Saudi Journal of Biological Sciences (2015) xxx, xxx–xxx King Saud University Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com ORIGINAL ARTICLE Development of an efficient callus proliferation system for Rheum coreanum Nakai, a rare medicinal plant growing in Democratic People’s Republic of Korea Song-Chol Mun a,*, Gwan-Sim Mun b a Department of Pharmacy, Pyongyang Medical College, Kim Il Sung University, Pyongyang, Democratic People’s Republic of Korea b Department of Medical Plant Resources, Institute of Pharmaceutics, Academy of Medicine Sciences, Pyongyang, Democratic People’s Republic of Korea Received 6 March 2015; revised 25 May 2015; accepted 25 May 2015 KEYWORDS Abstract A clonal mass propagation to obtain mountainous sources of Rheum coreanum Nakai, a Anthraquinone; rare medicinal plant in Democratic People’s Republic of Korea was established by rhizome tissue Plant growth regulators; culture. Whole plants were selected and collected as a vigorous individual free from blights and Rheum coreanum Nakai; harmful insects among wild plants of R. coreanum grown on the top of Mt. Langrim (1.540 m above Rhizome; the sea) situated at the northern extremity of Democratic People’s Republic of Korea. Induction of Rhubarb; the callus was determined using four organs separated from the whole plant and different plant Tissue culture growth regulators. The callus was successfully induced from rhizome explant on MS medium con- taining 2.4-D (0.2–0.3 mg/l). In the MS medium supplemented with a combination of BAP (2 mg/l) and NAA (0.2 mg/l), single NAA (0.5 mg/l), or IBA (0.5 mg/l), a higher number of shoot, root and plantlets was achieved. The survival rate on the mountainous region of the plantlets successfully acclimatized (100%) in greenhouse reached 95%, and yields of crude drug and contents of active principles were higher than those obtained by sexual and vegetative propagation. This first report Abbreviations: 2,4-D, 2,4-dicholorophenoxy acetic acid; BAP, 6-benzylaminopurine; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; KT, kinetin; MS, Murashige and Skoog culture medium; NAA, a-naphthalene acetic acid. * Corresponding author at: Department of Pharmacy, Pyongyang Medical College, Kim Il Sung University, St. Sungri, Pyongyang, Democratic People’s Republic of Korea. E-mail address: [email protected] (S.-C. Mun). Peer review under responsibility of King Saud University. Production and hosting by Elsevier http://dx.doi.org/10.1016/j.sjbs.2015.05.017 1319-562X ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Mun, S.-C., Mun, G.-S. Development of an efficient callus proliferation system for Rheum coreanum Nakai, a rare medicinal plant growing in Democratic People’s Republic of Korea. Saudi Journal of Biological Sciences (2015), http://dx.doi.org/10.1016/j.sjbs.2015.05.017 2 S.-C. Mun, G.-S. Mun of R. coreanum tissue culture provides an opportunity to control extinction threats and an efficient callus proliferation system for growing resources rapidly on a large scale. ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction sea, 10–18 °C in summer), which adapt to the growth condi- tion of R. coreanum. The aim of this work is to study the pos- Rheum coreanum Nakai is a perennial plant indigenous to sibility of rhizome tissue culture technology for the clonal mass DPR of Korea (Hoang et al., 1997). The rhizome and root propagation of R. coreanum on mountainous regions. of this plant have been generally used a stomachic, antidote, cathartic and hemostatic in the traditional medicine of DPR 2. Materials and methods of Korea. On the other hand, Rheum species is useful in the treatment of hemorrhoid (C¸akılcıog˘ lu and Tu¨ rkog˘ lu, 2007), 2.1. Materials inflammation (Hu et al., 2014), pass kidney stones (C¸akılcıog˘ lu and Tu¨ rkog˘ lu, 2008), and diabetes disease (Kaval et al., Whole plants were selected and collected as vigorous individu- 2014; Raafat et al., 2014). It also shows anti-allergic als free from blights and harmful insects among wild plants of (Matsuda et al., 2004), antidyslipidemic (Mishra et al., 2013), R. coreanum grown on the top of Mt. Langrim (1.540 m above antifungal (Agarwal et al., 2000), antimicrobial (Kosikowska the sea) in the northern extremity of Province of Jagang, DPR et al., 2010), antioxidant (O¨ ztu¨ rk et al., 2007), anti-platelet of Korea and were used as initial materials in this study. (Aburjai, 2000), antiviral (Chang et al., 2014), cytotoxic (Kubo et al., 1992), hepatoprotective (Ibrahim et al., 2008), 2.2. Preparation of an explant segment and hypoglycemic (Naqishbandi et al., 2009) activities, and immuno-enhancing (Kounsar et al., 2011), and renal function Stem tip, young leaf, rhizome and root tip were separated from improving (Azhar Alama et al., 2005) effects. Its demand has the whole plants. After washing the separated parts of the increased in connection with the antivirus and anticancer activ- plant under running tap water, the explants were surface- ities of anthraquinone derivatives, which have recently been sterilized by washing with 75% ethanol for 30 s and undiluted known (Li et al., 2009; Martens et al., 2014). The contents of sodium hypochlorite with the pH adjusted to 6.8–7.0 using free anthraquinones, total anthraquinones and combined hydrochloric acid (HCl) for approximately for 3 min. anthraquinones in roots were higher than in other organs, that Subsequently, the Stem tip, young leaf, rhizome and root tip is, their contents in the underground part were much more were washed five times with sterile distilled water and cut it than those of the aerial parts. A previous study reported the into the size of 10–15 mm3 in capacity. content of anthraquinone derivatives in the rhubarb processed with R. coreanum was higher than the rhubarb processed with 2.3. Induction of callus on the explant segments of different other Rheum species such as Rheum palmatum, Rheum officiale organs and Rheum undulatum.(Ohshima et al., 1988) However, natu- ral resources of R. coreanum were exhausted due to the lower propagation rate of this plant distributed at limited areas of Explants were placed to germinate in Petri dishes containing high mountain tops (more than 1.500 m above the sea) and MS medium with half strength of macronutrients, 0.8% agar over collection for a long time. The plant, a polar plant, is and 3% sucrose (pH 6). The medium also contained different damaged by insects (Kurosaki et al., 1992; Walkey and kinds and concentrations of plant growth regulators. The pH Matthews, 1979) and changed into metamorphosis when culti- of the media was adjusted to 5.6 prior to autoclaving for vated in lower regions. Rheum species may be propagated by 15 min at 120 °C. Subsequently, they were incubated at sexual reproduction with seeds that produce hereditary non- (25 ± 1 ) °C and 60–70% relative humidity under a 14 h pho- À2 À1 uniformity generations since it is the cross-fertilization plant toperiod by cool-white fluorescent lights (36.0 lmol m s ) and by vegetative reproduction with single lateral buds that for 25 days. are propagated very slowly (Ohshima et al., 1988; Schulte et al., 1984). Plant tissue culture techniques offer a powerful tool for Table 1 Comparison of inducing rate of the callus on germplasm conservation and mass multiplication of many different organs of Rheum coreanum (n = 30). threatened plant species, and also offer an alternative to manufacture economically important secondary metabolites Parts of plant Inducing rate of callus according to kinds and concentrations of plant growth regulators (%) such as flavors and pharmaceuticals within controlled labora- tory environments (Dakah et al., 2014; Guo et al., 2007; 2.4-D/mg/l NAA/mg/l Kinetin/mg/l Palacio et al., 2012; Ozel et al., 2015; Wang et al., 2011). An 0.2 0.5 0.2 0.5 0.5 1.0 Rheum franzenbachii efficient callus proliferation system for Stem tip 85 80 58 65 12 7 Munt., a rare medicinal plant, has been developed because of Leaf 36 25 92 30 5 3 its demand (Palacio et al., 2012; Wang et al., 2011). Rhizome 95 82 75 80 15 13 Therefore, we designed that resources of R. coreanum would Root top 60 73 42 55 16 10 be created at mountainous regions (1.000–1.500 m above the Please cite this article in press as: Mun, S.-C., Mun, G.-S. Development of an efficient callus proliferation system for Rheum coreanum Nakai, a rare medicinal plant growing in Democratic People’s Republic of Korea. Saudi Journal of Biological Sciences (2015), http://dx.doi.org/10.1016/j.sjbs.2015.05.017 Callus proliferation system for Rheum coreanum Nakai 3 2.4. Callus multiplication culture of rhizome segments 3. Results and discussion The rhizome segments were incubated in MS liquid medium 3.1. Induction of callus from different organs of the plant containing various kinds and concentrations of plant growth regulators. Cultures were kept in a growth chamber at Rhizome among various organs of the whole plant is an excel- (25 ± 1) °C and 30 rotations/min under a 14 h photoperiod lent explant for callus induction. A combination of plant À2 À1 by cool-white fluorescent lights (36.0 lmol m s ) for growth regulators had successfully induced the formation of 28 days in order to form the callus.