Kor. J. Hort. Sci. Technol. 31(1):117-122, 2013 DOI http://dx.doi.org/10.7235/hort.2013.12150 잇꽃종자의발아에서제 (Hilum) 의형태적특성과기능 안석현ㆍ정남진 * 전북대학교농업생명과학대학작물생명과학과 Morphological Characteristics and Function of Hilum in Safflower Seed Germination Seok-Hyeon Ahn and Nam-Jin Chung * Department of Crop Science & Biotechnology, Chonbuk National University, Jeonju 561-756, Korea Abstract. This research was carried out to clarify the germination characteristics with reference to hard seed coat in safflower. Morphologically, seed coat surface has hilum and micropyle which were evident during seed development stage. In the flower of developing seeds, the hilum area is connected with placenta of maternal tissue while the micropyle area is connected with the style of pistil. When the seeds imbibed, the hilum surface began to crack and the embryo protrudes through the hilum. To investigate the route for moisture absorption and gas exchange on the seed coat, the hilum and the micropyle were artificially sealed by paraffin. The seeds whose hilum were sealed could not germinate, which indicates that the exchange of moisture and oxygen takes place through hilum in safflower seeds. The germination was tested at 15, 20, and 25 by three substrates with different moisture conditions; top of paper method (hilum submerged in water), between-paper method, and soil seeding. The germination percentages were 31.3% at 15, 15.7% at 20 and 6.0% at 25 in the top of paper method; and 45.5% at 15, 30.0% at 20 and 14.0% at 25 in the between-paper method; and 80.0% at 15, 77.0% at 20 and 78.0% at 25 in the soil seeding, respectively. When the internal structure of hilum was investigated through SEM, it was found out consisting of vascular bundle element. In conclusion, the hilum of safflower seed was closely related with water absorption and gas exchange during initial germination process. Additional key words: hard seed, micropyle, gas and water exchange 서언잇꽃 (safflower, Carthamus tinctorious L.) 은국화과 (Compositae) 에속하는 1년생초본으로홍람 ( 紅藍 ) 홍화 ( 紅花 ) 잇나물이라고칭하며원산지는아프가니스탄의산악지대또는이집트및에티오피아가원산이며, 한나라시대에중국에유입되었고, 우리나라에는신라시대이전부터약용이나염료작물로재배되어온것으로알려져있다 (Choi, 1992; Choi et al, 2011; RDA, 1990). 줄기의길이는약 1m 내외이고, 꽃은 6월중순경에피고넓은피침형의가장자리에는가시가돋쳐있다 (Ahn and Yuk, 1975; Lee, 1980). 잇꽃은천연염료, 식용유, 곡식, 한방약제, 화장품원료등으로다양하게사용되고있다. 꽃에함유되어있는수용성의황색색소와불용성의적색색소는식품의착색염료등천 연염료사용되고있으며, 잇꽃씨는혈액, 심장, 자궁, 비뇨기와관련된약용으로사용된다 (Choi et al, 2011; Lee et al., 2008). 잇꽃기름은불포화지방산인 linoleic acid나 oleic acid 함량이높아서식용유로이용되고있고, 앨러지를유발하지않기때문에화장품의원료로도쓰이고있다 (Noh and Park, 1992). 또한, 잇꽃종자에는단백질및식이성섬유가풍부할뿐만아니라 lignin flavonoid와 serotoinin 그리고이들의배당체같은항암, 항염증및항산화성페놀화합물을다량함유하고있어최근기능성신소재로써의활용이활발히연구되고있다 (Lee et al., 2008; Shimomura et al., 1990; Stobart et al., 1997; Takeuchi et al., 1995; Zhou et al., 1992). 잇꽃종자의발아에관해서는아직체계적인연구가미흡한상태이나그동안보고된내용을보면, 잇꽃종자는 2-5 부 *Corresponding author: njchung@jbnu.ac.kr Received 27 July 2012; Revised 17 October 2012; Accepted 24 October 2012. Kor. J. Hort. Sci. Technol. 31(1), February 2013 117
터발아가가능하며 (Johnston et al., 2002), 발아적온은 Ryu et al.(2005) 은 25 라하였고, Jang(2009) 은 5-10 로보고하여서로상이한연구결과를나타내고있다. 종자에서제 (hilum) 는배병또는태좌에붙어있던흔적으로제의색과모양은종자에따라다양한형태를이루고있으며, 발아공 ( 주공, micropyle) 은화분관이수정을위하여배낭으로침입하는통로로써배주의한쪽끝에있던구멍이종자에남은흔적이다 (Copeland and McDonald, 2001; Hong et al., 2009). 잇꽃종자에서발아공및제의구조와기능에대해서는보고된바없으나, 마편초종자에서는종자의수분흡수에제가주요한역할을한다고하며 (Maekawa and Carpenter, 1991), 대부분종자에서수분흡수가종피보다는발아공을통하여왕성하게이루어진다고보고되고있다 (Bewely and Black, 1982). Hyde(1954) 는 Vicia속과팥속 (Phaseolus) 식물의종자는건조할경우제부분이열리며, 이를통해수분이빠져나와종자내부가건조되며, 강낭콩은제, 주공, 봉선 (raphe) 을통해서전체수분의 97-98% 를흡수한다고한다. 종피가두껍고단단한경실종자는등숙후일정기간동안수분흡수및공기교환이불가능하여종피휴면을나타내는것이대부분이다 (Bewley, 1988; Copeland and McDonald, 2001). 그러나잇꽃은경실종자임에도불구하고종자휴면이없으며, 수확기인 7-8월경의장마로수발아피해가발생하기도한다 (Choi et al., 2005). 따라서본연구에서는잇꽃종자의구조와경피와관련된발아특성을조사하였으며, 연구과정중제가종자발아에밀접한관련이있어주사전자현미경으로제부위의형태적특성을관찰하였다. 재료및방법식물재료및형태관찰본연구에서사용한잇꽃품종은청수이며, 전북대학교실험포장의비닐하우스내에서재배하여사용하였다. 잇꽃종자의표면에있는특수조직의형성과정을확인하기위하여재배중인식물체 (2011년 3월 14일파종 ) 에서개화후약 14일 (7월 14일 ) 에꽃을수집한후종축으로절단하여해부현미경 (SZ61, Olympus, Japan) 으로화기조직에서종자의위치를관찰하였다. 미숙종자의단면구조는꽃에서채취한종자 ( 개화후 14일 ) 를종축방향으로메스로절단하여 2,3,5-triphenyl tetrazolium chloride (TTC) 1% 용액으로 30 항온기에서 2시간동안염색한다음해부현미경 (SZ61, Olympus, Japan) 으로관찰하였다. 또한, 성숙종자의외부형태와종축단면, 그리고발아시유아의돌출부위도 위와같은방법으로관찰하였다. 종자의형태적특성으로종자의길이, 두께, 폭, 천립중을측정하였고, 종자외형의특수기관인제와주공의길이와폭을측정하였다. 또한종피의두께, 경도, 천립중을측정하였다. 종자의파라핀처리와발아율검정종자파라핀처리및발아율검정전에 70% ethanol에서 1분, 2% sodium hypochlorite solution에서 15분동안 shaking 하여소독한후증류수로수세하였다. 소독종자는상온에서 24시간자연건조시킨후용해된파라핀을제와주공에각각떨어뜨렸다. 종자위에서파리핀이굳은후에손끝으로부드럽게문질러서표면이완전히파라핀으로코팅되어밀봉되도록하였다. 파리핀처리종자의발아율검정은파종상자 (15.5cm 6cm 10cm) 에상토 (Arichan, Sang-lim Co., Korea) 를 7cm 정도채우고종자를 1.5 1cm 간격으로 50립씩파종후상토를채워파종심도가 3cm가되도록하였다. 파종상자는저면관수로수분이공급되도록수분공급용트레이에항상 0.5-1cm 의증류수가유지되도록하였다. 파종상자는 25 incubator (VS-1203PI-LN, Vision Scientific Co., Korea) 에서완전임의배치 4반복으로 14일간처리후발아율을조사하였다. 온도와수분공급조건에따른잇꽃종자의발아율을조사하기위하여다음의 3가지파종방법을이용하여발아율을검정하였다 ; top-of-paper method(tp), between-paper method(bp), soil seeding method. TP 는 90mm 의 perti-dish 에여과지 (Quantitative filter papers No. 2, Advantec, Japan) 를깔고종자를치상하고증류수를공급 (10mL/petri-dish) 하여제부분이증류수에침수되도록하였다. BP는 seed testing paper(whatman) 에증류수를스프레이하여적신후종자를 paper 상부에 1cm 간격으로 4줄로파종하였다. 파종후다른한장의젖은 paper를덮고느슨하게종이를말아서고무줄로고정시킨후수분공급용트레이에종자가위쪽으로올라오도록세우고트레이에는 3cm 정도의증류수가유지되도록하였다. Soil seeding은파라핀처리종자의발아율검정과같은방법으로상토에파종하여발아율을측정하였다. 온도는 15, 20, 25 의 3처리로하였으며, 발아율은반복당 100립씩 4반복으로, 파종후 14일후에조사하였다 (ISTA. 2005). 종자제조직횡단면의미세구조관찰무처리종자와 15 에서 48시간동안침종시킨종자의제조직횡단면을 SEM으로비교관찰하기위하여, 종자를전처리액인 isomyl acetate로치환하고임계점건조기내에서액체이산화탄소로치환한다음임계점 ( 약 40, 90-110kg cm -2 ) 118 Kor. J. Hort. Sci. Technol. 31(1), February 2013
에서건조시켰다. 그후금속증착 (gold coating) 과정을거쳐 SEM (JSM-5600LV, JEOL, Japan) 을이용하여관찰하였다. 결과및고찰화기내부에서종자의형성과정은 Fig. 1에서보는바와같다. 종자의하부는모식물체의자실벽의태좌 (placenta) 와연결되어있고상부는암술의화주 (style) 와연결되어있다 (Figs. 1A and 1B). 종자발달이완료되면하부의자실벽과연결되었던흔적은제 (hilum) 가되고암술대와연결되었던흔적은주공 (micropyle) 으로형성되는것을확인하였다 (Fig. 1D). 잇꽃의꽃에서발달과정에있는종자의종축단면을 tetrazolium으로염색하여관찰한사진 (Fig. 1C) 을보면, 종피조직은암술대와연결되어있는주공부위를포함하여종자의외곽부위에발달되어있고제부위는덮고있지않으며, 제는종피와는다른조직으로발달되어있다. 종피내부에는자엽이발달되어있고자엽사이에배가위치하고있다 (Fig. 1E). 종자의외관을보면, 종자모양은방추형이며종피색은흰색에가까운옅은회색빛을띄고있다 (Fig. 1D). 방추형의뾰족한부분에제조직이있으며제의반대편에암술의화주부분이연결되어있던주공이위치하고있다 (Fig. 1D). 종자의발아시제를통하여배가출현하며이때종피조직이두쪽으로벌어지며발아된다 (Fig. 1F). 잇꽃종자의크기는 Table 1에나타낸바와같이, 길이는 6.54 ± 0.33mm, 두께는 3.70 ± 0.22mm, 종자의폭은 4.53 ± 0.38mm이었고, 천립중은 34.5 ± 0.09g이었다. 잇꽃종자에서종피의천립중은 20.9 ± 0.09g으로종자무게의 60.5% 에해당되며, 두께는 0.42 ± 0.08mm, 경도는 2.06 ± 0.47kg mm -2 으로매우단단한경실종자이다 (Table 2). 종자의외형에나타난특수기관인제의크기는길이 1.07 ± 0.14mm, 폭 0.97 ± 0.09m였고, 표면적의 4.6% 를차지하고있으며, 주공은지름이 1.28 ± 0.16mm인원형으로표면적의 8% 에해당된다 (Table 3). 잇꽃종자의흡습과정에서제와주공의형태적변화를관찰한결과는 Fig. 2와같다. 제는종자침종후 2-3시간부터중심부를기점으로타원형으로표면이갈라졌으며, 발아공은수분흡수와종자가발아되는과정에서형태적변화가나타나지않았다. 잇꽃종자의발아에서수분흡수와가스교환이이루어지는부위를알아보기위하여제와주공을파라핀으로봉합하여발아율을검정한결과는 Table 4와같다. 주공을파라핀으로봉합한종자의발아율은 81% 로무처리종자의발아율 (80%) 과차이가없었으나, 제부위를봉합한종자는 2% 의발아율을보였다. 따라서, 종자의제부분을통하여수분흡수와가스교환이이루어지는것으로판단된다. 주공과제 Table 2. Characteristics of seed coat in safflower seed. Hardness (kg mm -2 ) Thickness Weight of 1000 seed coat (g) 2.06 ± 0.47 0.42 ± 0.08 20.9 ± 0.09 Table 3. Size of hilum and micropyle on seed surface of safflower. Hilum Micropyle Length Width Diameter 1.07 ± 0.14 0.97 ± 0.09 1.28 ± 0.16 Fig. 1. Pictures of safflower seed at developing stage (A, B, and C) and matured stage (D, E, and F). A, Longitudinal section of flower; B, Developing seed located between placenta and style in flower; C, Longitudinal section of developing seeds (stained with tetrazolium); D, Seed exterior; E, Longitudinal section of seed; F, Sprouting seed. Table 1. Seed size and weight in safflower. Length Width Thickness Weight of 1000 seeds (g) 6.54 ± 0.33 4.53 ± 0.38 3.70 ± 0.22 34.5 ± 0.09 Fig. 2. Pictures of hilum and micropyle surface before and after imbibitions. Kor. J. Hort. Sci. Technol. 31(1), February 2013 119
등종자표면에있는특수구조가종자수분흡수과정에서의역할에대하여잇꽃종자에서는보고된바없으나다른종의종자에서보고된바에의하면, Phaseolus vulgaris (great northern bean) 종자는발아공의개폐가수분흡수와관련이있으며 (Kyle, 1955), 홍초 (Cannaceae) 종자의경우수분흡수가봉선 (raphe) 을통하여이루어지는것으로보고되었다 (Grootjen and Bouman 1988). 마편초 (Verbena) 에서는제의구조가종자의발아와밀접한관련이있으며 (Maekawa and Table 4. The effect of paraffin sealing of hilum and micropyle on germination in safflower seed. Treatments Germination percentage (%) Control Hilum sealing Micropyle sealing 80.0 a z 2.0 b 81.0 a z The same letters are not significantly different at 5% level of DMRT. Fig. 3. Germination percentage of safflower seeds at different temperature (15, 20, and 25 ) and seeding methods (top of paper, between-paper, and soil seeding). Carpenter, 1991), Vicia속과 Phaseolus속식물의종자가건조한상태에놓이면제부분이열리고, 이를통해수분이빠져나와종자내부가건조되며, 강낭콩은제, 주공, 봉선 (raphe) 을통해서전체수분의 97-98% 를흡수하는것으로보고되었다 (Hyde, 1954). 온도와발아기질에따른발아율검정결과는 Fig. 3과같다. 제부분이물에잠기도록처리한 top of paper method에서종자의발아율은 15 에서 31.3%, 20 에서 15.7%, 25 에서 6.0% 로, 전반적으로저조한발아율을보였고온도가높아지면서발아율은감소되었다. 발아용흡습지를이용한 between-paper method에서는 15 에서 45.5%, 20 에서 30.0%, 25 에서 14.0% 로 top of paper method에의한발아율보다는다소높게나타났으며온도가증가할수록발아율이낮아지는경향은같게나타났다. 한편, 토양에종자를파종하고파종상자의하부를통하여수분을공급한 soil seeding 처리에서는온도에관계없이 80% 내외의정상적인발아율을보였다. 이러한결과로볼때, 잇꽃종자의발아에수분공급방법과온도가밀접한관련이되어영향을미치는것으로보인다. 즉, 종자의발아시수분이이동되는경로가제 (hilum) 로한정되어있기때문에과도한수분이공급될경우제입구가물로막히어산소의출입이차단될수있다. 이경우높은온도에서호흡장해로인하여발아율이낮아지는것으로추측된다. 그동안잇꽃종자의발아적온이연구자에따라저온 (5-10 ) 또는고온 (25 ) 으로결과가다르게보고되었던것은 (Jang, 2009; Ryu et al., 2005) 수분공급방법의차이에의한것으로판단된다. 잇꽃종자의수분공급과산소교환통로인제의미세구조를확인하고자종자의흡습전후의형태를주사전자현미경 Fig. 4. SEM pictures of longitudinal sectioned hilum before and after imbibitions in safflower seed. 120 Kor. J. Hort. Sci. Technol. 31(1), February 2013
으로관찰한결과는 Fig. 4와같다. 제의두께는약 750μm 였으며, 제표피의두께는 10-30μm 정도였다. 제의내부조직은도관요소를이루는세포로구성되어있었다. 도관요소의상층부는공문상비후조직 (foraminate thickening tissue) 으로이루어져있었으며 (Fig. 4, 2-1) 나머지부분은망상비후조직 (reticulate thickening tissue) 으로구성되어있었다 (Fig. 4, 2-3 and 2-4). 수분흡수전 후의제의표면사진을보면, 식물체잎의기공이변형된모습으로중심부위에돌기 (micro protuberance) 가있고돌기를중심으로주변조직이수분이흡수되면열리는것을확인할수있었다. 기공내부의도관조직은수분흡수전후형태적변화는나타나지않았다. 이러한결과를종합해보면, 잇꽃종자의제 (hilum) 는발아과정에서수분흡수와가스교환의통로가되는것으로판단된다. 초록본실험은경실종자인잇꽃의종자구조와발아특성을밝히고자수행하였다. 잇꽃종자의외형적특징은단단한종피표면에제 (hilum) 와주공 (micropyle) 이있으며, 제는종자의발달과정에서태좌와연결되어있었고, 주공은암술대와연결되어있었던부분이다. 종자침종후 2-3시간후에제의표면이열렸으며, 발아시유아 (embryo) 가제를뚫고돌출되었다. 잇꽃종자의흡습전에제와주공에파라핀을처리하여밀봉한결과, 제부위를밀봉한처리에서종자의발아가이루어지지않아잇꽃종자의수분과산소교환이제를통하여이루어진다는것을확인하였다. 잇꽃종자의제가물에잠기도록수분을공급한발아실험 (top of paper method) 에서는 15 에서 31.3%, 20 에서 15.7%, 그리고 25 에서 6.0% 가발아하였으며, between-paper method에의한실험에서는 15 에서 45.5%, 20 에서 30.0%, 25 14.0% 로발아율이나타났고, 토양에파종한종자는온도에관계없이 80% 내외의발아율을보여, 잇꽃종자의발아에수분공급조건과온도가밀접하게연관되어있었다. 제 (hilum) 의내부구조를 SEM(scanning electron microscope) 을이용하여관찰한결과도관요소를구성하는세포로이루어져있었다. 이러한결과로볼때, 잇꽃종자의제 (hilum) 는발아과정에서수분흡수와가스교환통로가되는것으로판단된다. 추가주요어 : 경실종자, 주공, 공기및수분교환 인용문헌 Ahn, D.G. and C.S. Yuk. 1975. Modern materia medica. Komoon Ltd., Seoul, Korea. Bewely, J.D. and M. Black. 1982. Physiology and biochemistry of seeds in relation to germination. Springer-Verlag, New York, U.S.A. Bewley, J.D. 1988. Seed dormancy and germination. Champman & Hall, New York, U.S.A. Choi, C.H., H.D. Kim, and E.B. IM. 2011. Reviews of research trends on safflower seed (Carthamus tinctorius L.). J. Oriental Medicinal Classics 24:63-90. Choi, S.Y., J.C. Kim, and K.J. Kim. 2005. Effect of cultivation methods and harvesting time on yield and quality of Safflower (Carthamus tinctorious L.) seeds. Korean J. Crop Sci. 50: 187-190. Choi, Y.J. 1992. Perfume medicinal taste spice encyclopedic of plants. Osung, Ltd., Seoul, Korea. Copeland, L.O. and M.B. Mcdonald. 2001. Principles of seed science and technology. Champman & Hall, New York, U.S.A. Grootjen, C.J. and F. Bouman. 1988. Seed structure in cannaceae: Taxonomic and ecological implications. Ann. Bot. 61:363-371. Hong, B.H., K.H. Kang, B.H. Choi, J.G. Kim, S.H.Kim, and T.G. Min. 2009. New seed science. Hyangmoon Ltd., Seoul, Korea. Hyde, E.O.C. 1954. The function of the hilum in some papilionaceae in relation to the ripening of the seed and the permeability of the testa. Ann. Bot. 18:241-256. International Seed Testing Association (ISTA). 2005. International rules for seed testing edition 2005. ISTA, Bassersdorf, SH-Switzerland. Jang, S.J. 2009. Determination of antioxidative activity and phenolic compounds in germinated safflower seeds. MS Thesis, Chonbuk Natl. Univ., Jeonju, Korea. Johnston, A.M., D.L. Tanaka, P.R. Miller, S.A. Brandt, D.C. Nielson, G.P. Lafond, and N.R. Riveland. 2002. Oilseed crops for semiarid cropping systems in the northern great plains. Agron. J. 94:231-240. Kyle, J.H. 1955. A study of the relationship of the micropyle opening to hard seeds in the Great Northern bean. MS Thesis, Univ. of Idaho, Moscow, U.S.A. Lee, C.B. 1980. Picture book of Korean plants. Baekyang Ltd., Seoul, Korea. Lee, K.S., Y.H. Kim, and N.J. Chung. 2008. Determination and isolation of antioxidative serotonin derivatives, N-(p-Coumaroyl) serotonin and N-feruoylserotonin from safflower seeds. Korean J. Crop Sci. 53:171-178. Maekawa, S. and W.J. Carpenter. 1991. Verbena seed hilum morphology contributes to irregular germination. Hort. Sci. 26:129-132. Noh, W.S. and J.S. Park. 1992. Lipid composition of Korean safflower seeds. J. Korean Agric. Chem. Soc. 35:110-114. Rural Development Administration (RDA). 1990. Color atlas. Native plants of Korea (herbaceous plant). RDA, Suwon, Korea. Ryu, J.H., J.W. Seo, and C.S. Kang. 2005. The effect of some stress on the germination and the growth of seedling in safflower (Carthamus tinctorious L.). Bul. Agricultural College, Chonbuk Natl. Univ. 36:49-64. Kor. J. Hort. Sci. Technol. 31(1), February 2013 121
Shimomura, Y., T. Tamura, and M. Suzuki. 1990. Less body fat accumulation in rats fed a safflower oil diet than in rats fed a beef tallow diet. J. Nutr. 120:1291-1296. Stobart, K., M. Mancha, M. Lenman, A. Dahlqvist, and S. Stymne. 1997. Triacylglycerols are synthesised and utilized by transacylation reactions in microsomal preparations of developing safflower (Carthamus tinctorius L.) seeds. Planta 203:58-66. Takeuchi, H., T. Matsuo, K. Tokuyama, Y. Shimomura, and M. Suzuki. 1995. Diet-induced thermogenesis is lower in rats fed a lard diet than in those fed a high oleic acid safflower oil diet, a safflower oil diet or a linseed oil diet. J. Nut. 125:920. Zhou, Q. H., V. Klekner, and N. Kosaric. 1992. Production of sophorose lipids by Torulopsis bombicola from safflower oil and glucose. J. Amer. Oil Chemists Soc. 69:89-91. 122 Kor. J. Hort. Sci. Technol. 31(1), February 2013