J Plant Biotechnol (2018) 45: DOI: ISSN (Print) ISSN (Online) Research Article 층층둥

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1 J Plant Biotechnol (2018) 45: DOI: ISSN (Print) ISSN (Online) Research Article 층층둥굴레 (Polygonatum stenophyllum Maxim.) 의캘러스유도및기내식물체분화 박민완 류시현 이윤영 송재모 김진호 안영희 배기화 Callus induction and in vitro plant regeneration of Polygonatum stenophyllum Maxim. Min Wan Park Shi Hyun Ryu Yoon Young Lee Jae-Mo Song Jin Ho Kim Young-Hee Ahn Kee Hwa Bae Received: 10 August 2018 / Revised: 23 August 2018 / Accepted: 23 August 2018 c Korean Society for Plant Biotechnology Abstract APolygonatum stenophyllum Maxim. is an important endangered plant belonging to the family Liliaceae. A method was developed for the rapid micropropagation of P. stenophyllum through plant regeneration from rhizome (1-year, 3-years, and 5-years) explant-derived calli. The rhizome segments were cultured in Murashige and Skoog (MS) medium supplemented with varying concentrations of 2,4-D (0, 0.5, 1.0, 1.5 mg L -1 ) for callus induction. In media supplemented with 0.5 mg L -1 of 2,4-D, 87% of 3-years rhizome produced callus. Subsequently, the callus was transferred to 1/2MS medium supplemented with various concentrations of IAA, IBA, NAA, and 2,4-D (0, 0.1, 0.5 and 1.0 mg L -1 ) for adventitious shoot formation. The highest percentage of adventitious shoot induction (57%) was observed in 1/2MS medium containing 0.5 mg L -1 of NAA. Elongation of the adventitious shoot was achieved in 1/2MS medium supplemented with 0.1 mg L -1 of BA. Rooting was achieved in 1/2MS medium without any hormones. It is hypothesized that the stated in vitro propagation protocol will be useful for conservation and mass propagation of the endangered Polygonatum stenophyllum Maxim. for bioresources. Keywords Adventitious shoot, Rhizome, Root induction M. W. Park S. H. Ryu K. H. Bae ( ) 국립낙동강생물자원관 (Nakdonggang National Institute of Biological Resources, Sangju, 37242, Republic of Korea) khbae7724@nnibr.re.kr Y. Y. Lee 황학산수목원 (Hwanghaksan Arboretum, Yeoju, 12653, Republic of Korea) J.-M. Song 강원도산림개발연구원 (Forest Science Institute of Gangwon Province, Chuncheon 24207, Republic of Korea) M. W. Park J. H. Kim 경북대학교 (Kyungpook National University, Sangju, 37224, Republic of Korea) Y.-H. Ahn 중앙대학교 (Department of Plant System Science, Chung-Ang University, Anseong 17546, Republic of Korea) 서론 층층둥굴레 (Polygonatum stenophyllum Maxim.) 는백합과 (Liliaceae) 둥굴레속 (Polygonatum) 에속하는다년생식물이다. 본연구의대상종인층층둥굴레가속한둥굴레속은북반구온대지방에 30 ~ 40 여종이자라며, 우리나라에는 10 종, 4 변종이분포하고있는것으로알려져있다. 우리나라에서는경기도, 강원도, 충청도일대의강이나모래가퇴적된하천주변에자생하며, 야생생육형태중지하부의특징으로는수평방향으로지하경이신장한다. 줄기는 30 ~ 90 cm 로털이없어표면이매끈하며잎의앞면은녹색이고뒷면은연한녹색을띠며대생한다. 층층둥굴레는마디사이가위로올라갈수록넓어지는것이다른둥굴레와다른특징이다. 꽃은 6 월에피고황색이며잎겨드랑이에달린다. 열매는장과 ( 漿果 ) 로 9 월에검게익는다 (Jang et al. 1998). This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 J Plant Biotechnol (2018) 45: 예로부터층층둥굴레가포함된둥굴레속은자양, 강장의효능이있다고전해지며말린근경은황정 ( 黃精 ), 편황정 ( 片黃精 ), 옥죽 ( 玉竹 ) 이라는이름의한방약재로사용되어왔다 (Kim 1996; Ahn 2003). 이러한효능을검증하기위해 10 여년전부터둥굴레속식물의천연물연구와관련하여여러연구자들에의해항비만 (Ko et al. 2015), 항산화와항피로 (Kim et al. 2016), 지방세포분해억제 (Jang and Jung 2010), 항암효능 (Jeong and Jeong 2011), 미백효과 (Kim 2008) 등이연구되었다. 또한둥굴레속의근경은점액질이풍부하고전분질, 아미노산, 알카로이드등의성분을많이함유하고있어식품학적으로도우수한것으로보고되었다 (Lee et al. 2000). 말린근경을볶았을때구수한풍미가더해져예로부터전통차로많이음용되었다 (Park et al. 2000). 이러한이유로근래에는전통차중둥굴레차가녹차다음으로많이음용되고또한근경의수요도늘어날것으로전망하고있다 (Ryu et al. 1995). 그리고본연구의대상종인층층둥굴레의새순과여린줄기는식약청식품원재료서비스에등록된식용가능식물자원이여서앞으로다양한생물소재로도활용이가능할것으로보여진다 (Lee et al. 2000; Jang et al. 2004). 이러한식 의학적가치에비해둥굴레속의증식에관한연구는매우미비한실정이다. 둥굴레종자의발아에미치는저장기간, 온도및 priming 처리의영향에관한연구 (Jang and Lee 2007) 만수행되었을뿐조직배양등개체증식에관한전반적인연구는국내외적으로전무한실정이다. 한편, 층층둥굴레는경제개발에따른급속한환경파괴와전지구적기후변화등으로인해자생지가교란되면서법정보호식물 ( 멸종위기야생식물 2 급 ) 로지정되어보호를받았다. 하지만층층둥굴레는환경부에서 2017 년멸종위기야생생물목록재개정시자생지내개체수증가등의이유로목록에서제외가되어관심종으로분류되었다. 우리는층층둥굴레가멸종위기야생생물에서해제되기전부터그들의증식과보전을위하여자생지조사와더불어생리적특성에대해면밀히조사를하고있었다. 또한증식과보전연구의일환으로종자번식, 기내증식등의지속적인연구도수행하고있다. 따라서본연구는층층둥굴레의세포에서유래된재분화개체의증식법을연구하여또다시멸종위기에처하지않도록복원에대한안전한가이드라인을제시하고, 생물소재활용을위한대량증식매뉴얼개발의기초자료를확보하고자본연구를수행하였다. 재료및방법 식물재료및배양조건 본실험에사용된층층둥굴레 (Polygonatum stenophyllum Maxim) 는여주에위치한황학산수목원에서종자와 3 년생, 5 년생개 체를분양받았고, 3 년생, 5 년생개체는근경을분리하여살균후기내도입하였다. 층층둥굴레근경의살균방법은분리한 3 년생, 5 년생근경에뭍은이물질을흐르는물에서수세하고멸균된증류수로 1 차세척한다음 70% ethanol 에 1 분간표면살균한후, 1% NaOCl 용액에침지하여 20 분간흔들어주었다. NaOCl 용액을제거하기위해멸균된증류수로 5 회수세한후배지에치상하였다. 종자도위와동일한방법으로살균하여 1/3MS 배지에올린후발아된개체의근경을실험에사용하였다. 모든배양은온도 22± 1 C, 광주기 16/8 시간, 광도 46 µmol m -2 s -1 의배양실에서배양하였다. 사용된배지는 MS (Murashige and Skoog 1962) 배지에 30 g L -1 sucrose (Duchefa, Netherland), 4.0 g L -1 gelrite (Duchefa, Netherland), ph 5.7) 를기본으로제조하고옥신류및사이토키닌호르몬을첨가하여페트리디쉬 (12 5 cm) 에 50 ml 씩, 4 각 plastic 배양병 (SPL, Korea) 에각각 100 ml 씩분주하였다. 층층둥굴레연생별근경의캘러스유도 층층둥굴레의 1 년, 3 년, 5 년생근경절편으로부터캘러스를유도하기위한배지는 MS 기본배지에옥신류호르몬인 2,4-D (2,4-dichlorophenoxy acetic acid, Duchefa, Netherland) 를각각 0, 0.1, 0.5, 1.0, 1.5 mg L -1 을첨가하고 sucrose 30 g L -1, gelrite 4.0 g L -1, ph 5.8 로조정하고 121 C, 20 분간고온고압의증기멸균을한다음, 페트리디쉬에 50 ml 씩분주하여제조하였다. 준비된배지위에 1 1 cm 로절단한 1 년생, 3 년생, 5 년생층층둥굴레근경을배지별로 30 개씩 3 반복하여치상한다음 28 암조건에서 5 주간배양하고 2 주간동안은 1 회계대배양을하였다. 배양 5 주후에연생별근경절편으로부터캘러스유도율을조사하였다. Auxin 류호르몬처리농도별부정아유도 층층둥굴레캘러스유래의부정아유도조건을조사하기위해호르몬이첨가되지않은 MS 배지에서 2 회에걸쳐계대배양한캘러스를절단하여 100 ~ 200 mg 취해옥신류호르몬인 IAA (Indole acetic acid, Duchefa, Netherland), IBA (Indole butiric acid, Duchefa, Netherland), 2,4-D, NAA (Naphthalene acetic acid, Duchefa, Netherland) 를각각 0, 0.1, 0.5, 1.0 mg L -1 씩처리한 1/2MS 배지 (20 g L -1 sucrose, 4.0 g L -1, gelrite, ph 5.7) 에 20 개씩 5 반복치상하여 4 주간배양한후처리구당부정아유도율을조사하였다. BA 와 Kinetin 처리에따른부정아신장 층층둥굴레캘러스유래부정아의줄기신장을조사하기위해부정아를호르몬이첨가되지않은 MS 배지에서 2 주간전배양하였다. 그런다음 BA (Benzylaminopurine, Duchefa, Ne-

268 J Plant Biotechnol (2018) 45:266 272 therland) 와, Kinetin (Duchefa, Netherland) 을각각 0.5, 1.0, 3.0 mg L -1 씩처리한 1/2MS 배지 (20 g L -1 sucrose, 4.0 g L -1 gelrite, ph 5.

3 268 J Plant Biotechnol (2018) 45: therland) 와, Kinetin (Duchefa, Netherland) 을각각 0.5, 1.0, 3.0 mg L -1 씩처리한 1/2MS 배지 (20 g L -1 sucrose, 4.0 g L -1 gelrite, ph 5.7) 에 20 개씩 3 반복치상하여 4 주간배양한후처리구당신초신장에따른길이를측정하였다. 광주기 16/8h, 온도 22 ± 1 C 에서 4 주간배양한후부정아줄기의길이를조사하였다. 발근및기내증식 층층둥굴레캘러스유래의부정아의신초가자란후뿌리의신장을살펴보기위하여옥신류호르몬인 NAA, IBA, 2,4-D 를각각 0, 0.1, 0.5, 1.0 mg L -1 씩처리한 1/2MS 배지 (20 g L -1 sucrose, 4.0 g L -1 gelrite, ph 5.7) 에 4 개씩 5 반복치상하여 4 주간배양한후처리구당뿌리신장길이를측정하였다. 통계분석 모든데이터는평균 ± 표준편차로표시하였고, 집단간변이차이를알아보기위해서 one-way ANOVA 를실시하여, 유의성이있는경우 Duncan s multiple range test 로사후검증을실 시하였다. 통계적유의성은 P<0.05 로설정하여분석하였다. 결과및고찰 연근경별, 호르몬종류및농도별층층둥굴레캘러스유도 대량재배조건이확립되지않은야생식물의조직배양을통한대량증식체계확립을위해서는초기배양재료의선택이중요하다. 층층둥굴레는야생에서채취가어렵고 2 년발아를한다고알려져있어조직배양을통한야생복원과생물소재로활용하기가매우까다롭다. 따라서조직배양을통한원활한식물체생산을위해본연구에서는우선근경의노화정도에따라캘러스유도율을조사하였다. 층층둥굴레의연근경별캘러스유도에미치는각각의호르몬종류와농도를알아보기위해 1 년 (Fig. 1A) 생, 3 년 (Fig. 1B) 생, 5 년 (Fig. 1C) 생근경을절단하여옥신류호르몬이첨가된 MS 배지에배양한결과, 호르몬무처리구에서는캘러스가전혀유도되지않았다 (Fig. 1D, Table 1). 하지만근경의생육기간별캘러스 Fig. 1 Callus induction of P. stenophyllum. A: 1-year rhizome (scale bar, 2 cm), B: 3-years rhizome (scale bar, 4 cm), C: 5-years rhizome (scale bar, 4 cm), D: Without 2,4-D (scale bar, 50 mm), E: 1-year rhizome in the presence of 0.5 mg L -1 2,4-D (scale bar, 30 mm), F: 2-years rhizome in the presence of 0.5 mg L -1 of 2,4-D (scale bar, 20 mm), G: 5-years rhizome in the presence of 0.5 mg L -1 of 2,4-D (scale bar, 20 mm), H: Yellowish callus induction in the presence of 0.5 mg L -1 of 2,4-D after 6 weeks of culture (scale bar, 2 cm), I: Development of greenish callus in the presence of 1.0 mg L -1 of BA and 0.1 mg L -1 of NAA after 6 weeks of culture (scale bar, 2 cm)

4 J Plant Biotechnol (2018) 45: Table 1 Effect of varying concentrations of 2,4-D and age of rhizome on callus induction of 5 weeks old culture of P. stenophyllum in MS medium supplemented with 30 g L -1 of sucrose and 4.0 g L -1 of gelrite Concentration of 2,4-D (mg L -1 ) Callus induction (%) 2,4-D concentrations (mg L -1 ) 1-year rhizome 3-years rhizome 5-years rhizome ±5.2b 44.8±3.4b 21.6±7.1b ±6.4a 87.6±7.2a 50.6±12.1a ±7.2c 38.4±5.3c 21.4±7.8b ±4.5d 12.2±2.1d 6.4±2.2c 유도는 2,4-D 호르몬이첨가하여배양하면 1 년생 (Fig. 1E), 3 년생 (Fig. 1F), 5 년생 (Fig. 1G) 근경모두에서노란색의캘러스가유도되었다. 캘러스유도율은 0.5 mg L -1 2,4-D 가첨가된배지에 3 년생근경을배양할경우 87% 로가장높게조사되었다 (Table 1). 한편, 2,4-D 의농도가높아지면캘러스유도율이감소하는경향을보였다 (Table 1). 각각의층층둥굴레근경으로부터유도된캘러스를핀셋으로절취하여 0.5 mg L -1 2,4-D 가첨가된배지에 6 주간배양한결과구형의노란색캘러스덩어리가다량으로증식된다 (Fig. 1H). 6 주후증식된캘러스를 1.0 mg L -1 BA 와 0.1 mg L -1 NAA 가혼합처리된배지에치상하면경화된초록색의캘러스가유도되고 (Fig. 1I) 이렇게유도된캘러스를절단하여부정아유도의실험재료로사용하였다. 층층둥굴레를비롯한둥굴레속식물의근경을이용한캘러스유도는현재까지연구된바가없다. 동속은아니지만백합과의조경수종인소엽맥문동의캘러스유도를통한식물체재분화연구에서도 1.0 mg L -1 2,4-D 처리구에근경을배양하면 91% 의캘러스유도율을보인다고하여본연구와비슷한결과를보였다 (Bae and Yoon 2013). 층층둥굴레캘러스유래의부정아기내증식 경화된층층둥굴레캘러스 (Fig. 1I) 를절단하여부정아를유도하기위해옥신류호르몬종류와농도가다르게처리된배지에서 8 주간배양한결과, 옥신류호르몬이첨가되지않은대조구 (1/2MS 배지 ) 에서는부정아가유도되지않았지만 (Table 2), 0.5 mg L -1 NAA 처리구에서는배양 4 주후부터절단부위에캘러스가만들어지고 6 주가경과되면부정아가유도되었다 (Fig. 2A). 한편, NAA 를제외한다른옥신류처리구에서는부정아유도율이 5% 미만으로조사되었고 (Table 2), 사이토키닌류인 BA 와 Kinetin 을처리구는전혀부정아유도가이루어지지않았다 ( 데이터미제시 ). 본실험결과층층둥굴레캘러스로부터부정아를유도하기위해서는 NAA 의처리가가장효과적인것을확인할수있었다. Matsuoka 와 Hinata (1979) 에의해 8.0 mg L -1 NAA 처리는 Solarium melongena 의배축절편으로부터부정아유도의최적조건이라고보고 한바있다. 이후종마다다르지만 BA 또는 Kinetin 의조합처리가부정아유도에효과적인것을많은연구자들에의해연구되고있다 (Meyer and van Staden 1998; Shibli and Ajlouni 2000; Koh 2005; Madubanya et al. 2006). 층층둥굴레의캘러스로부터유도된부정아의신장과발근에적합한호르몬의종류와농도를조사하였다. 부정아의신장에적합한호르몬처리를알아보기위해 1/2MS 배지에 0.1, 0.5, 1.0 mg L -1 BA 와 Kintin 을단용처리하여 4 주간배양한결과는 Table 3 과같다. 층층둥굴레부정아를 0.1 mg L -1 BA 가첨가된배지에치상하여배양할경우, 4 주후에신초길이는 3 cm 로신장하였고 (Fig. 2B), BA 의농도가높아질수록감소하는경향을보였다. 신초가신장된후호르몬이첨가되지않는 1/2MS 배지로옮겨 4 주간배양하면본엽이신장되었다 (Fig. 2C). 그리고기내에서무균적으로온전한개체를생산하기위해 shoot 부위가신장된부정아에서발근에적합한배지를조사하였다. 발근에적합한호르몬처리농도를알아보기위해신장된부정아를옥신류호르몬이농도별로첨가된배지에치상하여 4 주간배양한결과, 호르몬을처리하지않은대조구에서뿌리의길이가 11 cm 로조사되었다 (Table 4). 층층둥굴레부정아유래의뿌리는배양 2 주가경과하면 4~5 개가배지의아래부분으로길게 1 차신장을한다. 신장이후근단부분부터곁뿌리가유도된다 (Fig. 2C). Malabadi 등 (2004) 은 Vanda coerulea 에서, Emst(1994) 는 Doritaenopsis 와 Phalaenopsis 에서식물생장조절제의농도가높은배지에서생장, 분화된신초일수록발근율이감소한다고하였다. 일반적으로기내배양체 ( 초본, 목본류 ) 의발근은토양순화시토양활착을양호하게하기위한전단계로정상적인기내발근이이루어져야토양순화후배양체가생존할확률이높아지는것이다. 하지만내생옥신 (endogenous auxin) 이많은식물의경우식물생장조절물질이첨가되지않은배지에서도발근이잘이루어지는것으로알려져있다. 특히목본류인개느삼 (Echinosophora koreansis) 과팔손이 (Fatsia japonica), 초본류인고추나물 (Hypericum erectum) 의경우옥신류식물생장조절물질무첨가배지에서도 20 ~ 50% 이상의발근률을보였다 (Choi et al. 2005; Kim et al. 2006; Moon and Kim 2008). 층

270 J Plant Biotechnol (2018) 45:266 272 Fig. 2 Plant regeneration from callus derived from P. stenophyllum. A: Adventitious shoot induction from callus in 1/2MS medium supplemented with 0.

5 270 J Plant Biotechnol (2018) 45: Fig. 2 Plant regeneration from callus derived from P. stenophyllum. A: Adventitious shoot induction from callus in 1/2MS medium supplemented with 0.5 mg L -1 of NAA (scale bar, 80 mm), B: Shoot elongation from adventitious shoot in 1/2MS medium supplemented with 0.1 mg L -1 of BA (scale bar, 2 cm), C-D: Shoot conversion and rooting in 1/2MS without PGR's, after 4 weeks of culture (scale bar, 3 cm) Table 2 Effect of varying concentrations of auxin on adventitious shoot induction of 4 weeks old culture of P. stenophyllum callus in 1/2MS medium supplemented with 20 g L -1 of sucrose and 4.0 g L -1 of gelrite Concentration of auxin (mg L -1 ) Adventitious shoot induction (%) IAA IBA NAA 2,4-D Adventitious shoot induction (%) ±0*g ±0.2g ±0.2g ±0.1g ±0.8e ±0.6d ±0.6f ±2.2c ±1.5a ±2.1b ±0.2g ±0.1g ±0.3g

6 J Plant Biotechnol (2018) 45: Table 3 Effects of varying concentrations of BA and kinetin on adventitious shoot elongation in 4-weeks old culture of P. stenophyllum in 1/2MS medium supplemented with 20 g L -1 of sucrose and 4.0 g L -1 of gelrite Concentration of BA (mg L -1 ) Concentration of KT (mg L -1 ) Length of the adventitious shoot (cm) 0 0 0±0*g ±0.2a ±0.1b ±0.1c ±0.1d ±0.1e ±0.1f Table 4 Effect of varying concentrations of auxin on adventitious root elongation in 4-weeks old culture of P. stenophyllum in 1/2MS medium supplemented with 20 g L -1 sucrose and 4.0 mg L -1 gelrite Concentration of auxin (mg L -1 ) Length of the adventitious root (cm) IAA IBA NAA 2,4-D Length of adventitious root (cm) ±1.8*a ±0.1b ±0.1b ±0.1b ±0.1b ±0.1b ±0.1b ±0.1b ±0.1b ±0.1b ±0.1b ±0.1b ±0.1b 층둥굴레의발근도호르몬무첨가배지에서이루어졌다. 이는위의결과들과비교하여수종은다르지만층층둥굴레도내생옥신이많아외부호르몬처리없이도발근이이루어졌음을알수있었다. 따라서본연구결과에서층층둥굴레연생별근경, 옥신종류및농도에따른최적의캘러스유도조건을조사하였고, 유도된캘러스를이용하여분화에최적조건을조사하였다. 또한발근에필요한최적의조건을조사하여성공적인기내배양을통한재분화시스템을확립하였다. 이러한결과는향후층층둥굴레의개체보전과생물소재활용을위한소재확보에중요한기초자료로사용될것으로생각된다. 적요 본연구는백합과에속하며환경부에서는멸종위기종리스트중관심종으로분류된층층둥굴레의기내증식에미치는연근별, 호르몬종류와농도별배양환경을조사하기위해수행되었다. 효과적인캘러스유도조건을알아보고자 0.1, 0.5, 1.0, 1.5 mg L -1 의 2,4-D 가첨가된 MS 배지에 1, 3, 5 년생층층둥굴레근경을치상한후에캘러스유도율을조사한결과, 0.5 mg L -1 2,4-D 배지에 2 년생근경배양한배지에서 87% 의캘러스유도율을보였다. 1 년생근경은 67%, 5 년생근경은 50% 로조사되었다. 층층둥굴레캘러스유래의부정아유도는 0.5 mg/l NAA 처리구에서 57% 로조사되었다. 호르몬이첨가되지않은대조구에서는부정아가유도되지않았다. 층

7 272 J Plant Biotechnol (2018) 45: 층둥굴레부정아의신장과발근에적합한호르몬의종류와농도를조사하였다. 층층둥굴레부정아신장의최적조건은 0.1 mg L -1 BA 호르몬이첨가된배지이고 BA 의농도가높아질수록감소하는경향을보였다. 그리고기내에서무균적으로온전한개체를생산하기위해 shoot 부위가신장된부정아에서발근에적합한배지를조사하였다. 발근에적합한조건은호르몬이첨가되지않은대조구이며뿌리의길이는 11 cm 로조사되었다. 본연구결과에서층층둥굴레연생별근경, 옥신종류및농도에따른최적의캘러스유도조건을조사하였고, 유도된캘러스를이용하여분화에최적조건을조사하였다. 또한발근에필요한최적의조건을조사하여성공적인기내배양을통한재분화시스템을확립하였다. 이러한결과는향후층층둥굴레의개체보전과생물소재활용을위한소재확보에중요한기초자료를제공할것으로생각된다. 사사 본논문은환경부국립낙동강생물자원관 2018 년 서식지외보전기관운영 사업의지원을받아수행되었습니다. References Ahn DK (2003) Illustrated Book of Korean Medicinal Herbs. Kyo-Hak Publishing Co., Ltd. Seoul pp Bae KH, Yoon ES (2013) Callus induction and plant regeneration from Ophiopogon japonicus (L.f.) Ker Gawl in ornamental species. Kor J Nature Conservation 7(1):35-41 Chang YD, Lee CH (2007) Effect of storage duration, temperature and priming treatment on seed germination of Polygonatum odoratum var. pluriflorum. Kor J Plant Res 20(5): Choi KM, Hwang SJ, Ahn JC, Lee HY, Kim JH, Hwang B. (2005) In vitro propagation from axillary bud explants of Fatsia japonica Deene. et Planch. Kor J Med Crop Sci 13(6): Emst R (1994) Effects of thidiazuron on in vitro propagation of Phalaenopsis and Doritaenopsis (Orchidaceae). Plant Cell Tiss Org Cult 39: Jang JS, Jung JC (2010) Anti-adipogenic effect of kaempferol. a component of Polygonati rhizoma. J Kor Oriental Med 31(2): Jang KH, Park JM, Kang JH, Lee ST (1998) Growth and flowering characteristics of Polygonatum spp. Kor J Medicinal 6(2): Jang KY, Song GW, Chung JI, Kang JH (2004) Analysis of genetic relationships of Korean native Polygunatum spp. Kor J Medicinal Crop Sci 12(3): Jeong YS, Jeong JC (2011) Anti-tumor effect of kaempferol, a component of Polugonati rhizoma, in lung cancer cells. Kor J Oriental Physiology & Pathology 25(5): Ko JH, Jeon UJ, Kwon HS, Yeon SU, Kang JH (2015) Anti-obesity effects of ethanolic extract of Polygonatum sibiricum rhizome in high-fat diet-fed mice. Kor J Food Sci Technol 47(4): Koh JC (2005) Callus culture and plant regeneration from shoot apex of Iris Sanguinea Donn ex. Horn. J Plant Biotechnol 3(1):93-98 Kim IC (2008) Antioxidative properties and whitening effects of the Polygoni Multiflori Radix, Polygonati Rhizoma and Ephedrae Herba. Kor Oil Chemists' Society 25(4): Kim JS, Lee AR, Roh SS, Kwon OJ, Seo YB (2016) Antioxidant and anti-physical fatigue effects of Polygonati rhizoma and steamed Polygonati rhizoma. Kor J Herbol 31(3):49-57 Kim ML, Nam DW, Ahn JC, Hwang B (2006) Micropropagation of Hypericum erectum by axillary bud culture. Kor J Med Crop Sci 14:23-26 Kim SD, Park SY, Kim TJ, Cheong IM, Kim SM (1996) Studies on the promoting of seed germination of Adenophora triphylla var. japonica Hara. Kor J Plant Res 9: Lee GD, Lee JE, Kwon JH (2000) Application of response surface methodology in food industry. Food and Industry 33:33-45 Madubanya LA, Makunga NP, Fennell CW (2006) Dierama luteoalbidum : liquid culture provides an efficient system for the ex situ conservation of an endangered and horticulturally valuable plant. S Afr J Bot 72: Malabadi RB, Mulgund GS, Nataraja K (2004) Efficient regeneration of Vanda coerulea, an endangered orchid using thiadiazuron. Plant Cell Tiss. Org Cult 76: Matsuoka H, Hinata K (1979) NAA-induced organogenesis and embryogenesis in hypocotyl callus of Solarium melongena L. J Exp Bot 30: Meyer HJ, van Staden J (1998) In vitro multiplication of Ixia fkexuosa. Hort Sci 23: Moon HK, Kim YW (2008) In vitro propagation of a rare and endangered species, Echinosophora koreensis Nakai. by axillary bud culture. Kor J Plant Biotechnol 35: Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: Park NY, Jeong YJ, Lee GD, Kwon JH (2000) Monitoring of maillard reaction characteristics under various roasting conditions of Polygonatum odoratum root. Kor Soc Food Sci Nutr 29(4): Ryu KC (1995) Optimization of the roasting conditions for high-quality Polygonatum odoratum tea. Kyungbuk National University, pp 39 Shibli RA, Ajlouni MM (2000) Somatic embryogenesis in the endermic Black Iris. Plant Cell Tiss Org Cult 61:15-21

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<31335F28C0CFB9DD29B9E8B1E2C8AD5FC3D6C1BE2E687770> J Plant Biotechnol (2017) 44:325 329 DOI:https://doi.org/10.5010/JPB.2017.44.3.325 ISSN 1229-2818 (Print) ISSN 2384-1397 (Online) Research Article 멸종위기수생식물인매화마름 (Ranunculus kazusensis Makino) 의기내신초증식 박민완