CNU Journal of Agricultural Science Vol. 38, No. 4, pp. 613-618, December 2011 교배육종및소포자배양에의한뿌리혹병 race4 저항성배추계통육성 박수형 1 ㆍ윤무경 1 ㆍ임용표 2 * 1 농촌진흥청국립원예특작과학원, 2 충남대학교원예학과 Development of clubroot race4 resistant inbreds using conventional breeding and microspore culture method in Chinese cabbage Suhyoung Park 1, Moo-Kyoung Yoon 1, Yong Pyo Lim 2 * 1 National Institute of Horticultural and Herbal Science, RDA, Suwon 440-706, Korea 2 Department of Horticulture, Chungnam National University, Daejeon 305-764, Korea Received on 2 December 2011, revised on 12 December 2011, accepted on 18 December 2011 Abstract : To develop clubroot resistant Chinese cabbage inbreds, IT 033820, a clubroot resistant turnip, was cross pollinated with a Chinese cabbage inbred of BP 079. From 2005, conventional breeding and microspore culture method performed using these F1 plants as parental materials. In 2007, conventional breeding method resulted in 21 F3 inbreds. After inoculation of clubroot race 4, one inbred showing 83% resistant was selected and registered as Wonkyo 20036ho in 2008. From 2005, we scanned hybrid cultivars using micro spore culture and developed many doubled haploid (DH) lines in Chinese cabbage. Using Chinese cultivar of Zoong-baek 2ho, we developed 26 DH inbreds in 2007. After inoculation of clubroot race 4, one DH inbred showing 77% resistant and yellow inner leaf color was selected and registered as Wonkyo 20034 in 2008. We found conventional breeding method was effective using introduced germplasm showing low germination. However, when using hybrid cultivar as starting material, microspore culture method was powerful for developing various inbred in short time. Key words : Chinese cabbage, Clubroot, Breeding, Microspore culture, Inbred I. 서론 뿌리혹병은절대활물기생균인 Plasmodiophora brassicae 에의해발생하는토양전염성병으로 (Braselton, 1995), 처음엔진균 (fungi) 으로분류되었으나 DNA 염기서열분석결과진균과는연관이없는것으로밝혀졌으며 (Castlebury 와 Domier, 1998; Down 등, 2002), ribosomal RNA 의발생학적분류결과원생동물 (protozoa) 로분류되었다 (Van de Peer 등, 2000). 우리나라에서는 1928 년수원등경기지역에서최초로발병이보고되었으나문제를일으키지않았고 (Kim 과 Oh, 1997), 1990 년대중반고령지지역에서보고된이후급속하게발병지역이증가하여 (Lee 등, 2001) 현재는전국의봄, 여름, 가을및월동재배작형에서발병하고있다. 병이발생하면수량의 30~70% 정도를감소시킴 *Corresponding author: Tel: +82-42-821-5739 E-mail address: yplim@cnu.ac.kr 으로배추재배에큰위협을주고있다. 따라서뿌리혹병의방제방법및저항성품종개발관련연구가지속적으로추진되고있다. 뿌리혹병방제를위해화학약품, 농약및길항균을이용하는방법이개발되었으나 (Datnoff 등, 1987; Cheah 등, 2000; Yeoung 등, 2003), 처리비용 ( 약제비및노동비 ) 이많이소모되는문제가있으며방제효과도처리한연도의재배에는효과가있으나이듬해재배할경우휴면포자상태로생존한병원균의밀도가높아지면서병이다시발생하는문제가발생한다. 이러한문제점해결을위해육성된저항성품종의경우초기육성된 CR계저항성품종 (Jang등, 2001) 은병에는저항성을보이나, 절임특성등가공적성이적합하지못하여시장에서사용을하지않게되었다. 이후원예적가공적성및속잎특성을개선하여새롭게육성된품종들은품질이우수하여시장에서반응이좋았으나, 한지역에서 2년이상연작할경우병원성이무너지는경우가발생하였다. CNU Journal of Agricultural Science 38(4), 2011. 12 613
임용표 / 교배육종및소포자배양에의한뿌리혹병 race4 저항성배추계통육성 뿌리혹병저항성품종육성을위해서는우선적으로병저항성재료가필요한데, 초기 CR계품종은신속한품종육성을위해일본에서도입된품종을활용하였으며, 이후엔각회사에서다양한저항성자원을도입하여품종개발에이용되어왔다. 그러나저항성품종의이병화가신속하게발생함에따라다양한저항성자원의개발이필요하게되었다. 그런데배추에는뿌리혹병저항성자원이없어서순무등에서저항성유전자를도입해야하므로저항성계통의육성에는많은노력과시간이소모된다. 전통적방법인교배육종의경우저항성유전자고정을위해최소한 4세대를전개해야활용가능하며이를위해 4년정도가소요된다. 그런데배추과채소는소포자배양 (Lee 와 Nam, 1995) 이비교적잘됨으로이를활용할경우저항성유전자가고정된계통을 1~2 년만에육성이가능하다. 따라서국립원예특작과학원에서는다양한뿌리혹병저항성배추품종개발을돕고자병원성이높은단포자유래균주 race4(kang 등, 2000) 를이용하여유용계통을육성코자하였다. 이를위해원예원보유중인저항성자원등과배추를교배하여그후대를뇌수분으로세대진전시키는방법과소포자배양방법을활용하여계통으로육성코자하였다. 1. 재료 II. 재료및방법 에서약 6주간재배하면서춘화처리를실시하였다. 춘화처리이후지름 20 cm 화분에상토와흙을반반섞어담은후이식하였다. 이식후추대가발생하면처음추대는제거하여여러개의추대를유도한후지지대를세워서추대가꺾이지않도록관리하면서교배를실시하였다. 뇌수분은꽃봉오리의길이가 5 mm 정도로아직개화하지않은봉오리의윗부분을제거하고봉지안에서개화된동일한개체의화분을이용하여수분한후다시일주일간봉지를씌워오염수분을방지하였다. 뇌수분후 50~60 일이지나종자가노랗게익으면꼬투리가벌어지기전에채종하였다. 3. 소포자배양방법 (Lee와 Nam, 1995) 개화한꽃봉오리에서 2-3 mm 정도크기의화뢰를 30 개수집하여거즈로싼후, 95% EtOH 2초, 차아염소산나트륨 (2%) 15분소독후멸균수로 3분간 3회세척하였다. 이후 B 5 배지에서막자사발로갈아 45 μm체로거른후동일한배지로 3회세척 ( 세척은 35.7 ml의 B 5 배지를넣고 1000 rpm, 3분간원심분리후상징액버림 ) 한다. 이후 75 ml 의 NLN 배지로바꾼후 2.5 ml씩을지름 6 cm petridish에분주하여 parafilm 으로막아준다. 32.5 에서 24시간처리한후 25 암상태에서약 2주처리후명실 shaker 위에서배양하면서발생한배상체를 1/2 MS 배지로옮겨키웠다. 4. 뿌리혹병접종법 뿌리혹병저항성자원으로원예원에서선발한순무 (Brassica rapa L.) 도입자원번호 IT033820을이용하였다. 순무의저항성을배추로도입하기위해원예원에서육성한바이러스병저항성배추계통인 BP079 와교배하여이를육종재료로이용하였다. 또한배추에효과적인소포자배양조건구명을위해중국및한국시판품종을소포자배양을위한재료로이용하였다. 2. 교배육종방법 ( 뇌수분에의한세대진전 ) 저항성순무와이병성배추교배후대종자 300 립을뇌수분하기위해 50공프러그트레이를이용하여온실에파종하였다. 파종후본엽이 3매정도전개될때까지온실에서키운후 5 가유지되며태양광이공급되는춘화처리실 각계통및자원의종자 25~30 립을 9 cm petridish 등에여과지 2장을깔고파종한후물이마르지않도록관리하였다. 파종온도에따라떡잎전개시기가다르지만대체로 7~9 일이되어떡잎이완전하게펼쳐지면단포자분리로획득된 race4(kang 등, 2000) 를증류수에갈은후약 5 10 5 농도로계산된접종액으로접종하였다. 접종 1일이후상토를채운 50공프러그트레이에정식한후접종액이나관주양액이밖으로흘러나가주변을오염시키지않도록트레이물받침을받혀주었다. 육묘용한방양액으로 4~6주간재배후뿌리를조사하였다 (Park 등, 2008). 5. 원예적특성평가 ( 원예원가을재배 ) 육성된계통의원예적특성을평가하기위해수원시장 614 농업과학연구제 38 권제 4 호, 2011. 12
Yong Pyo Lim / Development of clubroot race4 resistant inbreds using conventional breeding and microspore culture 안구이목동소재국립원예특작과학원채소과노지포장에서가을에재배하였다. 파종은 2008 년 8월 13일, 정식은 8월 29일이었으며재배방법은표준영농교본배추재배법에준하여재배하였다. 11월 20일생육조사를실시하였다. 계통을육성하였다. 저항성순무의경우 20 이상에서는발아가잘되지않았는데, 그후대들중일부는발아력이매우낮아식물체를획득하지못하였고, 교배를하더라도 III. 결과및고찰 뿌리혹병저항성자원으로이용하고있는순무 IT033820 은국립원예특작과학원채소과에서 2003 년에농업유전자원센터에서분양받은순무자원 300 점을병접종하여선발한자원으로, 뿌리혹병에는저항성이나액아가많이발생하고결구를전혀형성하지못하여국내배추품종에바로이용하기는어렵다. 따라서채소과육성계통인배추 BP079 와교배하여그후대를이용하여뇌수분과소포자배양을병행하였다. 뇌수분을위하여 300 개의 F 1 종자를 2005 년에파종하여 2007 년까지세대를진전시킨결과 42점의 F 3 Fig. 1. The shape of clubroot resistant inbred of Wonkyo 20036ho. Table 1. Result of clubroot inoculation using F 3 lines derived from cross pollination between turnip (IT033820) and Chinese cabbage (BP079). Label Rsistance Medium sensitive Sensitive Total Disease grade z (%) 07-R78 2 3 0 5 40.0 07-R79 7 3 0 10 70.0 07-R81 10 10 0 20 50.0 07-R82 0 22 1 23 0.0 07-R83 4 21 0 25 16.0 07-R84 4 19 2 25 16.0 07-R85 6 15 4 25 24.0 07-R86 3 20 2 25 12.0 07-R87 1 19 2 22 4.5 07-R88 4 6 0 10 40.0 07-R89 9 12 3 24 37.5 07-R90 9 14 0 23 39.1 07-R91 4 15 0 19 21.1 07-R92 15 9 2 26 57.7 07-R93 19 4 0 23 82.6 07-R94 1 19 0 20 5.0 07-R95 5 18 0 23 21.7 07-R96 4 20 0 24 16.7 07-R97 6 16 0 22 27.3 07-R98 0 10 5 15 0.0 07-R99 0 12 1 13 0.0 z Number of resistance plants/number of total plants x 100, 30 seeds were sown in each line, inoculated with race 4 isolates in 5 10 5 concentration after 7 days, cultivated for 8 weeks using hydroponic solution in a restricted place. CNU Journal of Agricultural Science 38(4), 2011. 12 615
임용표 / 교배육종및소포자배양에의한뿌리혹병 race4 저항성배추계통육성 채종이되지않는경우가많아획득된계통의숫자가적었다. 따라서저항성유전자의유실여부를조사하기위해 2007 년에채종량이 100 립이상인 21 개 F 3 계통의뿌리혹병 접종실험실시결과저항성이높은계통을선발할수있었다 (Table 1). 이들의후대를진전하여 2008 년에접종액의농도를높여 2차접종하였을때도저항성을보여저항성 Table 2. Result of clubroot inoculation using F 4 inbreds derived from cross pollination between turnip (IT033820) and Chinese cabbage (BP079). Label Parental Rsistance Medium sensitive Sensitive Disease grade z Line (%) 08-RU11 y 07-R90 1 3 4 13 08-RU12 07-R91 3 1 4 38 08-RU13 07-R92 0 2 4 0 08-RU14 07-R93 4 0 2 67 z Number of resistance plants/number of total plants x 100, 20 seeds were sown in each inbred, inoculated with race 4 isolates in 1 x 10 8 concentration after 7 days, cultivated for 8 weeks using hydroponic solution in a restricted place. y Among 21 lines, 8 lines produced seeds and only 4 lines produced enough seeds for clubroot inoculation test. Table 3. Result of clubroot inoculation using Doubled haploid lines derived from Chinese cabbage Zoong-baek 2 ho. Label Rsistance Medium sensitive Sensitive Total Disease grade z (%) 07-R52 0 19 0 19 0 07-R55 0 5 0 5 0 07-R59 0 25 0 25 0 07-R60 0 23 1 24 0 07-R63 0 24 0 24 0 07-R70 0 17 0 17 0 07-R64 2 17 6 25 8 07-R56 3 22 0 25 12 07-R68 2 13 0 15 13 07-R61 3 18 1 22 14 07-R67 3 14 4 21 14 07-R71 2 5 3 10 20 07-R66 5 17 2 24 21 07-R51 6 16 1 23 26 07-R65 7 17 0 24 29 07-R62 8 17 0 25 32 07-R47 9 16 0 25 36 07-R46 9 13 1 23 39 07-R58 10 12 1 23 43 07-R49 11 13 1 25 44 07-R57 14 11 0 25 56 07-R45 15 10 0 25 60 07-R53 16 10 0 26 62 07-R48 14 6 0 20 70 07-R54 17 4 1 22 77 07-R69 20 4 0 24 83 z Number of resistance plants/number of total plants x 100, 30 seeds were sown in each line, inoculated with race 4 isolates in 5 10 5 concentration after 7 days, cultivated for 8 weeks using hydroponic solution in a restricted place. 616 농업과학연구제 38 권제 4 호, 2011. 12
Yong Pyo Lim / Development of clubroot race4 resistant inbreds using conventional breeding and microspore culture Fig. 2. The shape of clubroot resistant inbred of Wonkyo 20034 ho and Zoong-baek 2 ho. A, Outer shape of Zoong-baek 2 ho ; B, Inner shape of Wonkyo 20034ho ; C, Outer shape of Wonkyo 20034ho. 계통으로선발하였으며 (Table 2) 원교 20036 호 로품종등록하였다. 그러나육성계통은결구를형성하지않으며액아도많아원예적형질은좋지못하였다 (Fig. 1). 2005년봄부터뿌리혹병저항성순무자원을소포자배양을실시하였으나배양을성공하지못하여배양법개발을위해국내및중국수집배추품종의배양을병행하였다. 2005년겨울에다양한배추시판종유래배상체를획득하였으며저항성순무와교배된자원의배양도성공하였다. 그러나저항성순무와교배된자원의경우다른품종에비교하여배상체를얻기가어려웠다. 배추의배발생률이품종간차이가크다는보고 (Kim 과 Lee, 1995) 에의해동일한조건에서다양한국내, 외시판품종을배양한결과몇몇품종에서는많은배상체를획득하였다. 중국품종인 북경소잡 61호 의경우 1년배양으로 150 여점의배상체를획득하였으나저항성순무는 2년간더많은노력을기울여배양하여배상체를약 40여점을획득하였다. 순무교배자원유래배상체는채종도어려워 2년간증식하여 2계통에서 100 립이상종자를수확할수있었다. 중국품종인 북경소잡61호 를 2년간배양하여 311 점의배상체유래계통을육성하였으며이중채종량이 200 립이상인 26계통의뿌리혹병접종결과저항성비율 0~72% 까지다양한결과를보였다 ( 자료미제시 ). 또다른중국품종인 중백2 호 의소포자배양결과 69점의배상체유래계통을육성하였으며이중채종량이 100 립이상인 26계통의병접종결과저항성비율 0~83% 의결과를보였다 (Table 3). 중백 2호 유래계통중뿌리혹병 race4 저항성 77% 이며속잎이노랗고느슨한결구를형성하는원예적형질이양호한계통을선발하여 원교 20034 호 로품종등록하였다 (Fig. 2). 육성된계통들 은추후국내종묘회사에분양되어뿌리혹병저항성일대잡종품종의육종에이용될계획이다. IV. 결론 Hong 등 (2005) 이소포자배양법으로뿌리혹병에저항성인다양한계통을육성하였다는보고가있어신속한병저항성계통의육성을위해소포자배양을우선적으로활용코자하였으나, 소포자배양결과획득되는배상체유래계통의 bias 현상에관한우려가제기되어교배육종도함께진행하였다. 동일한시기에전통교배육종법과소포자배양법을병행하여계통을육성한결과발아력및채종력이낮은유전자원을활용할경우교배육종법이오히려소포자배양법보다계통육성에는효과적이었다. 그러나일대잡종품종을소포자배양재료로이용하였을때전통적인교배육종법으로는 3년내에수백점의계통육성이불가능하지만소포자배양법으로는가능하였으며, 뿌리혹병 race4 에저항성인계통의선발도가능하였다. 따라서육종에사용할재료가발아력, 채종력등이우수한일대잡종품종일경우소포자배양법은단기간에다수의계통을육성하기에매우효과적인방법으로판단되었다. 참고문헌 Braselton JP. 1995. Current status of the plasmodiophorids. Critical Reviews in Microbiology. 21: 263-275. Castlebury LA, Domier LL. 1998. Small subunit ribosomal RNA gene phylogeny of Plasmodiophora brassicae. Mycologia. 90: 102-107. Cheah LH, Veerakone S, Kent G. 2000. Biological control of CNU Journal of Agricultural Science 38(4), 2011. 12 617
임용표 / 교배육종및소포자배양에의한뿌리혹병 race4 저항성배추계통육성 clubroot on cauliflower with Trichoderma and Streptomyces. Spp. New Zealand Plant Protection 53: 18-21. Datnoff LE, Kroll TK, Lacy GH. 1987. Efficacy of chlorine for decontaminatining water infected with resting spores of Plasmodiophora brassicae. Plant Disease 71: 734-736. Down GJ, Grenville LJ, Clarkson MJ. 2002. Phylogenetic analysis of Spongospora and implications for the taxonomic status of the plasmodiophorids. Mycological Research. 106: 1060-1065. Hong SY, Cho KS, Moon JY, Ryu SY, Lee HC, Yoon HK. 2005. Microspore Culture and its Progeny Test for Resistant Line to Clubroot (Plasmodiophora brassicae) in Chinese Cabbage. Kor. J. Hort. Sci. Technol. 23(3): 45-45. Jang CS, Piao Z, Park YJ, Lim YP. 2001. Development of anther-derived lines resistant to clubroot disease in Chinese cabbage. J. Kor. Soc. Hort. Sci. 42: 689-694. Kang WJ, Yoon MK, Kim DH, Kim JS, Harn JS, Om YH. 2000. Classification and selection for resistance to clubroot in Cruciferous vegetables. Kor. J. Hort. Sci. Technol. 18(2): 187. Kim DW, Oh JH. 1997. Incidence, pathogenicity of club root fungus (Plasmodiophora brassicaea) and viral resistance in Chinese cabbage. Korean J. Plant. Pathol. 13: 95-99. Kim YH, Lee SS. 1995. Culture of microspore of B. campestrisssp. Kor. J. Hort. Sci. Technol. 10: 372-373. Lee JT, Ru GL, Lee JN, Kweon HJ, Lee EH, Kim WB, Park HY. 2001. Selection of alternative crops on Chinese cabbage in alpine. Kor. J. Hort. Sci. Technol. 19(suppl II): 63. Lee SS, Nam SC. 1995. Microspore culture of Broccoli. J. Kor. Soc. Hort. Sci. 36(5): 635-640. Park S, Kwak JH, Yoon MK. 2008. Development of an effective inoculation method for large quantaties of clubroot disease using hydroponics in Chinese cabbage. Kor. J. Hort. Sci. Technol. 26(4): 449-453. Van de Peer Y, Baldauf SL, Doolittle WF, Meyer A. 2000. An updated and comprehensive rrna phylogeny of eukaryotes based on rate-calibrated evolutionary distances. J. Mol. Evol. 51: 565-576. Yeoung YR, Kim JH, Kim BS, Jeon JY, Yoon CS. 2003. Effects of beneficial antagonists (Bacillussp., Pseudomonassp., and Trichodermasp.) on control of club root of Chinese cabbage. 2003. Kor. J. Hort. Sci. Technol. 21(3): 194-198. 618 농업과학연구제 38 권제 4 호, 2011. 12