Triterpenoid Production and Phenotypic Changes in Hairy Roots of Codonopsis Lanceolata and the Plants Regenerated from Them

Plant Biotechnol Rep (2011) 5:255–263 DOI 10.1007/s11816-011-0180-5 ORIGINAL ARTICLE Triterpenoid production and phenotypic changes in hairy roots of Codonopsis lanceolata and the plants regenerated from them Ji-Ah Kim • Yun-Soo Kim • Yong-Eui Choi Received: 9 April 2011 / Accepted: 23 April 2011 / Published online: 13 May 2011 Ó Korean Society for Plant Biotechnology and Springer 2011 Abstract Codonopsis lanceolata (Campanulaceae) has Introduction been used in traditional medicines, as its roots contain several kinds of triterpenoid saponin with high medicinal Codonopsis lanceolata Trautv. (Campanulaceae) is mainly values. In this work, we induced transgenic hairy roots of distributed in East Asia, including Korea, Japan, and C. lanceolata and analyzed triterpenoid saponins from the China. The roots of C. lanceolata have been used in tra- hairy roots and hairy root-derived transgenic plants. Hairy ditional medicines as an anti-inflammatory agent, for roots were obtained from leaf explants by the transforma- bronchitis, and for cough (Lee et al. 2002; Xu et al. 2008). tion of Agrobacterium rhizogenes R1000. Transgenic hairy The root extracts of this plant can inhibit the reduction of root lines were confirmed by the transcriptional activities blood testosterone levels caused by immobilization stress of rolA, B, C, and D genes by RT-PCR. Transgenic root in mice (Sekita et al. 2005). Furthermore, a supplement lines actively proliferated on hormone-free medium but not containing the root extracts of C. lanceolata functions to in nontransformed roots. Hairy roots contained richer trit- alleviate partial androgen deficiency of the aging male erpenoids (lancemaside A, foetidissimoside A, and aster (PADAM)-like symptoms (Morales and Lunenfeld 2002; saponin Hb) than nontransformed roots. Transgenic plants Ushijima et al. 2007). The roots contain triterpenoids and were regenerated from the hairy roots via somatic phenylpropanoid glycosides (Li et al. 2007; Shirota et al. embryogenesis. They showed phenotypic alterations such 2008). To date, seven 3,28-bidesmosidic triterpenoid sap- as shortened shoots and an increased number of axillary onins have been identified from the C. lanceolata roots buds and adventitious roots. The transgenic plants also (Sekita et al. 2005; Shirota et al. 2008; Ichikawa et al. contained higher triterpenoid levels than wild-type plants. 2009). Among them, lancemaside A is a major compound, These results suggest that hairy roots and transgenic plants and this aids the recovery of decreased testosterone levels of C. lanceolata could be used as medicinal materials for in the blood (Sekita et al. 2005; Komoto et al. 2010). the production of triterpene saponins. Recently, the demand for C. lanceolata roots has been accelerating, along with its increasing reputation in Keywords Agrobacterium rhizogenes Á medicinal plant markets of Asia (Li et al. 2003). However, Aster saponin Hb Á Codonopsis lanceolata Trautv. Á the plants are mostly cultivated on mountains and wild Foetidissimoside A Á Lancemaside A hills, and they are harvested 3–4 years after planting them in the soil. These cultivation traits are a major obstacle to obtaining useful compounds from their roots. As described above, lancemaside A is considered a major compound of J.-A. Kim and Y.-S Kim contributed equally to this work. C. lanceolata, and this compound has been reported to have anti-inflammatory and anti-tumor effects (Joh and J.-A. Kim Á Y.-S. Kim Á Y.-E. Choi (&) Kim 2010; Joh et al. 2010). These facts imply that the Division of Forest Resources, College of Forest Sciences, demand for lancemaside A is set to grow rapidly. There- Kangwon National University, Chuncheon 200-701, South Korea fore, an efficient method of producing lancemaside A e-mail: [email protected] production is very desirable. 123 256 Plant Biotechnol Rep (2011) 5:255–263 Hairy roots are tumorous outgrowths that are induced on for 15 min. After rinsing 5 times with sterilized water, the many dicots by infection with Agrobacterium rhizogenes, explants were cultured on MS (Murashige and Skoog 1962) and are characterized by fast and high growth rates (Tepfer medium supplemented with 30 g l-1 sucrose and 3 g l-1 1990; Giri and Narasu 2000). They can also synthesize a gelrite (Duchefa Biochemie, The Netherlands). New shoots variety of secondary metabolites in their tissues (Bulgakov that emerged from the lateral buds were used for further et al. 1998; Palazoń et al. 1998; Sevoń and Oksman-Cal- works. All media were adjusted to pH 5.8 before auto- dentey 2002; Choi et al. 2005; Kumar and Gupta 2009). claving at 121°C for 20 min. Cultures were placed under Furthermore, hairy roots can be regenerated into plants 16 h of artificial light (40 lmol m-2 s-1) at 24–26°C. either spontaneously or by transferring the roots to hormone-containing medium. The regenerated plants often Induction of hairy roots exhibit several phenotypic changes, including wrinkled leaves, increasing branching, shortened internodes, reduced Leaf explants (0.5 cm2) were immersed in a diluted apical dominance, increasing rooting, and altered flowering A. rhizogenes strain R1000 (OD = 1.0) for 15 min. The (Tepfer 1990; Christey 2001). In some cases, it has been explants were blotted on sterile filter paper, and then placed reported that transgenic plants that were regenerated from onto a hormone-free 1/2 strength WPM (Lloyd and McC- hairy roots produced higher levels of secondary metabo- own 1980) medium for co-cultivation. After 3 days of lites in their plant bodies (Pellegrineschi et al. 1994; Pa- culture, the explants were transferred to fresh medium lazoń et al. 1998). Genetic transformation of C. lanceolata containing 30 g l-1 sucrose and 300 mg l-1 cefotaxime by a special A. tumefaciens strain led to the production of a (Duchefa Biochemie, The Netherlands) to suppress transgenic C. lanceolata that overexpressed c-tocopherol A. rhizogenes. Putative hairy roots (about 15 lines) were methyltransferase (Ghimire et al. 2008). However, there is induced from the excised margins of leaf segments, and still no report on the production of transgenic hairy roots in these roots were detached from explants and subcultured C. lanceolata by Agrobacterium rhizogenes. onto the same medium containing sucrose and cefotaxime. In the present work, we first generated hairy roots from C. lanceolata infected with A. rhizogenes, and then ana- Reverse transcription (RT)-PCR analysis lyzed the production of triterpenoid saponin in both the hairy roots and the regenerated transgenic plants derived Total RNAs were extracted from hairy root lines using an from the hairy roots. RNeasy Plant Mini Kit (Qiagene, USA), and were then reverse-transcribed by the Improm-II Reverse Transcrip- tion system (Promega, USA). First-strand cDNA was used Materials and methods as a template for the RT-PCR reaction. The primer sets that were used to amplify the rolA, B, C, D, and 18S Plant materials rRNA genes are detailed in Table 1. PCR was carried out for 30 cycles of denaturation at 94°C for 30 s, annealing Codonopsis lanceolata plants were collected from Ho- at 55°C for 30 s, and extension at 72°C for 1 min. After engseong, Korea. The stem explants, including lateral fractionation on agarose gel electrophoresis, products buds, were surface-sterilized by 70% ethanol for 1 min, were identified by visualization with ethidium bromide and disinfected by immersion in 2% sodium hypochlorite staining. Table 1 Primers used for RT- Genes Primers PCR product Accession PCR analysis of rol genes in size (bp) number hairy roots of Codonopsis lanceolata rolA50-ACGGTGAGTGTGGTTGTAGG-30 403 K03313 50-GCCACGTGCGTATTAATCCC-30 rolB50-TCAGGTTTACTGCAGCAGGC-30 696 GU182968 50-AACCTATTCGAGGGGATCCG-30 rolC50-TGTGACAAGCAGCGATGAGC-30 480 X03433 50-AAACTTGCACTCGCCATGCC-30 rolD50-CCTTACGAATTCTCTTAGCGGCACC-30 477 DQ852612 50-GAGGTACACTGGACTGAATCTGCAC-30 18S rRNA 50-GACGTGAACAACATCGGAGA-30 330 AY548195 50-CCCGAACCATTGAGTCTT-30 123 Plant Biotechnol Rep (2011) 5:255–263 257 Growth of hairy roots on different sucrose and IBA mobile phase comprised a mixture of 0.1% formic acid and contents acetonitrile (67:33, v/v), and it was delivered at a flow rate of 0.2 ml/min. The injection volume was 2 ll. The mass To optimize the growth of hairy roots, different concen- spectrometer was operated in the negative ion and selected trations of sucrose (30 or 50 g l-1) and indole-3-butyric ion monitoring (SIM) modes. Electrospray ionization (ESI) acid (0 or 0.2 mg l-1 IBA) were added to half-strength was conducted using a spray voltage of 4.5 kV. The cap- WPM liquid medium. The nontransformed adventitious illary voltage and the tube lens offset were fixed at -40 and roots were used as a control, and were cultured on the same -130 V, respectively. The heated capillary temperature strength WPM containing IBA (0 or 0.2 mg l-1) and 30 or was fixed at 350°C. 50 g l-1 sucrose. All cultures were accomplished in 250 ml flasks containing 100 ml liquid medium, and incubated in darkness at 25°C on a gyratory shaker at 120 rpm. Production of transgenic plants through regeneration from hairy roots via somatic embryogenesis For the induction of embryogenic callus, 10 mm root segments of wild-type and transformed hairy roots were cultured on MS medium supplemented with 2.0 mg l-1 2,4-D, 20 g l-1 sucrose, and 3 g l-1 gelrite. The culture was maintained at 25 ± 2°C in the dark for 6 weeks, and then the induced embryogenic calli were transferred to 1/2MS medium supplemented with 50 g l-1 sucrose and 3gl-1 gelrite for somatic embryo development. Cotyle- donary somatic embryos were transferred to 1/2MS medium supplemented with 20 g l-1 sucrose and 3 g l-1 gelrite for germination.

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