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 cell284 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