Indi an Journal of Experimental Biology Vol. 39, June 200 I, pp. 564-57 I

Permeabilization and in situ adsorption studies during growth and coumatin production in hairy root cultures of Cichorium intybus L.

Harsh Pal Bais, G Sudha, B Suresh & GA Ravishankar*

Department of Plant Cell Biotechnology,Central Food Technological Research Institute, Mysore 570 013, India Received 18 August 2000; revised 22 January 2001

Effect of addi tion of a penneabilizing agent dimethy l sulfox ide (DMSO) and a solid adsorbent, XAD -7, on growth and coumarin production in hairy root cultures of C. intybus was studied . Continuous permeabilization of the hairy root cultures of C. inrybus with DMSO has been shown to be an effecti ve strategy for enhanced release o f coumarins wh ile preserv ing the root viabi lity. DMSO at 0.2 % (vlv) level showed the maximum growth and coumarin production but was less as compared to cont rol on day 28. Treatment of cell s with increasi ng concentrati ons of DMSO (0.3 - 0.6 % vlv) to hairy root cu ltures of C. intybus. showed an inverse relationship with growth and coumarin production. Growth and production o f coumarins increased with I % medi a filtrate (MF) of cultures of Phytopthora parasirica var. nicoriana treatment. It was observed that treatment with DMSO (0.2 % vlv) and I % MF of P. parasitica showed the better growth and coumarin production with an increased re lease of coumarins as compared to the control and o ther treatments. It was observed that treatment of hairy root cu ltures with XAD -7 resulted in lesser growth and coumarin production as compared to cont ro l during the cu lture period . Addition of XAD -7 along with I % MF of P. parasirica showed enhanced growth , coumarin producti n and increased adsorpti on as compared to control and lone XAD-7 treatment. Combined add ition of DMSO I XAD-7 wi th fungal e li c itor showed sy nergistic response in terms of biomass and coumarin production. Excretion of coumarins in both the cases was dependent on the presence of DMSO I XAD-7. These results showed that continuous permeabilization of hairy root cultures of C. it1tybus by using DMSO at 0.2 % (vlv) level coupled with I % MF of P. parasirica maintained viabi lity of ti ssues and produced cou marins at hi gher level.

Production of secondary metabolites with plant cell cu ltures is a very promising field. Application of plant cell cultures for production of these compounds is still at an early phase. Although some commercial processes exist, there are a large number of po sibly commercial compounds that have not attained the commercial success yet and the reason being low productivity of plant cell cultures. It is not unusual that productivity drops dramatically when call us is formed from the plant parts , and again when a suspension culture is started from the callus culture. Another drawback is slow growth of plant cell cultures, along with the fact that most of the cell products are stored intracellularly. This makes it inevi table to harvest the biomass for extraction of the product. If it is possible to force the cells to excrete the metabolite while preserving the cell viability, and production capacity, the chances for plant cell culture­based products to be economically feasible would have become much better.

Plant cell culture is a promising method for production of some secondary metabolites. One of the limitations observed with plant cell cultures is poor

·correspondent author - Fax: + 9 J -821-5 J 7 233; Emai l: pcbt @cscftri .ren.nic.in

productivity and low secretion capabi lity of secondary metabolites. Majority of the favoured metabolites remain within cell vacuoles and a small portion is secreted into the supported medium. Thi s intracellular accumulation of product restrains use of the grown cells repeatedly because the cells have to be macerated to obtain the product. This also limits the use of cell immobilization technology, which was used only for the products secreted into the medium. However, it IS difficult to release secondary metabolites without reducing the cell viability . Various methods have been studied to release these intracellular products into the medium and these techniques either alter or permeabilize the cell membranes by using organic solvents 1-

4, changes in

. . h5 H h'f 6 I . 7 d tontc strengt , p s 1 ts , e ectroporatton an iontophoresis8

. Among the organic solvents used, dimethyl sulfoxide (DMSO) has been reported to be most successfu l agent for permeabili zat ion9

. Lu ndberg et al. 10 have shown that 5 % of DMSO treatmen t resulted in 90 % release of total ajmalicine from Ca1haranthus roseus cells. However the use of DMSO higher than 5 % concentration in ca~c of certain alkaloids releasf' has shown deleterious effects3

· 7

. Another ex perimental approach to product release is to use DMSO co.1centrations at lower

BAIS eta/.: PERMEABILIZATION & IN SITU ADSORPTION I CICHORIUM 565

levels, on a long-term basis to cause the cells to release the products slowly while maintaining the cell viabilit/1. 4 . In literature there are several interesting examples of the combination of increased excretion and production by the addition of another organic

12- 15 0 . 1. .d dd d h phase or adsorbent . rgamc tqut s a e to t e medi a as a second phase in pl ant cell cultures, are hexadecane and miglyol , wherei n Kim and Chang 13

have obtai ned many fold increase in shikonin producti on in cell cultures of Litlwspermum ervth rorhizon upon addition of hexadecane. As a solid phase, the amberl ite resin XA D-7 is the one that is used most. It has got few attract ive hydrophilic

. 11 R b. d properties, in contra t to other resms -. o ms an Rhodes ( 1986) 16 have shown 15 fo ld increase of anthraquinone production by adding XAD-7 to the cell cu ltures of Cinchona ledgeriana.

Development in the field of elicitation has opened a new avenue fo r production of secondary metaboli tes 17

• Elicitors used for enhancement of secondary metabolites have been well investigated 18

.

Elicitors from Fusarium conglutinous enhanced thiophene production in hairy root cultures of Tagetes Spp.19

, and solaveti vone production has been increased by Rhizoctonia so/an i in hairy roots of Hyocya111us nntticus20

. Increased levels of ajmalicine and catharanth ine has been observed in hairy root

21 15 d . . cul tures of Catlwranthus roseus · an capsatcm production in Capsicum frutescens callus cultures by fungal elicitors has been reported22

•

The work reported here is ai med to study the influence of addition of a permeabilizing agent DMSO coupled with the fungal elicitors on growth of hairy roots of C.intybus and formation of coumarins viz., esculin (6,7 dihydroxy coumarin, 6- glucoside) and esculetin (6,7 dihydroxy coumarin) in hairy root cultures. Esculin is used as a marker in microbiological media23 and esculetin is used widely as a UV filter in cosmetics24

• Another objective was to check the synergistic effect of XAD-7 coupled with fungal e licitors on growth and production of coumarins in hairy root cultures of C. intybus.

Materials and Methods Plant material - Seeds from ten plants of

Cichorium intybus L.cv .Lucknow local were obtained from Banthara Research Station, (NBRI), Lucknow in August 1994.

Culture condition-Seeds were washed by immersing them in water containing Tween 20 (5 %) for 15 min . and sterilized with ethanol (70 %) and

sodium hypochlorite (5 %) for 10 min . These were finally rinsed thrice with sterile distilled water. Sterile

. "5 seeds were placed on 40 mL of MS basal medtum -containing sucrose (3 %) and 0.8 % of agar (w/w) for germination . The seedlings obtained were grown at 25° ± 2°C under 16 hr light (37.6 ± lO. lj.lmole m·2 s·1

;

Phillips, India) and 8 hr dark cycle.

Induction of /wily roots -Hairy roots were initiated by inoculating wounded stems of 4 week old seedlings with Agrobacterium rhizogenes LMG 150 (mannopine type; obtained fro m P.l.J Hooykaas Clusius Laboratorium, Rijks Universiteit, Leiden , The Netherlands). Bacterial colonies were cultured for 3 days on solid YEB medium prior to inoculation 26

under same conditions for seed ling growth. Transformed roots appeared within 10 ± 2 days of infection. Roots segments of 3 ± 0 .2 em were excised and immediately transfen ed to 40 ml of MS basal liquid media containing carbenicillin (500 mg mL-1

) in 150 mL Erlenmeyer fl asks. Roots were periodically subcultured thrice at intervals of 3 days each in antibiotic containing MS basal liquid media. The roots were subsequently transferred to MS basal liquid medium and incubated in dark on a rotary shaker at 90 rpm and maintained at 25° ± 2°C to obtain axenic hairy root cultures. Transformed nature of the hairy roots was ascertained by PCR as reported earlier by Bais et al. 27

. An inoculum size of 100 ± 0.075 mg was used for the experiments. The hairy roo: cultures were harvested at regular intervals, and the root mat was washed twice in sterile distilled water. After pressing between the folds of filter paper, fresh weight of the mat was recorded as g L-1

• Whole experiment was repeated twice with each treatment having five replicates, and average weight was recorded.

DMSO experiments -Dimethyl sulfoxide was obtained from (Sigma Co. Ltd. St. Louis USA) and was administered toMS basal liquid media containing hairy roots of C. intybus, after filter sterilization using 0.22 J-iM filters (Sartorius Co. Ltd).

Second phase-Solid adsorbent used was XAD-7, a neutral polycarboxylic ester (Sigma Chemical Co. St. Louis USA). Amberlite XAD-7 was washed in methanol (1 00 % ), rins~d in Millipore water, and dried. Prior to use, the desired amount (5 g L- 1

) of XAD-7 was weighed into dialysis bags (Spectrum Lab Inc. USA, Spectra Pore membrane) and autoclaved in medium before use to prevent undesired adsorption of medium components when XAD-7 was

566 INDINA J EXP BIOL, JUNE 200 1

added to the medium with hairy roots. Diameter of XAD-7 beads was 0.3-0.78 mm.

Estimation of coumarins-Estimation of esculetin and esculin was carried28 out using spectrophotometry, HPLC and later confirmed by 1 H NMR. Root samples (1 g) were extracted in 5 mL of solvent (ethanol: water, 70: 30) and centrifuged at 5000 g for 15 min. The supernatant was made up to a fina l volume of 5 mL by adding the extraction solvent. Aliquots (20 f1L) were injected into HPLC after pass ing through a filter (0.45 - 11m). HPLC was performed28 by gradient elution maintained with an UV detector at 340 nm. The separation was performed on a 10-f,lm-f,l-Bondapak C 18 (30 em x 3.9 mm inner diameter) column at ambient temperature. A mixture of acetic acid and water (I: 99 v/v) and pure acetonitrile was used as a solvent system. Flow rates of 1.5 mL min·1 and column pressure of 1200 psi were maintained. Retention time was recorded and compared with those of standards, and by calculating the peak area.

Analyses of coumarins upon adsorption-Extraction and analyses of coumarins from hairy roots and medium has been described elsewhere 29

· 30

. Coumarin content of XAD-7 was determined by extracting coumarins by adding 4 mL of ethanol (80 %), the sample was concentrated into a tube and evaporated under nitrogen flushing in a water bath at 40°C. This was used for HPLC as described above.

XAD-7 experime11.ts-Shake flasks with 40 mL of MS basal media were inoculated with hairy roots of C. i11.tybus. Apart from the controls, all flasks were supplied with dialysis bags (obtained from Spectrum Lab Inc. USA, Spectra Pore membrane) containing XAD-7 (5 g L-1

). After every 7 days, five flasks were removed of each treatment and analyzed for fresh weight and coumarin content in roots, media and in XAD-7.

Preparation and administration of elicitor-Fungi used for elicitation were Phytopthora parasitica var. nicotiana and Pythium aphanidermatum. They were obtained from SS Shenoi, Tobacco Research Center, Hunsur, India. Both of these fungi were grown in 250 mL flasks containing 50 ml of potato dextrose media and were incubated at room temperature. The medium was prepared by boiling potatoes (200 g) in di still ed water (200 mL), filtered through four layers of muslin cloth and added dextrose (20 g) . The volu me was made up to I 000 mL by adding di st illed water, the pH of the medium was then adjusted to 3.5 with I 0 % of tartaric acid. At stati onary phase (generally 20 days

old), flasks with the cultures were autoclaved and the fungal mat was separated by filtration . The fungal mat was then washed several times with disti lled water and homogenized in a mortar and pestle using acid­washed neutralized sand to have an aqueous extract. This extract was filtered through muslin cloth and the volume was made equal to that of filtrate. The extracts and filtrates were sterilized by autoclaving before use.

Mediafiltrate (MF) and mycelial extracts (ME)­Tn the pilot experiments performed in our laboratory we have used three concentrations (0.5, 1.0, and 3.0 % v/v) of medi a filtrate (MF) and three concentrations of (0.5, 1.0, and 3.0 % w/v) mycelial ex tracts (ME) of Phytopthora parasitica var. nicotiana and Pythium aphanidernzatum to study their effect on growth, coumarin production, and morphological responses in terms of branching patterns. Admini stration of I % MF of P. parasitica var. nicotiana to the hairy root cultures of C.intybus resulted in enhanced growth and coumarin production. It was envisaged to administer the same concentration (1 % MF of P. parasitica) with DMSO and XAD-7 to study their synergistic activity and release of coumarins.

Results and Discussion Effect of DMSO treatments-Influence of DMSO

treatments (0.1 - 0 .6 % v/v) on growth and production of coumarins in hairy root cultures of Cichorium intybus was studied. Higher concentrations of DMSO (0.3 - 0.6 % v/v) showed an inhi bitory effect on growth and production of coumarins as compared to control (Fig. 1). However, DMSO at 0.1 - 0.2 % (v/v) did not affect much growth (205 ± 15 .3 g L-1

; 224 ± 16.8 g L-1

) and coumarin production in these treatments as compared to control (27 4 ± 20.5 g L-1

;

Figs. 1, 2). Growth and coumarin production decreased at an accelerated rate in hairy roots, which were treated with DMSO with increasing concentration (0.3- 0.6 % v/v).

Effect of fungal elicitor and DMSO treatments on biomass-Supplementation of 1 % MF of P. parasitica to the hai ry root cultures of C. intybus resulted in enhanced growth and coumarin production. This concentration was chosen for addition with DMSO (0.2 % v/v) to hairy root cultures of C. intybus. to check its effect on growth, coumarin production and prod uct release (Fig. 3). It was observed that when DMSO (0.2 % v/v) with I % MF of P. parasitica was added to the hairy roo; cultures, it resulted in increased biomass (329.9 ± 24.7 g L-1

) as compared to

BAIS eta/.: PERMEABILIZATION & IN SITU ADSORPTION IN CICHORIUM 567

the control (274 ± 20.5 g L-1 ;Fig. 3), which was 1.2 fold higher over the control on day 28. This was supported by enhanced esculin (19.86 ± 1.4 mg L ' 1

)

and esculetin ( 13 .96 ± 1.04 mg L ' 1) production as

compared to control (7.9 ± 0.59 mg L' 1; 5.65 ± 0.42

mg L' 1), which was 2 .5 and 2.4 fold more than in

control on day 28 (Fig. 4) . Effect of fungal elicitor and DMSO treatments on

release of cou111arins - It was found that the mcrease in DMSO concentration resulted in increased release of coumarins, with a maximum esculin (95 .6 %) and esculetin (94.4 %) with DMSO

300 r ----e- Control j <~ OI% 0MSO //" I

250 ·-.- 0 2~/o DMSO

--'\/'- 03% DMSO ~I : ,= ~ ~~ ~~~~ ~ - +- 06% OMSO / ......4---

j _ _)f----._y

c~-~ .;:) t-·-- ---- --9

~~ -t

:

:._, 200 : .::? :E m 150 'iii ~

-"' "' " 100 .::

50

0 --,-----~ --~ ---~---'

0 14 2 1 26

Time (days)

Fi g. I - Effect o f DMSO treatment on growth o f hairy root cultures o f C. intybus. (Values are mean± SD, of 5 replica tions

:- 250 :_, E! 200 :;: -~ 150

" ~ .r:; 100

"' " .t 50

-- 6 :_,

"' 5 E - 4 .s B 3

.1! 2

0

1 Conlfol 2 0 1% 0MSO 3 02%DMSO 4 OJ% OMSO 5. 0 41.1. OMSO 6 05% DMSO 7 06%0MSO

6,--~--------------,

---' "' . .§. ~ 3

;; 2

" .. w

Cooc~ntration of DMSO (0.1.)

Fig. 2 - Effect of DMSO treatment on growth and producti on o f coumarins in hairy roo! culture o f C. intybus on day 28 (Values are mean ± SD of 5 replications)

(0.6 % v/v) as compared to control and other treatments (Fig.5). This was supported by higher concentration of esculin (1.26 ± 0.09 mg L ' 1

) and esculetin (0.98 ± 0 .07 mg L- 1

) traced in the media with 0.6 % v/v DMSO treatment as compared to the control (0.68 ± 0.051; 0.52 ± 0.039 mg L-1

; esculin and esculetin respectively) on 28' 11 day respectively (Fig. 5). Treatment with 1 % MF of P. parasitica showed an increased production of coumarins as compared to the control and other DMSO treatmen ts (Fig. 5). Treatment with 1 % MF of P. parasitica with 0 .2% DMSO resulted in increased

500

___...__ Control ---0--- 0 2'4 DMSO

- ... --:l 400

:._,

.::? 300

100

_,_ 0 2% OMSO • 1% MF Pparasll;ca --9-- 1% MF P paraS,ICB

14

Time (days)

2 1 26

Fig. 3-Effect of DEMO and fungal e licitor treatments on growth and hairy root cultures of C. intybus. (Values are mean ± SD, o f 5 replications).

_ 1 Conlr~

500 l" --~ 2 0.2°!. OMSO

0 400 - 3 0.2% OMSO + 1_~ MF P.parasllica

- 4. 1% MF P.parssit~ea E 300 -~ I

! 200 1' e 100 u.

0 --

"t 30

:._, 25

"' .§. 20

.!: 15 :; " 10 ' "' w I

5 ;

0 .

25

-:.., 20

m .s 15

.!: c:; 10 ;; :;: w

Coaccotration of 01\-ISO (~•)

Fi g. 4 - Effect o f DMSO and fun gal elicitor treatments on growth and coumarin productio n in hairy root cultures of C. inrybus. on day 28. (Values are mean± SD, o f 5 replications).

568 I DINA J EXP BIOL, JUN E 200 1

release of both coumarins as compared to the treatment with lone l % MF of P. parasitica and control (Fig. 5), which was evident by enhanced esculin (6.86 ± 0.51 mg L-1

) and escu letin (5.96 ± 0.44 mg L-1

) levels in the media as compared to their respective controls (0.68 ± 0.051; 0.52 ± 0.039 mg L-1

respectively) on clay 28. However the treatment with I % MF of P. parasitica showed higher intracellular titers of coumarins as compared to control and other treatments (Fig. 5). The sy nergist ic activity of coupled treatment of DMSO (0.2 % v/v) with 1.0 % MF P.

parasitica to the hairy root cultures of C. intybus was ev ident by the higher excretion rate of coumarins, which was supported by the growth in terms of biomass, productivity and enhanced release (Figs. l-5) . Similar lines of studi es have been performed by Park and Martinez'', wherein they have shown that continued permeabilization of Coleus blu111ei cells with lower concentrations of DMSO, resulted in effective production of rosamarini c acid, wi th an enhanced product release, which is further enhanced by treating the cells with similar concentrations of

40 ~--------------------------------------~----~-------.- 100 - In media ~ 35 _j 00::~ In roots

Ol 30 - c=J Total E

-----% release

c 25 0 +-' ::::l .0 20

...... +-' (f)

15 -"0

-- 80

~ 60

! 40 •·

c 10

::::l 20 (.) (f)

5 w

0 ~--~u_~~L-~~--~~_J~L-~~--~~~~L_~~-LO

30 - 100

~

_j 25 80 Ol

E c 20 0 60 +-' ::::l .0 15 ...... +-' (f)

"0

c 10 +-' Cll

::::l (.) 5 (f)

UJ

0

0 2 3 4 5 6 7 8 9

Concentration of DMSO (%)

c ::l u <f)

<ll

<ll <f)

ro ~ <ll ._

::oe 0

c +J

<ll

:::l u <f)

<ll

<ll <f) ro <ll

<ll ._

::oe 0

Fig. 5- DislribUiion of coumarins in hairy root cu ltures of C. intybus upon DMSO and fungal eli citor treatments. (Values are mea n ± SO. of 5 replications).

BAIS et a/.: PERM EA BILIZATION & IN SITU ADSOR PTIO I CICHORIUM 569

5001 I

400 •

-+- Conlfol -<.>- XAD - 7 - • - XAD 7 • ICJ ... MF P rarasitica - v- · 1% Mf. P pnta~111ca

14 21

Time (days)

28

Fig. 6 - Effect of XAD-7 and fungal elicitor treatments on growth of hairy root cultures of C. i11 tybus. (Values are mean ± SD. of S replications).

DMSO coupled with fungal e li c itors. Parr et at.31 have shown the permeabi li zation of Cinchona ledgerina cell s using lower concentrations of DMSO to avoid the cell mortali ty.

Influence of XA D-7 and fungal elicitors on biolllass- Growth and coumarin production in hairy root cultures of C. intybus was examined in the presence and absence of XA D-7 and fungal e li cito rs. The biomass accumul ation was not affected by XAD-7 treatment as compared to contro l on day 28 (Fig. 6). However the treatment wherein XAD-7 was fed alon<> 0

with I % MF of P. parasitica showed an increase in biomass (339 .8 ± 25.4 g L-1

; Fi g. 6) , and coumarin

product ion (esculin , 12 .36 ± 0.92 mg L-1; and

esculetin , 9.86 ± 0.73 mg L-1) on day 28 as compared

to their respective controls (7.9 ± 0.59 mg L· ' ; 5.65 ± 0.42 mg L· '; Fig. 8). MF (I %) o f P. parasitica treatment resulted in the maximum growth and cou marin production as compared to the control, XA D-7 and XAD-7 with fungal elicitor treatments (Figs 6, 7). Payne et a/. 32 have shown a 40 % increase in alkalo id production in Catharanthus roseus cell s upon XAD-7 suppl ementation.

li!flu ence of XA D-7 and f ungal elicitors on adsorption of cownarins - Addition of XAD-7 to hairy root cultures of C. intybus resulted in maximum percentage adsorption of coumarins on XAD-7 res in as compared to contro l (Fi g. 8). However, treatment with XA D-7 in combination with I % MF of p_ parasitica , resulted in higher excretion of coumarins with an enhanced adsorption fo r esculin (32.7 %) and esculetin (38 .1 %) as compared to contro l, lone XAD-7 and I % MF of P. parasitica treatment (Fig. 8) . Buitelaar et a/.33 have reported that the total release of thiophenes is enhanced by 50 % upon XAD-7

35

.; 30 ...J

1 Control 2 XA0 - 7

2l 25 :E 20 Cl

3. XA0 - 7 .. 1% MF P parasrtiCB 4 1% MF P parasrtica

~ 15

.s;;; 10

"' e 5 u.

251 -

- 20 i

-~ ::L UJ 5

0

500 -.------- - - -

~ 400 J

"' _s JOO .!: ;; 200 :; " w 100

Treatments

Fig. 7 - Effect of XAD-7 and fun ga l eli citor treatments on growth and coumarin production in hairy root cultures of C. i111ybus on day 28. (Values are mean ± SD, of S replicati ons).

suppl ementati on, which is supported by increased adsorption, and is in accordance with the observation in our study, wherein the coupled treatment of XAD-7 and I % MF of P.parasitica to the hairy root cultures resulted in increase in growth , coumarin production and adsorption as compared to lone XA D-7 treatment and the control (Figs . 6-8).

Buitelaar et at. 33 have shown the use of an or<>an ic 0

second phase of hexadecane in hairy root cultures of Tagetes patula for plant cell culture, for a two step ex tractive production of thiophenes. Improved yield in terms of growth and coumarin production in hairy roots of C. in tybus, upon DMSO, XAD-7 and fungal e li citor treatment may be attributed to the lower rate of degradati on of the coumarins, which was released by the cell by DMSO induced permeabilization accompanied by enhanced production th rough eli c itor and simultaneous adsorption on XAD-7 . Continual product removal by the use permeabili zing agents has been found benefi cial in other sys tems also, vi:., coniferyl alcohol in Matricaria chanwmilla 34

,

lipophillic products in other plant cell cultures 35 and azadirachtin in cell cultures of Azadirachta indica 36

It may be noted that use of XAD-7 in hairy root cultures of C. intybus resulted in 3 1.1 % increase in product excretion rate, with release of esculin and esculetin up to 32.7% of the total production. Thus the

570 INDINA 1 EXP BIOL, JUNE 200 1

40 .------------------------- 35

35

30

~ 25 Ol

E 20

c

3 15 ~ ~ I

10 1

- Inroots ~ lnmedia ~ Inbags c:::J Total _._ % Adsorption

30

25

20

15

10

5

c Q e-0

"' "0 «: ~

5 1 0 +-----~~~~--~~~--~~~W-----~~~~ 0

30.--------------------------------------------,- 50 I) control 2) XAD-7

25 3) XAD-7+ I% MF P.parasitica

~ 20 Ol

E ~ 15 Q)

:J

~ 10 w

5

4) 1% MF P.parasitica 40

30 c 0

E-0

"' -c

20 «: -:.!2 0

10

0 -r-----_.~~~--~~~----~~~--~Ri.-LL-LQ

0 2 3 4

Treatments Fi g. 8- Coumari ns adsorpt ion patterns in hairy root cu ltures of C. intybus upon XAD-7 and fun ga l elicitor treatment s. (Values are mean ± SD. of 5 rep lications).

cell s are ab le to maintain a maximum threshold level, whi ch is ev ident from the fact that there was 32.7% increase in the production, and also 31.1 % increase in the excretion of the products. This implies that these treatments facilitated higher levels of production of the secondary metabolites by releasing the overproduced compounds out of the cell. This study can be applied to the scaling up of the process m bioreactors with an ex tractive mode.

Acknowledgement Authors HPB, GS and BS g ratefully acknowledge

CS!R New Delhi for awarding Senior Research Fellowships. This work was supported by the grant

from Department of Biotechnol ogy (DBT), New Delhi .

References I Bro<,lelius P & Ni lsson K, Permeabilization of immobili zed

plant cells, resulting in release of intrace llularly stored products while preserving cell viability. Eur 1 Microbial Biotechno/, 17 (1983) 275 .

2 Felix H, Permeabi lized cell s. Anal Biochem. 120 ( 1982) 2 11 . 3 Parr A J. Smith J I, Robins R J & Rhodes M J C, Apparent

free space and cell volume estimation: a non destructi ve method for assess ing the growth and membrane integrity of immobili sed cells. Plant Cell Rep, 3 ( 1989) 161.

4 Schmidt A J, Lee J & An G, Media and environment effects on phenolics producti on from tobacco cell cultures. Biotechno/ Bioeng, 33 (1989) 1437.

BAIS et a/.: PERMEABILIZATION & IN SITU ADSORPTION IN CICHOR/UM 571

5 Tanaka H, Hirao C, Semba H, Tozawa Y & Ohmomo S, Release of intrace llularly stored 5' phosphodiesterase with preserving plant cell viability. Biotecluw l Bioeng, 27 ( 1985) 890.

6 Bouyssou H. Pareilleux A & Marigo A, The role of pH gradi ents across the pl asmalemma of Catharanthus roseus and its involvement in the release of alkaloids. Plant Cell Tissue Organ Cult, 10 (1987) 9 1.

7 Brodelius P, Funk C & Shillito R, Penneabili zation of culti vated pl ant cells by electroporation for release of intracellularly stored secondary products. Plant Cell Rep , 7 ( 1988) 186.

8 Pu T U. Yang R Y & Saus F L, Iontophoretic release and transport of alkaloids from Catharantluo roseus cell s in a ceramic hollow fibre reactor. Biotech no/ Lett , II ( 1989) 83.

9 Brodelius P, Deas R, Mosbach K & Zenk M H, Immobili zed plant cells for the producti on and transformati on of natural products. Plant Cell Rep, I 03 ( 1983) 93.

10 Lundberg P, Linsefors L, Voge l H J & Brodelius P, Permeabili zation of plant cell s: 31 P NMR studies on the permeability of the tonoplast. Plant Cell Rep, 5 ( 1986) 13

II Park P & Martinez B C. Enhanced release of rosmarinic acid from Coleus blumei permeabili zed by dimethyl sulfox ide (DMSO) while preserving cell viability and growth . Biotechnol Bioeng, 40 ( 1992) 459.

12 Buitelaar R M, Leenen E J T M. Geurtsen G, Groot E & Tramper J, Effects of the addition of XAD-7 and of eli citor treatment on growth , thiophene production , and excretion by hairy roots of Tagetes patula. Enzyme Microb Techno/, 15 ( 1993) 670.

13 Kim D J & Chang H N. En hanced shikonin prod uction from Li1lwspennum erythrorhizon by in situ extracti on and ca lcium alginate immobilizat ion. Bioteclmol Bioeng, 36 ( 1990) 460.

14 Bui telaar R M. Cesario C M & Tramper J. Elicitation of thi ophene production by hairy roots of Tagetes paw/a. Enzyme Microb Techno/. 14 ( 1990) 2.

15 Asada M & Shuler M. Stimulation of ajma li cine producti on and excretion from Catharanthus roseus: effects of adsorption in sill/ elicitors and alginate immobi li zati on. Appl Microb ial Biotecluwl, 30 ( 1989) 4 78.

16 Robins R J, Rhodes M J C, The stimulati on of anthraquinone producti on by Cil rchona ledgeriana cultures with polymeri c adsorbents. Appl Microbial Biotechno/24 ( 1986) 35.

17 Dicosmo F & Misawa M, Elicit ing secondary metabolites in plant cell cultures. Trends Biotechno/, 3 ( 1985) 319.

18 Gurmeet Singh. Application of fungal elici tati on for enhancing production of secondary metabolites by hai ry roots cultured in large-sca le bioreactors, in Hairy Roots: culture and applications. edited by PM Doran. Harwood Academic Publi shers. 1998, 209.

19 Mukundan U, & Hjortso H M. Effect of fu ngal elicitor on thiophene produc tion in hairy root cultures of Tagetes palllla. Appl Microbial Biotecluw l. 33 (1990) 145.

20 Ramakri shna S V, Reddy R G, Curti s W G, & Humphry A E, Stimulation of solave ti vone sy nthesis in free and immobili sed cell s of Hyocyamus 1nu1icus by Rhi~oc1onia so/ani fun ga l

components. Biotecluwl Lett , 15 ( 1993) 307. 2 1 Dicosmo F, Quesnel A, Misawa M & Tallevi S G, Increased

synthesis of ajmalicine and catharanthine by cell suspension cultures of Catharanthus roseus in response to fungal culture filtrates. App/ Biochem Biotechnol, 14 ( 1987) I 0 l.

22 Johnson T S, Ravishankar G A & Venkataraman L V. Eli citation of capsaicin production in freely suspended cells and immobilized cell cultures of Capsicum frutescen s Mill. Food Biotechnol, 5 (1991) 197.

23 Goodwin R H & Pollock B M, Ultrav iolet absorpti on spectra of coumarin deri va ti ves, Arch Biochem Biophys, 49 (1954) l.

24 ShahS, Chemistry of coumarins. Chem Rev. 36 ( 1945) I. 25 Murashi ge T & Skoog F, A rev ised medium for rapid growth

and bioassay of tobacco ti ssue cultures. Physiol Plant , 15 ( 1962) 473.

26 Verve liet G, Holsters M, Teuchy H, Van Montagu M & Schell , Characteristics of different plaque forming and defect ive temperate phages in Agrobacterium strai ns. J C en Viral, 26 ( 1975) 33.

27 Bais H P, Venkatesh R T, Chandrasekhar A & Rav ishankar G A, Agrobacteriwn rhizogenes- mediated transformat ion of Witloof chicory- in vit ro shoot regeneration and induction of fl owering, Curr Sci. 80 (200 I) I 0 l.

28 Tamma R V & Miller G C, Hi gh performance level chromatograph ic analysis of coumarin and fla vonoids from sections of Tridentatae of Artemisia. J Chromatogr. 322 ( 1985) 236.

29 Bais H P, George J & Ravi shankar G A, Influence of Polyam ines on growth and prod uction of coumarins in hairy root cultures of Cichoriwn intybus L cv. Jucknow local. J Plant C r01t'lh Reg, 18 ( 1999) 33.

30 Bais H P, Sudha G & Ravishankar G A, Putrescine in fl uences growth and production of coumarin s in hairy root cultures of Cichorium intybus, L. cv. Lucknow loca l (Witloof Chicory). J Plant Growth Reg, 18 (1999) 159.

31 Parr A J, Robins R J & Rhodes M J C. Permeabili zation of Cmchona ledgerianana cell s by dimethyl sulfox ide. Effects on alkaloid release and long-term membrane integrity. Plant Cell Rep, 5 ( 1984) 262.

32 Payne G F, Pay ne F F, Shuler M & Asada M, liz si/11 adsorption for enhanced alkaloid producti on by Catharanthus roseus. Biotech no/ Lett , I 0 ( 1988) 187.

33 Buitelaar R M, Langenhoff A A M, Heidstra R & Tramper J. Growth and thiophene production by hairy root cultures of Tagetes pant/a in va ri ous two- liquid-phase bioreactors. Enzyme Microb Techno/, 13 ( 199 1) 487

34 Knoop B & Beiderbeck R. Adsorbenskultur- ein Weg:ur Steigenuzg de Sekundarstoffprodukrion in pjlwdichen Suspensionskulturen. Z Naturfosch, 38 ( 1983) 484.

35 Becker H, Reichling J H, Bisson W & Herold S, Two phase culture- a new method to yield lipophilic secondary products from plant suspension cultures. in Proceedings of 3'J European Congress on Biotechnology I ( 1984) 209.

36 Kuruvill a T, Komaraiah P & Ramakrishna S V, Enhanced secretion of azadirachtin by permeabilized margosa (Azadirachta indica) ce ll s. In dian J Exp Bioi, 37 ( 1999) 89.