Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Elimination of Grapevine fleck virus from infected grapevines 'Kyoho' through meristem-tip culture of dormant buds

휴면아 경정 배양법을 통한 포도 '거봉' 에서 Grapevine fleck virus의 제거

  • Kim, Mi Young (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) ;
  • Chun, Jae An (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)
  • 김미영 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 조강희 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 천재안 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 박서준 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 김세희 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 이한찬 (농촌진흥청 국립원예특작과학원 과수과)
  • Received : 2017.10.06
  • Accepted : 2017.11.07
  • Published : 2017.12.31
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Abstract

Herein, we report the meristem-tip culture from dormant buds of grape 'Kyoho' single-infected with Grapevine fleck virus (GFkV), which is phloem-limited and transmitted by graft inoculation. We produced GFkV-free shoots without thermo- or chemotherapy using meristem-tip explants approximately 0.3 mm (73 explants) and 0.8 mm long (five explants) including shoot apical meristem, 2-5 leaf primordia, and 1-4 uncommitted primordia from dormant buds of the infected woody cuttings (stored at $4^{\circ}C$). Explants were cultured 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). After 16 weeks of culture, shoot (10-mm long) regeneration frequency achieved from 0.3-mm explants was 4.1% and that obtained from 0.8-mm explants was 40.0%. Virus-free efficiency (expressed as the percentage of RT-PCR negative shoots regenerated) from 0.3- and 0.8-mm explants was 100% and 50%, respectively. Following in vitro multiplication, RT-PCR assays revealed identical results to assays of the first regenerated shoots. Our new methodological approach could be applied for eliminating other viruses in grapevines, as well as for producing virus-free plants in many other deciduous tree species, including fruit trees.

본 연구진은 세계 최초로 GFkV에 단독 감염된 포도 품종 거봉의 휴면아로부터 직접 경정 분열조직을 절취하여 배양함으로써 별도의 열처리나 화학처리 없이 GFkV가 제거된 신초 재생에 성공하였다. GFkV는 포도 식물체의 체관에만 한정적으로 존재하고 접목으로 감염되는 포도 바이러스이다. 경정조직은 $4^{\circ}C$에서 일정기간 저장된 1년생 경화가지의 휴면아로부터 0.3 mm (73 절편체)와 0.8 mm (5절편체)를 절취하였는데, 절취 부위는 생장점(apical meristem)을 포함하여 2 ~ 5개의 엽원기(leaf primordia)와 미분화 원기(uncommitted primordia) 1 ~ 4개를 포함하였다. 절취된 경정조직을 BA 3.0 mg/L와 IBA 0.1 mg/L가 혼합된 배지에 16주간 배양한 결과, 0.3 mm와 0.8 mm의 경정조직에서 각각 4.1%와 40.0%의 신초 재생률이 관찰되었고, 재생된 신초에서의 바이러스 제거율(재생된 신초수에 대한 RT-PCR negative 신초수의 백분율)은 0.3 mm의 경정조직에서는 100%를, 0.8 mm에서는 50%를 나타내었다. 신초를 증식시킨 후 감염바이러스를 다시 진단한 결과, 신초가 처음 재생되었을 때의 진단결과와 동일하였다. 휴면아로부터 분열조직을 배양하여 바이러스가 제거된 신초를 확보한 예는 세계적으로 보고된 바가 없는 것으로서, 본 연구진의 새로운 바이러스 제거방법은 향후 포도 뿐만 아니라 과수를 포함한 낙엽성 수목의 무병묘 생산에 매우 유용하게 활용될 수 있을 것이다.

Keywords

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