Lithospermum We show that in our culture system the hairy roots erythrorhizon were established by transformation of in continuously produced shikonin and released it to the vitro grown shoots with Agrobacterium rhizogenes 15834. culture medium.Rhodes et al.(1986)reported the Hairy roots cultured on Murashige and Skoog solid medium adsorption of nicotine to Amberlite XAD-4 in hairy root did not produce any red pigments.However,the hairy roots cultures of Nicotiana rustica.Here we describe a culture cultured in Root Culture solid or liquid media produced a system equipped with an XAD-2 column,which allows the large amount of red pigments,which were released to the long term culture of L.erythrorhizon hairy roots and the medium.The addition of adsorbents to the culture medium continuous shikonin production. stimulated shikonin production by ca.3-fold.Using this method an air-lift fermenter system was established, equipped with a XAD-2 column,which continuously Materials and methods produced ca.5 mg/day of shikonin during a period of more than 220 days. Plant material.Hairy roots of Lithospermum erythrorhizon were induced by direct infection of axenic shoot cultures with Agrobacterium rhizogenes strain 15834.The bacteria were eliminated Key words:Lithospermum erythrorhizon-Hairy root on Murashige and Skoog (MS.Murashige and Skoog 1962)solid culture-Shikonin-Secretion-Adsorbent medium containing 1 g/I carbenicilline.The hairy roots were subcultured onto MS solid medium and later to Root Culture [RC. EMBO course 1982 as follows (mgA):Ca(NO3)2+4H20 (288),KNO3 (80),KC1(65),MgS047H20(370),Na2S0410H20(226.7), Introduction NaH2P044H20(21.5),H3BO3(1.5).ZnS047H20(2.65).KI(0.75). Na2Mo042H0(0.25)nCuS045H20(0.02),MnC24H20(6.0),FeC3 Lithospermum erythrorhizon Sieb.et Zucc.(Boraginaceae) (3.2),EDTA-2Na (7.4),thiamine-HCI (0.1),nicotinic acid (0.5), contains a large quantity of shikonin derivatives in its roots pyridoxin-HCI (0.1).glycine (3.0)]liquid medium containing 3% (Kyogoku et al.1973).These naphthoquinone pigments sucrose (pH 5.7,30 ml medium /100 ml flask).The cultures were kept possess antibacterial activity and stimulate the formation of on a rotary shaker at 100 rpm,at 25C in the dark.The transformation granulation tissue,which makes them useful in medicinal was proven by the detection of the opines using paper electrophoresis according to the method of Petit et al.(1983).Voucher specimens are and cosmetic applications.Production of shikonin in high deposited at Tsukuba Medicinal Plant Research Station. yield from callus and cell suspension cultures has been Culture experiments.Different amount of sterilized adsorbents reported by several researchers (Deno et al.1987;Fujita et (charcoal,liquid paraffin,XAD-2,XAD-4 and XAD-7)were aseptically al.1983 and ref.cited therein).However,the production of added to the hairy root cultures in RC liquid medium (30 ml /100 ml flask).After 35 days of culture growth of the hairy roots and shikonin shikonin derivatives in cell suspension cultures requires two content of roots,medium and adsorbents were determined.Three flasks different kinds of media,one for cell growth and one for were used for each experiment and cultured at 25C in the dark on a shikonin production.An addition problem is that shikonin rotary shaker at 100 rpm. particle produced by cultured cells are located between the Fermenter system.A2I air-lift type fermenter was used and connected plasma membrane and the cell wall (Tsukada and Tabata to a XAD-2 column(25 g)through a peristatic pump.The hairy roots 1984),and they may inhibit the growth and further (200 mg,obtained from RC solid medium after 10 days)were shikonin Droduction (Deno et al.1987). subcultured into 2/of RCmedium,containing3%sucrose at pH 5.7,at 25C in the dark.Medium and XAD-2 column were changed every 4-6 In this report we describe the transformation of L. weeks.The XAD-2 was washed with water and extracted with CHCl3 for erythrorhizon with Agrobacterium rhizogenes strain 15834. the determination of shikonin. Offprint requests to:K.ShimomuraPlant Cell Reports (1991) 10:282-285 Plant Cell Reports 9 Springer-Verlag 1991 Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon Koichiro Shimomura 1, niroshi Sudo 2, Hitoshi Saga 2 and Hiroshi Kamada 2 1 Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki, 305 Japan 2 Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305 Japan Received March 1, 1991/Revised version received June 17, t991 - Communicated by E Constabel Summary Hairy root cultures of Lithospermum erythrorhizon were established by transformation of in vitro grown shoots with Agrobacterium rhizogenes 15834. Hairy roots cultured on Murashige and Skoog solid medium did not produce any red pigments. However, the hairy roots cultured in Root Culture solid or liquid media produced a large amount of red pigments, which were released to the medium. The addition of adsorbents to the culture medium stimulated shikonin production by ca. 3-fold. Using this method an air-lift fermenter system was established, equipped with a XAD-2 column, which continuously produced ca. 5 mg / day of shikonin during a period of more than 220 days. Key words: Lithospermum erythrorhizon - Hairy root culture - Shikonin - Secretion - Adsorbent Introduction Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) contains a large quantity of shikonin derivatives in its roots (Kyogoku et al. 1973). These naphthoquinone pigments possess antibacterial activity and stimulate the formation of granulation tissue, which makes them useful in medicinal and cosmetic applications. Production of shikonin in high yield from callus and cell suspension cultures has been reported by several researchers (Deno et al. 1987; Fujita et al. 1983 and ref. cited therein). However, the production of shikonin derivatives in cell suspension cultures requires two different kinds of media, one for cell growth and one for shikonin production. An addition problem is that shikonin particle produced by cultured cells are located between the plasma membrane and the cell wall (Tsukada and Tabata 1984), and they may inhibit the growth and further shikonin production (Deno et al. 1987). In this report we describe the transformation of L. erythrorhizon with Agrobacterium rhizogenes strain 15834. We show that in our culture system the hairy roots continuously produced shikonin and released it to the culture medium. Rhodes et al. (1986) reported the adsorption of nicotine to Amberlite XAD-4 in hairy root cultures of Nicotiana rustica. Here we describe a culture system equipped with an XAD-2 column, which allows the long term culture of L. erythrorhizon hairy roots and the continuous shikonin production. Materials and methods Plant material. Hairy roots of Lithospermum erythrorhizon were induced by direct infection of axenie shoot cultures with Agrobacterium rhizogenes swain 15834. The bacteria were eliminated on Murashige and Skoog (MS, Murashige and Skoog 1962) solid medium containing 1 g / l carbenicilline. The hairy roots were subcultored onto MS solid medium and later to Root Culture [RC, EMBO course 1982 as follows (mg/1) : Ca(NO3)2o4H20 (288), KNO3 (80), KC1 (65), MgSO4,7HzO (370), Na2SO4,10H20 (226.7), NaH2PO4o4H20 (21.5), H3BO3 (1.5), ZnSO4oTH20 (2.65), KI (0.75), NazMoO4,2HzO (0.25), CuSO4o5H20 (0.02), MnC12,4H20 (6.0), FeCh (3.2), EDTA-2Na (7.4), thiamine-HC1 (0.1), nicotinic acid (0.5), pyridoxin-HC1 (0.t), glycine (3.0)] liquid medium containing 3% sucrose (pH 5.7, 30 ml medium / 100 ml flask). The cultures were kept on a rotary shaker at 100 rpm, at 25~ in the dark. The transformation was proven by the detection of the opines using paper electrophoresis according to the method of Petit et al. (1983). Voucher specimens are deposited at Tsukuba Medicinal Plant Research Station. Culture experiments. Different amount of sterilized adsorbents (charcoal, liquid paraffin, XAD-2, XAD-4 and XAD-7) were aseptically added to the hairy root cultures in RC liquid medium (30 rnl / 100 ml flask). After 35 days of culture growth of the hairy roots and shikonin content of roots, medium and adsorbents were determined. Three flasks were used for each experiment and cultured at 25r in the dark on a rotary shaker at 100 rpm. Fermenter system. A 2 l air-lift type fermenter was used and connected to a XAD-2 column (25 g) through a pedstatic pump. The hairy roots (200 mg, obtained from RC solid medium after 10 days) were subcultured into 2 l of RC medium, containing 3% sucrose at pH 5.7, at 25~C in the dark. Medium and XAD-2 column were changed every 4-6 weeks. The XAD-2 was washed with water and extracted with CHCI3 for the determination of shikonin. Offprint requests to: K. Shimomura