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Evaluation of Tissue Culture Efficiency in a Speed Breeding System for Stable and Sustainable Supported Wheat (Triticum aestivum) Immature Embryogenesis

안정적이며 지속적 밀(Triticum aestivum) 미성숙배 조직배양을 위한 스피드 브리딩 조건의 배양 효율 검정

  • Lee, Geon Hee (Department of Plant Resources, College of Industrial Science, Kongju National University) ;
  • Kim, Tae Kyeum (Department of Plant Resources, College of Industrial Science, Kongju National University) ;
  • Choi, Chang Hyun (National Institute of Crop Science, Rural Development Administration) ;
  • Kim, Jae Yoon (Department of Plant Resources, College of Industrial Science, Kongju National University)
  • 이건희 (국립공주대학교 식물자원학과) ;
  • 김태겸 (국립공주대학교 식물자원학과) ;
  • 최창현 (국립식량과학원 밀연구팀) ;
  • 김재윤 (국립공주대학교 식물자원학과)
  • Received : 2020.11.05
  • Accepted : 2020.11.13
  • Published : 2020.12.01

Abstract

Immature embryogenesis is a useful process in wheat tissue culture, including transgenic technology, because of its high regeneration efficiency compared to that in other tissues. However, it is a very labor-intensive and time-restrictive method, because the preparation of immature embryos is limited to the optimal time after flowering. In this experiment, 'Speed Breeding', a breeding technique that accelerates breeding generation advancement by extending the photoperiod, was applied to the wheat variety 'Bobwhite'. A controlled growth room was constructed by adjusting the photoperiod (22-hour light/2-hour dark) using LED lights at temperature of 22℃. After vernalization of the Bobwhite seeds at 4℃ for 4 weeks, the seedlings were grown in a controlled growth room and a greenhouse to compare the heading date. In both conditions, calli were induced from immature embryos on the 11th day after flowering. After 4 weeks, the calli were transferred to a regeneration medium. Regeneration efficiencies under greenhouse conditions and Speed Breeding conditions were determined as 45.05% and 43.18%, respectively. Antioxidant enzyme activity and reference gene expression analysis were performed to confirm the presence of stress due to an extremely long-day photoperiod. As a result, the antioxidant enzyme activity was not distinguished from that of the greenhouse condition. The reference gene expression analysis revealed that the PsaA and CDC genes were highly expressed under the Speed Breeding condition. However, expression of PsbA was similar expression in both conditions. These results will provide useful information for the application of immature embryogenesis to the wheat transformation system.

미성숙배를 이용한 조직배양은 높은 재분화율로 인하여 밀 형질전환에 적용하기 위한 가장 좋은 방법이지만 매우 한정적인 수집시기와 생육조건으로 인해 이용하기 위한 시간의 제한과 많은 노동력이 집약되어있다. 따라서 안정적이고 지속적인 미성숙배 수집을 위해 스피드 브리딩 조건을 이용하여 출수가 촉진된 밀 이삭을 이용한 미성숙배 조직배양의 효율을 검정하였고, 연구결과는 다음과 같다. 1. 스피드 브리딩 조건의 출수기는 정상조건과 비교하여 36일에서 54일까지 단축되었으며, 개화 후 11일 된 영과에서 수집된 미성숙배 조직배양의 재분화율과 소식물체 출현 비율은 정상조건과 비교하여 유의한 차이가 없다는 것을 확인하였다. 2. 두 배양 조건에서 항산화 효소 활성 분석 결과 차이가 나타나지 않았으며, 연관 표지 유전자 발현 검정을 통하여 일장과 세포분열과의 관계를 검정하여 스피드 브리딩 조건에서 수집된 이삭의 안정성을 검정하였다. 3. 본 연구 결과는 스피드 브리딩 조건을 활용한 미성숙배 조직배양에 활용될 것이며 관련 유전자 발현 및 항산화 활성 검정 결과를 통해 검증된 안정적 미성숙배는 밀 형질전환 기술 개발에 활용될 수 있을 것으로 사료된다.

Keywords

Acknowledgement

본 연구는 농촌진흥청 연구사업(세부과제명 : 유전자 교정 기술을 이용한 내재해성 국내 밀 개발, 세부과제번호 : PJ014823012020)에 의해 이루어진 것임. 김재윤: 이 논문은 2018년 공주대학교 학술연구지원사업의 연구지원에 의하여 연구되었음.

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