Kor. J. Hort. Sci. Technol. 31(3):359-365, 2013 DOI http://dx.doi.org/10.7235/hort.2013.12219 국화화색돌연변이품종 ARTI-purple 및 ARTI-queen 꽃잎조직의재분화와신초형성에미치는식물생장호르몬의영향 이유미 1 ㆍ강은정 2 ㆍ성상엽 1 ㆍ김상훈 1 ㆍ하보근 1 ㆍ김동섭 1 ㆍ김진백 1 ㆍ강시용 1* 1 한국원자력연구원첨단방사선연구소, 2 한국생명공학연구원연구안전관리지원센터 The Effects of Plant Growth Regulators on Plant and Direct Shoots Formation of Petal Explants of Chrysanthemum Flower Color Mutants Varieties, ARTI-purple and ARTI-queen Yu-Mi Lee 1, Eun Jeong Kang 2, Sang Yeop Sung 1, Sang Hoon Kim 1, Bo-Keun Ha 1, Dong Sub Kim 1, Jin-Baek Kim 1, and Si-Yong Kang 1* 1 Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Korea 2 Research Safety Management Center, Korea Research Institute of Bioscience and Biotechnology, Ochang 363-883, Korea Abstract. Chrysanthemum is one of the most popular ornamental plants worldwide. Recently, lots of new and novel chrysanthemum varieties have been developed using mutagenesis. However, there was no study for comparison of tissue culture condition among the mutant varieties derived from one original variety, until now. This study was conducted to compare the efficient regeneration condition of the two chrysanthemum mutant varieties, ARTI-purple and ARTI-queen. Two different flower parts (disk and ray florets) at the unopened and early blooming stages were used for comparison of regeneration condition on MS medium supplemented with combinations of three growth regulators (BA, NAA, and IAA). The highest regeneration rate was identified on the NAA and BA combination when the disk florets at unopened blooming stage are used. The best optimum combinations of growth regulators were identified as NAA 1.0 mg L -1 and BA 0.5 mg L -1 at ARTI-purple, which displayed 47.9 regeneration. However, regeneration of ARTI-queen was the highest as 25.6 at NAA 2.0 mg L -1 and BA 1.0 mg L -1. There results indicate that there is a difference for the optimum regeneration condition between the mutant varieties derived from one original variety. These results will be useful for construction of efficient regeneration system of diverse chrysanthemum mutants developed by mutation breeding. Additional key words: auxin, cytokinin, disk floret, radiation, ray floret 서언국화는세계시장에서화훼작물중장미다음으로중요한부분을차지하고있으며, 절화및분화작물로많이이용되고있다 (Jaime and Silva, 2003; Kumar et al., 2006;). 국내에서도재배면적및생산액이국내 1-2위를차지하고있는중요화훼작물로서, 현재까지품종육성은주로교배육종에의존하고있다 (Kang et al., 2011). 반면, 네덜란드등의화훼선진국은교배육종을통해육성한우수품종을돌연변이육 종을통해시리즈화하여상품가치를극대화하고있으며, 품종육성또한주로민간회사중심으로이뤄지고있다. 이러한돌연변이육종법이국화등의화훼류육종에최근많이시도되고있으며 (Kumar et al., 2012; Park et al., 2007a; Yamaguchi et al., 2008), 국화에서는방사선을이용하여화색, 화형, 및꽃의크기등의형질을개선한보고가있다 (Broertjes, 1996; Datta et al., 2001; Mandal and Datta, 2005). 국내에서는 2005년한국원자력연구원에서아네모네형 *Corresponding author: sykang@kaeri.re.kr Received 21 November 2012; Revised 22 February 2013; Accepted 3 March 2013. 본연구는교육과학기술부지원한국원자력연구원주요사업및원자력연구개발사업에의해수행되었으며이에감사드립니다. 359
스프레이국화 Argus 의줄기배양유식물체에각각 30Gy, 40Gy를조사하여 2009년 11월에원품종보다설상화의색상이선명하고진해진 ARTI-queen 과, 설상화와통상화가모두적자색으로변한 ARTI-purple 을육성하였다. 국화의방사선돌연변이육종은삽수나어린식물체에방사선을조사하여방사선처리당대인 M 1 세대에서변이체의선발을하게된다. 이러한돌연변이체의발생은고정된형태의돌연변이체 (solid type) 가발생하기도하지만적지않게꽃의일부에서돌연변이가발생하는주연구분키메라 (mericlinal chimera) 나구분키메라 (sectorial chimera) 가발생하기도한다. 이러한키메라돌연변이체의경우삽목을하게되면다음세대에서분리가일어나거나변이가소실될수있기때문에꽃잎배양법을통해돌연변이체를고정된계통으로육성한다. 국화의조직배양은잎 (Himstedt et al., 2001), 꽃자루또는꽃 (Mandal and Datta, 2005; Petty et al., 2003), 원형질체 (Malaure et al., 1989; Sauvadet et al., 1990), 정아 (Waseem et al., 2009; Zalewska et al., 2007) 와줄기 (Annadana et al., 2000; Himstedt et al., 2001; Jevremović and Radojević, 2004; Park et al., 2007a) 등다양한조직들에대한연구가이루어졌다. 또한조직배양시사용되는식물생장호르몬은사용한품종과조직에따라상당한차이를보이고있다 (Hoque et al., 1998; Tripepi, 1997). 특히국화꽃잎배양의경우 NAA와 BAP의혼용처리에서높은재분화율이보고되었다 (Chakarbarty et al., 2000; Nahid et al., 2007). 하지만하나의국화품종에서유래한돌연변이품종간의배양조건 A 이나원품종과돌연변이품종간의배양조건에대한보고는전무한실정이다. 따라서본연구는국화 Argus 에서유래한화색변이품종 ARTI-purple 과 ARTI-queen 의최적꽃잎배양조건을탐색하고품종간의차이유무를검증하기위해수행되었다. 재료및방법실험재료한국원자력연구원첨단방사선연구소시험포장에재배중인스프레이국화화색돌연변이품종인 ARTI-purple 과 ARTI-queen 의꽃잎을채취하여흐르는수돗물에깨끗하게수세한다음증류수로 1차세척을하였다. 이후 70(v/v) 에탄올에 20초간침지후멸균수로 1-2회수세하였고, Tween 20을첨가한치아염소산나트륨 (NaOCl) 0.2 에서 20분소독한후멸균수로 4회세척하였다. 세척된조직은멸균된필터페이퍼를이용하여물기를완전히제거한후꽃잎의아래쪽을배지에밀착시켜치상하였다. 적절한배양부위를정하기위하여배양재료는꽃잎의설상화와통상화를구분하였고, 개화시기는꽃봉오리시기 (S1 단계 ) 와통상화개화이전시기 (S2 단계 ) 로구분하여사용하였다 (Fig. 1). 재분화배양조건배지조성은 MS(Murashige and Skoog, 1962) 기본배지에 3 sucrose 및 0.3 gelrite, 식물생장호르몬을혼용하였으며, ph는 5.8로조정하였다. 식물생장호르몬이국화 B C D Fig. 1. Stages of chrysanthemum florets used for tissue culture. (A) unopened flower stage of ARTI-queen, S1; (B) early blooming stage of ARTI-queen, S2; (C) unopened flower stage of ARTI-purple, S1; (D) early blooming stage of ARTI-purple, S2. 360
재분화에미치는영향을알아보기위해 auxin 계열인 NAA, IAA와 cytokinin 계열인 BA를혼용으로처리하였고, 각각 0.5-2.0mg L -1 농도로첨가되었다. 또한배지는직경 100mm 높이 15mm 일회용 petri-dish에 40mL씩분주하였으며, 꽃잎은 petri-dish당 16개씩 3반복으로수행하여 8주간배양하였다. 배양 4주마다재분화율을조사하였고, Duncan 다중검정법을이용하여통계적유의성을검정하였다. 기내배양조건은온도 25 ± 1 C, 일장주기 16/8시간 ( 명 / 암 ), 광도 27μmol -2 s -1 형광조명을유지해주면서실험을수행하였다. 발근및순화배양 10주후재분화된신초는 1/2 MS 배지로옮겨신초의신장과발근을유도하였다. 성공적으로발근된소식물체는인공토양 (Vermiculite:Perlite = 1:1) 이들어있는분에정식하여플라스틱필름으로덮어습도를유지하면서생육시켰으며, 상대습도를점진적으로낮추면서기외순화시켰다. 결과및고찰국화 ARTI-purple 및 ARTI-queen 의꽃잎절편체로부터재분화를유도하기위해 IAA와 BA를혼합하여농도별로처리하여배양한후 4주와 8주후에각각재분화율을조사하였다. 그결과 ARTI-purple 의경우설상화, 통상화모두개화시기 S1, S2 단계에서배양 4주에는높은재분화초기반응률을보였으나, 8주후에는대부분고사하였다 (Table 1). ARTI-queen 의경우 ARTI-purple 과동일하게배양 4 주에는개화시기 S1, S2 단계의설상화, 통상화모두높은재분화초기반응률을보였으나, 배양 8주후에는대부분고사하였다. IAA와 BA 혼합처리에서정상적인식물체가유기되는가장높은재분화율은 ARTI-queen 의 S1 단계통상화를배양하였을때 IAA 1.0mg L -1, BA 1.0mg L -1 조합에서 6.3 로확인되었으나적정재분화조건은아닌것으로판단된다 (Table 1). 이러한결과는 IAA를단일처리하였을때재분화가이루어지지않았고, BA를단일처리하였을때는대부분의품종에서재분화율이저조하였다는기존연구결과와유사하다 (Song et al., 2011). 그러나 cytokinin과 auxin의혼합처리는국화조직배양시재분화에효과적이라는여러연구보고가있다 (Jeong et al., 2002; Seiichi et al., 1995; Tanaka et al., 2000; Waseem et al., 2009). 특히국화품종 Klondike 와 Orlando 의꽃잎배양에서 IAA 57μM, BA 44μM, kinetin 0.4μM을혼합하여처리하였을때가장높은재분화율을보인연구보고가있다 (Park et al., 2007b). 그러나본연구결과에서는 IAA와 BA를혼합한호르몬을 처리하였을때 4주까지는비교적높은재분화초기반응률을보였지만, 8주후에는대부분고사하여 ARTI-purple 과 ARTI-queen 의경우 IAA와 BA 혼합조합은재분화조건에적합하지않은것으로판단된다. 본연구에사용된두개품종의원품종인 Argus 의경우잎과절간을사용하여 IAA 0.5mg L -1 와 BAP 1.0mg L -1 를혼용처리한 1/2 MS 배지에배양한결과잎과절간모두재분화가이루어지지않았다 (Han et al., 2009). 본연구결과를통해원품종인 Argus 와유래돌연변이품종 ARTI-purple 과 ARTI-queen 모두 IAA와 BA의혼합조합은재분화조건으로적합하지않은것으로판단된다. 다른식물호르몬조합을사용하여국화두품종의꽃잎절편체로부터재분화를유도하기위해 NAA와 BA를혼합하여농도별로처리하여배양한후 4주와 8주후에각각재분화율을조사하였다. 그결과두품종의 S1, S2 단계설상화, 통상화모두 IAA, BA 혼합처리시와유사하게배양 4주에는높은재분화초기반응률을보였으나, 8주에는큰차이를보였다. 배양후 8주에 ARTI-purple 의경우개화시기 S1 단계의통상화및설상화모두 NAA 1.0mg L -1, BA 0.5mg L -1 혼합처리구에서각각 47.9 와 23.5 로가장높은재분화율을보였다 (Table 2). 그러나개화시기 S2 단계에서는통상화의경우 NAA 1.0mg L -1, BA 2.0mg L -1 처리구에서 16.7 의재분화율을보였고, 설상화의경우대부분고사하였다. 반면배양후 8주에 ARTI-queen 의경우 S1 단계의통상화에서 NAA 2.0mg L -1 와 BA 1.0mg L -1 의혼합처리시 25.6, 설상화의경우 NAA 1.0mg L -1, BA 2.0mg L -1 처리구에서 21.3 의재분화율을보였다. 개화시기 S2 단계에서는통상화의경우 NAA 1.0mg L -1, BA 0.5mg L -1 처리구에서 18.8 의재분화율을보였고, 설상화의경우대부분고사하였으나 NAA 1.0mg L -1, BA 2.0mg L -1 처리구에서 4.2 의재분화율을보였다. 국화 ARTI-purple, ARTI-queen 두품종의개화시기에따른재분화율을비교한결과각각의호르몬조건에서 S1 단계의조직이 S2 단계에비해상대적으로더높은재분화율을보였고, S2 단계의설상화의경우상대적으로낮게확인되었다. 따라서본연구에서는 ARTIpurple 의경우개화시기 S1 단계에서 NAA 1.0mg L -1 과 BA 0.5mg L -1 의혼용처리를하였을때가장높은재분화율을보이는것을확인할수있었으며, ARTI-queen 의경우에도개화시기 S1 단계에서 NAA 2.0mg L -1 과 BA 1.0mg L -1 의혼용처리시가장좋은재분화율을확인할수있었다. 본연구에서사용된 2개품종의원품종인 Argus 의경우줄기를사용하여 shoot의증식정도를관찰해본결과 NAA 0.2mg L -1 와 BA 0.1mg L -1 에서는신초가형성되고, NAA 361
Table 1. Effect of growth regulator combination (IAA and BA) on regeneration of petal florets of chrysanthemum ARTI-purple and ARTI-queen at 4 and 8 weeks after tissue culture. Growth regulator (mg L -1 ) IAA BA DPS1 z RPS1 DPS2 RPS2 DQS1 RQS1 DQS2 RQS2 y x 0.0 0.0 81.3 ab w 0.0 97.9 a 0.0 64.2 ab 0.0 66.3 a 0.0 100.0 a 0.0 100.0 a 0.0 93.8 ab 0.0 31.0 bc 0.0 0.5 76.5 ab 0.0 72.9 cd 0.0 79.2 a 0.0 71.3 a 0.0 85.4 abc 0.0 68.2 abcdef 0.0 70.8 cde 0.0 77.1 ab 0.0 1.0 70.3 b 0.0 93.8 ab 0.0 70.5 ab 0.0 65.8 a 0.0 54.2 ef 0.0 39.6 ef 0.0 83.3 abcd 0.0 69.8 abc 0.0 2.0 67.8 b 0.0 88.1 abc 0.0 77.1 a 0.0 66.0 a 0.0 50.0 f 0.0 84.8 ab 0.0 85.4 abcd 0.0 60.7 abc 0.0 0.5 0.0 81.3 ab 0.0 85.4 abc 0.0 77.0 a 0.0 100.0 a 0.0 81.3 abc 0.0 90.6 ab 0.0 100.0 a 0.0 90.6 a 0.0 0.5 79.2 ab 0.0 80.8 abcd 0.0 45.8 bc 0.0 64.4 a 0.0 68.8 cde 0.0 47.9 def 0.0 93.8 ab 0.0 28.3 c 0.0 1.0 39.6 c 0.0 92.5 ab 0.0 72.9 ab 0.0 79.1 a 0.0 79.2 bcd 0.0 97.6 a 0.0 91.7 ab 0.0 89.3 a 0.0 2.0 31.3 c 0.0 77.1 bcd 0.0 64.6 ab 0.0 88.4 a 0.0 83.1 abc 2.2 b 71.1 abcde 0.0 85.4 abcd 0.0 92.9 a 0.0 1.0 0.0 83.3 ab 0.0 78.1 bcd 0.0 69.3 ab 0.0 72.6 a 0.0 92.5 ab 0.0 82.6 abc 0.0 97.9 a 0.0 86.7 a 0.0 0.5 75.0 ab 0.0 91.7 abc 0.0 64.6 ab 0.0 88.2 a 2.1 a 81.3 abc 0.0 37.5 f 0.0 93.8 ab 0.0 96.7 a 0.0 1.0 89.6 a 0.0 80.6 abcd 0.0 49.1 abc 2.0 a 79.0 a 0.0 72.9 cd 6.3 a 50.7 cdef 0.0 70.8 cde 0.0 91.7 a 0.0 2.0 31.3 c 0.0 77.1 bcd 0.0 56.3 abc 0.0 100.0 a 2.1 a 61.7 def 0.0 85.0 ab 0.0 60.6 e 0.0 85.4 abc 0.0 2.0 0.0 81.3 ab 0.0 95.8 ab 0.0 76.0 ab 0.0 91.9 a 0.0 85.4 abc 0.0 87.1 ab 0.0 89.6 abc 0.0 71.7 abc 0.0 0.5 79.2 ab 0.0 95.8 ab 0.0 80.1 a 0.0 97.2 a 0.0 54.5 ef 0.0 76.8 abcd 0.0 75.8 bcde 0.0 87.5 a 0.0 1.0 72.9 ab 0.0 85.4 abc 0.0 33.3 c 0.0 100.0 a 0.0 54.2 ef 0.0 63.7 bcdef 0.0 39.6 f 0.0 95.8 a 0.0 2.0 43.8 c 0.0 62.7 d 0.0 62.1 abc 0.0 100.0 a 0.0 75.0 bcd 0.0 83.8 ab 0.0 68.2 de 0.0 70.0 abc 0.0 z DPS1: disk florets of ARTI-purple (S1), RPS1: ray florets of ARTI-purple (S1), DPS2: disk florets of ARTI-purple (S2), RPS2: ray florets of ARTI-purple (S2), DQS1: disk florets of ARTI-queen (S1), RQS1: ray florets of ARTI-queen (S1), DQS2: disk florets of ARTI-queen (S2), RQS2: ray florets of ARTI-queen (S2). y Data were recorded at 4 weeks after culture initiation. x Data were recorded at 8 weeks after culture initiation. w Mean separation within columns by Dunkan s multiple range test at P = 0.05. Table 2. Effect of growth regulator combination (NAA and BA) on regeneration of petal florets of chrysanthemum ARTI-purple and ARTI-queen at 4 and 8 weeks after tissue culture. Growth regulator (mg L -1 ) NAA BA DPS1 z RPS1 DPS2 RPS2 DQS1 RQS1 DQS2 RQS2 y x 0.0 0.0 81.3 ab w 0.0 97.9 ab 0.0 64.2 c 0.0 66.3 abc 0.0 100.0 a 0.0 100.0 a 0.0 93.8 a 0.0 31.0 b 0.0 0.5 91.2 ab 0.0 66.7 abcd 0.0 87.5 abc 0.0 60.8 bc 0.0 70.8 d 0.0 95.8 ab 0.0 87.5 a 0.0 64.6 ab 0.0 1.0 92.1 ab 0.0 93.8 ab 0.0 84.4 abc 0.0 65.8 abc 0.0 91.7 abc 0.0 89.6 ab 0.0 100.0 a 0.0 69.8 ab 0.0 2.0 87.5 ab 0.0 97.9 ab 0.0 78.0 abc 0.0 93.8 ab 0.0 91.7 abc 0.0 100.0 a 0.0 93.8 a 0.0 60.7 ab 0.0 0.5 0.0 85.4 ab 0.0 100.0 a 0.0 97.9 ab 0.0 91.7 ab 0.0 95.8 ab 0.0 97.2 a 0.0 93.8 a 0.0 95.1 a 0.0 0.5 91.7 ab 37.5 ab 97.9 ab 0.0 96.5 ab 12.6 ab 87.5 ab 2.1 a 76.4 cd 9.0 bc 97.9 a 2.1 b 100.0 a 5.4 bc 100.0 a 0.0 1.0 70.8 b 16.7 cd 54.2 cd 2.1 b 94.9 ab 8.1 abc 87.8 ab 0.0 81.3 bcd 6.3 bc 79.9 b 4.2 b 97.9 a 0.0 67.9 ab 2.2 a 2.0 100.0 a 22.2 bc 43.8 d 10.4 b 97.9 ab 6.3 bc 94.4 ab 0.0 100.0 a 17.2 ab 100.0 a 4.2 b 100.0 a 15.6 ab 90.7 ab 0.0 1.0 0.0 77.1 ab 0.0 71.7 abcd 0.0 100.0 a 0.0 100.0 a 0.0 100.0 a 0.0 100.0 a 0.0 93.8 a 0.0 100.0 a 0.0 0.5 87.5 ab 47.9 a 67.3 abcd 23.5 a 97.8 ab 8.5 abc 100.0 a 0.0 75.0 cd 16.7 ab 97.9 a 21.2 a 97.9 a 18.8 a 90.0 a 0.0 1.0 89.6 ab 37.5 ab 81.3 abc 10.4 b 73.1 bc 7.6 bc 41.0 c 0.0 82.2 bcd 15.6 ab 100.0 a 2.1 b 100.0 a 8.3 abc 100.0 a 3.3 a 2.0 81.9 ab 20.8 bcd 85.8 abc 2.1 b 100.0 a 16.7 a 83.3 ab 0.0 100.0 a 23.4 a 82.4 ab 21.3 a 97.9 a 10.4 abc 69.9 ab 4.2 a 2.0 0.0 83.3 ab 0.0 97.9 ab 0.0 91.7 ab 0.0 68.8 abc 0.0 100.0 a 0.0 100.0 a 0.0 96.7 a 0.0 100.0 a 0.0 0.5 87.5 ab 37.9 ab 85.4 abc 6.3 b 100.0 a 3.6 c 100.0 a 0.0 100.0 a 18.8 ab 100.0 a 8.3 b 100.0 a 14.6 ab 72.9 ab 0.0 1.0 89.6 ab 12.5 cd 100 a 0.0 82.9 abc 0.0 69.2 abc 0.0 100.0 a 25.6 a 100.0 a 10.4 b 89.9 a 5.6 bc 87.5 ab 0.0 2.0 77.1 ab 22.9 bc 62.5 bcd 4.2 b 93.8 ab 8.3 abc 57.6 bc 0.0 93.9 ab 9.4 bc 100.0 a 9.4 b 93.8 a 10.0 abc 71.1 ab 0.0 z DPS1: disk florets of ARTI-purple (S1), RPS1: ray florets of ARTI-purple (S1), DPS2: disk florets of ARTI-purple (S2), RPS2: ray florets of ARTI-purple (S2), DQS1: disk florets of ARTI-queen (S1), RQS1: ray florets of ARTI-queen (S1), DQS2: disk florets of ARTI-queen (S2), RQS2: ray florets of ARTI-queen (S2). y Data were recorded at 4 weeks after culture initiation. x Data were recorded at 8 weeks after culture initiation. w Mean separation within columns by Dunkan s multiple range test at P = 0.05. 362
0.2mg L -1 와 BA 1.0mg L -1 에서는줄기생장이이루어진다고보고하였다 (Park et al., 2007a). 개화시기에따른배양조건의재분화율을비교한결과최적 NAA, BA 호르몬조합에서 ARTI-purple 의경우 S1 단계가 S2 단계에비해최대 3배정도의재분화율차이 (47.9:16.7) 를보이고있으며, ARTI-queen 의경우 S1 단계가 S2 단계에비해다소높은것으로 (25.6:18.8) 확인되었다 (Table 2). 기존연구에서는개화시기에따라설상화만개전 2-3일과통상화개화 7-8일로나누어비교한결과설상화만개전 2-3일에꽃잎에서더많은재분화식물체를얻을수있었다 (Park et al., 2007b). 또한발육단계에있는조직을사용하였을때체세포배발생과부정지를유도하는데유리하다는연구보고도있다 (Gilissen et al., 1996; Lu et al., 1982). 이러한결과는세포변화가왕성히일어나는어린조직이재분화에유리한것으로추정된다. 꽃잎의조직 ( 설상화, 통상화 ) 에따른재분화율을비교한결과최적 NAA, BA 호르몬조합에서 ARTI-purple 의경우통상화가설상화에비해 2배이상의재분화율차이 (47.9:23.5) 를보이고있으며, ARTI-queen 의경우통상화가설상화에비해다소높은것으로 (25.6:21.3) 확인되었다 (Table 2). 본실험에서 8주간배양한후에재분화된신초는 1/2 MS 배지로옮겨신초의신장과발근을유도하였고 (Fig. 2E), 성공적으로발근된소식물체는인공토양 (Vermiculite: Perlite = 1:1) 이들어있는분에정식하여온실에서순화처리하여정상적인식물체를획득할수있었다 (Fig. 2F). 결론적으로 ARTI-purple, ARTI-queen 모두꽃잎배양에는 S1 단계의통상화를 NAA, BA를혼용하여사용하는것이재분화에유리한것으로판단되며, 비록두품종이동일한 Argus 에서유래되었으나, 최적재분화조건에는다소차이가있음을확인할수있었다. 이는국화방사선돌연변이육종시최초변이체선발및변이체고정에중요한정보를제공할것으로기대된다. 초록국화는전세계적으로가장대중적인화훼류중의하나로 A B C D E F Fig. 2. The process of shoot regeneration and acclimation using disk and ray florets of chrysanthemum ARTI-queen. (A) culture initiation; (B) adventitious shoot formation from petal segments; (C) shoot regeneration; (D) shoot elongation; (E) rooting; (F) acclimation. 363
써, 최근새로운국화품종들이돌연변이육종을통해개발되고있다. 그러나현재까지하나의국화원품종에서유래한돌연변이품종들간의조직배양조건차이에대한보고는전무하다. 따라서본연구에서는 2개의국화화색돌연변이품종 ARTI-purple 과 ARTI-queen 의효율적인재분화조건을비교하기위해수행되었다. 실험재료로꽃봉오리시기와통상화개화이전시기의꽃잎을이용하였고, 꽃의조직에따른차이를확인하기위해통상화및설상화를구분하여실험을수행하였다. 국화재분화를위한식물생장호르몬의적정조합을찾기위해 3 sucrose, 0.3 gelrite를포함한 MS 배지에 BA, NAA, IAA 중 2가지호르몬을조합별로첨가하여실험을수행하였다. 절편체는일장 16시간, 온도 25 ± 1 C 조건으로배양하였으며, 재분화율조사는배양후 4주및 8주차에실시하였다. 결과적으로, 가장높은재분화율은두품종모두최적 NAA와 BA의호르몬조합에서꽃봉오리시기의통상화를사용할시가장높게확인되었다. 재분화를위한식물생장호르몬의최적조합은 ARTI-purple 의경우 NAA 1.0mg L -1 와 BA 0.5mg L -1 로 47.9 의재분화효율을보였으며, ARTI-queen 의경우 NAA 2.0mg L -1 와 BA 1.0mg L -1 로 25.6 의재분화효율을나타내었다. 재분화된지상부는 1/2 MS배지에서발근시켰으며, 기내소식물체는유리온실에서성공적으로순화되었다. 본연구결과는감마선을사용한돌연변이육종법에의해개발된다양한국화품종의효율적인재분화시스템을구축하는데유용한정보를제공할것이다. 추가주요어 : 옥신, 사이토키닌, 통상화, 방사선, 설상화 인용문헌 Annadana, S., W. Rademaker, M. Ramanna, M. Udayakumar, and J. de Jong. 2000. of stem explants to screening and explant source as a basis for methodical advancing of regeneration protocols for chrysanthemum. Plant Cell Tissue Organ Cult. 62:47-55. Broertjes, C. 1966. Mutation breeding of chrysanthemums. Euphytica 15:156-162. Chakrabarty, D., A.K.A. Mandal, and S.K. Datta. 2000. SEM and light microscopic studies on direct shoot regeneration from ray florets of chrysanthemum. Isr. J. Plant Sci. 48:105-107. Datta, S.K., D. Chakrabarty, and A.K.A. Mandal. 2001. Gamma ray-induced genetic manipulations in flower colour and shape in Dendranthema grandiflorum and their management through tissue culture. Plant Breed. 120:91-92. Gilissen, L.J.W., M.J. van Staveren, J.C. Hakkert, and M.J.M. Smulders. 1996. Competence for regeneration during tobacco internodal development: Involvement of plant age, cell elongation stage, and degree of polysomaty. Plant Physiol. 111:1243-1250. Han, B.H., S.Y. Lee, and B.M. Park. 2009. Comparison of chrysanthemum cultivars based on direct shoot regeneration rates in tissue culture. J. Plant Biotechnol. 36:275-280. Himstedt, J.P., H.J. Jacobsen, and G. Fisher-Kluver. 2001. Shoot regeneration from stem and leaf explants of chrysanthemum (Dendranthema grandiflorum). Acta Hortic. 560:421-424. Hoque, M.I., M.T. Jahan, and R.H. Sarker. 1998. In vitro shoot regeneration and ex vitro rooting in Chrysanthemum morifolium Ramat. Plant Tissue Cult. 8:157-164. Jaime, A. and T. da Silva. 2003. Chrysanthemum: Advances in tissue culture, cryopreservation, postharvest technology, genetics and transgenic biotechnology. Biotechnol. Adv. 21:751-766. Jeong, J.H., D. Chakrabarty, S.J. Kim, and K.Y. Paek. 2002. Transformation of chrysanthemum (Dendranthema grandiflorum Kitamura cv. Cheonsu) by constitutive expression of rice OsMADS1 gene. J. Kor. Soc. Hort. Sci. 43:382-386. Jevremović, S. and L.J. Radojević. 2004. Mass production of different chrysanthemum (Chrysanthemum morifolium) cultivars by culture in vitro. J. Sci. Agric. Res. 65:47-54. Kang, C.H., S.J. Yun, B.S. Han, G.J. Lee, K.H. Choi, J.S. Park, and Y.K. Shin. 2011. Development of salt-tolerant transgenic chrysanthemum (Dendranthema grandiflorum) lines and bioassay with a change of cell specificity. J. Plant Biotechnol. 38:1-8. Kumar, B., S. Kumar, and M. Thakur. 2012. In vitro mutation induction and selection of chrysanthemum (Dendranthema grandiflora Tzelev) lines with improved resistance to Septoria Obesa Syd. Int. J. Plant Res. 2:103-107. Kumar, S., K.V. Prasad, and M.L. Choudhary. 2006. Detection of genetic variability among chrysanthemum radiomutants using RAPD markers. Current Sci. 90:1108-1113. Lu, C., I.K. Vasil, and P. Ozias-Akins. 1982. Somatic embryogenesis in Zea mays L. Theor. Appl. Genet. 62:109-112. Malaure, R.S., M.R. Davey, and J.B. Power. 1989. Isolation and culture of protoplasts of Chrysanthemum cinerariaefolium Vis. Pyrethrum Post 17:90-94. Mandal, A.K.A. and S.K. Datta. 2005. Direct somatic embryogenesis and plant regeneration from ray florets of chrysanthemum. Biol. Plant. 49:29-33. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-479. Nahid, J.S., S. Shyamali, and H. Kazumi. 2007. High frequency shoot regeneration from petal explants of Chrysanthemum morifolium Romat. in vitro. Pak. J. Biol. Sci. 10:3356-3361. Park, I.S., G.J. Lee, D.S. Kim, S.J. Chung, J.B. Kim, H.S. Song, D.H. Goo, and S.Y. Kang. 2007a. Mutation breeding of a spray chrysanthemum Argus by gamma-ray irradiation and tissue culture. Flower Res. J. 15:52-57. Park, S.H., G.H. Kim, and B.R. Jeong. 2007b. Adventitious shoot regeneration from cultured petal explants of chrysanthemum. Hort. Environ. Biotechnol. 48:387-392. Petty, L.M., N.P. Harberd, I.A. Carré, B. Thomas, and S.D. Jackson. 2003. Expression of the Arabidopsis gai gene under its own promoter causes a reduction in plant height in chrysanthemum by attenuation of the gibberellin response. 364
Plant Sci. 164:175-182. Sauvadet, M.A., P. Brochard, and J. Boccon-Gibod. 1990. A protoplast-to-plant system in chrysanthemum: Differential responses among several commercial clones. Plant Cell Rep. 8:692-695. Seiichi, F., J. de Jong, and W. Rademaker. 1995. Efficient genetic transformation of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura) using stem segments. Breed. Sci. 45:179-184. Song, J.Y., N.S. Mattson, and B.R. Jeong. 2011. Efficiency of shoot regeneration from leaf, stem, petiole and petal explants of six cultivars of Chrysanthemum morifolium. Plant Cell Tiss Organ Cult. 107:295-304. Tanaka, K., Y. Kanno, S. Kudo, and M. Suzuki. 2000. Somatic embryogenesis and plant regeneration in chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura). Plant Cell Rep. 19:946-953. Tripepi, R.R. 1997. Adventitious shoot regeneration, p. 45-71. In: R.L. Geneve, J.E. Preece, and S.A. Merkle (eds.). Biotechnology of ornamental plants. CAB International, Wallingford, U.K. Waseem, K., M.S. Jilani, and M.S. Khan. 2009. Rapid plant regeneration of chrysanthemum (Chrysanthemum morifolium L.) through shoot tip culture. Afr. J. Biotechnol. 8:1871-1877. Yamaguchi, H., A. Shimizu, K. Degi, and T. Morishita. 2008. Effects of dose and dose rate of gamma ray irradiation on mutation induction and nuclear DNA content in chrysanthemum. Breed. Sci. 58:331-335. Zalewska, M., J. Lema-Rumińska, and N. Miler. 2007. In vitro propagation using adventitious buds technique as a source of new variability in chrysanthemum. Sci. Hortic. 113:70-73. 365