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.Shimomura
Plant 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
283 a Fig.1.a)Hairy roots cultured on hormone-free RC solid medium containing 3%sucrose.b)Hairy roots cultured in Determination of shikonin.Culture media,adsorbents and lyophilized hormone-free RC liquid medium and shikonin secretion. hairy roots were extracted with CHCl3.After drying over Na2S04 the c)Microscopic observation of hairy roots and shikonin extract was evaporated to dryness and the shikonin content was secretion.d)Shikonin adsorption on a XAD-2 column. determined by its UV absorption at 520 nm (Fujita et al.1983). light.Two-3 weeks after the inoculation,hairy roots (ca 5/ shoot)appeared at the infected sites.They were cut off and Results and discussion subcultured on MS solid medium containing carbenicillin. Within one week the hairy roots formed calli on the Transformation antibiotic medium.After the elimination of the bacteria the Axenic shoots of L.erythrorhizon were cultured for 3 weeks white calli were transferred to hormone-free MS solid at low light intensity (100 lux).The etiolated shoots were medium,where the formation of hairy roots with red spots infected directly with A.rhizogenes 15834 and kept at low in the older parts of the tissue was observed.However
283 Determination ofshikonin. Culture media, adsorbents and lyophilized hairy roots were extracted with CHCI3. After drying over Na2SO4 the extract was evaporated to dryness and the shikonin content was determined by its UV absorption at 520 nm (Fujita et al. 1983). Results and discussion Transformation Axenic shoots of L. erythrorhizon were cultured for 3 weeks at low light intensity (100 lux). The etiolated shoots were infected directly with A. rhizogenes 15834 and kept at low Fig. 1. a) Hairy roots cultured on hormone-free RC solid medium containing 3% sucrose, b) Hairy roots cultured in hormone-free RC liquid medium and shikonin secretion. c) Microscopic observation of hairy roots and shikonin secretion, d) Shikonin adsorption on a XAD-2 column. light. Two-3 weeks after the inoculation, hairy roots (ca 5 / shoot) appeared at the infected sites. They were cut off and subcultured on MS solid medium containing carbeniciUin. Within one week the hairy roots formed calli on the antibiotic medium. After the elimination of the bacteria the white calli were transferred to hormone-free MS solid medium, where the formation of hairy roots with red spots in the older parts of the tissue was observed. However
284 these reddish roots tumned white after a second subculture to shikonin was produced and the cell growth was decreased the same medium.Therefore,it was presumed that MS with the addition of liquid paraffin. medium was not suitable for the production of shikonin in the hairy roots.The high NH4+content of the MS T138制 medium is thought to inhibit shikonin production (Fujita et 【1] al.1981a,b).Hairy roots cultured on the NH4+-free RC 【10) solid and liquid media showed fast growth and produced ☑hairy roots medium large amounts of shikonin derivatives,which were released adsorbent to the medium (Fig.la,b).After two weeks of culture in 且25 the RC liquid medium,shikonin production by the hairy 20 roots increased and reached its maximum after 4 weeks (ca. [721 77 15 5.5 mg/flask)(Fig.2).During the entire culture period ca. 25%of the shikonin derivatives produced were released to 【3 【351 the culture medium 6 8 789 contro 5 mg 10 mg 20 mg 10 ml 20 ml 30 ml 40 ml addition of charcoal addition of liquid paraffin Fig.3.Shikonin production by hairy roots of L.erythrorhizon cultured 4 in RC liquid medium with different adsorbents for 35 days. The numbers in the parentheses show the dry weight [mgl. 2 As liquid paraffin is not very useful in a continuous 「341 culture system,we examined the influence of different macroreticular adsorbents such as Amberlite XAD-2,4,and -7 (Rhodes et al.1986).The maximum production of 2 time [weeks】 shikonin,2-fold the amount of the control,was observed when XAD-2 was added to RC liquid medium (Fig.4). Fig.2.Shikonin production in hairy roots of L.erythrorhizon cultured in RC liquid medium. The numbers in the parentheses show the dry weight [mg]. 13 111 XAD-2,47 The microscopic observation of the hairy roots 25 medium ☑hair灯root 1 showed,that only the older parts of the tissue contained red [10】 pigments,while in the root tips no shikonin was detected 2.0 (Fig.la).The red pigments were located mainly as 5 [731 granules on or near the epidermis of the hairy roots (Fig. 1c),while in the normal roots shikonin derivatives were found only in the periderm (Tsukada and Tabata 1984). Liquid cultures 0.0- Deno et al.(1987)reported the production of shikonin by 501005001000 501005001000501005001000 control cell suspension cultures in two layer culture systems.We XAD-2 XAD-4 XAD-7 [mg/flask] applied this culture method to the hairy roots.The addition Fig.4.Shikonin production by hairy roots of L.erythrorhizon of charcoal (5-20 mg flask)inhibited the shikonin cultured in RC liquid medium (30 ml/100 ml flask)for 35 days production in the hairy roots cultured in RC liquid medium with different resin adsorbents (only 10 the level of shikonin was produced)(Fig.3). The numbers in the parentheses show the dry weight [mg]. We suggest that this inhibition might be caused by the complexation of essential nourishing elements in the Ca.85-90 of the shikonin produced was trapped in the medium(mainly the organic elements),because the growth resin,while only 1-5 was found in the medium and 10- was also inhibited. 15 in the hairy roots.XAD-4 and-7 adsorbed less On the other hand,the addition of liquid paraffin shikonin and lower productivity of the hairy roots was increased shikonin production in the hairy roots (Fig.3). observed.As has been observed with the addition of liquid In addition the growth of the hairy roots was enhanced by paraffin,increased growth of the hairy roots was obtained the two layer culture system (almost doubled growth and when the adsorbents were added to the culture medium. tripled shikonin production).These observations are in Thus,shikonin production by the hairy roots can be contrast to those of Deno et al.(1987).In cell suspension stimulated with the addition of adsorbents to the culture cultures of L.erythrorhizon only the same amount of medium,while this stimulation was not possible in cell
284 these reddish roots turned white after a second subculture to the same medium. Therefore, it was presumed that MS medium was not suitable for the production of shikonin in the hairy roots. The high NH4 + content of the MS medium is thought to inhibit shikonin production (Fujita et al. 1981a, b). Hairy roots cultured on the NH4+-free RC solid and liquid media showed fast growth and produced large amounts of shikonin derivatives, which were released to the medium (Fig. la, b). After two weeks of culture in the RC liquid medium, shikonin production by the hairy roots increased and reached its maximum after 4 weeks (ca. 5.5 mg/flask) (Fig. 2). During the entire culture period ca. 25% of the shikonin derivatives produced were released to the culture medium. 6. 5' r= 4. $3- ._= == 2" 1" [st] [] medium [] hairy root [S~ [34] 2 3 4 time [weeks] Us] iiiiiii Fig. 2. Shikonin production in hairy roots of L. erythrorhizon cultured in RC liquid medium. The numbers in the parentheses show the dry weight [mg]. The microscopic observation of the hairy roots showed, that only the older parts of the tissue contained red pigments, while in the root tips no shikonin was detected (Fig. la). The red pigments were located mainly as granules on or near the epidermis of the hairy roots (Fig. lc), while in the normal roots shikonin derivatives were found only in the periderm (Tsukada and Tabata 1984). Liquid c ultures Deno et al. (1987) reported the production of shikonin by cell suspension cultures in two layer culture systems. We applied this culture method to the hairy roots. The addition of charcoal (5 - 20 mg / flask) inhibited the shikonin production in the hairy roots cultured in RC liquid medium (only 10 % the level of shikonin was produced) (Fig. 3). We suggest that this inhibition might be caused by the complexation of essential nourishing elements in the medium (mainly the organic elements), because the growth was also inhibited. On the other hand, the addition of liquid paraffin increased shikonin production in the hairy roots (Fig. 3). In addition the growth of the hairy roots was enhanced by the two layer culture system (almost doubled growth and tripled shikonin production). These observations are in contrast to those of Deno et al. (1987). In cell suspension cultures of L. erythrorhizon only the same amount of shikonin was produced and the cell growth was decreased with the addition of liquid paraffin. [138] 4.5' [1x4] 4.0. [t0Sl 3.5' ~ hairy roots I~~ ' ~ medium ~J~ 3.0" + adsorbent ~~ 2'01 [721 [771 i 1.0 [301 [3Sl 0.0 addition of charcoal addition of liquid paraffin Fig. 3. Shikonin production by hairy roots of L. erythrorhizon cultured in RC liquid medium with different adsorbents for 35 days. The numbers in the parentheses show the dry weight [mgl. As liquid paraffin is not very useful in a continuous culture system, we examined the influence of different macroreticular adsorbents such as Amberlite XAD-2, -4, and -7 (Rhodes et al. 1986). The maximum production of shikonin, 2-fold the amount of the control, was observed when XAD-2 was added to RC liquid medium (Fig. 4). r % ~ 2.0 ,'~ 715 9 '" % % .= " 0.5" ," 0.0 5O Fig. 4. [1O3l 11111 [] XAD-2, -4, -7 ~ H medium / hairy root t~] ...' [1011 ...' 11161 11041 ~ P 9 03 r149 P 9 ,..' ~ [921 ~ r " [~31 [10$ " 9 9 r149 ~. 9 s'~s" r f 9 tO0 500 1000 50 "i00"500 1000150 "100 "500 IOOG control XAD-2 XAD-4 i XAD-7 [mg/llask] Shikonin production by hairy roots of L. erythrorhizon cultured in RC liquid medium (30 ml/100 ml flask) for 35 days with different resin adsorbents. The numbers in the parentheses show the dry weight [mg]. Ca. 85 - 90 % of the shikonin produced was trapped in the resin, while only 1 - 5 % was found in the medium and 10 - 15 % in the hairy roots. XAD-4 and -7 adsorbed less shikonin and lower productivity of the hairy roots was observed. As has been observed with the addition of liquid paraffin, increased growth of the hairy roots was obtained when the adsorbents were added to the culture medium. Thus, shikonin production by the hairy roots can be stimulated with the addition of adsorbents to the culture medium, while this stimulation was not possible in cell
285 suspension cultures of L.erythrorhizon (Deno et al.1987). constant level of 5.2 mg day during a culture period of 220 days. Fermenter system From the results obtained above,we established a fermenter The data obtained in this study show that hairy roots system for continuous shikonin production. are a useful system for production of shikonin derivatives, Approximately 200 mg of 10 day old hairy roots that had as one single culture medium can be used which allowed been grown on RC solid medium were inoculated in a 2 both the growth of the hairy roots and shikonin production. air-lift type fermenter.The same liquid medium was used Using the fermenter system with the XAD-2 column the for the fermenter containing 3%sucrose at pH 5.7.The continuous and stable production of shikonin could be fermenter was connected with a column containing 25 g obtained. XAD-2.The medium was continuously pumped through the column by the peristatic pump (Fig.1d).Medium and Acknowledgements.The authors express their appreciation to Dr. XAD-2 column were exchanged periodically.After about Martina Sauerwein for preparing the manuscript and Dr.Darryl Macer, 20 days the shikonin production began and the level visiting Professor,University of Tsukuba,for critical reading of our manuscript. increased until ca.110 days of culture,when it reached 5.9 mg day (Fig.6).Shikonin derivatives were produced at a References Deno H,Suga C,Morimoto T,Fujita Y(1987)Plant Cell Reports 6: 197-199 EMBO course(1982)The use of Ti plasmid as cloning vector for genetic engineering in plants,August 4-23,p.109 Fujita Y,Hara Y,Ogino T,Suga C(1981 a)Plant Cell Reports 1:59-60 4.0 Fujita Y,Hara Y,Suga C,Morimoto T(1981 b)Plant Cell Reports 1: 61-63 Fujita Y,Maeda Y,Suga C,Morimoto T(1983)Plant Cell Reports 2.0 2:192-193 Kyogoku K,Terayama H,Tachi Y,Suzuki T,Komatsu M(1973) Syoyakugaku Zasshi 27:31-36 00 1090110140175193201211222 Murashige T,Skoog F(1962)Physiol.Plant.15:473-497 time [days] Petit A,David C,Dahl,GA,Ellis JG,Guyon P,Casse-Delbart F, Tempe J(1983)Mol.Gen.Genet.190:204-214 Fig.6.Shikonin production by hairy roots of L.erythrorhizon Rhodes MIC,Hilton M,Parr AJ,Hamill JD,Robins RJ(1986) cultured in 2 air-lift type femmenter with RC liquid medium Biotech.Lett.8:415-420 (3%sucrose)for 222 days. Tsukada M,Tabata M(1984)Planta Med.50:338-340
constant level of 5.2 mg / day during a culture period of 220 days. suspension cultures of L. erythrorhizon (Deno et al. 1987). Fermenter system From the results obtained above, we established a fermenter system for continuous shikonin production. Approximately 200 mg of 10 day old hairy roots that had been grown on RC solid medium were inoculated in a 2 l air-lift type fermenter. The same liquid medium was used for the fermenter containing 3% sucrose at pH 5.7. The fermenter was connected with a column containing 25 g XAD-2. The medium was continuously pumped through the column by the peristatic pump (Fig. ld). Medium and XAD-2 column were exchanged periodically. After about 20 days the shikonin production began and the level increased until ca. 110 days of culture, when it reached 5.9 mg / day (Fig. 6). Shikonin derivatives were produced at a The data obtained in this study show that hairy roots are a useful system for production of shikonin derivatives, as one single culture medium can be used which allowed both the growth of the hairy roots and shikonin production. Using the fermenter system with the XAD-2 column the continuous and stable production of shikonin could be obtained. Acknowledgements. ]'he authors express their appreciation to Dr. Martina Sauerwein for preparing the manuscript and Dr. Darryl Mater, visiting Professor, University of Tsukuba, for critical reading of our manuscript. References 6.0 e~ "4 4.0 r 2.0' 0.0 Fig. 6. J f 10 90 110 140 175 193 201 211 222 time [days] Shikonin production by hairy roots of L. erythrorhizon cultured in 2 1 fir-lift type fermenter with RC liquid medium (3% sucrose) for 222 days. 285 Deno H, Suga C, Morimoto T, Fujita Y (1987) Plant Cell Reports 6: 197-199 EMBO course (1982) The use of Ti plasmid as cloning vector for genetic engineering in plants, August 4-23, p.109 Fujita Y, Ham Y, Ogino T, Suga C (1981 a) Plant CeU Reports 1:59-60 Fujita Y, Hara Y, Suga C, Morimoto T (1981 b) Plant Cell Reports 1: 61 - 63 Fujita Y, Maeda Y, Suga C, Morimoto T (1983) Plant Cell Reports 2:192-193 Kyogoku K, Terayama H, Tachi Y, Suzuki T, Komatsu M (1973) Syoyakugaku Zasshi 27:31-36 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Petit A, David C, DaM, GA, Ellis JG, Guyon P, Casse-Delbart F, Temp6 J (1983) Mol. Gen. Genet. 190:204-214 Rhodes MIC, Hilton M, Parr AJ, Hamill JD, Robins RI (1986) Biotech. Lett. 8:415-420 Tsukada M, Tabata M (1984) Planta Med. 50:338-340