중앙대학교식물생명공학전공 박희승
발표순서 Ⅰ. 서언 Ⅱ. 광학현미경검경용시료준비 Ⅲ. 과수에서의연구분야예시 Ⅳ. 결론
Ⅰ. 서언 3/23 과수의각기관을구성하는조직이나세포들의연구에는실체현미경, 광학현미경, 전자현미경, 공초점레이저주사현미경등다양한현미경이이용되고있음 연구자가관찰하고자하는대상에따라위의현미경들은다시여러종류로세분되며, 각각의현미경을사용하기위한시료준비방법과염색법및절편을제작하기위한 microtome에도많은종류가있음 다양한현미경의용도는관찰하고자하는시료의크기와검경목적에따라다르며, 검경을위한준비과정과비용이적지않으므로관찰하고자하는대상과목적을명확히하여실험에임하여야함
Ⅱ. 광학현미경검경용시료준비 8/23 포매재료에따른장단점비교 Epon method Paraffin method 고정액 Glutaraldehyde + Osmium tetroxide Formaldehyde Microtome Ultramicrotome Rotary microtome 절편두께 1 μm or 60~70nm 10 μm 장점 단점 높은해상도하나의시료로광학현미경과전자현미경검경가능염색법이한정됨 보다큰크기의시료제작가능다양한염색법사용가능해상도가낮음전자현미경검경불가능
Ⅱ. 광학현미경검경용시료준비 4/23 시료채취 고정 ( 탈기 ) 탈수 포매 절편 염색 검경 1. 시료채취 (Sampling) 1 가능한한 20개정도의블록을만들수있는시료를준비한다. 2 투과전자현미경 (TEM) 뿐만아니라광학현미경용시료도가급적작게채취하는것이좋으며, 특히 TEM 에서는 1x1x1mm 정도로작게하는것이좋다. 3 방향성이요구되는경우에는 1x1x2mm 정도로한쪽방향을약간길게하여야후에 embedding 과정에서방향맞추기가용이해진다. 4 신속히고정액에담아야한다. 과육 과피
Ⅱ. 광학현미경검경용시료준비 2. 고정에서포매까지 1 고정 : 저온 ( 냉장고보관 ) 에서진행 - 1 차고정 Glutaraldehyde(2.5%) : 90 분, 4 ( 고정액에담근후바로공기제거 ) Washing : 0.1M Phosphate buffer(ph7.2), 15 분간격으로 4~5 회 - 2 차고정 Osmium tetroxide(1%) : 90 분, 4 Washing : 0.1M Phosphate buffer(ph7.2), 20 분간격으로 4~5 회 0.1M Phosphate buffer 에서 over night 5/23 2 탈수 : 상온에서진행 40% Ethanol(5 분 ) 60% Ethanol(5 분 ) 80% Ethanol(5 분 ) 90% Ethanol(5 분 ) 95% Ethanol(5 분 ) 100% Ethanol(5 분 ) 100% Ethanol(15 분 ) 100% Ethanol(15 분 ) 100% Ethanol(30 분 ) Ethanol & Propylene oxide(epon 의용매, 1 : 1, 15 분 ) Propylene oxide(15 분 ) Propylene oxide(15 분 ) Propylene oxide(30 분 ) Propylene oxide & Epon : 약 2:1 로섞은후최소 1 시간 Propylene oxide 와 Epon 을 1:1 로섞은후최소 1 시간 Epon(DMP30 을넣지않은것 ) 에서 overnight 3 포매 (embedding) : 상온 + 60 에서진행 Epon + DMP30(Epon 의 1.5%) : 60 에서 4 일동안 Hardening
Ⅱ. 광학현미경검경용시료준비 6/23 3. 절편제작 (Sectioning) 1 포매된 block을 trimming 한다. 2 Ultramicrotome을사용하여전자현미경검검용은 60~70nm, 광학현미경검경용은 1µm의두께로절편을제작한다. 4. 염색 (PAS 염색법 ) 1 Periodic acid(h 5 IO 6 ) 용액 : 30분 2 증류수로 10분간 2~3회 Washing 3 Schiff s Reagent : 15분 4 Sodium bisulfite 용액 : 15분 5흐르는물에 30분간세척 5. Histomount 를이용하여 cover glass 를씌움
6. 현미경검경 7/23 실체현미경 광학현미경
Ⅲ. 과수에서의연구분야예시 9/23 Sound axillary bud (A) and main bud necrosis of axillary bud (B) in Campbell Early grapevines. AB, accessory bud; IP, inflorescence primodium; LP, leaf primodium; MB, main bud, N, node. ( 최인명ㆍ이창후ㆍ홍윤표ㆍ박희승. 2007. Kor. J. Hort. Sci. Technol.) The necrosis types of bud observed by stereomicroscope in Hongisul grapevine. A: normal bud, B: whole bud necrosis, C: main bud necrosis, D: accessory bud necrosis. MB: main bud, AB: accessory bud. Arrows ( ) indicate the necrosis part. ( 김은주ㆍ이별하나ㆍ권용희ㆍ신경희ㆍ정규환ㆍ박서준ㆍ박희승. 2011. Kor. J. Hort. Sci. Technol.)
10/23 Fig. 3. Cross section of floral bud in Fuji apple trees at 75 (A), 150 (B), 210 (C), 240 (D), 270 (E), and 300 (F) days after full bloom. FM, floral meristem; S, scales; Fp, flower primordium; Scale bars = 500 μm. ( 이별하나ㆍ박요섭ㆍ박희승. 2015. Kor. J. Hort. Sci. Technol.) Fig. 4. Cross section of floral bud necrosis in Fuji apple trees at 255 (A, A ) and 300 (B, B ) days after full bloom. Arrows indicate necrotic spots. Scale bars = 500 μm. 7 월중순경부터분열조직이비대하여돌기모양으로솟아오른형태가관찰되어이시기에영양생장으로부터생식생장으로의전환이이루어지는것을알수있음 (Fig. 3A). 9 월에는중심화의화원기가뚜렷하게관찰됨 (Fig. 3B). 중심화의원기가관찰된후약 90 일정도지난 12 월에최소 3 개이상의화원기가형성되어있음 (Fig. 3D). 12 월은내재휴면심도가최대에도달한시기임이밝혀져내부의구조적인변화가거의없을것으로생각되었으나, 내부적인발달은지속되고있음을알수있음 (Fig.3F). 따라서 Fuji 사과의내재휴면은꽃의원시세포 (flower primordium) 가형성되는시기에시작되어수술을포함한꽃의내부구조물이형성된이후타파됨을확인함. 휴면기간동안대부분눈이건전하게생존해있으나일부에서화원기의괴사현상이나타남 (Fig. 4). 꽃눈괴사시냉각또는서리에의한피해는저온에직접적인영향을받기보다는주로얼음결정형성에의해발생하며, 얼음결정이발생하는위치에따라세포의피해유무가달라진다고알려져있음.
11/23 포도에서암술이형성되는과정. 포도는 2 개의심피가융합하여 1 개의자예를이루는합생자예로써 2 개의심피가융합하여 2 개의심실을형성하나융합되지않는부위에서는갈라진틈이나타난다. A ; 가장바깥쪽으로부터악편, 화판, 수술이발달하면서가장안쪽에 2 개의심피가형성되는초기단계. B : 심피가점점생장하면서안쪽으로배주의원기가형성된다. C : 심피가화축방향으로자라면서 2 개의심피가맞닿을듯이가까워짐. D : 안쪽으로자라던심피가융합하면서암술의아래쪽으로생장하여 2 개의심실을형성함. (BENHARBIT EL ALAMI Naïma. 1995)
12/23 Merlot. Très jeune baie quelques jours après la nouaison. Position des faisceaux conducteurs et importance relative des différents tissus pendant le développement de la baie (M. FOUGÈRE-RIFOT, H.-S. PARK, J. BOUARD. 1993. J. Int. Sci. Vigne Vin)
13/23 E H 포도품종의성숙기과실조직 ( 좌 : 캠벨얼리, 우 : 거봉 ) F 배품종의성숙기과실조직 ( 좌 : 화산, 중 : 신고, 우 : 황금배 )
T F E H S S E H F 14/23 감홍품종의반점성장해정상조직반점부위 T H E S E H E S T F F F 50µm 50µm Cross section of Tsugaru apple Cross section of the pitted tissue fruit in maturity in Tsugaru apple Abbreviation; E : Epidermis, F : Flesh, H : Hypodermis, S : Starch Grain, T : Tannin 50µm Cross section of the cork tissue in Tsugaru apple
15/23 Before the initiation of the disorder Initial stage Mid stage Final stage Fig. 3. Developing procedure of peeling-off disorder on Niitaka pear from harvest to 120 days of storage. A: harvest time on Niitaka pear fruit skin; B: initial stage on peeling-off disorder; C: mid stage on peeling-off disorder; D: final stage on peeling-off disorder. CC, cork cell; H, hypodermis; NTC, non-tannin cell; P, phellogen; P (PE), phellogen (elongated cell); SC, stone cell; TC, tannin cell. (Yoon-Pyo Hong, Seung-Koo Lee, Youn-Moon Park and Hee-Seung Park. 2008. J. Japan. Soc. Hort. Sci.)
Fig. 5. Transmission electron micrograph of the sound (A, x27,800), collapsed (B, x1,670) and the thinned cell wall (C, x1,670) in peeling-off disorder of Niitaka pear. CC, cork cell; CW, cell wall; ML, middle lamella; P, phellogen; P (EC), phellogen (elongated cell); TP: tonoplast. 16/23
17/23 A B Morphological structure at 90 days after full bloom of Heukboseok grape treated with GA 3 and TDZ. (A) non-treated; (B) primary and secondary 25 mg L -1 GA 3 + 2.5 mg L -1 TDZ F: flesh; H: Hypodermis; IS: Intercellular space; OE: Outer epidermis, TC: Tannin cell. ( 정명희, 이별하나, 박요섭, 오진표, 김희섭, 박희승. 2015. Kor. J. Hort. Sci. Technol.)
18/23 Fig. 2. Structural changes in flesh tissue as a cause of softening. A to C are respective pictures of fruit flesh at 30, 70, and 110 days after full bloom, which shows that the increase in intercellular space occurs primarily due to cell expansion. D to F show fruit flesh harvested 120, 130, and 140 days after full bloom, respectively. G to I show the flesh of fruits stored at room temperature for 20 days after harvesting at 120, 130, 140 days after full bloom. J to L show the flesh of fruit stored at low temperature for 70 days after harvesting at 120, 130, and 140 days after full bloom. (YoSup Park, ByulHaNa Lee, Hee-Seung Park. 2016. Scientia Horticulturae)
19/23 Fig. 1. Cracking on surface of Mansoo pears. Distribution of cracks through fruit surface (A), occurrence of shortlength-shaped cracking (SLSC) on the lenticel (B), enlargementof cracks (C), normal surface of the Mansoo pear (D) and normal surface of the Niitaka pear (E). Scale bar = 1 mm. Fig. 3. Surface and transverse section of Mansoo and Niitaka pear peal at maturity observed by SEM (A C) and LM (D F). Normal (A, D) and cracked region (B, E) of Mansoofruit and normal region of Niitaka (C, F). C, cork tissue; Cr, crack; F, filling tissue; H, hypodermis; IS, intracellular space; L, lenticel; P, parenchyma; SC, stone cell. Scalebar = 500 m. (YongHee Kwon, Hyun-Hee Han, Hee-Seung Park. 2016. Scientia Horticulturae)
20/23 Development of the pedicels of non-treated (A-D) and GA-treated fruits (E-H) as observed under an optical microscope. Exogenous GA was applied to the pedicel at 35 days after full bloom (DAFB). The images show development at 40 (A and E), 60 (B and F), 80 (C and G) and 100 DAFB (D and H). C, cork layer; Ct, cortex; Ft, fibrous tissue; P, phloem; Pi, pith; X, xylem. (Yosup Park, Hee-Seung Park. 2017. Scientia Horticulturae) The final size of the pedicels did not differ between treatments, but the GA-treated pedicels attained their final size 20 days earlier than the nontreated pedicels due to the accelerated enlargement of the GA-treated pedicels shortly after treatment. The increased size of the GA-treated fruits appeared to be dependent on increased cell expansion, whereas the accelerated pedicel development appeared to be dependent on increased rates of cell division. In particular, the increase in the diameter of the GA- treated pedicels was primarily due to improvements in the sizes of xylem and phloem tissues as a result of acceleration of cell division. In addition, the effect of exogenous GA on the acceleration of pedicel development was concentrated at the initial stage of active cell division, and during this period, the sizes began to differ between the GA-treated and non-treated fruits.
21/23 Merlot 포도과실의과피와과육의경계 ( 박희승 ) 청도반시단감에에틸렌발생제처리 7 일후세포벽분해 ( 박서준, 박희승, 김종천 ) 신고배의탈코르크과정에서의코르크형성층세포의세포벽분해 ( 홍윤표, 이승구, 박윤문, 박희승 )
Ⅳ. 결론 22/23 과수각기관의세포분열, 세포비대및조직분화등기본적인구조에대한연구는과수재배시시기별로발생하는여러가지현상에대한이해를가능하게함 수확전 후의다양한환경에서발생하는생리장해의원인구명과피해경감기술을개발하는데활용도가매우높음 과수재배시이용되는각종처리에대한식물체의반응을직관적으로구명할수있음 신품종육성시과수의해부학적특성파악은재배상에서나타나는다양한현상을예측가능하게해줌 따라서현미경을이용한연구는모든식물의구조와기능을파악하기위한연구에활용될수있는것과마찬가지로과수연구에서도다양한분야에활용할수있음 반면에과수는원예연구의한분야로써응용과학의특성상해부학적연구만으로모든문제를해결하기에는한계가있을수있으며현장연구나대사생리를포함한다양한연구와접목될때상승효과를낼수있음