Journal of Plant Biotechnology

  • 제44권4호
  • /
  • Pages.379-387
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  • 2017
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  • 1229-2818(pISSN)
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  • 2384-1397(eISSN)
한국식물생명공학회 (The Korean Society of Plant Biotechnology)
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사과 품종 홍로의 휴면아 분열조직 배양을 통해 형성된 캘러스에서의 바이러스 제거효율

Efficiency of virus elimination in apple calli (cv. Hongro) derived from meristem culture of dormant buds

  • 김미영 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 천재안 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 조강희 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 박서준 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 김세희 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 이한찬 (농촌진흥청 국립원예특작과학원 과수과)
  • Kim, Mi Young (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Chun, Jae An (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Cho, Kang Hee (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Park, Seo Jun (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Kim, Se Hee (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Lee, Han Chan (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration)
  • 투고 : 2017.09.18
  • 심사 : 2017.10.23
  • 발행 : 2017.12.31
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초록

Apple scar skin viroid (ASSVd), Apple chlorotic leaf spot virus(ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV)는 사과를 감염시키는 대표적인 바이러스로, 이러한 4종 바이러스에 복합 감염된 사과 '홍로'의 동절기 가지의 휴면아(측아)로부터 다양한 크기 (0.4 mm ~ 1.2 mm)와 발달단계 (이하 Stage 1, Stage 2, Stage 3라 칭함)의 경정 및 측부 분열조직을 절취하여 BA 3.0 mg/L 와 IBA 0.1 mg/L가 혼합된 배지에 배양하여 캘러스를 형성시킨 후, 감염 바이러스바이러스의 제거유무를 조사하였다. 직경 10 mm이상 증식된 캘러스 31 계통을 RT-PCR 분석으로 제거효율을 알아 본 결과, ACLSV는 100%, ASSVd와 ASPV는 93.5%의 높은 제거 효율을 보인 반면, ASGV는 25.8%로 상대적으로 제거효율이 낮았다. 4종 바이러스가 동시에 모두 제거되는 경우는 Stage 1의 휴면아의 분열조직을 배양하였을 때만 가능하였는데, 그 이유는 ASGV가 이 시기의 배양에서 주로 제거되었기 때문으로, ASGV는 다른 바이러스와는 달리 분열조직의 발달단계가 큰 영향을 미치는 것으로 나타났다. 휴면아의 분열조직을 배양하여 바이러스를 제거한 예는 세계적으로 보고된 바가 없는 것으로서, 본 연구결과는 향 후 사과의 무병묘 생산에 매우 유용한 자료로 활용될 것이다.

Various sizes (0.2 ~ 1.2 mm) and developmental stages (referred to as Stage 1 ~ 3) of apical and lateral meristems were excised, together or separately, directly from dormant buds of apple 'Hongro'. They were mixed infected by Apple scar skin viroid (ASSVd), Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV), which are major viruses attacking apples. A total of 31 callus lines (> 10 mm in diameter) were obtained by culturing the explants on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 3.0 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-butyric acid (IBA), and they were subjected to RT-PCR analysis for virus detection. A high rate of virus elimination (expressed as the percentage of calli that did not amplify during RT-PCR, i.e., RT-PCR negative calli per total number of calli obtained) was achieved for ACLSV (100%), ASSVd (93.7%), and ASPV (93.7%), whereas it was only 25.8% for ASGV. ASPV was detected in the presence of 2 ~ 3 bracts. Simultaneous virus elimination of ASSVd, ASPV, ACLSV, and ASGV occurred during the meristem culture, in which the early stages of the dormant buds (Stage 1) were used, because ASGV was mostly eliminated during that stage. The results of the present study will be valuable for the production of virus-free apple trees.

키워드

참고문헌

  1. Chen W, Tien P, Lin LP, Wang GP, Liu FC (1986) Study of viroid RNA isolated from apple scar skin diseased tissues. Chin J Virol 2:366-371
  2. El-Doungdoug KA, Osman ME, Abdelkader HS, Dawoud RA, Elbaz RM (2010) Elimination of Hop Stund Viroid (HSVd) from infected each and pear plants using cold therapy and chemotherapy. Aust J Basic Appl Sci 4:54-60
  3. Faccioli VC and Marani F (1998) Virus elimination by meristem tip culture and tip micrografting. In plant virus disease control. Hadidi A et al. (eds). American Phytopathological Society. pp. 346-380
  4. Gambino G, Perrone I, Gribaudo, I (2008) A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem Anal 19:520-525 https://doi.org/10.1002/pca.1078
  5. Grout BWW (1999) Meristem-tip culture for propagation and virus elimination. In: Hall RD (ed.). Methods in molecular biology. vol 111. Human Press Inc. Totowa, NJ. pp. 115-125
  6. Hansen A, Lane W (1985) Elimination of apple chlorotic leaf spot virus from apple shoot cultures by ribavirin. Plant Dis 69: 134-135
  7. Hu G, Dong Y, Zhang Z, Fan X, Ren F, Zhou J (2015) Virus elimination from in vitro apple by thermotherapy combined with chemotherapy. Plant Cell Tiss Cult 121:435-443 https://doi.org/10.1007/s11240-015-0714-6
  8. Kim DH, Kim HR, Heo S, Kim SH, Kim M, Shin IS, Kim JH, Cho KH, Hwang JH (2010) Occurrence of Apple scar skin viroid and relative quantity analysis using real-time RT-PCR. Res Plant Dis 16: 247-253 https://doi.org/10.5423/RPD.2010.16.3.247
  9. Kwon MJ, Hwang SL, Lee SJ, Lee DH, Lee JY (2002) Detection and distribution of the Apple scar skin viroid-Korean strain (ASSVd-K) from apples cultivated in Korea. J Plant Pathol 18: 342-344 https://doi.org/10.5423/PPJ.2002.18.6.342
  10. Lee G, Kim JH, Kim HR, Shin IS, Cho KH, Kim SH, Shin J, Kim DH (2013) Production system of virus-free apple plants using heat treatment and shoot tip culture. Res Plant Dis 19: 288-293 https://doi.org/10.5423/RPD.2013.19.4.288
  11. Lee JH, Park JK, Lee DH, Uhm JY, Ghim SY, Lee JY (2001) Occurrence of Apple scar skin viroid-Korean strain (ASSVd-K) in apples cultivated in Korea. Plant Pathol J 17:300-304
  12. Leisner SM, Turgeon R (1993) Movement of virus and photoassimilate in the phloem: a comparative analysis. BioEssays 15:741-748 https://doi.org/10.1002/bies.950151107
  13. Li BQ, Feng CH, Hu LY, Wang MR, Wang QC (2016) Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks 'M9' and 'M26'. Ann Appl Biol 168:142-150 https://doi.org/10.1111/aab.12250
  14. Menzel W, Jelkmann W, Maiss E (2002) Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. J Virol Methods 99:81-92 https://doi.org/10.1016/S0166-0934(01)00381-0
  15. Momma T, Takahashi T (1983) Cytopathology of shoot apical meristem of hop plants infected with Hop Stunt Viroid. Phytopath Z 106; 272-280 https://doi.org/10.1111/j.1439-0434.1983.tb00052.x
  16. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Phyiol Plant 15:473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  17. Paprstien F, Sedlak J, Polak J, Svobodova L, Hanssan M, Bryxiova M (2008) Results of in vitro thermotherapy of apple cultivars. Plant Cell Tiss Cult 94:347-352 https://doi.org/10.1007/s11240-008-9342-8
  18. Plopa C, Preda S (2013) Elimination of Apple mosaic virus by tissue culture of some infected apple cultivars. Acta Hortic 981:517-522
  19. Quak F (1987) Therapy of individual plants. In: Viruses of potatoes and seed-potato production. De Bokx JA and van der Want JPH (eds). Wageningen, Pudoc. pp. 151-161
  20. Shim H, Min Y, Hong S, Kwon M, Kim D, Kim H, Choi Y, Lee S, Yang J (2004) Nucleotide sequences of a Korean isolate of Apple stem grooving virus associated with Black necrotic leaf spot disease on pear (Pyrus pyrifolia). Mol Cells 2:192-199
  21. Ushirozawa K, Tojo Y, Takemae S, Sekiguchi A (1968) Studies on apple scar skin disease. 1. On transmission experiments. Bul Nagano Hort Expt Res Stn 7:1-12
  22. Wang QC, Cuellar WJ, Rajamaki ML, Hirata YM, Valkonen JPT (2008) Combined thermotherapy and cryotherapy for efficient virus eradication: relation of virus distribution, subcellular changes, cell survival and viral RNA degradation in shoot tips. Mol plant Pathol 9:237-250 https://doi.org/10.1111/j.1364-3703.2007.00456.x
  23. Wang QC, Valkonen JPT (2009) Cryotherapy of shoot tips: novel pathogen eradication method. Trends Plant Sci 14:119-122 https://doi.org/10.1016/j.tplants.2008.11.010