07-왜성(MJ)

Journal of Radiation Industry 5 (3) : 231~236 (2011) Review Paper 왜성변이품종 꼬마 무궁화의캘러스유도및지상부형성에식물생장조절물질이미치는영향 이지연 강은정 김상훈 김동섭 김진백 하보근 강시용 * 한국원자력연구원정읍방사선과학연구소 The Effect of Plant Growth Regulators on Callus Induction and Shoot Regeneration from the Dwarf Type Variety, Hibiscus syriacus L.var. Ggoma Ji Yeon Lee, Eun Jeong Kang, Sang Hoon Kim, Dong Sub Kim, Jin-Baek Kim, Bo-Keun Ha and Si-Yong Kang* Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Korea Abstract - Ggoma is a new Hibiscus dwarf type variety developed by gamma irradiation at the Korea Atomic Energy Research Institute (KAERI). This study was conducted to determine the best optimum cultural callus formation and shoot regeneration condition. Sterilized leaf tissues were cultured on MS (Marashige and skoog s) medium containing 3% sucrose, 0.8% agar with different concentration and combination of TDZ, 2, 4-D, KT, BA, and 2iP for 4 weeks in vitro culture. MS medium containing 2,4-D 0.1 mg l - 1 and BA 0.5 mg l - 1 were most effective on callus formation and growth. After 4 weeks, callus was transferred on BA (0.5, 1, 1.5 mg l - 1 ) and TDZ (0.1, 0.2, 0.3 mg l - 1 ) for shoot formation. The best condition for inducing the shoot from callus was BA 1.5 mg l - 1 and TDZ 0.3 mg l - 1. This result will be useful for the rapid multiplication of Hibiscus syriacus L.var. Ggoma. Key words : Callus formation, Dwarf, Hibiscus syriacus, Radiation, Shoot formation 서 무궁화는나라꽃이며아욱과에속하는낙엽활엽관목으로 7월부터꽃이피기시작하여늦가을까지계속적으로꽃이피며한국, 인도, 중국및시베리아등지에널리분포한다. 높이는 2~3m이며잎은난형으로밑은쐐기 론 * Corresponding author: Si-Yong Kang, Tel. +82-63-570-3310, Fax. +82-63-570-3329, E-mail. sykang@kaeri.re.kr 모양이고세갈래로갈라지며가장자리는톱니모양이다. 무궁화는재배면적이많이필요하고육종연한이 5~7 년정도소요되는점이신품종육성의장애가되고있기때문에광범위하고새로운연구가필요하다. 현재까지의무궁화육종은국내자생종의수집, 품종간교잡방법, 도입육종, 콜히친처리에의한배수체육종이주를이루었다 (Kim 1962; Kim 1979; Yu et al. 1979). 또한무궁화의자가불화합성특성과유전적이형성은교배육종을시도할경우분리가불규칙적으로나타나는특성이있다 231

232 이지연 강은정 김상훈 김동섭 김진백 하보근 강시용 (Song 2006). 이러한교배육종방법의제약을극복할수있는방안으로방사선을이용한돌연변이육종방법이있다. Staller (1928) 는방사선을보리에처리하면인위적으로돌연변이를일으킬수있는사실을밝혀내었고다양한작물로부터방사선을이용하여육종기술에적용한연구를보고하였다. 또한방사선을이용한돌연변이연구로는품종별방사선감수성, 급조사및완조사에따른방사선감수성의선량별비교, 변이율증대를위한방사선처리시기와재료선정및키메라생성등에관한연구가있다 (Van Harten et al. 1981; Shin et al. 1998). 타작물의경우국화에서방사선을이용하여화색, 화형, 꽃색깔등의형질을개선한보고가있다 (Broertjes 1996). 그러나무궁화의방사선돌연변이육종연구는현재까지미비한실정이다. 국내에서는 1991년한국원자력연구원에서화색, 화형등의형질개선및관상가치가향상된무궁화신품종개발을위해홍단심-2종자에 120 Gy 처리하여 2006년극왜성으로선발된개체 꼬마 품종을육성하였다 (Song 2006). 꼬마 의특징으로일반무궁화잎의크기는 1/2 ~1/3에불과하며꽃의색은보라색에자단심계통이며꽃의크기는일반무궁화꽃지름보다훨씬작은 4~5 cm로매우작은꽃으로앙증스럽다 (Song 2006). 꼬마는불임성으로종자번식이곤란하다. 또한, 왜성이기때문에마디길이가짧아삽수체에의한번식율도다른품종에비교하여떨어진다. 기내조직배양은신품종을개발했을때대량증식과품종유지를가능하게하는방법중하나이다. 조직배양에사용되는방법으로는캘러스를유도하여재분화시키는방법과바로신초를형성시키는방법등이있다. 무궁화조직배양은배배양, 원형질체배양, 삽수배양, 잎배양등이있다 (Chung and Jo 1986; An and park 1992; Kim 1998a; Brian 2008). 조직배양시식물생장조절제로는 BA, NAA, Kinetin, 2, 4-D 등이이용되고있다. 특히, 하와이무궁화삽수에서는 0 μm~5 μm BA 호르몬처리가최적의지상부를유도시켰다 (Sunil 2009). 홍화랑 잎배양에서는 TDZ 0.01 mg l -1 호르몬처리시높은캘러스형성률을보였고 TDZ와 BA의조합으로신초를형성시켰다 (Yoo 1996). 또한캘러스유도후 NAA를사용하여지상부를발생시켰고 IBA 0.5 mg l -1 이용하여재분화를유도한바있다 (Yang 1995). 본연구는왜성품종 꼬마 의대량증식시스템을구축하기위해어린잎을이용하여식물생장조절제의농도및조합이캘러스형성과신초형성에미치는영향을알아보고이를통해무궁화의산업적가치를증대시키는목적으로연구가수행되었다. 재료및방법 1. 실험재료유리온실에서재배중인 꼬마 (Hibiscus syriacus L.var. Ggoma) 의어린잎을채취하여 70% 에탄올에서 20초, Tween 20을첨가한차아염소산나트륨 (NaOCl) 0.8% 에서 20분소독후멸균수로 4회세척하였다. 잎은가운데엽맥을중심으로 0.5 cm 0.5 cm 크기로잘라사용하였다. 2. 캘러스배양조건배지조성은 MS 기본배지에 3% sucrose 및 0.8% agar 를사용하였다. 식물생장조절제가캘러스형성에미치는영향을알아보기위해 MS 기본배지에 TDZ, 2, 4-D, BA 를각각0.1, 0.5, 1, 1.5, 2 mg l -1 의농도로조합한배지를사용하였다. 또한 2, 4-D 0.1 mg l -1 에 KT, BA, 2iP를각각 0.1, 0.3, 0.5, 1, 1.5 mg l -1 의농도로조합하였고 TDZ 0.5 mg l -1 에 KT, BA, 2iP를각각0.1, 0.3, 0.5, 1, 1.5 mg l -1 의농도로조합한배지를사용하였다. 치상한배지는 90 mm 15 mm 일회용 petri-dish에 40 ml씩분주하였으며어린잎절편체는 petri-dish당 16개씩 5반복으로수행하여 4주간배양하였다. 배양 4주후에캘러스형성률과색깔및성장정도를조사하였다. 기내배양환경은온도 24 ±1 C, 일장주기 16시간, 광도는 27 μmol m -2 sec -1 형광조명을사용하여실험을수행하였다. 3. 지상부배양조건캘러스가유도된후잎의재분화조건을탐색하기위해 MS 기본배지에 BA (0.5, 1, 1.5 mg l -1 ) 와 TDZ (0.1, 0.2, 0.3 mg l -1 ) 를조합한배지를사용하였다. 진녹색을띄는단단한배양체캘러스를선택하여배지는 90 mm 15 mm petri-dish당 20개씩 3반복수행후 4주간배양하였다. 캘러스배양 4주후에신초형성률과성장정도를조사하였고, 캘러스에잎이 1개이상나온것을조사하였으며하나의캘러스에여러개의신초가형성된것도하나로간주하였다. 기내배양환경은온도 24±1 C, 일장주기 16시간, 광도는 27 μmol m -2 sec -1 형광조명을사용하여실험을수행하였다. 결과및논의 꼬마 의잎으로부터 callus 형성을유도하기위하여 TDZ, 2, 4-D, BA 단일호르몬처리를한결과 BA 0.5 mg

꼬마 캘러스유도및지상부형성 233 A B Fig. 1. Two different callus types on the treatments of single plant growth regulator. A: White+Yellow callus, B: Yellow+Green callus. Table 1. Effect of three different plant growth regulators at five combinations on callus formation rate from leaf disc culture of Hibiscus syriacus L.var. Ggoma at 4 weeks after culture Treatment Callus Callus (mg l -1 ) formation (%) growth a Callus color Control b 0.0 (0/80) - - TDZ 0.1 38.8 (31/80) ++ Yellow+Green 0.5 51.3 (41/80) +++ Yellow+Green 1.0 42.5 (34/80) ++ Yellow+Green 1.5 23.8 (19/80) + Yellow 2.0 12.5 (2/80) + Yellow 2,4-D 0.1 0.0 (0/80) - - 0.5 1.3 (1/80) + White+Yellow 1.0 2.5 (2/80) + White+Yellow 1.5 16.3 (13/80) ++ White+Yellow 2.0 35.0 (28/80) ++ White+Yellow BA 0.1 10.0 (8/80) + Yellow+Green 0.5 85.0 (68/80) ++++ Yellow+Green 1.0 57.5 (46/80) +++ Yellow+Green 2.0 0.0 (0/80) - - a -: none; +: low; ++: medium; +++: high; ++++: very high b Control: only MS l -1 에서 85.0% 의비교적높은캘러스형성률을보였고 TDZ 0.5 mg l -1 에서 51.3% 의캘러스형성률을보였으며 (Table 1), 캘러스색이노란색과녹색이혼합된것을확인하였다 (Fig. 1B). 2, 4-D의경우캘러스형성률이가장낮았으며캘러스색은흰색을나타냈다 (Table 1, Fig. 1A). 단일호르몬처리를한결과캘러스형성은되었으나재분화에적합한녹색캘러스를얻지못하였다. 이와같은결과는 홍화랑 품종이 TDZ 0.01 mg l -1 에서 100% 의캘러스형성률을나타냈고 (Kim 1998a), 4주동안어린잎배양결과 TDZ 0.1 mg l -1 에서캘러스형성률이높 Table 2. Combined effect of two different growth regulators at five combinations on callus formation rate from leaf disc culture of Hibiscus syriacus L.var. Ggoma at 4 weeks after culture Treatment (mg l -1 ) Callus Callus formation (%) growth a Callus color Control b 0.0 (0/80) - - TDZ 0.5+ KT 0.1 0.0 (0/80) - - 0.5 0.0 (0/80) - - 1.0 0.0 (0/80) - - BA 0.1 0.0 (0/80) - - 0.3 7.5 (6/80) + Green 0.5 25 (20/80) + Green 1.0 61.25 (49/80) +++ Green 1.5 62.5 (50/80) +++ Green 2iP 0.1 0.0 (0/80) - - 0.5 0.0 (0/80) - - 1.0 0.0 (0/80) - - 2,4-D 0.1+ KT 0.1 0.0 (0/80) - - 0.5 0.0 (0/80) - - 1.0 0.0 (0/80) - - BA 0.1 35.0 (28/80) ++ Green 0.3 90.0 (72/80) ++++ Yellow+Green 0.5 96.2 (77/80) ++++ Green 1.0 87.5 (70/80) +++ Green 1.5 0.0 (0/80) - Green 2iP 0.1 83.7 (67/80) ++++ Yellow+Green 0.5 45.0 (36/80) ++ Yellow+Green 1.0 32.5 (26/80) ++ Yellow+Green 1.5 17.5 (14/80) + Yellow+Green a -: none; +: low; ++: medium; +++: high; ++++: very high b Control: only MS

234 이지연 강은정 김상훈 김동섭 김진백 하보근 강시용 A B Fig. 2. Callus induction and callus formation from leaf disc of Hibiscus syriacus L.var. Ggoma on 2,4D 0.1+BA 0.5 mg l -1 medium. A: Callus induction from the leaf disc for 2 weeks; B: Development of a callus formation for 4 weeks. A B C Fig. 3. Callus induction and development formation from leaf disc of Hibiscus syriacus L.var. Ggoma on 2,4 D 0.1+BA 0.5 mg l -1 medium at 2 and 4 weeks after culture. A: Callus induction from the leaf disc at 2 weeks after culture; B: Development of the callus at 4 weeks after culture; C: Shoot development from callus. 은것으로보고된 (Yoo 1996) 기존연구결과와는차이가있다. 이경우방사선을이용하여만든돌연변이품종으로기존연구에서사용된무궁화와는다르게극왜성의특징을가지기때문인것으로사료된다. 이렇듯단일호르몬처리만으로는캘러스형성의어려움이있기때문에캘러스형성률이더좋은호르몬조합을찾기위하여 TDZ 0.5 mg l -1 에 KT, BA, 2iP (0.1, 0.3, 0.5, 1, 1.5 mg l -1 ) 조합을사용하였으며 2, 4-D 0.1 mg l -1 와 KT, BA, 2iP (0.1, 0.3, 0.5, 1, 1.5 mg l -1 ) 의조합을사용하였다. 그결과잎배양2주후2, 4-D 0.1 mg l -1 와 BA 0.5 mg l -1 를조합한배지에서 96.25% 로높은캘러 스형성률을보였으며, 캘러스의색은진녹색을띄고캘러스성장정도도가장높았다 (Table 2). 잎배양2주후녹색의캘러스가잎앞면에서형성되었고 4주후에는캘러스가더성장한것을볼수있었으며신초유기에적합한캘러스를얻을수있었다 (Fig. 2). 재분화유도를위한캘러스의상태는단단하며녹색을띄는것을선택하여배양해야한다고보고된바있다 (Kim 2000). 잎배양후유도된캘러스를재분화배지에옮길때잎배양2주경과된녹색캘러스가 4주경과된캘러스보다잎과분리가잘되어온전한캘러스만배양하는데더효과적인방법이라고할수있다 (Fig. 2).

꼬마 캘러스유도및지상부형성 235 Table 3. Shoot formation from callus of Hibiscus syriacus L.var. Ggoma on the BA and TDZ combination at 4 weeks after culture Treatment (mg l -1 ) Shoot formation (%) Shoot growth a Control b 0.0 (0/60) - BA 0.5+TDZ 0.1 0.0 (0/60) - 0.2 0.0 (0/60) - 0.3 1.7 (1/60) + BA 1+TDZ 0.1 0.0 (0/60) - 0.2 0.0 (0/60) - 0.3 0.0 (0/60) - BA 1.5+TDZ 0.1 0.0 (0/60) - 0.2 5.0 (5/60) + 0.3 18.3 (11/60) ++ a -: none; +: low; ++: medium; +++: high; ++++: very high b Control: only MS 배지에서는 96.25% 로높은캘러스형성률을보였다. 캘러스가형성된지 4주후신초를유도하기위해 BA와 TDZ를조합하여배양한결과 BA 1.5 mg l -1 와 TDZ 0.3 mg l -1 에서 18.3% 의신초가형성되었다. 이러한실험결과는기존의무궁화캘러스및신초형성조건과는차이가나는것을확인하였다. 본연구의결과는기내배양을통한 꼬마 의대량증식시스템을구축하는데중요한정보로활용될수있을것이며추후유도된캘러스에방사선조사를통하여돌연변이신품종을얻을수있는기초자료로활용될것으로기대된다. 사사 Agarwal (1992) 은목본류의경우캘러스에서신초를유도하는데 BA와 TDZ가효과적임을보고하였다. 따라서본연구에서도신초를유도하기위하여 BA (0.5, 1, 1.5 mg l -1 ) 와 TDZ (0.1, 0.2, 0.3 mg l -1 ) 조합을사용하였다. 그결과 BA 1.5 mg l -1 와 TDZ 0.3 mg l -1 에서 18.3% 의신초가형성되었다 (Table 3, Fig. 3). 본연구결과에서 꼬마 의신초형성률이높은수치는아니지만 Preece and Huetteman (1991) 는목본류에서는재분화가어렵다고보고하였으며기존무궁화연구에서도잎배양을이용하여캘러스유도후의신초형성률이낮은것으로보고되어있다 (Kim 1998b). 그리고 NAA 0.1 mg l -1 와 IBA 0.5 mg l -1 를이용하여재분화를유도한연구결과 (Yang 1995) 를바탕으로본연구에서도유도된캘러스에서신초형성을위해 IBA, NAA를추가실험하였으나지상부가유도되지않았으며캘러스색이갈색으로변하며고사하는것을확인할수있었다 ( 자료미제시 ). 이러한결과는기존연구에서사용된품종과의차이뿐만아니라 꼬마 의주요특성인극대성이배양조건의차이를야기시키는것으로사료된다. 결론본연구에서는왜성무궁화품종 꼬마 의대량증식시스템을구축하기위해잎을이용하여식물생장조절제의농도및조합이캘러스형성과신초형성에미치는영향을알아보고자하였다. 캘러스형성을위해 TDZ, 2, 4- D, BA 단일호르몬처리, TDZ와 KT, BA, 2iP를조합및 2, 4-D와 KT, BA, 2iP를조합한결과, 단일호르몬처리에서의캘러스형성률은 TDZ 0.5 mg l -1 에서 51.3% 를나타냈으나 2, 4-D 0.1 mg l -1 와 BA 0.5 mg l -1 의조합한 본연구는교육과학기술부지원한국원자력연구원기본사업에의해수행되었으며이에감사드립니다. 참고문헌 Agarwal B, Singh U and Banerjee M. 1992. In vitro clonal propagation of tea [Camelia sinensis (L) O. Kuntze]. Plant Cell Tissue. Org. Cult. 30:1-5. An YK and Park HG. 1992. Studies on vitro shoot regeneration from hypocotyl and cotyledon in Hibiscus syriacus L. Kor. Soc. Hort. Sci. 33:458-465. Brian C, Sridevy S, Margrethe S and Renate M. 2008. In vitro culture of Hibiscus rosa-sinensis L.: Influence of iron, calcium and BAP on establishment and multiplication. Plant Cell Tissue. Org. Cult. 93:151-161. Broertjes C. 1996. Mutation breeding of Chrysanthemum. Euphytica 15:156-162. Chung JD and Jo JD. 1986. Isolation and culture of protoplast of Hibiscus syriacus and Hibiscus rosa sinensis. Kor. J Plant Tissue Culture 13:71-83. Kim CS. 1962. Some characterstics of tetraploid Hibiscus syriacus for. purpureus induced by colchicine treatment. Ann. Rep. of Inst. of Forest Genetics 2:57-68. Kim CS. 1979. Review of genetics and breeding in Hibiscus genus. Kor. J. Forestry Sci. 43:64-73. Kim EK, Yoo YK and Kim KS. 1998a. Thidiazuron-induced shoot formation of Hibiscus syriacus L. Honghwarang by suspension culture. Kor. J. Hort. Sci. Technol. 16(4): 525-527. Kim EK, Yoo YK and Kim KS. 1998b. Effect of thidiazuron on callus and multiple shoot formation in shoot-tip culture of Hibiscus syriacus L. Honghwarang. Kor. J. Hort. Sci. Technol. 16(4): 520-524.

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