Acta Physiol Plant (2008) 30:849–853 DOI 10.1007/s11738-008-0190-2
ORIGINAL PAPER
Ethrel treatment enhanced isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa, a woody legume Shaily Goyal Æ K. G. Ramawat
Received: 16 January 2008 / Revised: 9 May 2008 / Accepted: 22 May 2008 / Published online: 10 June 2008 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2008
Abstract The cell cultures of Pueraria tuberosa, a perennial leguminous lianas, were maintained in modified MS medium (KNO3 475 mg l-1, thiamine 1 mg l-1, biotin 1 mg l-1, calcium pantothenate 1 mg l-1) containing 0.1 mg l-1 2,4,5-trichloroacetic acid and 0.1 mg l-1 kinetin. Isoflavonoids (puerarin, genistin, daidzein, genistein) accumulation in cell suspension cultures was increased by 14-fold to *12 mg l-1 after 48 h of adding 100 lM ethrel. Ethrel inhibitors (silver nitrate and silver thiosulfate) completely inhibited this effect in the presence of ethrel and isoflavonoids were not detected in the spent medium. The increase was dose dependent and can be explored to trigger high yield of isoflavonoids production. Keywords Cell culture Ethrel Isoflavonoids Pueraria tuberosa
Introduction Pueraria tuberosa DC (Fabaceae) is a perennial woody lianas, producing underground tubers up to 20 kg. Tuber contains isoflavonoids (puerarin, genistin, daidzein, genistein) and is highly valued in Ayurveda since the time of Samhitaas (Dev 2006). Epidemiological studies have demonstrated a link between isoflavones and reduced
Communicated by M. Stobiecki. S. Goyal K. G. Ramawat (&) Laboratory of Bio-Molecular Technology, Department of Botany, M. L. Sukhadia University, Udaipur 313001, India e-mail:
[email protected]
risk of breast and prostrate cancers in humans, chemoprevention of osteoporosis and cardiovascular diseases (Dixon and Ferreira 2002; Ren et al. 2001). The cell cultures of lupine, Glycyrrhiza echinata, Cicer arietinum and Pueraria lobata have been studied for elicitor-induced manipulation of isoflavonoids production (Luczkiewicz 2008). Fungal elicitor, Penicillium citrium, induced puerarin production in P. thomsonii (Maojun et al. 2006) and exogenous cork pieces, XAD-4 and methyl jasmonate induced seven to eightfold higher daidzein and genistien production in P. montana (Kirakosyan et al. 2006). Ethylene is known to stimulate phenylpropanoid metabolism (Saltveit 1999), promoted alkaloid production in suspension cultures of Coffea arabica and Thalictrum rugosum (Cho et al. 1988), but it had no effect on sanguinarine accumulation in Papaver somniferum (Songstad et al. 1989) and had adverse effect on anthocyanin production (Shibli et al. 1997). However low concentration of isoflavonoids in callus and cell cultures limit its commercialization (Vaishnav et al. 2006; Luczkiewicz 2008). Therefore, increased production in short duration is a major goal towards commercialization. The productivity of cell cultures may depend on the choice of medium, culture conditions and plant growth regulators (Ramawat and Mathur 2007; Kirakosyan 2006). We have previously reported occurrence of isoflavonoids in callus cultures (Vaishnav et al. 2006) and influence of nutrients (Goyal and Ramawat 2007) on isoflavonoid accumulation in cell cultures of P. tuberosa. About eightfold increase in isoflavonoid contents from *80 to *600 lg g-1 in cell cultures grown in MS medium modified with nitrogen and supplemented with 1 mg l-1 of kinetin (Goyal and Ramawat 2007). In the present communication, we report for the first time ethrel induced [14-fold increase in isoflavonoids accumulation in the cell cultures of P. tuberosa.
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Materials and methods Cultures and experimental setup The cell cultures were subcultured every fourth week with 10% v/v inoculum [*100 mg dry weight (DW)/100 ml medium]. Modified MS (Murashige and Skoog 1962) medium (KNO3 475 mg l-1, thiamine 1 mg l-1) containing biotin (1 mg l-1), calcium pantothenate (1 mg l-1), 2,4,5-T (0.1 mg l-1) and kinetin (0.1 mg l-1) with 3% sucrose, pH 5.8 at 25 ± 1°C (Vaishnav et al. 2006) was used (referred to as maintenance medium). The cell cultures were agitated at 100 rpm. These cultures were treated either with filter sterilized aqueous solution of ethrel (2-chloroethyl phosphonic acid, 20–400 lM) or 100 lM ethrel along with ethylene inhibitors (silver nitrate 120 lM or silver thiosulfate 20 lM) after 27 days of growth.
Acta Physiol Plant (2008) 30:849–853
performed at a flow rate of 1.0 ml min-1, and chromatographic peaks were monitored at 254 nm. Standard compounds puerarin (daidzein 8-C-glucoside), genistein (5,7,40 -trihydroxyisoflavone), genistin (genistein7-O-glucoside) and daidzein (7,40 -dihydroxyisoflavone) were purchased from Sigma Chemical Co. (St Louis, MO, USA), dissolved in methanol to yield a final concentration of 1.0 mg ml-1. The concentration of the other compounds was calculated on the basis of puerarin. A series of dilutions of the standard solutions of puerarin were made in 100% methanol (HPLC grade) with concentrations ranging from 7.4 to 250.0 lg ml-1 by diluting the stock solution. They were tested to determine the linearity. Method linearity was demonstrated by determining a calibration curve, calculating the regression coefficient (r2) and slope equation based on data obtained from injections of different concentrations. Statistical analysis
Sample preparation The cell cultures were harvested after 24, 48, 72, 96, 168 h for time course and 48 h for other ethrel treatments, washed with distilled water and filtered under mild vacuum. The cells were weighed to obtain the fresh weight per 100 ml medium (FW) and DW was then determined by drying the cells at 60°C in an oven to a constant weight. Dried homogenized cells weighing 100–150 mg were extracted in 5 ml methanol for 12 h (room temperature) on a test tube rotator, centrifuged at 2,000 rpm for 10 minutes and then the supernatant was collected and evaporated. HPLC analysis The HPLC system used for the separation of compounds was equipped with a pump (model L2130, Hitachi, Tokyo, Japan), auto sampler (model L-2200, Hitachi) and a UV detector (L-2400, Hitachi) controlled with ‘‘Lachrome Elite’’ software. The separation was performed on a 250 9 4 mm C18 [5 (m) reverse-phase column (LichroCART, Merck KGaA, Darmstadt, Germany)] protected by a guard column of the same material. The HPLC analysis was performed with little modifications, as described by Kirakosyan et al. (2003). The solvent system used was: solvent A, 0.0025% trifluoroacetic acid in water and solvent B, 80% acetonitrile (E. Merck, Mumbai, India) in solvent A. The mobile phase consisted of solvent (A) and (B). The step-gradient programme of solvent A was as follows: 0–2 min: 85%; 2–5 min: 85–80%; 5–15 min: 80–50%; 15–20 min: 50–40%; 20–30 min: 40–30%; 30–35 min: 30–20%; 35–45 min: 20–0%; 45–48 min: 0%; 48–50 min: 0–85%; 50–55 min: 85%. The separation was
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All results are averaged over two separate analyses for isoflavonoids estimation and two consecutive experiments with six replicate flasks in each treatment for growth value determination. The results are expressed as lg g-1 cell dry biomass.
Results and discussion Time course study for induction of isoflavonoids Isoflavonoids induction in cultures treated with 20 and 400 lM of ethrel is presented in Fig. 1. The maximum puerarin (226 lg g-1), genistin (203 lg g-1) and genistein (53.0 lg g-1) content recorded at 48 h of 20 lM ethrel treatment whereas in case of daidzein maximum content (78.7 lg g-1) was recorded after 72 h of treatment. After 96 h of treatment decrease in puerarin, genistin and daidzein content was recorded. In cells treated with 400 lM ethrel, maximum puerarin (241 lg g-1), genistin (345 lg g-1) and daidzein (126 lg g-1) content was recorded at 48 h of treatment while genistein (55.2 lg g-1) was maximum at 72 h of treatment. Increase in puerarin, genistin and genistein content was observed within 24 h of treatment while increased daidzein accumulation was recorded only after 24 h of treatment. In both 20 and 400 lM concentrations of ethrel tried, cells accumulated maximum amount of total isoflavonoids (Fig. 2) at 48 h of treatment (6,784 and 8,991 lg l-1, respectively). Moreover, there was a marked *8-fold and *11-fold increase in sum of analyzed isoflavonoids yield in cells treated with 20 and 400 lM ethrel, respectively.
Puerarin
Genistin
Daidzein
Fig. 1 Time course of ethrel a 20 lM b 400 lM, induced accumulation of puerarin, genistin, daidzein and genistein (lg g-1 DW) in cell suspension cultures of P. tuberosa (filled circle treated cultures, filled square control cultures)
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Genistein
Acta Physiol Plant (2008) 30:849–853
A
250 200 150 100 50 0
B
300 200 100 0
250 200 150 100 50 0
300 200 100 0
250 200 150 100 50 0
300 200 100 0
250 200 150 100 50 0
300 200 100 0 0
24
48
72
96
120
144
168
0
24
Hours of treatment Fig. 2 Total analyzed isoflavonoids yield (TIY) in lg g-1 DW in cell suspension of P. tuberosa. a 20 lM b 400 lM ethrel (filled circle treated cultures, filled square control cultures)
A
10000
48
72
96
120
144
168
144
168
Hours of treatment
B
8000
TIY
6000
4000
2000
0 0
24
48
72
96
120
Hours of treatment
Effect of different concentrations of ethrel Maximum growth (12.4 g l-1) was recorded in cells grown with 200 lM of ethrel. Maximum puerarin (507 lg g-1) and genistein (109 lg g-1) accumulated in the cells treated with 100 lM of ethrel while maximum genistin
144
168
0
24
48
72
96
120
Hours of treatment
(345 lg g-1) and daidzein (264 lg g-1) accumulated in the cells treated with 200 and 50 lM of ethrel, respectively. However maximum isoflavonoids yield was observed in the cultures treated with 100 lM of ethrel (11,684 lg l-1) which was *14-fold higher than that recorded in the control cultures and the yield increased
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Acta Physiol Plant (2008) 30:849–853
Table 1 Effect of different concentrations of ethrel on the isoflavonoids content in the cell suspension cultures of P. tuberosa Ethrel (lM)
Culture dry biomass (g l-1)
Isoflavonoids content lg g-1 DW ± SD Puerarin
Genistin
Daidzein
Yield lg l-1 Genistein
Total
Control
10.3
9.8 ± 0.35
44.7 ± 1.6
17.7 ± 0.9
8.9 ± 0.5
81.0
835
20
12.2
226 ± 7.2
203 ± 3.1
74.7 ± 1.7
53.0 ± 0.7
556
6,784
50
11.8
476 ± 3.4
149 ± 5.4
264 ± 6.3
77.8 ± 3.3
967
11,412
100
11.9
507 ± 3.6
194 ± 8.5
172 ± 2.6
109 ± 3.0
981
11,684
200
12.4
241 ± 2.9
345 ± 4.4
126 ± 2.3
12.9 ± 0.2
725
8,991
400
12.1
82.8 ± 5.8
72.2 ± 2.7
97.6 ± 4.6
96.4 ± 1.5
349
4,224
with the concentration of ethrel (Table 1). The use of ethylene inhibitors like silver nitrate and silver thiosulfate along with 100 lM ethrel completely inhibited the ethylene effect (sum of isoflavonoids analyzed was 780 and 590 lg l-1, respectively), thereby confirming that the effect was solely due to release of ethylene. Isoflavonoids were not detected in the spent medium (data not presented). The plant growth regulators influence the biomass and cell differentiation (Suri and Ramawat 1995) and the reduction in biomass is always correlated with increased accumulation of secondary metabolites (Ramawat and Mathur 2007). Plant growth regulator has been known to markedly influence the production of secondary metabolite in in vivo (Haque et al. 2007) and in vitro cultures (Taguchi et al. 2001). Earlier, we recorded similar effect of morphactin for the first time on guggulsterone accumulation in Commiphora wightii callus cultures (Tanwar et al. 2007). Marked increase in isoflavonoids accumulation by ethrel at 100 lM has been demonstrated in this work. Such marked effect of ethylene in such a short duration has never been observed in any other species (Shibli et al. 1997). Ethrel is known to cause morphological changes and inhibit the development (Basile and Basile 1984). Its action is based on ethylene production by its degradation on entering into plant cell. The resulting increased ethylene concentration inhibits cell extension or biomass production (Haque et al. 2007). This finding is also important as the sum of total isoflavonoid content achieved was at par with isoflavonoids content (400–950 lg g-1) of soybean seeds (Chiari et al. 2004). In the present work, it has also been demonstrated that the effect was solely due to ethylene and presence of ethylene inhibitors completely suppressed this effect. Similar enhancement of secondary metabolites by ethylene was also reported in C. arabica and Thalictrum rugosam (Cho et al. 1988). Generally ethylene inhibitors are known to inhibit ethylene production and improve secondary metabolite formation (Rengel and Kordan 1987) and shoot formation (Mathur et al. 1993). However, in the present study ethylene inhibition was associated with reduction in secondary metabolite. Therefore, such effect of ethrel can
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be explored for high yields of secondary metabolite production in short time in bioreactors. Reduction in time and increase in production have significant effect on cost effectiveness. Acknowledgments This work was supported by financial assistance from UGC-DRS under special assistance programme for medicinal plant research and DST-FIST programme for infrastructure development to Prof. KG Ramawat.
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