Establishment of Hairy Root Culture of Rubia Akane Nakai for Alizarin and Purpurin Production

Full Length Research Paper

Establishment of hairy root culture of Rubia akane Nakai for alizarin and purpurin production

Sang Un Park1, Yong Kyoung Kim1 and Sook Young Lee2*

1Division of Plant Science and Resources, Chungnam National University, 220 Gung-Dong, Yuseong-Gu, Daejeon, 305- 754, Korea. 2Research Center for Proteineous Materials, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju, 501-759, Korea.

Accepted 12 January, 2009

Alizarin and purpurin from Rubia akane have been used since ancient times as a natural dye in Asia. This study established a hairy root culture of Rubia akane Nakai by infecting leaf explants with Agrobacterium rhizogenes R1000 to investigate the growth and anthraquinones (alizarin and purpurin) content. Hairy roots were cultured in MS liquid medium. With increasing time, the growth as well as the content of alizarin and purpurin were increased significantly. Maximum growth (10.4 g/L) was achieved by 20 days of culture with content of alizarin (3.9 mg/g dry wt) and purpurin (4.6 mg/g dry wt). This result demonstrates that Rubia akane hairy culture can be an efficient approach for the production of anthraquinones.

Key words: Agrobacterium rhizogenes, alizarin, anthraquinones, hairy root culture, purpurin, Rubia akane Nakai.

INTRODUCTION

The plant genus Rubia contains about 60 species and the terium rhizogenes, a gram-negative soil bacte-rium, is best known species are Rubia tinctorum (common one of the most widely studied. It can easily infect plant madder) in Europe and Rubia akane in Asia used as a cell and leads to the formation of hairy roots. (Hamill et natural dye for thousands of years (Singh et al., 2004). al., 1987; Signs and Flores 1990). In many plant species, Rubia akane belonging to the Rubiaceae family is a hairy root cultures have widely proven to be an efficient perennial plant widely distributed in East Asian countries. alternative production system for secondary metabolites In Korea, the pigment (anthraquinones) from R. akane because of their genetic and biochemical stability, rapid has been used since ancient times as a vegetable red growth rate and ability to synthesize natural compounds dye. Anthraquinones are an important group of natural at levels comparable to in vivo grown plants (Giri & product used in the production of dyes, such as alizarin Narasu 2000; Guillon et al. 2006). and purpurin shown in Figure 1 (Shin, 1989). In addition, However, there have been no reports about anthraqui- anthraquinones have been reported to exhibit various nones production in the hairy root culture of R. akane. pharmacological and biological activities including anti- This study reports the production of anthraquinones and cancer (Zhang et al., 2007; Son et al., 2008), antimalarial the establishment of hairy root cultures of R. akane trans- (Bringmann et al., 2008), antimicrobial (Lenta et al., 2007; formed with A. rhizogenes. Xiang et al., 2008), antifungal (Singh et al., 2006), and antioxidant activities (Galindo et al., 2008). Diverse species of bacteria can transfer genes to higher MATERIALS AND METHODS plants (Broothaerts et al., 2005). Among them Agrobac- Plant material

Seeds of R. akane were kindly provided by the Seed Bank of Medicinal Plants (Dongsin University, Naju, Korea). Seeds were *Corresponding author. E-mail: [email protected]. surface-sterilized with 70% (v/v) ethanol for 1 min and 2% (v/v) Tel.: +82-622307567. Fax: +82-622278345. sodium hypochlorite solution for 10 min, then rinsed three times in Park et al. 095

white fluorescent tubes with a flux rate of 35 µmol s-1m-2 and a 16 h photoperiod. After 30 days of culture, hairy roots were harvested and the dry weight and anthraquinones content were determined. Each experiment was carried out with 3 flasks per culture condition and repeated twice.

Analysis of anthraquinones

Hairy roots were dried at -80oC for at least 48 h using a freeze dryer (Model FD-5508, Ilsin Engineering Co., Seoul, Korea). Dried samples were ground into a fine powder using a mortar and pestle. Samples (0.5 g) were extracted twice with 10 ml methanol at 50oC in a water bath for 1 h. After centrifugation, the supernatant was concentrated under reduced pressure and the residue dissolved in 1 mL methanol. The quantitative analysis of alizarin and purpurin produced in the hairy root cultures was done by Beckman Coulter DU 700 series UV/Vis spectrophotometers. The absorbances of alizarin and purpurin at 572 and 516 nm were measured and their amounts were calculated by reading on the calibration curves of the standard compounds. The standard chemicals, alizarin and purpurin were Figure 1. Chemical structures of (A) purchased from Wako Pure Chemical Industries, Ltd. (Osaka, alizarin and (B) purpurin. Japan) and Sigma-Aldrich Korea (Seoul, Korea). The analysis of alizarin and purpurin was performed using the method of Shin (1989). sterilized water. Four seeds were placed on 25 ml of agar-solidified culture medium in Petri dishes (100 x 15 mm). The basal medium consisted of salts and vitamins of MS (Murashige and Skoog, 1962) RESULTS AND DISCUSSIONS medium and solidified with 0.7% (w/v) agar. The medium was adjusted to pH 5.8 before adding agar and then sterilized by auto- To induce hairy roots from leaf explants of R. akane, A. claving at 121°C for 20 min. The seeds were germinated in a rhizogenes R1000 was tested for its ability. For removing growth chamber at 25°C under standard cool white fluorescent A. rhizogenes, explant tissues were transferred to an -1 -2 tubes with a flux rate of 35 mol s m and a 16 h photoperiod. agar-solidified MS medium containing 200 mg/l Timentin, 2 days after co-cultivation with A. rhizogenes R1000.

Preparation of Agrobacterium rhizogenes Wounded R. akane leaf explants were susceptible to infection by A. rhizogenes R1000. It infected more than Agrobacterium rhizogenes strain R1000 was kindly provided by Dr. 70% of the leaf explants 30 days after inoculation. Hairy Felipe Vázquez-Flota (Centro de Investigación Científica de root initials emerged from wound sites on leaf within 15 Yucatán, México). It was grown to mid-log phase (OD = 0.5) at 600 days after inoculation. After 30 days, it was shown that 28°C on a gyratory shaker at 180 rpm in liquid Luria-Bertani (LB) hairy roots of R. akane began to grow more rapidly. Ra- medium. The bacterial cells were collected by centrifugation for 10 pidly growing hairy roots were excised from the explant min at 250 g, and re-suspended at a cell density of A = 0.5 in 600 tissues and transferred on an agar-solidified MS medium liquid inoculation medium (MS salts and vitamins containing 30 g/l containing 200 mg/l Timentin every two weeks (Figure sucrose). 2A). After repeated transfer to fresh medium for three

months, hairy root clones were replaced to MS liquid cul-

Establishment of hairy root cultures ture medium (Figure 2B). Hairy roots were cultured in MS liquid medium for 25 Young leaves of R. akane were taken from in vitro grown plants and days where the growth and anthraquinones (alizarin and 2 were cut at the ends, in sections 7 mm . Excised leaves were purpurin) content were investigated. At 5 days intervals, dipped into the A. rhizogenes culture in liquid inoculation medium six flasks were harvested to measure the growth of hairy for 10 min, blotted dry on a sterile filter paper and incubated in the roots. During the period of 25 days in the liquid medium, dark at 25°C on an agar-solidified MS medium (Murashige & the dry weight of the hairy root increased from the initial Skoog, 1962). After two days of co-cultivation, the explant tissues inoculum of 0.5 g/l to 10.3 g/l (Figure 3A). With increasing were transferred to a hormone-free medium containing MS salts and vitamins, 30 g/l, 200 mg/l Timentin and 8 g/l agar. Numerous time, the growth as well as the content of alizarin and hairy roots were observed emerging from the wound sites within purpurin increased. The maximum growth (10.4 g/l) was three weeks. The hairy roots were separated from the explant achieved by 20 days of culture with contents of alizarin tissue and sub-cultured in the dark at 25°C on an agar-solidified MS (3.9 mg/g dry wt) and purpurin (4.6 mg/g dry wt) (Figures medium. After repeated transfer to fresh medium, rapidly growing 3B and C). After 25 days of culture, the hairy roots turned hairy root cultures were obtained. Isolated roots (200 mg) were transferred to 30 ml of MS liquid medium, containing 30 g/l sucrose, brown and the brown pigment released into the culture in 100 ml flasks. Root cultures were maintained at 25°C on a medium. gyratory shaker (100 rpm) in a growth chamber under standard cool Biotechnological productions of anthraquinone by plant 096 Sci. Res. Essays

Figure 2. Hairy root cultures of Rubia akane. Rapidly growing hairy roots on agar-solidified MS medium (A) and hairy root culture in MS liquid culture medium (B).

cell culture of R. akane Nakai have been reported (Endo et al., 1997; Mizutani et al., 1997; Shim et al., 1999). Also, hairy root culture of several Rubia species, R. cordifolia (Shin and Kim, 1996), R. peregrina (Lodhi & Charlwood, 1996; Lodhi et al., 1996), and R. tinctorum (Sato et al., 1991; Masahiro et al., 1994; Mantrova et al., 1999) for anthraquinone production have been studied. However, there have been no reports about anthraquinone production in hairy root culture of R. akane in Asia. This study has first developed an efficient A. rhizogenes-mediated protocol for the establishment of R. akane hairy root cultures using A. rhizogenes R1000 for the production of natural dyestuffs and medicinal compounds, alizarin and purpurin. In general, the growers can harvest roots of R. akane for anthraquinones production only once a year in Korea. However, this result indicates that hairy root culture of R. akane could be a valuable alternative approach for the continuous production of anthraquinones all the year round. Further investigations are recently being con- ducted for the improvement of anthraquinones production in hairy root cultures of R. akane.

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