Polyacetylenes in Lobelia sessilifolia hairy roots

Pergsmoa

POLYACETYLENES

Phyzoc~y, Vol. 35, No. 2, pp. 36%369.1994 copyri#hc Q 1994 Ekvicr z3d4!4m Ltd Rioted in otmt Britain All tl$hu raand 0031~9422/94s6.M + 0.00

IN LOBELZA SESSZLZFOLZA HAIRY ROOTS

KANJI ISHIMARU,*HIROKO ARAKAWA,MICHIKO YAMANAKAand KOICHIRO SHIMOMURA~ Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, 1 Honjo, Saga 840, Japan; tTsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan (Receiued in revised form 20 Juty 1993)

Key Word Index--Lobeli sessilifolia; Campanulaceae; hairy root; Agrobacterium rhizogenes; polyacetylene; chlorophyll; lobetyol; lobetyolin; lobetyolinin.

A~~~-~be~iu sessil~liu hairy roots (six clones: A-l -4, and J-l and 2), induced by Agro~tffiu~ rhizogenes ATCC 15834 and A. rhizogenes MAFF 03-01724, grew well and produced some polyacetylenes (lobetyol, lobctyolin and lobetyolinin) in various hormone-free media. J-l clone, transformed with A. rhizogenes MAFF 03-01724, produced the highest amount of lobetyolin (21.48 mg in 50 ml Murashige- Skoog liquid medium) under illumination. Cultured in Murashige - Skoog medium in the light, only the greenish hairy roots which produced chlorophylls a and b showed better growth and polyacetylene production.

INTRODUCTION

Lobelia sessilijklia is found in the temperate and subtropical regions in East Asian countries e.g. Japan, Korea, China., etc. It has been reported to contain the piperidine alkaloid lobeline [l, 21. However, no detailed chemical studies on the secondary metabolites of this plant have been reported. In the course of chemical studies on tissue cultures of campanulaceous plants [3 - 61, we have reported the production of three polyacetylenes flobetyol (I), lobetyolin (2) and lobetyolinin (3)3 by L. in&zta hairy roots [3. 4, 61. These ~lya~tyien~ are expected to be useful chemotaxonomic markers. We have now succeeded in the establishment of hairy root cultures of L. sessilfilia which produced high amounts of these polyacetylenes (especially 2). The effects of media and illumination on the growth and polyacetylene production of L. sessil~olia hairy roots are also reported. RESULTS AND DlSCUS!SION

For the induction of hairy roots, two types of soil bacteria, Agrobacterium rhizogenes ATCC 15834 and A. rhjzogenes MAFF 03-01724, both of which have a root-inducing (Ri) plasmid, were used. Stem segments of L. sessilifolia were infected with these bacteria and several hairy roots were induced from the infected sites. Six clones, A- 1 h 4 by A. rhizogenes ATCC 15834, and J-l and 2 by A. rhizogenes MAFF 03-01724, were selected and used for this study. Hairy roots (A-l) which had been cultured in hormonefree Woody Plant (WP) liquid medium [7l for eight

weeks in the dark were lyophilized and the polyacetylenes (1-3) extracted (using methanol), isolated and identified by ‘H NMR spectroscopy. Six clones (A-l -4, and J-l and 2) were cultured in ho~one-fry Mur~hige-Skoog (MS) [S] and WP liquid media for five weeks in the light or dark, and their growth (fresh wt) and polyacetylene production determined (Fig. I). Although four clones (A-l, 3.4 and J-2) grew well in both MS and WP media, A-2 did not show good growth in these media. On the other hand, J-l grew well only in MS medium. When these hairy roots were cultured under light conditions, some clones turned green (so called ‘green hairy root’). The content of chlorophylls a and b in these roots (A-l, 3, 4, J-l and 2) was determined by HPLC (Fig. 2). In keeping with the appearance (greenish colourationf of the roots, four clones (A-l, 3,4 and J-l) in MS m~ium and three clones {A-l, 3 and 4) in WP medium were shown to contain chlorophylls. The highest content of chlorophyll a (0.26% as dry wt) was observed in A-3 in MS medium. The clone J-2 did not produce any detectable level of chlorophylls in either media. It was very interesting that, in MS medium, only the green hairy roots (A-l, 3, 4 and J-l) showed enhanced growth and polyaoetylene production on ill~ination i 1 M(I-C=C-C~c-C~c-C-cH-c=c-cM&t+&M@ I!

A*(?(

!

: R=-H 2 (lobetyolin) : Fbglc 3 (lobetyollnln) : Rmqk’-‘gk 1 (Melyol)

*Author to whom correspondence should be addressed. 365

K. ISHIMARIJ

366

et al.

[ 1: Fresh weight (g / flask) 25

q q

WP (light)

lobetyol lobetyolln lobetyollnln

0 -15 f

[WI

14241

L4.w

g0 5

A-1

A-3

A-2

A-4

J-l

J-2

A-l 25

25

2.

-15 f

A.2

1

A-3

A-4

J-l

J-2

A-3

A-4

J-l

J-2

WP

(dark)

.

$O-

E

.

A-l

A-2

A-3

Fig. 1. Growth

A-4

J-l

A-l

J-2

and polyacetylene

production

A-2

in hairy roots of Lnbelia sessi/(/iilia.

MS

q chl a

WP

E/ chl b 02 I ,o t e 0 0

0.1

%

0.0

A-l

A-3

A-4

J-l

A-l

J-2

Fig. 2. Chlorophylls

A-3

A4

J-2

in Lobelia sessili&o/ia hairy roots.

(Fig. 1). On the other hand, in WP medium, the effect of illumination on growth was unrelated to chlorophyll production. Indeed, the growth of A-l, 3 and J-2 was retarded in the light. The highest amount of 2 (21.48 mg per flask, 4.36% as dry wt) produced by J-l hairy roots in MS medium in the light was larger than that (17.6 mg per flask, 4.01% as dry wt) obtained in the hairy root (Li-A) culture of L. infhta (in WP medium, in the dark) [63.

Two clones (A- 1and J-2) which showed good growth in both MS and WP media were transferred and cultured in four, hormone-free liquid media {MS, Gamborg BS (BS) [9], WP, Root Culture (RC) [lo]}, and their growth (Fig. 3) and polyacetylene production (Figs 4 and 5) determined. The fresh weight of A-l rapidly increased in the latter stage of the culture period (Fig. 3a). Particularly, in WP

Polyacetylenes in Lobelia sessilijolia

341

12

MS B!i WQ

6

_ 0 :

6

6

#:

Gi

9

f

5

3

6

P

4

5

4

t

2

1f

2

0 0

1

2

3

4

5

6

7

0

6

0

1

2

3

Wemka

4

5

6

7

6

Weak8

Fig. 3. Growth of A-l and J-2 in four basal liquid media.

10

10 1

6I

1

IMd IotMyolln lobetyollnin

6 .

i4-

$4 z 2

0 0

1

2

3

4

5

6

7

6

0

1

2

3

4

5

6

7

6

5

6

7

6

WOdl9

Wnk. 10

10

6

6 Rc i 6

%

0

1

2

3

4

5

6

7

6

1 0

1

2

3

Wnkm

4 Wwko

Fig. 4. Polyacetylene production in A-l.

medium, A-l showed good growth, the fresh weight (9.26 g per flask) at week seven being almost double that in either BS or MS media, and over four times larger than that in RC medium. In contrast, J-2 started to proliferate in the early stage of culture in MS, BS and WP media (Fig. 3b). Growth then continued until the end of the culture period with concomitant production of high

yields of biomass (7.99 g, 11.42 g and 9.90 g per flask in MS, B5 and WP media, respectively). RC medium was not so effective for the growth of A-l and J-2. Polyacetylene production in A-l (Fig. 4) roughly paralleled the growth of A-l in four media. In WP medium, the amount of 2 continuously increased throughout the culture period and showed the highest level (9.89 mg per

368

K. ISHIMARU

er al.

18

IobayOl

lobdyolln IobHyollnln

0

1

2

3

4

Wwk8

5

6

7

a

Weeks 16

18 1 14

WP

12 10

$6 s

4 2

0

8 0 I

d

0

7

2

3

4

5

8

7

8

WooLa

WoeLo

Fig. 5. Polyacetylene production in J-2.

flask) at week seven. Among the four media tested, WP me&urn was best for the production of polyacetylenes (especially for 2) in A-i. Although the growth of A-l in B5 medium was almost double that in RC medium (Fig. 3a), the amounts of polyacetylenes produced in these two media were not so different (Fig. 4). In the case of J-2, a high amount of polyacetylenes was observed in MS and WP media (Fig. 5). In particular, the increment of 2 in MS medium was excellent showing the highest amount (15.60 mg per flask) at week seven. Similar to the case of A-l, polyacetylene production by J-2 in BS medium did not reflect the good callus growth. Therefore, BS medium seemed to be unsuitable for the production of polyacetylenes in L. sessif~iia hairy roots (A-l and J-2). The content of polyacetyfenes in the intact plant was almost one-twentieth (2: 0.204%, 3: 0.017% as dry wt) of that in the hairy roots. This emphasized the usefulness of L. sessilifolia hairy roots for the production of these polyacetylenes (especially 2). Furthermore, from the experiments mentioned above, the selection of clones and optimal culture conditions (medium, illumination, etc.) are essential in the use of hairy roots for the production of these secondary metabolites. EXPERlMENTAL

Polyacetylenes were identified by comparison of their ‘H NMR spectral (270 MHz) data with those ofauthentic

samples isolated from L. incurs hairy roots 13, 4). All culture media, MS (containing 30 g I- ’ sucrose), BS (containing 20 gl- ’ sucrose), WP (containing 20 g I- ‘ sucrose) and RC (containing 15 g I- ’ sucrose) were adjusted to pH 5.7 before autoclaving at 121” for 15 min. All cultures were placed at 25”. Data shown here are the mean of 3 expts. Plant material. Lobelia ~~~i~~oli~ plants were collected on the mountainside in the north part of Saga Prefecture. The nodal segments were sterilized (2% NaOCl) and placed aseptically on hormone-free MS solid medium (solidified with 2.5 g I- ’ gelrite) to establish shoot cultures. The axenic plants in vitro were used for Agrobacterium infection. Hairy root cultures. Agrobacterium rhitogenes ATCC 15834 and A. rhizogenes MAFF 03-01724 subcultured on YEB agar medium [1 1] were inoculated by a needle on to the cut ends of the stems (ca 1 cm length) prepd from the axenic shoot cultures. Three-4 weeks after infection, several hairy roots appeared at the inoculated sites (in the dark condition). The hairy roots were cut off and placed on hormone-bee WP solid medium containing an antibiotic (Claforan 0.5 mgml- ‘) to eliminate the bacteria. The axenic hairy roots thus obtained were transferred to hormone-free WP liquid medium (50ml per lOOmI flask) and cultured on a rotary shaker (100 rpm) in the dark. Six clones (A-l -4 induced by A. rhizogenes ATCC 15834,

Polyaatylencs in Lobelia sessilijdia

and J-l and 2 by A. rhizogenes MAFF 03-01724) were selected and used for this study. The transformation of these hairy roots was proven by the detection of opines (agropine and mannopine from A- 1 m 4 [ 121 and mikimopine from J-l and 2 [13]) using paper electrophoresis. Voucher specimens are deposited at the Faculty of Agriculture, Saga University. Isolation of polyacetylenes. From lyophilized hairy roots (A-l, 20 g, dry wt) cultured in WP medium for 8 weeks in the dark, polyacetylena 1 (23 mg), 2 (130 mg) and 3 (3 mg) were isolated by a method described elsewhere [3, 41. Hairy root cultures of A-l -4 and J-l and 2 in MS and WP media. Fresh roots of 6 clones (lo- 20 mg) were

inoculated into hormone-free MS and WP liquid media (50ml per 100 ml flask) and cultured (100 rpm, on a rotary shaker) in the dark or light (4000 lux, continuous light). After 5 weeks of culture, the hairy roots were harvested, weighed (fresh and dry) and their contents of polyacetylenes and chlorophylls determined by HPLC. Hairy root cultures of A-l and J-2 in four different media. Hairy roots of A-l (5 20 mg, fr. wt) and J-2 (10 - 100 mg, fr. wt) were inoculated into 4 hormone-free liquid media (MS, BS, WP and RC, 50ml per 100 ml flask) and cultured (100 rpm, on a rotary shaker) in the dark. Growth (root wt) and polyacetylene production were determined periodically (1 - 7 weeks). HPLC analysis for polyacetylenes. Sample prepn and HPLC conditions for the determination of 1-3 were the same as described before [6]. HPLC analysis for chlorophylls. Lyophilized samples (lo- 20 mg) were ground by means of a pestle and extracted with Me&O (1 ml) for 16 hr at room temp. in the dark. The extract, after filtration through a Millipore filter (0.5 pm), was subjected (15 ~1) to HPLC: column, Shim-pack CLC-ODS (6.0 mm i.d. x 150 mm) mobile phase, MeOH; flow rate, 0.97 ml min- ‘; temp. 40”; detection, 420 nm; R, (min), chlorophyll b (11.2) and chlorophyll a (17.6).

369

Acknowledgements-This work was supported by a Ministry of Health and Welfare, Science Research Fund Subsidy granted to the Japan Health Science Foundation. REFERENCES

Wysokinska, H. (1977) Farmacja Polska 33, 725. Krajewska, A., Szoke, E. and Szarvas, T. (1988) Herba Polon. 34, 27.

Ishimaru, K., Yonemitsu, H. and Shimomura, K. (1991) Phytochemistry 30. 2255. Ishimaru, K., Sadoshima, S., Neera, S., Koyama, K., Takahashi, K. and Shimomura, K. (1992) Phytochemistry 31, 1577.

5. Ishimaru, K., Ikeda, Y., Kuranari, Y. and Shimomum, K. (1992) Shoyakugaku Zasshi 46,265. 6. Ishimaru, K., Arakawa, H., Sadoshima, S. and Yamaguchi, Y. (1993) Plant Tissue Culture Letters 10, 191. 7. Lloyd, G. B. and McCown, B. H. (1980) Int. Plant. Prop. Sot. 30,421. 8. Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15, 473. 9. Gamborg, 0. L., Miller, R. A., Ojima, K. (1968) Exp. Cell Res. 50, 151. 10. EMBO Course in The Use of n Plasmid as Cloning Vector for Genetic Engineering in Plants p. 109.4 - 23 August 1982. 11. Vervliet, G., Holsters, M., Teuchy, H., van Montague, M. and Schell, J. (1975) J. Gen. Viral. 26, 33. 12. Isogai, A., Fukuchi, N., Hayashi, M.. Kamada, H., Harada, H. and Suzuki, A. (1990) Phytochemistry 29, 3131.

13. Petit, A., David, C., Dahl, G. A., Ellis, J. G., Guyon, P., Casse-Delbart, F. and Temp& J. (1983) Molec. Gen. Genet. 190,204.