Journal of Integrative Plant Biology 2006, 48 (9): 1108−1114

Received 12 Dec. 2005 Accepted 15 Mar. 2006

Publication of this paper is supported by the National Natural Science

Foundation of China (30424813) and Science Publication Foundation of the

Chinese Academy of Sciences.

*Author for correspondence. Tel: +82 31 290 1164; Fax: +82 31 290 1020;

E-mail: <soypark7@foa.go.kr>.

Effects of Nitrogen Source and Bacterial Elicitor onIsoflavone Accumulation in Root Cultures of

Albizzia kalkora (Roxb.) Prain

So-Young Park*, Wi-Young Lee, Youngki Park and Jin-Kwon Ahn(Biotechnology Division, Korea Forest Research Institute, Suwon 441-350, Korea)

Abstract

Changes in cellular isoflavone (daidzein and genistein) contents were monitored in root cultures of Albizziakalkora (Roxb.) Prain after feeding different ratios of NH4

+/NO3– and treatment with a biotic elicitor (three

strains of Rhizobium sp.). The NH4+/NO3

– ratio appears to be positively correlated with daidzein content inthe roots and shows a negative correlation with genistein. Among the three different strains of Rhizobiumused, the strain ATCC 15834 caused a 35% increase in daidzein production by infection. In the case ofgenistein, maximum production (94%) was obtained when cultures were treated on Day 6 by the strainsATCC 15834 and KCTC 1541. The biosynthetic pathway of the two isoflavones apparently reacts differently tothe same culture conditions and the same strains of Rhizobium. Therefore, the present data suggest thatthe production of daidzein and genistein could be modulated by changing the NH4

+/NO3– ratio and the applica-

tion of Rhizobium.

Key words: Albizzia kalkora; infection; isoflavone; nitrogen; root culture.

Park SY, Lee WY, Park Y, Ahn JK (2006). Effects of nitrogen source and bacterial elicitor on isoflavone accumulation in rootcultures of Albizzia kalkora (Roxb.) Prain. J Integr Plant Biol 48(9), 1108−1114.

www.blackwell-synergy.com; www.jipb.net

Albizzia kalkora (Roxb.) Prain (syn. A. coreana), a memberof the family Leguminosae, is a medicinal and endangered treedistributed only in a few regions of Korea, China, and India.Extracts of roots and stem barks of the species contain somebioactive compounds and, thus, have been used in folk medi-cine as antibiotics, tonics and prophylactic herbal drugs (Saoand Yook 1988; Seo et al. 1988; Ikeda et al. 1995; Kang et al.2000).

Among the bioactive compounds present in plants,isoflavones are a group of naturally occurring heterocyclicphenols found mainly in Leguminosae. Both genistein anddaidzein constitute the major isoflavones and have been impli-cated as potential agents for preventing disorders such as

heart diseases, cancer, and diabetes (Phillips and Kapulnik1995; Dastidar et al. 2004; Peng et al. 2004). The two isoflavoneshave been reported to mimic the pharmacological actions of theendogenous steroid estrogen, with which they have structuralsimilarities (Sonee et al. 2004).

The simplest method to produce isoflavones is to extract thecompounds from field-grown plants (Canter et al. 2005).However, it is difficult to control environmental conditions andother factors to enhance the metabolites in the field. In recentyears, biotechnology has been suggested as an alternativemethod to produce these compounds (Kieran et al. 1997;Fedoreyev et al. 2000). Cell and tissue cultures offer an alter-native production system for these metabolites because it ispossible to control the factors that may affect the biosyn-thetic pathways. Many workers have reported isoflavone pro-duction from in vitro cultures of various plants, includingGenista (Luczkiewicz and Gold 2003), Maackia amurensis(Fedoreyev et al. 2000), and Pueraria lobata (Park et al. 1995).Despite the considerable potential of in vitro cultures inisoflavone production, low yields have hampered theircommercialization.

Isoflavone Accumulation in Root Cultures of A. kalkora 1109

The nitrogen source in the medium not only significantly af-fects the growth and development of plants, but also changessecondary metabolism (Lourenço et al. 2002; Wang and Tan2002). However, the effect of the nitrogen source on the accu-mulation of secondary metabolites may vary depending uponthe target compounds and the plant species. For example, alow NH4

+/NO3– ratio promoted the production of shikonin (Fujita

et al. 1981) and betacyanins (Bohm and Rink 1988), whereas ahigh NH4

+/NO3– ratio increased the production of berberine

(Nakagawa et al. 1984) and ubiquinone (Ikeda et al. 1977).However, there is limited evidence to suggest any relationshipbetween nitrogen and secondary metabolite accumulation inplants.

To enhance desirable metabolites, elicitation has been a use-ful strategy in in vitro cultures of plant cells and tissues (Dixonet al. 1996; Jung et al. 2003). Among the elicitors, the use ofbiotic elicitors, such as pathogen-derived compounds, can sub-stantially trigger defense responses that are linked to the syn-thesis of secondary metabolites (Habereder et al. 1989; Kunkel

and Brooks 2002). Therefore, numerous attempts have beenmade to enhance the production of secondary metabolites byelicitation from plant cell/tissue culture (Bednarek et al. 2001;Jung et al. 2003; Lozovaya et al. 2004).

Previously, we have successfully developed a culture sys-tem for A. kalkora using adventitious roots (Park et al. 2003;Figure 1). As a continuation of our previous work, the presentstudy focuses on the elucidation of the effects of nitrogensource and bacterial infection on the accumulation of daidzeinand genistein in adventitious root cultures of A. kalkora.

Results and Discussion

Effect of nitrogen source on growth and isoflavoneaccumulation

The effect of the nitrogen source on the biosynthesis of isoflavone,daidzein, and genistein in root culture was investigated by

Figure 1. Adventitious root cultures of Albizzia kalkora in woody plant medium containing 0.5 mg/L indole-3-butric acid.

(A) Seedling of A. kalkora.(B) Adventitious roots induction from radicle of seedling.(C) Adventitious roots after 4 weeks of culture.(D) Mass proliferation of adventitious roots.

1110 Journal of Integrative Plant Biology Vol. 48 No. 9 2006

altering the ratio of different nitrogen sources of NH4+ and NO3

–

to 3 : 0, 2 : 1, 1 : 1, 1 : 2 or 0 : 3. As shown in Table 1, theaverage growth rate reached a maximum of 0.26 at a ratio of2 : 1 ammonium to nitrate. Ammonium as the sole nitrogen sourcedid not support growth to a visible extent, whereas nitrate asthe only nitrogen source resulted in root growth over two foldscompared with ammonium only. Similar results have been ob-tained with other plant species (Lourenco et al. 2002). It ispostulated that ammonium uptake leads to acidification of themedium and, thus, increases the intracellular ammonium con-centration (Oksman-Caldentey et al. 1994; Lourenco et al. 2002),resulting finally in poor growth of the culture.

Although the 2 : 1 ratio of ammonium to nitrate gave the bestroot growth, it caused the poorest production of desirable me-tabolites (Figure 2). It seems very difficult to achieve maximumproduction of the target metabolite without sacrificing maximalbiomass (Hagendoorn et al. 1999; Luczkiewicz and Cisowski2001). Luczkiewicz and Glod (2003) reported a negative cor-relation between callus growth and isoflavone synthesis inGenista. They suggested that abundant nitrogen in the mediumcould have been used as a precursor in the bioconversion ofisoflavone, but not in cell growth.

Figure 2 shows the effects of the ammonium to nitrate ratioon the production of daidzein and genistein. Higher ratios ofNH4

+/NO3– promoted the production of daidzein, whereas a

lower ratio favored the higher production of genistein in thecultures (Figure 2). The results indicate that it may be possibleto modulate the production of daidzein and genistein by con-trolling the ammonium and nitrate ratio in the culture medium foradventitious root cultures of this species. Similar results, namelythat the ratio of NH4

+/NO3– markedly affect the production of

secondary plant products, have been reported for cultures ofvarious plants. In Artemisia annua hairy root cultures, a lowerNH4

+/NO3– ratio supported a high level of artemisinin accumula-

tion (Wang and Tan 2002). Complete elimination of nitrate in themedium induced a twofold increase in pyrethrin accumulationin cultures of Chrysanthemum cinerariaefolium (Rajasekaranet al. 1991). However, the exact mechanism by which suchmetabolic changes occur is not yet clearly understood.Nevertheless, it is interesting to note that changes in the ammo-nium and nitrate ratio in culture medium affect the cellulardaidzein and genistein content in cultures. This feature willcertainly offer us a way to control the production of secondarymetabolites in cultures.

Effect of bacteria elicitor on enhanced production ofdaidzein and genistein

The production of daidzein and genistein by adventitious rootcultures was analyzed after growing in liquid woody plantmedium (WPM) for 4 weeks, followed by cocultivating withthree strains of Rhizobium for 12 d. Figure 3 shows the effectof bacterial elicitors on the accumulation of daidzein.Rhyzobium rhizogenes ATCC 15834 increased the productionof daidzein by approximately 35% compared with untreatedroots on Day 9 after treatment, whereas R. radiobacter strainsKCTC 1541 on Days 9, and KCTC 1541 on Day 12 increasedthe production by approximately 12% and 10%, respectively.

The strain ATCC 15834 markedly increased the production ofgenistein on Day 6 by approximately 94% compared with non-treated roots (Figure 4). Similar stimulatory effects on this

Table 1. Effect of nitrogen source on the root growth of adventitious roots in Albizzia kalkora after 4 weeks of cultureConcentration of NH4

+ and NO3– (mmol/L)

Ratio of NH4+ : NO3

– Fresh weight Dry matter Average growth rate

NH4+ NO3

– (g) (%)14.7 0.0 3 : 0 2.7d 13.4 0.06 9.7 5.0 2 : 1 8.7a 11.5 0.26 7.4 7.4 1 : 1 7.1b 11.8 0.20 4.7 10.0 1 : 2 5.1c 12.1 0.14 0.0 14.7 0 : 3 5.8c 12.6 0.16

Dry matter was calculated as (dry weight/fresh weight) × 100.Average growth rate was calculated as (maximum culture density–initial culture density)/(initial culture density) (culture day)Mean values within a column followed by different superscript letters are significantly different at P 0.05 (Duncan’s multiple range test).

Figure 2. Accumulation of daidzein and genistein from Albizziakalkora adventitious roots treated with different ammonium/nitrateratios in culture medium.

Isoflavone Accumulation in Root Cultures of A. kalkora 1111

plant defense pathways in response to infection by a patho-gen are regulated through a complex network of signaling path-ways (Kunkel and Brooks 2002). Pathogen infection triggersoxidative reactions in the plant that contribute to induced re-sponses and symptoms. Then, endogenous plant signalmolecules, including salicylic acid, jasmonic acid, and ethylene,are produced (Szabo et al. 1999). Because these signalingpathways are closely linked to secondary metabolism, theisoflavone is subsequently produced as a result of the activa-tion of the signaling pathway (Dixon et al. 1996; Dixon andSteele 1999). Another explanation is that isoflavone biosynthe-sis may be stimulated following infection with symbioticmicroorganisms. Isoflavone biosynthesis is known to be closelyrelated to legume-specific phenomena (i.e. symbiotic nitrogenfixation; Shimada et al. 2000). Rhizobia play an important role inestablishing nitrogen-fixing root nodules in leguminous plants.These facts substantiate our results that the production of twoisoflavones, namely daidzein and genistein, increased mark-edly when infected with Rhizobium. However, the extent ofthe increase induced varies among bacterial strains.

Furthermore, the present experiments showed that genisteinaccumulation was more affected by Rhizobium infection thanwas the accumulation of daidzein. Similar results have beenreported for the roots of white lupin seedlings, where elicita-tion by the symbiont Rhizobium resulted in an increased ratioof genistein monoprenyls (Gagnon and Ibrahim 1997). Theseauthors speculated that the increase in this metabolite wasdue to symbiotic phytoalexin, which acts as a signal moleculein Rhizobium-legume symbioses.

In practice, a two-stage cultivation method is commonlyadopted to maximize the production of desired metabolites when

Figure 5. Production of daidzein and genistein in adventitious rootsafter coculture with three strains of Rhizobium.

Control, non-treated roots; K1541, R. radiobacter KCTC 1541;K1542, R. radiobacter KCTC 1542; K15834, R. rhizogenes ATCCK15834.

Figure 3. Changes in daidzein content in adventitious roots duringthe culture period after the addition of bacterial suspension.

Control, non-treated roots; K1541, Rhizobium radiobacter KCTC1541; K1542, R. radiobacter KCTC 1542; K15834, R. rhizogenesATCC K15834.

Figure 4. Changes in genistein content in adventitious roots duringthe culture period after the addition of bacterial suspension.

Control, non-treated roots; K1541, Rhizobium radiobacterKCTC 1541; K1542, R. radiobacter KCTC 1542; K15834, R.rhizogenes ATCC K15834.

metabolite were also observed following the addition of thestrain KCTC 1541 (89%) on Day 6. The total content of bothmetabolites reached a maximum level on Day 6 after treatment(Figure 5).

Isoflavones are common to leguminous plants, play someroles in root nodulation, and exert their activity against patho-gen attack (Dakora and Phillips 1996; Bednarek et al. 2001;Subramanian et al. 2004). These metabolites are not inducedby simple wounding, but are commonly produced in plant-mi-crobial pathogen interactions (Mithofer et al. 2004). Generally,

1112 Journal of Integrative Plant Biology Vol. 48 No. 9 2006

secondary metabolite production is independent of growth(Endress 1994; Choi et al. 2000). In the present study, the firststage (or growth stage) consisted of culturing roots in WPMcontaining 9.7 mmol/L ammonium and 5 mmol/L nitrate (2 : 1ratio of NH4

+/NO3–). The second stage (or elicitation stage) start

when maximum root growth was attained at the end of the firststage. The cultures were fed with different levels of ammo-nium or nitrate to stimulate biosynthesis of daidzein or genistein.In addition, the production of daidzein or genistein could bemaximized by cocultivation of adventitious roots with R.rhizogenes ATCC 15834. To the best of our knowledge, this isthe first study demonstrating the regulation of isoflavone con-tent by nitrogen and a biotic elicitor in root cultures of A. kalkora.

Materials and Methods

Establishment of adventitious root cultures

Seeds of Albizzia kalkora (Roxb.) Prain were obtained fromthe Chollipo Arboretum (Suwon, Korea). Seeds were surfacesterilized and placed on 1/2 MS (Murashige and Skoog 1962)medium in the dark maintained at 23 °C for germination. After 2weeks, roots were excised from the seedlings and cultured in100 mL WPM (Lloyd and McCown 1980) containing 0.5 mg/Lindole-3-butric acid (IBA) and 3% (w/v) sucrose on a gyratoryshaker (110 r/min). The culture was maintained by subculturingto the same medium every 4 weeks.

Effect of NH4+ : NO3

– ratio on growth and yield ofisoflavone

A 1-g sample of adventitious roots was inoculated in 100 mLWPM containing 0.5 mg/L IBA and 3% (w/v) sucrose in 300-mLErlenmeyer flasks and grown at 25 °C on a gyratory shaker(110 r/min) under darkness. The nitrogen content in the mediumwas modified by altering the ratio of NH4

+/NO3– (3 : 0, 2 : 1, 1 :

1, 1 : 2 or 0 : 3) at 14.7 mmol/L of total nitrogen. After 4 weeksof culture, roots were harvested and analyzed for isoflavonecontent.

Bacterial preparation and elicitation

Rhizobium radiobacter strains KCTC 1541 and 1542 and R.rhizogenes (NCBI syn. Agrobacterium rhizogenes) strainATCC 15834 were used as biotic elicitors. Bacteria were grownin Luria Broth liquid medium (LB medium) in darkness at 27 °C,120 r/min for 2 d. Then, the bacterial suspension was dilutedand adjusted to an optical density of 1.0 at 600 nm. This wasused as an elicitor without autoclaving.

For root culture, flasks (300 mL volume) were filled with 100mL WPM and inoculated with 1.0 g fresh weight roots. After 4

weeks of culture, 200 µL of three different strains of Rhizo-bium suspension were added and cultured for a further 12 d.The pH of the medium was adjusted to 5.6–5.8 before auto-claving at 101 kPa and 121 °C for 15 min. For all experiments,growth regulators were added prior to autoclaving.

Determination of daidzein and genistein content

Extraction and analysis of both daidzein and genistein (Figure6) were performed according to the methods of Park et al.(2004). Isoflavone fractions were analyzed using the ThermoSeparation Products HPLC system (TSP, San Jose, CA, USA)equipped with an ultraviolet (UV) detector (UV 3000 HR; TSP)on a 5-µm LiChrospher 100 RP-18 column (250 mm × 4.0 mmi.d., particle diameter 5 µm; Merck, Darmstadt, Germany). Thefollowing gradient elution was used for isoflavones: 10%−50%B in A from 0 to 35 min, 50%−100% B in A from 35 to 45 min,100% B from 45 to 50 min, then 100%−10% B in A from 50 to51 min, and finally 10% B in A from 51 to 61 min; with A: H2O/CH3COOH (90 : 10) and B: acetonitrile. The flow rate of themobile phase was 0.7 mL/min. Daidzein and genistein weredetected at 260 nm. Samples were identified and quantified bycomparing the retention times and peak areas with those ofexternal standards (daidzein and genistein; Sigma, St Louis,MO, USA).

Acknowledgements

The authors thank Dr EW Noh, Biotechnology Division, KoreaForest Research Institute (KFRI), Suwon, Korea, for his criticalevaluation and valuable suggestions during the preparation ofthe manuscript.

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