초록 ▼

□ 고추형질전환 기초 인프라 시스템을 개발
-고주 30 계통에서 재분화와 형질전환이 잘되는 4 계통을 선정하고 Agrobacterium의 virulence level을 분석해서 EHA105와 LBA4404를 선정함.
-고추형질전환 방법, 조건을 구축함.
-Explant에서 직접 발생하는 direct shoot은 전혀 형질전환이 안되는 경우라고 결론 내림 (direct shoot formation). 이와는 반대로 co-culture후 callus를 통해 유기 되는 shoot 발생에서 형질전환율이 높다는 것을 처음으로

□ 고추형질전환 기초 인프라 시스템을 개발
-고주 30 계통에서 재분화와 형질전환이 잘되는 4 계통을 선정하고 Agrobacterium의 virulence level을 분석해서 EHA105와 LBA4404를 선정함.
-고추형질전환 방법, 조건을 구축함.
-Explant에서 직접 발생하는 direct shoot은 전혀 형질전환이 안되는 경우라고 결론 내림 (direct shoot formation). 이와는 반대로 co-culture후 callus를 통해 유기 되는 shoot 발생에서 형질전환율이 높다는 것을 처음으로 확인함 (callus-mediated shoot 또는 indirect shoot formation).
-Mannose selection을 이용한 형질전환은 성공하지 못하였음.
□ 대량형질전환 시스템 개발
-형질전환한 후 callus-mediated shoot을 찾아가는 선발을 통해 선발과정이 용이해지고 시간 단축됨. 유전자 3점이 4개의 서로 다른 계통에 현질전환됨으로서 재현성이 확인. 형질전환율: 0.19% (tile highest).
-Callus-mediated shoot 선발을 근거로 callus를 유기해서 형질전환하는 방법 개발함(callus-induced transformation: CIT). GFP를 이용하여 callus가 고효율로 형질전환됨을 확인함.
-CIT는 모든 line에 가능하며 재현성이 있다.
-Callus에서 재분화조건에 따라 CIT 형질전환 효율을 0.5-0.1%까지 유지할 수 있음
-CIT는 시간이 callus-mediated transformation 방법보다 약 1.5배 단축된다.
□ 고주형질전환체 선발
-총 5점 의 유전자가 여러 계통에 형질전환되어 있음. 서로 다른 independent한 $T_{0}$ 개체는 약 40여점 있으며 총 $T_{0}$ 수는 100여점임. 현재 PP11, TMV-CP는 $T_1$세대 병리 검정을 하였으며 TMV-CP는 $T_2$세대까지 확보함.

Abstract ▼

Chili pepper is one of world's staple vegetables. Variable genetic sources have been developed, and classical breeding programs for pepper cultivation have been well established. However, approaches aimed at the genetic engineering of pepper have been protracted due to the difficulty of pepper trans

Chili pepper is one of world's staple vegetables. Variable genetic sources have been developed, and classical breeding programs for pepper cultivation have been well established. However, approaches aimed at the genetic engineering of pepper have been protracted due to the difficulty of pepper transformation by Agrobacterium. In fact, Pepper is known as a difficult plant to transform and has resisted the efforts of many laboratories worldwide for many years. At present, pepper regeneration itself is not an obstacle, and is performed routinely. However, the high level of effort invested into transferring genes into chili pepper explants has not yielded a successful and reproducible transformation method. Unfortunately, relatively few studies have been undertaken upon experimental methodology.
To obtain a successful transformation system for pepper plants, we have set out a series of research project so far. This program focused on the basic methodology pertinent to transformation and finally we developed two novel methods useful for pepper transformation with reproducibility, faster selection and a large scale. The first method is a transformation selection by callus-mediated shoot formation (CMSF) and the second is a transformation by callus induction (CIT). CMSF is a selection method that eliminated shoots grown directly from explant and saved shoots grown indirectly from calli. These indirect shoots grown from the callus showed a high probability of being transformed. Several genes such as TMV-CP, CMV-CP, PPI1 (pepper-PMMV interaction 1 transcription factor) and WRKY (disease defense related transcription factor) were used to transform commercially important chill pepper inbred lines by Agrobacterium co-culture. Transformation was confirmed by Southern and Northern blot analyses. Although the transformation rate was low (0.2%), this is the best rate ever obtained and this transformation proved reproducible. The second method, another advanced method called callus-induced transformation (CIT), was to induce callus by auxin before co-culture and the shoot was generated from the callus. To monitor the transformation efficiency obtained by CIT, GFP gene was transformed and the GFP expression levels in calli were observed. The transformation rate of the induced calli was increased up to 0.5-1.0%. This method works for all the inbred lines tested, therefore this becomes a very powerful tool for pepper biotechnology. Taken together, the pepper transformation is not a bottleneck anymore. Since, in Korea, there are groups of research scientist good at pepper breeding technique and many elite genetic sources and a solid market network, the successful transformation system should be grafted soon to lead the future pepper biotechnology market in world.

목차 Contents

• 표지...1
• 제출문...2
• 보고서 초록...3
• 요약문...4
• SUMMARY...7
• CONTENTS...9
• 목차...11
• 제1장 연구개발과제의 개요...13
• 제1절 연구개발의 목적...13
• 제2절 연구개발의 필요성...14
• 제3절 연구개발의 범위...15
• 1. 1차년도...15
• 2. 2차년도...16
• 3. 3차년도...16
• 제2장 국내외 기술개발 현황...18
• 제1절 국외 관련분야에 대한 기술개발현황...18
• 제2절 국내 관련분야에 대한 기술개발현황...18
• 제3절 본 연구결과의 국제적 위치...19
• 제3장 연구개발수행 내용 및 결과...21
• 제1절 형질전환조건 확립...21
• 1. 식물재료...21
• 2. Mannose를 이용한 selection 방법...21
• 3. Mannose selection 방법을 이용한 형질전환...27
• 4. 고추 계통의 재분화 효율조사...29
• 5. Agrobacterium의 virulence와 계통간의 상호관계...30
• 6. 형질전환을 위한 기초조건 확립...32
• 제2절 Callus-Mediated Shoot Formation (CMSF)...34
• 1 Direct shoot과 indirect shoot의 비교...34
• 2. 공동배양후 direct shoot formation을 통한 형질전환...37
• 3. 공동배양후 indirect shoot formation을 통한 형질전환...38
• 4. 형질전환 protocol...39
• 5. Callus type...40
• 6. Southern과 northern blot 분석...41
• 7. 삽입유전자의 특성분석...43
• 제3절 Callus-Induced Transformation(CIT)...44
• 1. 캘러스 유도 및 GFP expression...44
• 2. CIT 형질전환효율...47
• 3. CIT 방법에 의한 형질전환체의 Southern blot 분석...49
• 제4장 목표달성도 및 관련분야에의 기여도...51
• 제1절 연구개발목표의 달성도...51
• 제2절 기술발전에의 기여도...52
• 제5장 연구개발결과의 활용계획...53
• 제1절 추가연구의 필요성...53
• 제2절 타연구에의 응용...53
• 제3절 기업화 추진방안...54
• 제6장 연구개발과정에서 수집한 해외과학기술정보...55
• 제7장 참고문헌...56