초록

○ 연구결과
1. 안토시아닌 관련 유전자 클로닝 (data base 구축)
2. 검정콩 유전자원의 수집 및 특성 분석
3. 안토시아닌 유전자의 유전적 및 유전생리적 특성 확인
4. 콩 형질전환 체계 확립
5. 건강기능성 콩 신품종 개발을 위한 germplasm 개발

Abstract ▼

In order to achieve highly efficient soybean transformation method, genotype screen of Korean soybean cultivars for amenability to Agro-infection and diverse experimental trials have been introduced in the current study such as hormone pre-culture and various selection techniques. The active genome

In order to achieve highly efficient soybean transformation method, genotype screen of Korean soybean cultivars for amenability to Agro-infection and diverse experimental trials have been introduced in the current study such as hormone pre-culture and various selection techniques. The active genome projects from several academically and economically important plants have provided sequence data of many genes and accumulated in Gene Bank data base promptly. Some of those genes reported have high potentials to improve plant quality in agronomical and industrial uses. However, difficulties to achieve the successful introduction of those valuable genes into plant can be found in recalcitrant genetic transformation procedure of some agriculturally important crops.
Especially none of research group in Korea have succeeded to report any formal results on the production of genetically engineered soybean variety or research publication, even though there have been several GMO soybean varieties released from abroad soybean seed companies. To achieve long term goal for producing high-value soybean variety by genetic transformation, there are couple of procedures to be improved. First, genotype screen of Korean soybean varieties whether they are amenable to Agro-infection and tissue culture responsive has to be done. Second, efficiency for genetic transformation has to be increased. In this study various experiments have been carried to achieve both proposed goals.
To determine the amenability of Korean soybean cultivars to Agrobacterium infection most commonly grown 31 Korean cultivars were used for genetic transformation. Cotyledonary node was used as a explant and inoculated with Agrobacterium strain LBA4404 harboring pTOK233, super binary vector. After placement on selection media containing 10ppm of hygromycin, 4 to 6 week old shoots(0.5 - 1cm) were stained with GUS solution. Shoot formation rates were high in Muhankong(12%), Manrikong(10%), Eunhakomg(19%), Daewonkomg(23%), and high frequency of GUS positives were observed in Ilpumkumjeongkong, Muhankong, Eunhakomg, Daewonkomg. In addition that, strong GUS expression was found in Jangmikong and Baekwunkong.
A series of experiments were carried to determine optimal condition for soybean transformation. For washing reagent, timentin had chosen because of its positive effect on regeneration and clear removal of Agrobacterium over cefotaxime. Hygromycin was used as a selection agent. The result indicated that best concentration of hygromycin was 30ppm in shoot induction media and 10 or 15ppm in shoot elongation media. In another experiment, three different agar concentrations(0.4, 0.6, 0.8%) were tested. Among three tested, high shoot formation was observed in 0.6% and 0.8%, and overall transformation was great in 0.8% agar concentration. For better selection of transformants at the early stage, orientation of placement(flat side up or down) was investigated. The GUS result showed that higher transformation rate was obtain when flat side(adaxial) was placed down by directly contacting Agro-infected area on the selection media. In two different wounding methods, either using scalpel or using a bundle of needles, wonding by needles gave high frequency of GUS positive.
To increase transformation frequency hormone pre-culture during co-cultivation was applied. In shoot formation, the highest rate was observed from the non-treated(18%) and BA(6-benzylaminopurine) 5ppm(13%) and BA 10ppm(11%) followed, unexpectedly. And no clear difference in GUS positive ratio was confirmed among three treatments. However, strong GUS staining was found in hormone treated ones as well as better survival of transformed shoots in later shoot elongation(SE) media. No survival of shoots was observed in non-treated ones. After introducing hormone pre-culture during co-cultivation, hygromycin concentration for optimal selection was reestablished. Hygromycin 5 & 10ppm were effective for shoot formation and emergency of GUS positives than hygromycin 20ppm. Like the previous result, all the shoots from the non-treated ones died during later selection in SE media. In the experiment to determine proper orientation of placement, higher GUS positive were observed in the placement of flat side down direction. The results of hormone pre-culture during co-cultivation indicated that high frequency of early shoot formation did not always lead to high frequency of genetic transformation, as shown in non-treated shoots; high frequency of shoot formation but no survival in later selection. Therefore, much effective selection method to minimize early escapes was searched. We introduced liquid selection instead of selection in solid media with limited contact. The result showed that GUS positive ratio was nearly double in the hormone treated(80%) after liquid selection than in the non-treated(44%). And survival rate of transformed shoots was also high in the hormone treated(33%) than in the non-treated(10%).
Recent reports on soybean transformation revealed that addition of thiol compounds, L-cystein, dithiothreitol(DTT), and sodium thiosulfate, improved T-DNA delivery by inhibiting the activity of plant pathogen- and wound-response enzymes, such as peroxidases (PODs) and polyphenol oxidases (PPOs). We compared regeneration frequency of shoots with or without thiol compounds addition to co-cultivation media. The efficiency of regeneration significantly increased from an average of 14.7% to 23%. We also observed much higher survival of transformed shoots in long-lasting selection on hygromycin-containing media and enhanced proliferation of multiple shoots.

목차 Contents

• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 5
• CONTENTS ... 8
• 목 차 ... 9
• 제 1 장 연구개발과제의 개요 ... 10
• I. 연구개발의 필요성 ... 10
• 1. 연구의 배경 ... 10
• 2. 연구의 필요성 ... 12
• 제 2 장 국내외 기술개발 현황 ... 14
• I. 국내ㆍ외 관련기술의 현황과 문제점 ... 14
• II. 앞으로의 전망 ... 14
• III. 기술도입의 타당성 ... 15
• IV. 현기술의 취약점 ... 15
• 제 3 장 연구개발 수행 내용 및 결과 ... 17
• I. 연구개발 목표의 달성도 ... 17
• II. 연구수행 방법 ... 17
• 1. 콩에서 저온, 상처 등 스트레스 관련 유전자 및 종피 착색단계별 유전자 선발 ... 17
• 2. Sequecing ... 18
• 3. Full gene cloning ... 18
• 4. 검정콩 형질전환 기술개발 ... 18
• 5. 안토시아닌 양상 분석 및 안토시아닌 induction 조건설정 ... 19
• II. 연구수행 내용 및 결과 ... 20
• 1. 안토시아닌 관련 유전자 library 확보 및 database구축 ... 20
• 2. 형질전환용 벡터 제작 ... 28
• 세부과제명 : 검정콩 형질전환 기술개발 ... 31
• 제 1 장 연구개발과제의 개요 ... 31
• 제 1 절 연구개발의 목적 ... 31
• 제 2 절 연구개발의 필요성 및 범위 ... 31
• 제 2 장 국내외 기술개발 현황 ... 34
• 제 3 장 연구개발 수행 내용 및 결과 ... 36
• 제 1 절 hygromycin농도가 형질전환체 선발 및 재분화에 미치는 영향 ... 36
• 제 2 절 Agar 농도, 치상방향, 상처방법이 형질전환체 선발효율에 미치는 영향 ... 41
• 제 3 절 유전자총을 이용한 콩 종자배축(embryonic axes) 형질전환 ... 48
• 제 4 절 호르몬 BA (6-Benzylaminopurin) 전처리가 형질전환 효율증대에 미치는 영향 (고체선발 배지사용) ... 52
• 제 5 절 호르몬 BA (6-Bensylaminopurin) 전처리 후, hygromycin 농도가 형질전환 효율에 미치는 영향 (고체선발배지사용) ... 56
• 제 6 절 호르몬 BA(6-Bensylaminopurin) 전처리 후 치상방향에 따른 형질전환 효율비교 (고체선발배지사용) ... 59
• 제 7 절 호르몬 전처리 후 액체배양의 선발효과 ... 61
• 제 8 절 호르몬 전처리와 liquid배지 선발이 형질전환체의 생존율에 미치는 영향 ... 64
• 제 9 절 공배양시 황화합물 첨가가 콩 형질전환에 미치는 영향 ... 68
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 77
• 제 5 장 연구개발결과의 활용계획 ... 80
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 82
• 협동과제명: 검정콩 안토시아닌분석 및 관련 marker 개발 ... 83
• I. 연구목적 ... 83
• II. 재료 및 방법 ... 83
• III. 연구 수행내용 ... 90
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 131
• 제 5 장 연구개발결과의 활용계획 ... 133
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 135
• 제 7 장 참고문헌 ... 137
• 끝페이지 ... 140