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J Plant Biotechnol (2014) 41:94 99 DOI:http://dx.doi.org/10.5010/JPB.2014.41.2.94 ISSN 1598-6365 Research Article 희귀수종미선나무 (Abeliophyllum distichum Nakai.) 의기내증식및발근에미치는 LED (light emitting diode) 효과 이나념 최용의 문흥규 Effect of LEDs on shoot multiplication and rooting of rare plant Abeliophyllum distichum Nakai Na Nyum Lee Yong Eui Choi Heung Kyu Moon Received: 11 June 2014 / Revised: 14 June 2014 / Accepted: 25 June 2014 c Korean Society for Plant Biotechnology Abstract This study was conducted to elucidate the effect of light sources and explant types on in vitro shoot multiplication and rooting of a rare and endangered plant Abeliophyllum distichum. Both apical buds and axillary buds were used as explants under 4 different light sources, cool white florescent light (F), 100% blue light-emitting diode (LED) (B), 50% blue and 50% red LED mixture (BR), and 100% red LED (R). Clear difference was observed in terms of shoot proliferation by light sources types but not by position-dependent explant types. Multiple shoot induction rates were enhanced under both B and BR light sources. Spontaneous rooting was induced in shoot induction medium under B light source. Both the rates of rooting and numbers of roots per explant were higher in apical bud explants compared to axillary bud explants. Interestingly R light source stimulated shoot elongation but inhibited root development. Therefore, our results suggest that the use of apical bud explants under B or BR light sources is suitable for in vitro micropropagation of a rare and endangered plant species, Abeliophyllum distichum. Keywords Micropropagation, Lighting effect, Explants position, Conservation N. N. Lee H. K. Moon ( ) 국립산림과학원산림생명공학과 (Division of Biotechnology, Korea Forest Research Institute, Suwon 441-350, Korea) e-mail: hkmoon@forest.go.kr Y. E. Choi 강원대학교산림자원학과 (Depart. of Forestry, Kangwon Nat l University, Chuncheon 200-701, Korea) 서론 미선나무는물푸레나무과에속하며충북의진천과괴산에서자라는낙엽활엽관목으로높이가 1.5 m 에달한다. 세계적으로 1 속 1 종밖에없는식물로천연기념물제 147, 220, 221 호로지정, 보호되고있을정도로극히제한된지역에서만생육하는대표적인희귀수종중하나이다 (Lee 1976; 1990). 기내배양은삽목과같은전통적인무성번식법의대안으로유용한수단이되어왔으며 (Chen et al., 2006; Kartsonas and Papafortiou 2007). 액아 (axillary bud), 혹은정아 (apical bud) 를이용한미세번식 (micropropagation) 기술이가장보편적인방법으로사용되었다. 이러한기술은국외에서 Ceropegia candelabrum 등다수의희귀약용식물에서효과적인번식법으로사용되었으며 (Beena and Martin, 2003), 국내에서도망개나무등여러희귀식물의미세번식기술이개발되었다 (Youn et al. 1992; Moon et al. 2006; Park et al. 2003; Han et al. 2004). 광 (light) 은식물광합성의에너지원으로써식물의생존및물질생산에필수적이며, 생장과형태형성및색소형성등에관여하는조절인자로써기능을가진다. 특히광질, 광량그리고광주기등이식물의형태, 기관생장및물질생성에밀접하게관여하는것으로알려져있다 (Kozai et al. 1995; Han et al. 2001; Lee et al. 2007). LEDs (light-emitting diode) 는형광등의대체광원으로최근조직배양에응용하여좋은결과를얻고있다 (Shin et al. 2008; Gupta and Jatothu 2013). 여러가지장점으로는, 1) 청색과적색의방사최고점이엽록소 a, b 의최대흡수점과밀접하게일치하고, 파장이최대의광합성효율을지니는점, 2) 내구성이매우높아장기간사용이가능하여

J Plant Biotechnol (2014) 41:94 99 95 비용이절감되는점, 3) 거의열을발산하지않아배양실냉방으로인한전기료가절약되고, 4) 대량번식실용화에적합하며, 5) 소형이어서다루기가쉽다는장점등이있다 (Nhut et al. 2003). 이러한 LED 의장점으로인해메리골드와살비아 (Heo et al. 2002) 등여러식물에서다양하게적용되어왔다 (Choi 2003; Lian et al. 2002; Han et al. 2000; Eun et al. 2000). 또한, 광질에따른연구로광합성기구의발달, 전분생합성, 엽록소및엽록체의발달, 기공세포의개폐, 광형태형성등에미치는영향도밝혀진바있다 (Gupta and Jatothu 2013). 미선나무의조직배양은액아배양의선행연구 (Moon et al. 1999) 가있으나, 기내식물의생장과발근에미치는광질의연구는아직까지수행되지못했다. 본연구는새로운식물배양용광원으로부각되고있는 LED 광원이미선나무의기내생장에미치는영향을조사하였다. LED system 은 GFPR-1600C (Good Feeling ( 주 )) 로 100% 적색광 LED (R), 50% 적색광 LED+50% 청색광 LED 청색 (BR) 및 100% 청색광 LED (B) 로사용하였다. 분광특성은각각적색광 660 nm, 청색광 450 nm 의파장영역에서광합성유효광량자유입밀도 (PPFD, Photosynthetic Photon Flux Density) 가최대치를나타내는단색 LED 광원이며, 대조광으로는냉백색의형광등 (F) 을사용하였다. 이들광원의 PPFD 는모두 40 µmol/m 2 /s 로조정하였으며, 배양환경은 24±1 C, 16h 일장조건에서 4 주간배양하였다. 통계분석 본실험의모든데이터는통계프로그램 SPSS 12.0 을이용하여산출하였다. 각처리간유의성검정을위해서는 one-way ANOVA 를실시하였고, 유의성이있는경우 Duncan s multiple range test 로차후검증을실시하였다. 통계적유의성은 P < 0.05 로설정하여분석하였다. 재료및방법 선행연구 (Moon et al., 1999) 를통해기내에서약 1 개월의배양주기로 MS 기본배지에서유지하고있는미선나무배양체를실험재료로사용하였다. 절편체는정아가있는줄기와정아가없는줄기로나누어마디는 2 ~3 개, 잎은 3~4 개가붙도록 2.5 cm 길이로절단하였다. 줄기유도는 MS (Murashige and Skoog 1962) 배지에 BA 1.0 mg/l 처리하였고, 발근유도는 1/2 GD (Gresshoff & Doy 1972) 배지에 IBA 0.5 mg/l 처리하였다. 탄소원으로는 3% sucrose, 배지의경화는 0.3% gelrite 로하였다. 산도 (ph) 를 5.7 로맞추고고압멸균하였다. 250 ml 배양병 (64 110 mm) 에 30 ml 씩분주한다음처리당 20 개씩 5 반복으로실험하였다. 결과 줄기증식정아와액아절편체를각각의광질하에서 4 주간배양한결과는 Table 1 및 Figure 1 과같다. 증식에있어절편에따른뚜렷한차이는관찰되지않았으나광질에따라서는차이가있었다. 두가지절편에서청색광 (B) 및혼합광 (BR) 에서증식이촉진되는것으로나타났으며, 정아절편에서는형광등과적색광보다유의적인차이를보였다 (Table 1). 액아절편에서는청색광에서만유의적인차이를보였고, 절편당 4.1 개의줄기가유도되어가장좋은결과를보였다. Table 1 Effect of explant position and LEDs on shoot multiplication and elongation. Cultures were maintained on MS medium supplemented with 1.0 mg/l BA Explants Lighting X No. of shoots/explant Shoot length (cm) Rooting (%) No. of roots/explant Apical bud F 1.8±1.2 a Y 2.4±0.9 a - - B 3.4±1.9 b 2.2±0.7 a 10.0 3.0 BR 3.6±1.3 b 2.1±0.9 a - - R 2.0±0.8 a 3.7±0.8 b - - Axillary bud F 2.4±0.8 a 2.9±0.7 a - - B 4.1±1.5 b 2.9±0.7 a 5.0 1.0 BR 2.6±1.0 a 3.0±0.8 a - - R 2.5±0.8 a 3.4±0.8 b - - X F- Fluorescent light; B- 100% blue LED; BR- 50% blue + 50% red LED; R- 100% red LED. Y Mean( ± SD) separation within column by Duncan s multiple range test (P=0.05)

96 J Plant Biotechnol (2014) 41:94 99 Fig. 1 Shoot growth pattern under different lightings : F - cool white fluorescent light; B-100% blue LED; BR - 50% blue LED + 50% red LED ; R- 100% red LED Table 2 Effect of explant position and LEDs on in vitro rooting. Cultures were maintained on half-strength GD medium supplemented with 0.5 mg/l IBA Explants Lighting X Rooting (%) No. of roots/explant Fresh weight of root (mg) Root length (cm) No. of shoots/explant Shoot length (cm) Apical bud F 100.0 b Y 3.1±0.9 b 26.4±9.1 b 5.28±0.6 c 1.0±0.0 1.8±0.2 ab B 100.0 b 3.3±1.3 b 27.7±5.8 b 5.92±1.2 d 1.0±0.0 1.8±0.4 ab BR 85.0 a 2.3±1.0 a 26.4±6.5 b 3.35±0.5 b 1.0±0.0 1.7±0.4 a R 100.0 b 3.0±0.9 b 9.90±2.0 a 2.70±0.5 a 1.0±0.0 2.1±0.6 b Axillary bud F 95.0 a 1.9±0.9 a 22.5±7.3 b 4.97±1.3 c 1.2±0.4 b 1.8±0.2 a B 85.0 a 2.0±0.9 a 24.2±6.5 b 3.75±0.9 b 1.1±0.2 a 2.2±0.4 b BR 90.0 a 1.9±0.7 a 35.7±9.3 c 3.76±0.8 b 1.0±0.0 a 2.1±0.5 ab R 95.0 a 2.8±1.0 b 9.80±2.3 a 2.30±0.6 a 1.0±0.2 a 3.3±0.7 c X F- Fluorescent light; B- 100% blue LED; BR- 50% blue + 50% red LED; R- 100% red LED. Y Mean( ± SD) separation within column by Duncan s multiple range test (P=0.05) 줄기의생장은적색광에서촉진되었으며다른광질과유의적인차이를보였다. 전반적으로형광등, 청색광및혼합광에서줄기의생장과함께잎의발달이정상적으로이루어진반면, 적색광에서는줄기의신장이촉진되나잎의발달은저조하게나타났다. 어느절편에서나절편의기부에서는약 0.3 cm 의캘러스가형성되었고특히청색광에서는일부절편에서발근이이루어졌다. 발근유도 대체적으로정아지절편이액아지절편보다양호한발근을보였다 (Table 2 및 Fig. 2). 그러나정아지의혼합광에서는다른광질보다저조한발근을나타내어유의적인차이를보였다. 뿌리수에있어서도광질에관계없이정아지절편이액아지절편보다양호하게나타났으나정아지의청색광에서는뿌리수가저조하고반면액아지의적색광에서는뿌리수가양호하게나타났다. 적색광에서 유도된뿌리는생장이매우저조하여뿌리생중량및길이생장에있어다른광질보다매우저조하였다. 뿌리의발달은혼합광의액아절편에서가장양호하여유의적인차이를나타내었고, 뿌리의생장은형광등하에서가장좋아다른광질과유의적인차이를보였다. 한편발근배지에서줄기의생장은주로 1 개의줄기로자랐고, 적색광에서줄기신장이촉진되었다. 다만적색광에서는증식시험에서관찰된것처럼잎의발달이저조하고일부절편에서는정단괴저가생기는단점이있었다. 이상의결과로볼때미선나무의기내발근은정아지를절편으로청색광에서배양함이좋을것으로나타났다. 고찰 기내배양에서 LED 를이용한광질의효과는식물에따라매우다르며주로초본식물을재료로많은연구가수행

J Plant Biotechnol (2014) 41:94 99 97 Fig. 2 Root induction under different lightings : F - cool white fluorescent light; B-100% blue LED; BR - 50% blue LED + 50% red LED ; R- 100% red LED 되었다. 감자의기내배양에서청색광은액아수를증가시켰는데이는광도에따라차이가있으며, 적색광은정아우세 (apical dominance) 를감소시켰으나광도와는무관하였다 (Seabrook 2005). Muleo 등 (2001, 2006) 은 Prunus cerasifera 에서대체적으로청색광은액아의형성을촉진한반면, 적색광은액아에서줄기의발달을촉진한다고하였다. 또한청색광은마디수를증가시켰으나절간신장을억제하였고, 적색광은절간신장이촉진되는결과를보였다고하였다. 도라지에서청색광은형광등과비슷한생장을보인반면적색광은초장을 3 배이상증가시켜도장된상태를보였다 (Eun et al. 2000). Budiarto (2010) 은적색광보다는청색광에서 Anthurium 의더많은줄기를얻었고, Lin 등 (2010) 은청색광에서 Dendrobium officinale PLBs 줄기형성을촉진함을, Heo 등 (2002) 은청색광에서 marigold 의최고줄기신장을얻었다. 그러나포도에서청색광처리는생중량혹은건증량의유의적인증가가없어식물에따른차이를보여주었다 (Heo et al. 2006). Kim 등 (2004c) 은적색광에서절간길이의촉진효과를보인반면, Hahn 등 (2000) 은 Rehmannia glutinosa 의줄기생장에서적색광의억제효과를얻었다. 본실험에서청색광은줄기유도를촉진하는유의적인효과를보여절편당 3 ~4 개의줄기가유도되었다. 줄기신장은광질에따른차이가없었다 (Table 1). 혼합광의효과는절편에따라다르게나타나정아절편은혼합광 (BR) 하에서최대줄기수를얻은반면, 액아절편에서는청색광하에서가장좋은결과를보였다. 대체적으로미선나무의건전한줄기생장및잎의발달은청색광및혼합광에서관찰되었다. 단일 LED 광원보다는혼합광의효과가많이발표되고있는데, Nhut 등 (2000, 2003) 은청색광과적색광의혼용처리로생중량및건중량의증가를가져왔으며, Lin 등 (2010) 은혼합광에서 Upland cotton 의줄기생장촉진으로최대생중량, 건중량을나타냈다. Shin 등 (2008) 도혼합광하에서잎의발달, 뿌리형성, 생중량, 건중량, 잎의면적이커짐을관찰했다. Kim 등 (2004a; 2004b; 2004c) 은이러한차이는청색과적색의광수용체 ( 크립토크롬혹은피토크롬 ) 의서로다른종류의시너지상호작용으로인해줄기신장의촉진혹은억제가나타나며, 이것은식물종에따라달라지는것으로추론하였다. 광질에따른발근및식물생장역시식물에따라반응이다르게보고된다. 덩굴용담에서청색광은발근을억제한반면적색광은발근을촉진하였고 (Moon and Park 2008), 도라지에서는적색광에서뿌리의생장이억제되었다 (Eun et al. 2000). 정등 (2009) 은가시오갈피의배양에서적색, 청색 LED 및형광등하에서발근율이 90% 이상으로높고, 특히청색광에서는뿌리가굵고솜털같은세근이발달되나, 원적색광 (far-red LED) 에서는발근율이 48% 로낮고, 뿌리의생육또한저조하여광질에따른차이를보여주었다. 본실험에서광질에따른발근효과는대체적으로정아지절편에서양호하여미선나무의발근시에는고려해야될내용으로생각되며, 특히적색광단일 LED 하에서는뿌리의형성및발달이저조하여피해야할것으로나타났다. Morrow (2008) 는 LED 의광파장이식물의광수용체에일치할때형태형성의적정반응을가져올수있다고하였는데, 그러나광질의효과는식물종, 식물의발달단계, PPF 와같은환경조건, 배지의조성, 환기조건에따라달라지기때문에기내배양의형태형성을조절하는광질의효과는여전히모호하다고볼수있다 (Gupta and Jatothu 2013). 이상의결과를볼때미선나무의기내배양은단일청색광 LED 혹은청색광및적색광의혼합광 LED 에서증식효과를가져올수있으며, 발근은형광등및청색광

98 J Plant Biotechnol (2014) 41:94 99 단일 LED 하에서효과적임을보여주었다. 그러나이러한효과는절편에따라반응이다르게나타나서광원의선택과함께절편체도고려해야함을시사하며미선나무는증식및발근에서액아지보다는정아지를절편으로하고특히발근유도시단일적색광이부적합한것으로나타났다. 한편 Nhut 등 (2003) 은혼합광 LED 에서생장이촉진된식물이차후토양이식후에도생장이좋은것으로관찰하였는데이러한결과는앞으로미선나무에서도검토해야될내용이다. 적요 희귀멸종위기식물인미선나무를재료로기내증식및발근에미치는광질및절편체 ( 정아지및액아지 ) 효과를조사하였다. 광질은냉백색형광등 (F), 100% 청색광 LED (B), 혼합광 (BR; 50% blue LED+50% red LED) 및 100% 적색광 (R) 을사용하였다. 줄기의증식은절편체종류에따른뚜렷한차이를보이지않았으나광질에따른차이를보였다. 전반적으로줄기의증식은청색광및혼합광에서촉진되는것으로나타났으며, 특히청색광에서는일부절편에서발근이이루어졌다. 줄기의생장역시광질에따른현저한차이가없었다. 적색광에서줄기신장이촉진된반면잎의발달은저조하였다. 발근은전반적으로광질에관계없이정아지가액아지보다양호하였고, 뿌리수도증가하였으나혼합광에서는발근이억제되었다. 한편적색광에서는뿌리의발달이매우부진하여다른광질과유의적인차이를보였다. 발근유도시줄기는주로단일줄기로자랐다. 이상의결과를볼때 LED 를이용한미선나무의기내번식은정아지를절편으로청색광및혼합광 LED (BR) 하에서배양함이좋은것으로나타났다. References Beena MR, Martin KP. 2003. In vitro propagation of the rare medicinal plant Ceropegia candelabrum L. through somatic embryogenesis. In Vitro Cell Dev Biol-Plant 39:510-513 Budiarto K. 2010. Spectral quality affects morphogenesis on Anthurium plantlet during in vitro culture. Agrivita 32:234-240 Chen UC, Hsia CN, Yeh MS, Agrawal DC, Rsay HS. 2006. In vitro micropropagation and ex vitro acclimation of Bulpleurum Kaoi an endangered medicinal plant native to Taiwan, In Vitro Cell Dev Biol-Plant 42:128-133 Choi YW. 2003. Effect of red, blue and far-red LEDs for night break on growth, flowering, and photosynthetic rate in Perilla ocymoides. J Kor Soc Horticul Sci 44:442-446 Eun JS, Kim YS and Kim YH. 2000. Effects of light emitting diodes on growth and morphogenesis of in vitro seedlings in Platycodeon grandiflorum. Korean J Plant Tiss Cult 27:71-75 Gresshoff PM, Doy CH. 1972. Development and differentiation of haploid Lycopersicon exculentum (tomato). Plant (Berl.) 107:161-170 Gupta SD, Jatothu B. 2013. Fundamentals and applications of light-emitting diodes (LEDs) in in vitro plant growth and morphogenesis. Plant Biotechnol Rep 7:211-220 Hahn EJ, Kozai T., Paek KY. 2000. Blue and red light-emitting diodes with or without sucrose and ventilation affect in vitro growth of Rehmannia glutinosa plantlets. J Plant Biol 43: 247-250 Han EJ, Kozai T and Paek KY. 2000. Blue and red light emitting diodes with or without sucrose and ventilation affects in vitro growth of Rehmania glutinosa plantlets. J Plant Biol 43:247-250 Han JS, Kim SK, Kim SW and Kim YJ. 2001. Effects of shading treatments and harvesting methods on the growth of Eleutherococcus senticosus Maxim. Korean J Medi Crop Sci 9:1-7 Han MS, Moon HK, Kang YJ, Kim WW, Kang BS, Byun KO. 2004. Micropropagation of an endangered species, Stellera rosea N. by tissue culture. Korean J Plant Biotechnol 31(1):31-35 Heo JW, Lee CW, Chakrabarty D and Paek KY. 2002. Growth responses of marigold and salvia bedding plants as affected by monochromic or mixture radiation provided by a Light- Emitting Diode (LED). Plant Growth Regul 38:225-230 Heo JW, Shin KS, Paek KY. 2006. Light quality affects in vitro growth of grape Teleki 5BB7. J Plant Biol 49:276-280 Jeong JH, Kim YS, Moon HK, Hwang SJ, Choi YE. 2009. Effects of LED on growth, morphogenesis and eleutheroside contents of in vitro cultured plantlets of Elutherococcus senticocus Maxim. Korean J Medicinal Crop Sci 17(1):39-45 Kartsonas E, Papafotiou M. 2007. Mother plant age and seasonal influence on in vitro propagation of Quercus euboica Pap., an endemic, rare and endangered oak species of Greece. Plant Cell Tiss Org Cult 90:111-116 Kim HH, Goins GD, Wheeler RM, Sager JC. 2004a. Green light supplementation for enhanced lettuce growth under red and blue light emitting diodes. HortSci 39:1617-1622 Kim HH, Goins GD, Wheeler RM, Sager JC. 2004b. Stomatal conductance of lettuce grown under or exposed to different light qualities. Ann Bot 94:691-697 Kim HH, Goins GD, Wheeler RM, Sager JC. 2004c. Effects of LEDs on net photosynthetic rate, growth and leaf stomata of Chrysanthemum plantlets in vitro. Sci Hortic 101:143-151 Kozai T, Watanabe K and Jeong BR. 1995. Stem elongation and growth of Solanum tuberosum L. in vitro in response to photosynthetic photon flux, photoperiod and difference in photoperiod and dark period temperatures. Sci Horticul 64:1-9 Lee TB. 1976. Studies on conservation of endemic species- Abeliophyllum distichum N. Nature Conservation 12:6-10 Lee TB. 1990. Conservation of threatened plants in Korea. Bull Kwanak Arboretum 3:190-196 Lee SW, Kim GS, Lee MJ, Hyun DY, Park CG, Park HK and Cha SW. 2007. Effect of blue and yellow polyethylene shading net

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