Korean J. Plant Res. 27(1):051-059(2014) http://dx.doi.org/10.7732/kjpr.2014.27.1.051 Print ISSN 1226-3591 Online ISSN 2287-8203 Original Research Article 조송미 1 *, 오상아 1, 최용수 1, 박상빈 2 1 전남과학대학교화훼원예과, 2 아시아종묘 ( 주 ) 생명공학육종연구소 Effect of Plant Growth Regulators on Regeneration from the Cotyledon Explants in Watermelon (Citrullus lanatus (thunb.) Matsum. & Nakai) Song Mi Cho 1 *, Sang A Oh 1, Yong Soo Choi 1 and Sang Bin Park 2 1 Department of Horticulture, Chonnam Techno University, Gokseong 516-911, Korea 2 R&D Center, Asia Seed Co. Ltd., Icheon 467-906, Korea Abstract - In this study, we developed a high frequency watermelon regeneration system using three breeding lines ( B02, B05 and D04 ) of watermelon (Citrullus lanatus (thunb.) Matsum. & Nakai) which are differed in their fruits in shape, color of pericarp and flesh. The highest frequency of explants with callus was observed by using explants that consist of cotyledon proximal part end in all breeding lines, and the highest rate of callus induction was obtained on MS medium containing 1.0 mg /L BAP + 0.5 mg /L IAA for B02 (94%), 3.0 mg /L BAP for B05 (95%), 3.0 mg /L BAP + 0.1 mg /L IAA for D04 (90%). The highest shoot regeneration rates from derived callus were obtained on MS medium containing 1.0 mg /L BAP + 0.5 mg /L IAA for B05 (94%), and then a B02 (81%) with a same culture conditions, and the lowest regeneration was obtained on MS medium containing 1.0 mg /L BAP for D04 (56%). Regenerated plants showed the best rates of root formation on MS containing 0.1 mg /L IBA for B02 (67%), 0.1 mg /L NAA for B05 (87%), 0.5 mg /L IAA for D04 (88%). The regenerated plants showed a 100% survival rate in soil condition. The tissue culture and regeneration conditions obtained from this study will be useful for regenerating plants in breeding applications, and will be a useful tool for further genetic transformation studies on watermelons. Key words - Tissue culture, Callus, Plant breeding 서언 수박은전세계에서 3번째로많이생산되고있는중요한원예작물중하나로서그재배역사가 4,000년이상이된것으로보고되고있다 (Mustafa et al., 2010). 수박의원산지는남아프리카중앙부칼라하리사막과주변의사바나지대로추정되고있으며, 지역에따라서일부야생종을음료, 사료또는약용등으로다양하게이용되고, 재배종은대부분여름철에생식으로많이이용된다. 특히수박열매는비타민C 와비타민 A 뿐만아니라칼륨, 철분, 칼슘과같은다양한영양분을공급해주고과육에풍부한라이코펜은항산화물질로서다양한암예방효과가있는 * 교신저자 (E-mail) : csmempal1@empas.com 것으로보고되고있다 (Comptom et al., 2004). 수박품종에있어서열매는크기나모양, 줄무늬, 과육, 과중, 종자의색깔및수확시기등이다양하다. 일반적으로널리알려진수박열매는줄무늬가있고붉은과육색을띠며검은색또는갈색종자를지니고있다 (Compton et al., 2004). 그러나상업적수박생산보고서에따르면과피의경우줄무늬가있거나없는경우도있으며색깔또한밝은색에서짙은녹색이될수도있고과육의색깔또한옅은붉은색에서부터짙은붉은색이거나노란색인경우까지다양하다고하였다 (Boyhan et al., 1999). 이러한수박열매의외형적인차이와더불어수박품종은크게종자의발달방법에따라자연수분형, F 1 교배형, 3배체또는씨없는형으로나눌수있다. 그러나수박육종가들에게있어서품종선택의기준은생산성과품질의우수성뿐만아니라 c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons -51- Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
韓資植誌 Korean J. Plant Res. 27(1) : 051~059(2014) 환경이나병에대한저항성을지니고있는것을중요하게여기고있으며무엇보다구매자인소비자의요구에따른시장흐름에맞추어변해가고있다. 이와더불어세계종자시장은그규모가거대해지고또한글로벌화되고있는추세이다. 이와같은국외종사산업의변화양상은국내에서도우수하고다양한종자유전자원에대한연구개발및보급에대한위기를확산시키고있다. 현재수박의생산량은토양전염성덩굴쪼김병균 (Fusarium oxysporum) 이나, 세균성과실썩음병균 (Acidovorax avenae subsp. citrulli), 쥬키니모자이크황화바이러스 (Zucchini yellow mosaic virus, ZYMV) 등과같은병원균에의해심각한영향을받고있다 (Hopkins et al., 2003; Compton et al., 2004; Ling et al., 2013). 이러한문제를해결하기위해기내조직배양을원천기술로활용한생명공학기술과환경이나병원균에내성을갖는형질전환체들이개발되어보고되고있다 (Akashi et al., 2005; Park et al., 2005). 또한이러한육종학적기술은씨없는수박과같은유용한품종을개발하는데이용되고있다 (Compton, 1999; Compton et al., 2004). 수박유묘자엽을이용한신초재분화기술에관한연구는신초기관형성, 체세포배형성, 원형질체배양, 형절전환체개발에효율성증진및방법의용이성뿐만아니라 4배체의상업적품종의종자증식시간의단축때문에많은관심을받고있다 (Choi et al., 1994; Compton and Gray, 1994; Tabei, 1997). 그러나이러한형질전환체개발이나유용한품종육성은기내재분화기술의실질적인적용방법과효율성때문에제한된품종에국한되고있다 (Wang et al., 2013). 그리고기내재분화효율은유전자형이나생장조절제의농도및종류, 절편체의사용시기및발근과순화조건에영향을많이받고있다 (Compton and Gray, 1993; Chaturvedi et al., 2001; Wang et al., 2013). 이러한연구중수박유전자형이나품종에따른자엽절편을이용한신초형성율이나그형성수에있어서생장조절제가미치는요인은 BAP (6-benzylaminopurine) 와같은사이토키닌의농도보다 IAA (Indol-3-acetic acid) 나 NAA (1-Naptalene acetic acid ) 와같은옥신의혼용처리유무에따라영향을받는다고알려져있으며 (Compton and Gray, 1993), 특히특정수박품종에있어서는 IAA 나 NAA 의농도에따라신초형성이억제되는연구도보고되고있다 (Srivastava et al., 1989). 국내에서대부분의수박을이용한형질전환체개발이나 4배체와같은배수체육종등을위한조직배양에대해계속적인연구에도불구하고그품종은주로국내시판용인녹색과피의원 형이면서적색과육을지닌대과종에한정되어있다. 따라서본연구에서는상업적으로이용가치가확대되고있는과형과과피및과육색이다양하면서과중이 4~8 kg에달하는새로운 3가지육종품종에대하여자엽절편을이용한기내재분화조건에영향을미치는생장조절제의농도조건을구명하여기내배양조건을확립하고자하였다. 재료및방법 식물재료및배지수박육종계통 B02, B05 그리고 D04 의종자를아시아종묘 ( 주 ) 에서분양받아시험재료로이용하였다 (Table 1). B02 계통의경우열매의특성이 Jubilee 타입에타원형이면서블랙과피와오렌지색과육을지닌평균 4~5 kg이며, B05 계통의경우열매의특성이 Stripe 타입에타원형이면서황색과피와오렌지색과육을지닌평균과중이 6~7 kg이며, D04 계통의경우열매의특성이 Stripe 타입에원형이면서녹색과피와적색과육을지닌평균과중이 7~8 kg이다 (Fig. 1). 기본배지는 MS (Murashige and Skoog, 1962) 기본배지에 3% sucrose 와 0.5 g/l 2-(N-Morpholino) ethanesulfonic acid (MES, Duchefa, Netherland), 그리고배지의고체화를위해 3.0 g/l 의 phytagel (Sigma, USA) 를넣었으며, ph는 1N NaOH 를이용하여 5.8로조정하였고, 이후 121, 1.5기압에서, 15 분간고압멸균하였다. 발아율검정수박종자는미리 12시간동안증류수에침지한후종피를제거하였고이후 70% ethanol 에 1분간침지후 2% NaOCl 에 5분간멸균하여표면살균하였다. 이를멸균증류수로 5회세척한후 10분간멸균한여과지 (110 mm Dia, Whatman, USA) 위에서수분을제거하였다. 수분이제거된종자를 MS 기본배지에 3% sucrose 와 0.5 g/l MES, 그리고 3.0 g/l의 phytagel을첨가하여배양실내암 ( 광차단 ) 조건과광조건 ( 광주기, 16h light/8h Table 1. Morphological traits of fruit in watermelon (Citrullus lanatus) breeding lines, B02, B05 and D04 Traits Breeding lines Fruit shape Oval Oval Circle Color of pericap Back Yellow Green Color of flesh Yellow Yellow Red -52-
신초형성자엽절편들로부터형성된캘러스를이용하여신초를형성시키기위해 MS기본배지에 30 g/l sucrose, 3 g/l phytagel을첨가하였으며생장조절제로옥신과사이토키닌을각각 1.0, 2.0, 3.0 mg /L BAP 와 0.1, 0.2, 0.3 mg /L IAA 를조합하여혼합하였다. 각육종계통별로 30개이상의캘러스를신초형성용배지조합별배지로계대배양하였다. 배양 4주후에분화된신초의수를조사하였으며이와같은과정을 3반복수행하였다. 배양조건은캘러스형성조건과동일하게하였다. 신초길이생장신초형성 4주이후분화된식물체의부정아를개개신초로분리하여길이신장을위한새로운배지로계대배양하였다. 길이신장을위해생장조절제로 1 mg /L BAP 를단용한 MS 기본배지에서신초를신장시켰으며, 배양조건은신초형성조건과동일하게배양하였다. 배양 4주후에각육종계통별로 30개이상의신초의길이를측정하였으며, 본과정을 3회이상수행하였다. Fig. 1. Difference of pericap and flesh color and shape on fruit of watermelon breeding lines B02, B05 and D04 (Scale bar=1 cm ). dark, 2,500lux, 26 ± 2 ) 하에서발아를유도하였다. 발아는종자로부터유근의출현으로발아여부를판단하였다 (Li et al., 2011). 캘러스유도발아후 5~7 일된유묘의자엽을기부 (proximal) 로부터정단부 (distal) 까지 1~0.5 cm 크기로 3등분횡절단된절편체를캘러스유도에사용하였다. 절편은식물생장조절제의처리별로 Petri-dish (SPL, South Korea) 당각각 10개씩치상하였고, 배지는 MS기본배지에 30 g/l sucrose, 3 g/l phytagel을첨가하였으며, 생장조절물질의조합은 BAP (6-benzylaminopurine, Duchefa, Netherland) 1.0, 2.0, 3.0 mg /L와 IAA (Indol-3- acetic acid, Sigma, USA) 0.1, 0.2, 0.3 mg /L 또는 2,4-D (2,4-Dichlorophenoxy, Duchefa, Netherland) 0.1, 0.2, 0.3 mg /L를각각혼합하여사용하였다. 배양 2~3 주후에부위별로형성된캘러스를육안으로관찰하여그수를조사하였고, 조사는각처리구마다 3반복으로실시하였다. 배양은 26 ± 2, 16 시간광주기, 2,500 Lux 광도의배양실조건에서수행하였다. 발근율검정생장조절제인 IAA, IBA (Indole-3-butyric acid, Sigma, USA) 및 NAA (1-Naptalene acetic acid, Duchefa, Netherland) 를각각 1.0, 2.0, 3.0 mg /L이첨가된 MS 배지에신초가 3 cm 이상증식된개체를배양하여뿌리생성을유도하였다. 뿌리가형성된개체는 giffy-pot (No. 7, Korea) 에옮겨 1주일간 100% 습도를유지하면서순화를유도한후멸균된인공배양토 (perlite: 부토, 3:1 v/v) 에이식하였다. 발근유무는발근배지로이식한 2주후에각육종계통당 30개이상의식물체에대하여조사하였으며, 본과정을 3회이상수행하였다. 통계분석본연구의각시험에대해 3회반복으로수행하였으며, 통계처리는 SPSS (version 12.0) 을이용하였고, 발아율에대한결과는 T-검정법 (Student s t-test) 을캘러스및신초형성율과발근율결과에대한다중범위검정 (Duncan s multiple range test) 을실시하여 5% 수준에유의성을검정하였다. 결과및고찰종자발아조건발아일수가조직배양시기관형성능에영향을준다는보고 -53-
韓資植誌 Korean J. Plant Res. 27(1) : 051~059(2014) Table 2. Germination of watermelon breeding lines B02, B05 and D04 on light and dark condition on Murashige & Skoog medium y Treatments Days to germination Germination rate (%) Days to germination Germination rate (%) Days to germination Germination rate (%) Light 5-7 89 ± 7.9 4-5 84.2 ± 8.5 4-5 98 ± 1.7 z Dark 5-7 96 ± 2.9 z 5-6 90.8 ± 4.2 z 5-7 90 ± 4.1 y Values are means of five replicates ± standard error. Each replicate includes ten explants per a petri dish. z Significant at P=0.05. A B C Fig. 2. Callus induction from cotyledon explants of watermelon breeding lines B02, B05 and D04. Calli were best highly induced from proximal part of leaf explants on the medium containing 0.5 mg /L IAA and 1 mg /L BA for B02 breeding line (A), 3 mg /L BA for B05 breeding line (B) and 0.1 mg /L IAA and 3 mg /L BA for D04 breeding line (C) (Scale bar=1 cm). A B C D E F Fig. 3. Germination and plant regeneration from cotyledon explants of watermelon breeding lines B02, B05 and D04. A, Seed germination. B, Adventitious callus induction from proximal part of cotyledon cultured on MS medium supplemented with 1 mg /L BA and 0.5 mg /L IAA or 2,4-D. C, Shoot induction from calli cultured on MS medium supplemented with 1 mg /L BA and 0.1 or 0.5 mg /L IAA D, Elongated shoot cultured on MS medium supplemented with 1 mg /L BA. E, Rooted plantlet on MS medium supplemented with 0.1, 0.5 mg /L IAA or IBA or NAA. F, Acclimatized plants grown were in plastic Mazenta box. (Srivastava et al., 1989) 가있기때문에각각육종라인을이용하여발아율을향상시키는조건을알아보고자하였다. 수박종자의발아는일정한온도조건하에서암처리시최적으로발아 가된다고하였으며외피를제거하면발아가더촉진된다고하였다 (Thanos and Mitrakos, 1992). 본연구에서는각계통별로동일한온도조건하에서외피를물리적으로제거한후광처리 -54-
Table 3. Comparison in the efficiency of adventitious callus induction among different plant parts excised from cotyledon of watermelon breeding lines B02, B05 and D04 y,z Percent of callus induction z Hormonal supplements ( mg L) y Part of explants Part of explants Part of explants Prox. end Prox. Distal. Prox. end Prox. Distal. Prox. end Prox. Distal. 2,4-D 0.1 38 ± 2.0cd 20 ± 2.0defg 0.0 ± 0.0g 20 ± 2.0bcd 10 ± 2.0cde 0.0 ± 0.0e 25 ± 2.0cde 10 ± 2.0ef 0.0 ± 0.0f 0.2 41 ± 1.5bcd 25 ± 1.5def 5.0 ± 1.0fg 35 ± 1.8b 15 ± 1.5cde 0.0 ± 0.0e 35 ± 1.5bcd 25 ± 1.0cde 5.5 ± 1.2ef 0.5 60 ± 2.3ab 30 ± 2.3de 10.0 ± 2.0efg 55 ± 2.2a 35 ± 2.3b 5.0 ± 2.0de 60 ± 2.0a 35 ± 2.0bcd 10.0 ± 2.0ef 1.0 65 ± 2.3a 55 ± 2.3abc 12.0 ± 1.5efg 70 ± 2.5a 25 ± 2.5bc 10.0 ± 1.5cde 50 ± 2.5ab 45 ± 2.5abc 15.0 ± 1.5def IAA 0.1 20 ± 1.5efg 25 ± 2.3ef 0.0 ± 0.0g 75 ± 1.5a 40 ± 2.0bcd 0.0 ± 0.0f 80 ± 1.5a 45 ± 2.3cd 0.0 ± 0.0e 0.2 35 ± 2.5de 25 ± 2.5ef 0.0 ± 0.0g 40 ± 2.5bcd 35 ± 2.5cd 0.0 ± 0.0f 65 ± 2.0ab 35 ± 2.0d 5.5 ± 1.3e 0.5 85 ± 2.5a 50 ± 1.3cd 10.0 ± 2.5fg 55 ± 2.5abc 30 ± 2.0de 16.0 ± 2.0f 50 ± 1.5bcd 55 ± 1.8bc 7.3 ± 1.5e 1.0 75 ± 1.2ab 60 ± 1.5bc 30.0 ± 2.3def 60 ± 1.2ab 30 ± 1.8de 10.0 ± 2.5ef 55 ± 1.8bc 50 ± 1.0bcd 16 ± 1.6e BAP+IAA 1/0.0 75 ± 3.2abc 25 ± 2.8def 0.0 ± 0.0f 88 ± 1.9ab 82 ± 2.3abc 25 ± 2.8bfgh 59 ± 1.6bc 75 ± 0.0ab 13 ± 2.0ef 1/0.1 50 ± 0.0bcde 25 ± 2.8def 0.0 ± 0.0f 58 ± 2.1bcde 67 ± 3.1abcd 25 ± 0.0fgh 88 ± 2.0a 25 ± 2.9def 0.0 ± 0.f 1/0.5 94 ± 1.0a 63 ± 3.3abcd 0.0 ± 0.0f 75 ± 2.0abc 83 ± 2.1abc 13 ± 2.0gh 50 ± 0.0bcd 50 ± 0.0bcd 13 ± 2.0ef 3/0.0 50 ± 3.4abcd 33 ± 3.3def 0.0 ± 0.0f 95 ± 1.3a 58 ± 2.1bcde 13 ± 2.0gh 63 ± 3.5abc 25 ± 2.9def 25 ± 0.0def 3/0.1 63 ± 2.9abcd 63 ± 0.9abcd 0.0 ± 0.0f 92 ± 1.9a 33 ± 2.1efg 42 ± 1.7efg 90 ± 1.8a 25 ± 2.9def 0.0 ± 0.0f 3/0.5 38 ± 3.5bcde 63 ± 3.0abcd 13 ± 2.9aef 75 ± 0.0abcd 67 ± 2.1bcde 50 ± 0.0def 50 ± 0.0bcd 50 ± 0.0bcd 13 ± 2.0ef 5/0.0 63 ± 2.9abcd 29 ± 1.1def 0.0 ± 0.0f 88 ± 2.9a 38 ± 2.0efg 25 ± 0.0fgh 63 ± 2.0abc 25 ± 2.9def 13 ± 2.0ef 5/0.1 38 ± 3.5bcde 25 ± 0.0def 0.0 ± 0.0f 90 ± 1.3a 38 ± 2.0efg 13 ± 2.0gh 75 ± 0.0ab 25 ± 2.9def 0.0 ± 0.0f 5/0.5 88 ± 2.0ab 50 ± 3.5bcde 0.0 ± 0.0f 75 ± 2.8abcd 83 ± 2.1abc 0.0 ± 0.0h 63 ± 2.0abc 38 ± 2.0cde 0.0 ± 0.0f 7/0.0 50 ± 3.4cdef 38 ± 3.1cdef 0.0 ± 0.0f 88 ± 2.0ab 50 ± 3.8def 25 ± 0.0fgh 63 ± 3.5abc 38 ± 2.0cde 8.5 ± 1.7f 7/0.1 84 ± 3.4ab 17 ± 2.3ef 0.0 ± 0.0f 75 ± 2.7abcd 55 ± 1.8cde 29 ± 1.2efgh 25 ± 0.0def 13 ± 2.0ef 0.0 ± 0.0f 7/0.5 50 ± 2.9abcd 17 ± 2.3ef 0.0 ± 0.0f 88 ± 2.0a 39 ± 1.8efg 0.0 ± 0.0h 50 ± 0.0bcd 50 ± 0.0bcd 0.0 ± 0.0f y Each explants were cultured on MS medium supplemented with 1, 3, 5 mg /L BA and 0.1, 0.5 mg /L IAA or not 0.1, 0.2, 0.5, 1.0 mg /L 2,4-D or IAA, only, and the callus induction frequency was evaluated 4 weeks after cultivation. z Values are means of five replicates ± standard error. Each replicate includes ten explants per a petri dish. Means followed by different letters within a column are significantly different by Duncan s multiple range test, P=0.05 level. 유무에따른종자의발아율을검토하였다. B02 계통의경우발아일수는광조건에영향을받지않았으며암처리조건에서발아율이 96% 로높게나타났고, B05 계통과 D04 계통의경우오히려광처리조건에서각각 98% 의높은발아율을보여주었다. 또한두계통 (B05, D04) 모두암처리보다광처리조건에서발아시기도더빨라지는것이확인되었다 (Table 2). 이와같은결과를통하여계통에따라발아에미치는광의영향이달라짐을확인할수있었다. 또한계통에따라자엽절편을이용한신초형성에있어서발아소요일수가짧은유묘를이용할수록신 초형성율이 6배이상높게나타난보고도있으며 (Compton and Gray, 1994), 발아시키는방법에있어서암처리가차후신초등과같은기관형성능을향상시킨다는보고 (Compton, 1999) 가있으므로계통에따라최적의발아조건을구명하는것이중요한것으로판단되며추가적으로광처리조건별로캘러스신초형성율에어떻게달라지는지연구가필요할것으로사료된다. 캘러스형성 B02, B05 그리고 D04 모두 BAP 와 IAA 가첨가된배지조 -55-
韓資植誌 Korean J. Plant Res. 27(1) : 051~059(2014) 건에서자엽기부 (proximal) 끝부분을절편화하여배양하였을때, 녹색빛깔의캘러스가가장많이형성되었다. 캘러스형성율은 B02 의경우1.0 mg /L BAP 와 0.5 mg /L IAA 를혼합한처리구에서, B05 의경우 1.0 mg /L BAP 단독처리구에서, D04 의경우 3.0 mg /L BAP 와 0.1 mg /L IAA 를혼합한처리구에서각각 94%, 95%, 90% 로높게나타났다 (Fig. 2, Table 3, Fig. 3A-B). 이와같은결과는자엽의기부 (proximal) 의끝부분을이용하였을때캘러스형성및재분화율이높다는결과와는일치하였다 (Gambley and Dodd, 1990; Mohiuddin et al., 1997; Compton et al., 2000; Lee et al., 2003; Han et al., 2005; Khatun et al., 2010). 그러나동일한수박에서도 BAP 와 IAA 를혼용처리하는것보다 1 mg /L의 2,4-D 를단독으로처리하였을때캘러스형성이가장높게나타난다는보고가있으며 (Khatun et al., 2010), 또다른박과채소인오이자엽절편을이용한캘러스형성에있어서도 0.5 mg /L의 kinetin 과 2 mg /L의 2,4-D 를혼용처리하였을때최적의캘러스가형성된반면그이하나그이상의농도를처리하였을때는오히려캘러스가거의형성되지않았다 (Chee et al., 1990). 이는계통별로또는식물종류별로캘러스형성을위한생장조절물질처리조건이달라질수있음을보여준다. 또한박과채소의기내기관형성에있어서유묘의이용 연령기가중요함이제시되고있어 (Compton and Gray, 1993) 향후본육종계통을이용하여유묘생육단계별로캘러스형성에관한효율성을추가적으로검토해보는것이필요한것으로판단된다. 신초재분화각계통별로신초형성율은 B02 와 B05 의경우 1.0 mg /L BAP 와 0.5 mg /LIAA 를혼합한처리구에서 D04 의경우 1.0 mg /L BAP 단독처리구에서각각 81%, 94%, 56% 로높게나타났다. 또한 MS 배지에 BAP 를단독으로처리한것보다 IAA 를추가하였을때캘러스로부터신초형성수가증가하였다 (Fig. 3C, Table 4). 그러므로 B02, B05 그리고 D04 계통별신초형성이나분화에있어사이토키닌의역할이중요하며계통과농도에따라옥신의역할이다르게나타남을보여주고있다. 이와유사하게수박자엽절편에서 BAP 단독처리구가눈의형성을높게한다는연구보고가있으며 (Li et al., 2011), 사이토키닌과옥신을혼합처리하면오히려옥신이신초분화를억제하여사이토키닌의신초형성기능을억제시킨다는보고도알려지고있다 (Srivastava et al., 1989; Compton and Gray, 1993). 또다른수박재분화에관한연구보고에따르면사이토키닌으로 BAP 대 Table 4. Effect of plant growth regulators on adventitious shoot regeneration of watermelon breeding line B02, B05 and D04. Via callus derived from cotyledon explants of 7-day-old seedlings were cultured on MS medium, and the efficiency was evaluated 4 weeks after culture initiation y,z Hormonal supplements ( mg /L) y BAP/IAA Regeneration frequency (%) Breeding lines z No. of shoots per callus Regeneration frequency (%) No. of shoots per callus Regeneration frequency (%) No. of shoots per callus 1/0.0 50 ± 0.0ab 1.0 ± 0.8ab 69 ± 3.6ab 1.7 ± 0.6abc 56 ± 2.1a 1.3 ± 1.3bcd 1/0.1 50 ± 0.0ab 2.3 ± 0.6b 63 ± 1.2ab 2.5 ± 1.2c 38 ± 0.0ab 3.5 ± 0.6a 1/0.5 81 ± 3.0a 3.3 ± 1.0a 94 ± 1.9a 3.5 ± 0.6a 50 ± 0.0ab 2.8 ± 0.5abc 3/0.0 42 ± 2.4ab 1.8 ± 0.5b 88 ± 0.0ab 2.0 ± 0.8bc 44 ± 3.6ab 2.0 ± 0.8bcd 3/0.1 63 ± 0.0ab 2.0 ± 0.8ab 63 ± 0.0ab 2.8 ± 1.0abc 44 ± 2.1b 3.0 ± 0.8ab 3/0.5 50 ± 0.0ab 3.0 ± 0.8ab 56 ± 0.7b 3.0 ± 0.8ab 50 ± 0.0ab 3.5 ± 0.8bcd 5/0.0 40 ± 4.0ab 1.0 ± 0.8b 63 ± 1.6ab 1.8 ± 0.5abc 44 ± 3.6ab 1.8 ± 0.5cd 5/0.1 31 ± 3.6b 3.0 ± 0.8b 69 ± 2.1ab 2.0 ± 1.0abc 50 ± 3.0ab 2.8 ± 0.5abc 5/0.5 63 ± 0.0ab 2.8 ± 1.0b 88 ± 2.6ab 2.5 ± 0.8bc 31 ± 2.1ab 3.5 ± 0.6d y Each explants were cultured on MS medium supplemented with 1, 3, 5 mg /L BA and 0.1, 0.5 mg /L, and the callus induction frequency was evaluated 4 weeks after culture initiation. z Values are means of five replicates ± standard error. Each replicate includes ten callus per a culture dish. Means followed by different letters within a column are significantly different by Duncan s multiple range test, P=0.05 level. -56-
신 5 40 μm kinetin 이나zeatin 또는 2-isopentenyladenine이나 0.1-10 μm thidiazuron 을첨가하면신초형성율이높아진다는보고도있다 (Dong and Jia, 1991; Compton and Gray 1993; Compton et al., 2004). 그러나이와같은결과들을보면신초형성및신초수발달에미치는사이토키닌과옥신의종류와농도는수박유전자형이나계통에따라달라질수있다고하였다 (Compton and Gray, 1994; Wang et al., 2013). 또한 thidiazuron 와같은사이토키닌의경우는계통에따라차이가있지만일정농도이상에서는신초가두껍고생장이저해되어비정상적인신초가만들어지기도하였다 (Compton and Gray 1993). 본실험에서는지금까지다양한수박계통에서신초형성율에영향을주었던 BAP 를생장조절제로이용하였다. 그결과농도에따라캘러스형성및재분화율이달라졌고계통에따라서는옥신으로 IAA 를혼용처리하는것이캘러스및신초형성에더효과적임을확인하였다. 본연구에이용된육종계통을활용하여신품종육성을위한재분화시험에적용할수있을것으로사료된다. 길이신장및발근재분화되어형성된신초를 1.0 mg /LBAP 가단독으로처리된 MS 배지에서계대배양하면신초의생장이일어남을관찰할수있었다 (Fig. 3D). 수박에서신초의길이증식에관한연구는생장조절물질을첨가하지않은배지조건에서길이생장이가능한경우 (Compton and Gray, 1993) 와 0.92 μm의 kinetin 이첨가된조건에서길이생장이잘되는경우 (Dong and Jia, 1991) 가보고되어있으며계통에따라차이가있음을알수있었다. 신장된신초의계통별로계대배양 2주경과후부터 B02 경우 0.5 mg /L IBA, B05 경우 0.1 mg /LNAA, D04 경우 0.5 mg /L IAA 조건에서각각 66.7%, 86.7%, 88% 의발근율을나타내었다 (Fig. 3E, Table 5). 수박재분화개체에대한발근조건에대한연구는 MS 기본배지에옥신을첨가하면신초에뿌리형성이촉진된다는보고가있으며 (Compton and Gray, 1993; Wang et al., 2013), 생장조절물질을첨가하지않고 1/2 농도의 MS 배지조건에서발근율이높다는보고도있다 (Zhang et al., 2011). 발근된소식물체를 giffy-pot(no. 7, Korea) 에습실처리후 2주간순화를유도하였고이후멸균된인공배양토에이식하여생장시키며관찰하였던결과뿌리발달이충분히된식물체는 100% 모두의생존하였다 (Fig. 3F). 본연구에서도과형과과피및과육색이다른 3가지육종계통을이용하여기내재분화조건을실험해본결과, 발아율에있 Table 5. Effect of different concentration of auxin on rooting from regenerated shoot from cotyledon derived callus of watermelon breeding line B02, B05 and D04 y,z Hormonal Percent of Rooted plants z supplements ( mg /L) y IAA 0.1 23 ± 2.0ns 23 ± 2.0ns 54 ± 1.1b 0.5 45 ± 2.3ns 22 ± 1.8ns 88 ± 0.8a 1.0 25 ± 1.2ns 23 ± 0.9ns 50 ± 1.3b IBA 0.1 33 ± 1.5b 36 ± 2.0c 63 ± 0.0b 0.5 67 ± 2.6a 55 ± 1.2b 80 ± 1.6a 1.0 43 ± 1.5ab 73 ± 0.9a 63 ± 1.1b NAA 0.1 32 ± 0.9b 87 ± 1.9a 61 ± 0.7b 0.5 40 ± 1.2a 23 ± 1.9b 75 ± 1.2a 1.0 33 ± 0.9b 32 ± 1.5b 66 ± 1.6ab y Each regenerants were cultured on MS medium supplemented with 0.1, 0.5, 1.0 mg /L IAA or IBA or NAA, and the rooting frequency was evaluated 2 weeks after cultivation. z Values are means of five replicates ± standard error. Each replicate includes ten plants per a culture dish. Means followed by different letters within a column are significantly different by Duncan s multiple range test, P=0.05 level. Ns, means were not significantly according to analysis of variance. -57-
韓資植誌 Korean J. Plant Res. 27(1) : 051~059(2014) 어서는계통별로광처리와암처리가발아율과발아시기에영향을미치는것을확인하였다. 또한이육종계통모두유묘자엽의기부 (proximal) 끝부분이다른부위보다캘러스형성율이높았고이에따라신초형성및재분화효율도높아졌다. 육종계통별로최적의캘러스와신초형성및발근조건에미치는생장조절물질의종류와농도범위를확립하였으며최종적으로토양순화를거쳐외부포장에서활용할수있는식물체들을본연구를통해확보하였다. 앞선연구에따르면이러한수박재분화기술의연구는형질전환에의한다양한물리적환경내성이나내병성품종개발뿐만아니라씨없는수박등과같은다양한배수체품종개발에잠재적인활용도가높다고알려져있다 (Choi et al., 1994; Tabei, 1997; Compton et al., 2004). 그러나이러한품종개발에있어서신초재분화시스템은생장조절제의조합이나함유농도그리고유묘의자엽이용부위나이용시기, 발근조건과순화조건등의많은문제들이선행적으로확립되어야상업적품종에적용될수있다고하였다 (Compton et al., 2004). 따라서본연구에서다루어지고있는다양한과형과과피및과육색을지닌유색수박이면서과중이 5~8 kg중소과종인육종계통의유전자원을이용한기내재분화조건의확립은이들유전자원을활용한다양한품종육성에기초자료로활용할수있을것으로사료된다. 적요수박에서과형, 과피, 과육색이서로다른중소과종인3 가지계통의재분화조건을확립하고자각계통의유묘의자엽절편을배양하여캘러스및신초형성에미치는생장조절물질의효과를조사한결과는다음과같다. 각발아된유묘자엽의캘러스형성은모든계통에서유묘의자엽부위중기부 (proximal) 의끝부분에서가장높은효율을나타내었다. 또한캘러스형성에미치는생장조절제의영향은 B02 의경우1.0 mg /L BAP 와 0.5 mg /L IAA 를혼합한처리구에서, B05 의경우 1.0 mg /L BAP 단독처리구에서, D04 의경우 3.0 mg /L BAP 와 0.1 mg /L IAA 를혼합한처리구에서가장높게나타나계통에따라생장조절제의조건이달라짐을확인하였다. 신초형성율은 B02 와 B05 의경우 1.0 mg /LBAP 와 0.5 mg /LIAA 를혼합한처리구에서 D04 의경우 1.0 mg /LBAP 단독처리구에서가장효과적인것으로확인되었다. 재분화된식물체의길이신장은 MS 기본배지에생장조절제로 1 mg /LBAP 가첨가된배지에서가장효과적이었으며, 이들식물체의최적발근은 B02 경우 0.5 mg /LIBA, B05 경우 0.1 mg /LNAA, D04 경우 0.5 mg /L IAA 를첨가된배지조건에서이루어졌다. 사사본연구는농림축산식품부 해양수산부 농촌진흥청 산림청 GSP 사업단연구지원금에의해서수행되었으며이에대해심심한감사를표하는바이다. References Akashi, K., K. Morikawa and A. Yolota. 2005. Agrobacteriummediated transformation system for the drought and excess light stress-tolerant wild watermelon (Citrullus lanatus). Plant Biotechnol. 22:13-18. Boyhan, G.E., D.M. Granberry and W.T. Kelley. 1999. Commercial Watermelon Production. Bulletin. 996. Cooperattive Extension Service, University of Georgia. http: www. Ces. Uga, edu/ pubcd B996-w.htm. Chaturvedi, R. and S.P. Bhatnagar. 2001. High-frequency shoot regeneration from cotyledon explants of watermelon cv. Sugar baby. In Vitro Cell. Dev. Biol. Plant. 37:255-258. Chee, P.P. 1990. High frequency of somatic embryogenesis and recovery of fertile cucumber plants. HortScience 25:792-793. Choi, P.S., W.Y. Soh, Y.S. Kim, O.J. Yoo and J.R. Liu. 1994. Genetic transformation and plant regeneration of watermelon using Argrobacterium tumerfaciens. Plant Cell Rep. 13:344-348. Compton, M.E. and D.J. Gray. 1993. Shoot organogenesis and plant regeneration from cotyledons of diploid, triploid and tetraploid watermelon. J. Am. Soc. Hort Sci. 118:151-157. Compton, M.E. and D.J. Gray. 1994. Adventitious shoot organogenesis and plant regeneration from cotyledon from tetraploid watermelon. HortScience 29:211-213. Compton, M.E. 1999. Dark pretreatment improves adventitious shoot organogenesis from cotyledons of diploid watermelon. Plant Cell Tiss. Organ Cult. 58:185-188. Compton, M.E. 2000. Interaction between explant size and cultivar affects shoot organogenic competence of watermelon cotyledons. HortScience 35:749-750. Compton, M.E. D.J. Gary and V.P. Gaba. 2004. Use of tissue culture and biotechnology for the genetic improvement of watermelon. Plant Cell Tiss. Organ Cult. 77:231-243. -58-
Dong, J.Z. and S.R. Jia. 1991. High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad.) Plant Cell Rep. 9:559-562. Gambley, R.L. and W.A. Dodd. 1990. An in vitro technique for the production de novo of multiple shoots in cotyledon explants of cucumber (Cucumis sativus L.). Plant Cell Tiss. Organ Cult. 20:177-183. Han, J.S., C.K. Kim, S.H. Park, K.D. Hirschi and C.G. Mok. 2005. Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl). Plant Cell Rep. 23:692-698. Hopkins, D.L. and C.M. Thompson. 2003. Wet seed treatment with peroxyacetic acid for the control of bacterial fruit blotch and other seedbone disease of watermelon. Plant Disease 87:1495-1499. Khatun, M.M., M.S. Hossain, M. Khalekuzzaman, A. Rownaq and M. Rahman. 2010. In vitro plant regeneration from cotyledon and internodes derived callus in watermelon (Citrullus lanatus Thumb.). Int. J. Sustain. Crop Prod. 5:25-29. Lee, Y.K., W.I. Chung and H. Ezura. 2003. Efficient plant regeneration via organogenesis in winter squash (Cucurbita maxima Duch.). Plant Sci. 164:413-418. Li, J., X.M. Li, Y.G. Qin, Y. Tang, L. Wang, C. Ma and H.X. Li. 2011. Optimized system for plant regeneration of watermelon (Citrullus lanatus Thumb.). Afr. J. Biotechnol. 10:9760-9765. Ling, N., W. Zhang, D. Wang, J. Mao, Q. Huang, S. Guo and Q. Shen. 2013. Root exudates from grafted-root watermelon showed a certain contribution in inhibiting Fusarium oxysporum f. sp. niveum. PLoS ONE 8: 1-8 Mohiuddin, A.K.M., M.K.U. Chowdhury, C. Abdullah-Zaliha and S. Napis. 1997. Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration. Plant Cell Tiss. Organ Cult. 51:75-78. Mustafa, P., A. Cevat, B. önder Türkme and S. Musa. 2010. Modeling of some physical properties of watermelon (Citrullus lanatus (Thumb.) Mansf.) seed depending on moisture contents and mineral compositions. Pak. J. Bot. 42:2775-2783. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. Park, S.M., J.S. Lee, S. Jegal, B.Y. Jeon, M. Jung, Y.S. Park, S.L. Han, Y.S. Shin, N.H. Her, J.H. Lee, M.Y. Lee, K.H. Ryu, S.G. Yang and G.H. Han. 2005. Transgenic watermelon rootstock to CGMMV (Cucumber green mottle mosaic virus) infection. Plant Cell Rep. 24:350-356. Srivastava, D.R., V.M. Andrianov and E.S. Piruzian. 1989. Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad. cv, Melitopolski). Plant Cell Rep. 8:300-302. Tabei, Y. 1997. Study on breeding of cucurbitaceae using biotechnology. Bull. Natl. Inst. Argobiol. Resource 11:1-107. Thanos, C.A. and K. Mitrakos. 1992. Watermelon seed germination. Seed Science Reseach 2:163-168. Wang, X., L. Shang and F. Luan. 2013. A highly efficient regeneration system for watermelon (Citrullus lanatus Thunb.). Pak. J. Bot. 45:145-150. Zhang, H., G. Peng and L. Feishi. 2011. Efficient plant regeneration from cotyledonary node explants of Cucumis melo L.). Afr. J. Biotechnol. 10:6757-6761. (Received 22 July 2013 ; Revised 13 December 2013 ; Accepted 15 January 2014) -59-