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1 J Plant Biotechnol (2014) 41:65 72 DOI: ISSN Review 원형질체융합을이용한감자육종 조광수 박태호 Potato breeding via protoplast fusion Kwang-Soo Cho Tae-Ho Park Received: 16 June 2014 / Revised: 20 June 2014 / Accepted: 26 June 2014 c Korean Society for Plant Biotechnology Abstract Plant cells from which the cell walls have been enzymatically or mechanically removed are called protoplasts. The protoplasts are theoretically totipotent and can be used as sources of somatic cell fusion in practical breeding programs. Wild Solanum species have often been used as sources of important agricultural traits including diverse disease resistance. However, they cannot often be directly applied to breeding programs due to their sexual incompatibility with S. tuberosum. Somatic hybridization via protoplast fusion is one of the ideal methods to overcome this limitation and to introgress certain traits into S. tuberosum. This technique has still widely been used in potato since the first fusion was reported in 1970s. Therefore, this review highlights general perspectives of protoplast fusion and discusses the application of protoplast fusion in potato breeding. 서론 원형질체 (protoplast) 는세포벽을기계적으로또는효소에의해제거한식물세포를말한다. 이론적으로원형질체는탈분화할수있는능력을가지며, 세포주기로진입하여반복적인유사분열을진행함으로써증식또는다양한기관으로재분화하는등의전형성능을갖는다. 이러한성 K.-S. Cho 국립식량과학원고령지농업연구센터 (Highland of Agriculture Research Center, National Institute of Crop Science, RDA, Pyongchang, , Republic of Korea) T.-H. Park ( ) 대구대학교원예학과, 대구대학교생명환경연구소 (Department of Horticulture and Institute of Life and Environment, Daegu University, Gyeongsan , Republic of Korea) thzoo@daegu.ac.kr 질은다양한식물종을대상으로원형질체융합을이용한실용적인육종방법을제공하며, 생식세포의수정을통해이루어지는접합자의형성전또는형성후, 정상적인접합자를형성하는데문제를발생시키는근연이종식물체간의교잡능력장벽을피해서로다른동형핵체 (homokaryon) 또는이핵접합체 (heterokaryon) 와이형세포질잡종 (alloplasmic hybrids, cybrids) 을창출할수있는기회를제공한다. 4 배체 (2n=4x=48) 인재배종감자 (Solanum tuberosum L.) 는전세계적으로생산량이네번째에이르는가장중요한작물중하나이다. 재배종감자는역병, 풋마름병, 더뎅이병등다양한병에대해감수성을보이며, 이러한병들은결과적으로감자의생산성에영향을줄뿐만아니라품질에도큰영향을미친다. 따라서다양한감자유전자원을이용한병저항성감자품종의육성은다양한병에대해저항성을달성할수있는가장현실적이며이상적인방법으로고려되고있다 (Jansky 2000). Solanum 속의야생종은이러한생물학적인요인뿐만아니라비생물학적요인과다양한농업형질에대한가치를지닌중요한유전자원으로가치를인정받고있으며 (Cardi et al. 1993; Helgeson and Haberlach, 1999), 다양한야생종들이실제감자의병저항성을증대시키기위한육종프로그램에이용되고있다. 하지만많은수의야생종들이재배종감자와생리적으로불화합성이란제한된조건에서육종에활용되고있다. 이는교배양친의배유의게놈구성비 (endosperm balance number, EBN) 에의해결정되며, 이에따른정상적인배와배유형성의여부가이종및이배수체간교잡의성공을결정하는요인이되고있다 (Ortiz and Ehlenfeldt 1992; Cho et al. 1997). 감자의육종에다양한야생종을활용하는데발생되는이러한불화합성을극복하기위한방법으로는도입유전자원의 EBN 을고려하여교배가가능한유전자원들을선재적으로교배하여재배종감자와교배가가능한새로운

2 66 J Plant Biotechnol (2014) 41:65 72 계통을육성하는방법 (bridge crosses), 재배종감자가보유하고있지못한유용형질과관련된유전자를근연야생종에서찾아클로닝하여형질전환방법을이용하여재배종감자로도입하는방법 (genetic modification, GM), 그리고앞서언급한원형질체융합을이용한체세포잡종을유도하는방법 (somatic hybridization) 이있다 (Ahn and Park 2013). 이중 bridge cross 와 GM 방법의경우, 전자는재배종감자와교배가가능한계통을육성하기위하여교배조합을구성하여후대식물체를전개하고도입하고자하는형질에대한선발과정이수차례반복되어야하며, 후자는우선적으로도입하고자하는유용형질과관련된유전자를클로닝하는작업이필수적인과정인이유로시간과노력이많이들뿐만아니라, 특히 GM 방법의경우여전히사회적으로거부감을나타내고있어, 실질적인감자육종에활용하는데많은어려움이있다. 반면에체세포잡종을이용한감자육종은 1970 년대후반감자의원형질체로부터식물체의재분화가성공적으로이루어지고체세포잡종육성프로그램에적용된이래로현재까지도여전히불화합성감자근연야생종으로부터농업적으로중요한형질을재배종감자로도입하는시도가이루어지고있다 (Shepard and Totten 1977; Binding et al. 1978; Melchers et al. 1978). 이에본논문에서는식물원형질체융합에대한전반적인연구현황과감자의근연야생종이가지고있는유용형질을원형질체융합을통하여재배종감자에도입하고자이루어진감자육종연구의실례를대상으로실제육종에이용된근연야생종, 목표형질, 선발방법등을조사하여검토한후국내에서이러한육종방법의적용가능성을논의하고자한다. 식물원형질체융합및선발방법 원형질체융합에대한이론은이미약 100 년여전에보고가되었으며, 작물을이용한체세포잡종의경우에는담배를이용한종간체세포잡종이첫번째성공사례로보고되었다 (Carlson et al. 1972). 이후다른많은식물종에서원형질체를이용하여체세포잡종창출이시도되었고이를통해성공적인체세포잡종식물체재분화가이루어졌으며, 최근까지도다양한식물종을대상으로지속적으로연구가진행되고있다 (Gamborg 2002; Eeckhaut et al. 2013; Shankar et al. 2013). 원형질체융합의방법으로는식물의종류에따라화학적융합법과전기적융합법이이용되고있으며거의유사한비율로적용되고있다. 두가지방법의장점을결합한새로운방법이개발되기도했으나, 이는운향과 (Rutaceae) 식물에서만성공적으로적용된사례로거의이용되고있 지는않다 (Olivares-Fuster et al. 2005). 또한융합대상식물들의유전적기여방식에따라대칭적원형질체융합 (symmetric fusion) 과비대칭적원형질체융합 (asymmetric fusion) 으로구분할수있다. 대칭적원형질체융합은융합대상양친간의완전한게놈이융합된형태를말하며, 이경우특정염색체간에서로거부반응을나타냄에따라유전자충돌 (gene conflict) 현상이증가하게된다. 뿐만아니라, 원하지않는유전적특성이도입되어융합식물체가비정상적으로또는매우느린속도로생장하거나거의불임인식물체로의재분화, 그리고비정상적으로다루기힘든캘러스로의분화등부정적효과가나타나기도한다 (Eeckhaut et al. 2006). 이에반해비대칭적원형질체융합은게놈분절 (genome fragmentation) 이후하나의제한된게놈이융합체로전달되게된다 (Xia 2009). 이러한비대칭융합은세포질 DNA 와핵 DNA 의융합에적용될수있으며, 특히세포질웅성불임 (cytoplasmic male sterility, CMS) 와같은형질의도입에많이이용되고있다 (Liu et al. 2005). 원형질체융합을육종에이용하기위한초기융합계통육성과정은일반적으로원형질체의분리, 분절 ( 비대칭융합의경우적용 ), 융합, 재분화그리고선발의과정을거친다 (Razdan 2003; Liu et al. 2005). 이중융합이후, 융합하고자하는서로다른식물체간의게놈이실제융합이되었는지를확인하고융합체를선발하는과정은육종을위해서가장중요하다고할수있다. 이를위해 DNA 분자표지의이용, 세포유전학적기법등다양한생명공학적기술이이용되고있다. 많이이용되는분자표지로는 RAPD (Randomly Amplified Polymorphic DNA), SSR (Simple Sequence Repeats), AFLP (Amplified Fragment Length Polymorphism), RFLP (Restriction Fragment Length Polymorphism), CAPS (Cleaved Amplified Polymorphic Sequence), SCAR (Sequence Characterized Amplified Region) 등이있으며, 이러한분자표지를적용하는데사용되는 DNA 정보는핵내 DNA 의서열뿐만아니라세포질의미토콘드리아나엽록체 DNA 의서열이활용되고있다 (Thieme et al. 2010; Patel et al. 2011; Saker et al. 2011; Wang et al. 2011a; Yu et al. 2012a). 이에더해최근에는 real time PCR, microarray, transcriptome, reverse transcription PCR, real-time reverse transcription PCR 분석등과같은전사체기반의분석기술과 (Liu et al. 2012; Yu et al. 2012b; Wang et al. 2011b, 2013) proteome 분석과같은단백질체기반의분석기술 (Gancle et al. 2006; Wang et al. 2010) 또한이용되고있다. 세포유전학적기법으로는염색체수검사, 유동세포분석법 (flow cytometry), GISH (Genomic in situ hybridization) 등이이용되고있으며 (Xu et al. 2007; Lian et al. 2011; Wang et al. 2011a; Jiang et al. 2012), 기타방법으로동위효소분석법 (isozyme analysis), 식물조직학적분석법 (histological analysis) 등이이용되고있다 (Wang et al. 2012; Yu et al. 2012a)

3 J Plant Biotechnol (2014) 41: 원형질체융합을이용한감자의육종 생식세포의융합 ( 유성생식잡종, sexual hybridization) 에반대적개념이라고할수있는체세포융합은종내또는종간두개의서로다른전체게놈을합치기위한시도로체세포로부터분리된두가지의원형질체를무성적으로융합 ( 무성생식잡종, asexual hybridization) 하는방법이다. 이러한방법은감자의육종에서유성적융합 ( 교잡에의한고전적육종 ) 과정에서나타나는불화합성과주요한형질의도입을위해이용되는이배체야생종과재배종감자의 EBN 차이에의한유성적융합의제한을극복하 기위한방법으로이용되고있다. 즉, 원형질체융합은이배체와재배종감자의이배성반수체 (dihaploid) 로부터새로운사배체 (tetraploid) 유전자원을새롭게창출하는과정에서많이이용된다. 감자에서의원형질체융합 감자의원형질체융합에는약 3-4 주간기내에서배양된식물체가이용되고있으며, 원형질체분리를위해서는효소처리에의한세포벽제거방법이주고이용되고있다. 융합의방법에는앞서언급한바와같이전기적방법 Table 1 Progress in protoplast fusion in potato during the last decade ( ) Species Aim Fusion tool Reference S. acaule Glycoalkaloid aglycone EF Rokka et al. (2005) S. berthaultii Tolerance to salinity EF Bidani et al. (2007) S. brevidens Resistance to soft rot, early blight and PLRV PEG Tek et al. (2004) S. brevidens Resistance to common scab EF, PEG Ahn & Park (2013) S. bulbocastanum Resistance to late blight PEG Boltowicz et al. (2005) S. bulbocastanum - EF Iovene et al. (2012) S. bulbocastanum S. pinnatisectum S. bulbocastanum, S. cardiophyllum, S. chacoense, S. pinnatisectum Resistance to late blight, soft rot, nematode, heat and drought EF Greplova et al. (2008) Resistance to late blight, Colorado potato beetle EF Chen et al. (2008) S. cardiophyllum Resistance to late blight EF Shi et al. (2006) S. cardiophyllum Resistance to Colorado potato beetle, PVY and late blight EF Thieme et al. (2010) S. chacoense Resistance to bacterial wilt EF, PEG Chen et al. (2013) S. chacoense - EF, PEG Cai et al. (2004) S. circaeifolium Resistance to late blight PEG Espejo et al. (2008) S. commersonnii Resistance to bacterial wilt EF Kim-Lee et al. (2005) S. etuberosum Resistance to PVY EF Tiwari et al. (2010) S. melongena Resistance to bacterial wilt EF, PEG Yu et al. (2013) S. michoacanum Resistance to late blight PEG Szczerbakowa et al. (2010) S. michoacanum Resistance to late blight EF Smyda et al. (2013) S. phureja - EF Lightbourn & Veilleux (2007) S. pinnatisectum Resistance to late blight EF Polzerova et al. (2011) S. pinnatisectum Resistance to late blight EF Sarkar et al. (2011) S. stenotomum Resistance to bacterial wilt EF Fock et al. (2007) S. tarnii Resistance to PVY and late blight EF Thieme et al. (2008) S. tuberosum - PEG Przetakiewicz et al. (2007) S. tuberosum Resistance to PVY and Pythium aphanidermatum EF Nouri-Ellouz et al. (2006) S. vernei - PEG Trabelsi et al. (2005) S. villosum Resistance to late blight PEG Tarwacka et al. (2013) * All species were fused with S. tuberosum. * Aim indicated with - was not precisely reported in the reference. * For fusion methods, EF and PEG indicate using electrofusion and polyethylene glycol as fusion agent, respectively.

4 68 J Plant Biotechnol (2014) 41:65 72 과 PEG 를이용한화학적방법이이용되고있으나, Table 1 에서제시된바와같이전기적방법이더욱보편적으로이용되고있다. 전기적방법에의한실험법 (Thieme et al. 2008; Symda et al. 2013) 과 PEG 를이용한화학적방법에의한실험법 (Trabelsi et al. 2005; Espejo et al. 2008) 은각각의문헌에제시된바와같이비교적자세히기술되어있으며, 그방법에있어서연구자에따라약간의차이를보이나대다수의연구에서보편적으로이용되고있다. 감자체세포융합의가장중요한목표는재배종감자가가지고있지않은병저항성, 환경스트레스내성과같은주요형질을야생종으로부터도입하는것이다. 이를달성하기위한가장효과적인방법으로는이배체의야생종과이배성반수체감자의대칭적융합이가장많이이용되다. 이러한방법은추가적인염색체의배가과정을거치지않고바로사배체를육성할수있게한다. 이후야생종으로부터유래된원하지않는유전적특성을배제시키기위해서는추가적인여교배 (backcross) 가요구된다. 하지만여기서요구되는여교배세대는 2-3 세대로제한된반면고전육종에의해융합이이루어질경우에는 6-7 세대까지이루어져야하는것을감안하면매우효율적이라할수있다 년대초 Chase (1963) 는감자의육종에서사배체를대신하여이배성반수체를이용하는것을제안하였다. 이후이는감자육종프로그램에서체세포융합기술이새로운품종을육성하기위한효율적수단이될수있게하였다 (Frei et al. 1998). 앞서서론에서언급했던바와같이, 감자에서원형질체를이용한체세포잡종생산이 1970 년대에성공적으로이루어진이후다양한야생종을대상으로여러유용형질들을감자에도입하고자하는시도가이루어지고있다. 하지만최근약 10 년여간이루어진감자원형질체융합사례를보면 (Table 1), 유용형질도입을위해이용된야생종의수가제한적이며, 대다수의도입형질이역병저항성을대표적으로한병저항성에국한되어있음을확인할수있다. 또한이러한방법을적용하여새로운감자품종을육성하는것이감자육종가들과상업적인감자육종기업에의해받아들여지기까지는많은시간이걸렸으며, 유럽과미국의감자육종회사에서원형질체융합을통하여성공적인감자품종육성이이루어지고있음에도불구하고, 최근한국에서체세포잡종법을이용하여감자품종을육성한예가있으나 (Kim et al. 2013), 한국을포함한중국, 인도등과같은대다수의개발도상국국가에서는그다지많이이용되고있지않다. 따라서원형질체를이용한체세포잡종의육성과고전육종방법의조합은여전히성공적인감자품종육성의기회가되고있으며, 이를통한지속적인감자의생산성증가와재배종감자의유전적다양성을확보하는기회를제공할것이다. 감자체세포융합의문제점및전망 체세포융합은다양하고폭넓은유전자원을대상으로유전적기능을결합하고자하는매우정교한방법이라할수있다. 여기에는원형질체의분리, 융합, 배양, 재분화, 선발, 체세포잡종의형질평가등을필요로하며이를이용한감자육종에는육성된체세포잡종재료를육종프로그램으로도입하는것이필요하다. 이를위해무엇보다도효율적이고효과적인체세포융합을위해서는많은비용과시간이필요할뿐만아니라장비와기술을필요로한다. 가장중요한제한적인요인으로는원형질체의분리와배양및재분화과정에서의유전자형효과 (genotype effects) 이다 (Rokka 2009). 감자의원형질체융합에대한다양한정보가생산되고있으나, 여전히그방법적인측면에서는경험에의존하는경우가많다. 지금까지많은수의계통들에반복적으로적용할수있는유전자형에독립적으로실험이가능한융합법이나재분화방법이확립되어있지않다. 융합법의경우최근까지도여전히 PEG 를이용한화학적융합법과전기적융합법이거의이용되고있으나 (Table 1), 이중전기적융합법의사용빈도가더많으며, 이는두가지의방법을비교해볼때융합효율적인측면에는큰차이를보이지않으나, 융합이후의세포성장과캘러스의분화등에서전기적융합법이더나은결과를보이기때문인것으로사료된다 (Cai et al. 2004). 결론적으로원형질체의분리, 융합, 배양, 재분화실험방법에대해서는유전자형에따라여전히최적화과정이필요하며, 많은노동력과자원을필요로한다. 추가적인문제점으로는원형질체융합체에대한빠르고효율적인선발에관한것으로대다수의융합실험에서표면적으로쉽게구별이가능한형태적표지인자가없어, 실질적인육종프로그램에서각각의목적에따른제한적인방법이적용되고있다. Table 2 에나타난바와같이, DNA 분자표지를이용한방법과세포유전학적방법이많이이용되고있으며, 대부분의연구에서는복수의방법을선택하여활용하고있다. 다만최근 10 여년전체적인흐름을고려해볼때, 감자의전체유전체해독이완료되고 (The Potato Genome Sequencing Consortium 2011) 체세포잡종선발과특성구명에많은이점이있는 DNA 기반의분자표지기술의활용빈도가더많은것을확인할수있다. 그러나이또한분석을위해서는체세포잡종개체들을대상으로재분화하는데소요되는시간이길다는점과분자표지분석에필요한충분한재료의양적확보를위하여잡종개체들을무균상태에서어느정도일정기간지속적인유지및증식이필요하다는것이다. 마지막으로원형질체를이용한체세포융합, 잡종은고전육종이나유전자변형을통한 GM 품종육성에대해

5 J Plant Biotechnol (2014) 41: Table 2 Approaches for somatic hybrid selection or characterization used during the last decade ( ) Tools References Isozyme analysis Trabelsi et al. (2005), Nouri-Ellouz et al. (2006) Flow cytometry analysis Cai et al. (2004), Tek et al. (2004), Trabelsi et al. (2005), Bidani et al. (2007), Greplova et al. (2008), Thieme et al. (2010), Tiwari et al. (2010), Polzerova et al. (2011), Sarkar et al. (2011), Ahn & Park (2013), Yu et al. (2013) Chromosome counting Tek et al. (2004), Boltowicz et al. (2005), Nouri-Ellouz et al. (2006), Shi et al. (2006), Przetakiewicz et al. (2007), Chen et al. (2008), Espejo et al. (2008), Szczerbakowa et al. (2010), Ahn & Park (2013), Tarwacka et al. (2013), Yu et al. (2013) SSR marker Cai et al. (2004), Tek et al. (2004), Trabelsi et al. (2005), Nouri-Ellouz et al. (2006), Bidani et al. (2007), Lightbourn & Veilleux (2007), Thieme et al. (2010), Tiwari et al. (2010), Polzerova et al. (2011), Sarkar et al. (2011), Iovene et al. (2012), Ahn & Park (2013), Chen et al. (2013), Smyda et al. (2013), Yu et al. (2013) AFLP marker Tek et al. (2004), Thieme et al. (2010), Ahn & Park (2013) RAPD marker Cai et al. (2004), Boltowicz et al. (2005), Rokka et al. (2005), Trabelsi et al. (2005), Shi et al. (2006), Przetakiewicz et al. (2007), Chen et al. (2008), Espejo et al. (2008), Greplova et al. (2008), Szczerbakowa et al. (2010), Tiwari et al. (2010), Polzerova et al. (2011), Sarkar et al. (2011), Ahn & Park (2013), Smyda et al. (2013), Tarwacka et al. (2013) RFLP marker Tek et al. (2004), Przetakiewicz et al. (2007) CAPS/SCAR marker Nouri-Ellouz et al. (2006), Sarkar et al. (2011), Smyda et al. (2013), Yu et al. (2013) DNA sequence analysis Bidani et al. (2007) Fluorescence in situ hybridization (FISH) Tek et al. (2004) Genomic in situ hybridization (GISH) Tek et al. (2004), Iovene et al. (2012), Tarwacka et al. (2013), Yu et al. (2013) 대체가되는방법을의미하는것은아니다. 앞서언급한바와같이, 원형질체융합을통하여얻어진체세포잡종개체는지속적인선발과여교잡과같은고전적육종방법이적용되어야한다. 이뿐만아니라, 앞으로는형질전환방법까지도여기에함께적용하여유전자집적 (gene pyramiding, gene stacking) 의목표를달성하는데도움이되어다양한형질에대해우수한형질을보유한우량한감자품종을육성할수있을것이다. 색체배수성화에따른불안정한임성, 융합이후의낮은식물체재분화효율등과같은문제점을여전히가지고있다. 따라서앞으로의연구는원형질체융합을통하여발생하는이러한문제점들을개선할뿐만아니라, 불화합성을이유로형질도입이어려웠던유전자원을활용하여농업적으로중요한더욱더다양한형질들을도입하고선발할수있는육종이이루어져야할것이다. 결론 원형질체를이용한체세포잡종육성은다양한유전체적조합을통해새로운계통을육성할수있는육종의한방법이라할수있다. 이는다른다양한육종방법과비교해보았을때매우다른특성을지니고있다. 기본적으로는불화합성유전자원의좋은형질을한개체로모을수있다는장점을가지고있으며, 특히형질전환방법을이용한육종과비교했을때클로닝되지않은다양한유전자를도입할수있는다는측면과형질전환에의해생산된 GM 작물에대한사회적, 정치적반감을회피할수있다는등의다양한장점을가지고있다. 하지만체세포잡종에의해일반적으로나타나는현상이라고할수있는불화합성유전자원간의융합에따른염색체재배열, 염 사사 본연구는농림축산식품부, 해양수산부, 농촌진흥청, 산림청 Golden Seed 프로젝트 ( 세부과제명 : 수출용감자품종육성효율증진을위한분자표지및유용유전자개발, 세부과제번호 : SB540) 및농촌진흥청연구사업 ( 세부과제명 : 감자육종능력증진을위한감자유전자원관리, 세부과제번호 : PJ ) 에의해이루어진것임 References Ahn YK, Kim HY, Choi, HS, Kim K-T, Park HG (2001a) Production of interspecific somatic hybrids between the cultivated potato (Solanum tuberosum) and the wild species

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