초록 ▼

○ 연구결과: 자색고구마의 잎과 괴근조직으로부터 캘러스를 유도한 후, 배발생 캘러스의 형성과 이어 식물체의 재분화 방법을 확립하였다. 색소 생합성은 3000 lux의 광조건에서 2배, blue광 2시간 조사를 통해 2.2∼2.4 배의 생합성이 상승되었다. 현탁 배양의 경우 계대배양 10주 이후에는 생장이 급속히 저하되면서 괴사하였다. 대처방안으로 변이체 캘러스를 선발한 후, 세포 집합체클론화에 의해 지속적으로 세포주를 선발하였다. 세포 생장은 접종 후 13일에 대수생장기를 거쳐 다음의 계대 배양기까지 접종량의 19배까지 성장하였다.

○ 연구결과: 자색고구마의 잎과 괴근조직으로부터 캘러스를 유도한 후, 배발생 캘러스의 형성과 이어 식물체의 재분화 방법을 확립하였다. 색소 생합성은 3000 lux의 광조건에서 2배, blue광 2시간 조사를 통해 2.2∼2.4 배의 생합성이 상승되었다. 현탁 배양의 경우 계대배양 10주 이후에는 생장이 급속히 저하되면서 괴사하였다. 대처방안으로 변이체 캘러스를 선발한 후, 세포 집합체클론화에 의해 지속적으로 세포주를 선발하였다. 세포 생장은 접종 후 13일에 대수생장기를 거쳐 다음의 계대 배양기까지 접종량의 19배까지 성장하였다. 변이체 캘러스의 배양조건에서 2,4-D 혹은 BA는 자색 변이체 캘러스의 생장과 안토시아닌 생합성을 억제하며, ABA는 생장률을 증가시키고 jasmonic acid는 안토시아닌 생합성량을 증가시켰다. 캘러스에 축적된 안토시아닌은 HPLC의 분석 결과 peonidin으로 분석되었다. 시험장에서의 실험결과는 한 자색고구마는 괴근 형성과 동시에 안토시아닌 색소가 축적되며 수확시기에 따른 변화는 차이가 없으며, 상대적 다수확을 위해서는 120일 이상 재배하는 것이 안토시아닌 색소함량의 제고를 위한 적정 재배조건으로 생각된다.

Abstract ▼

Anthocyanins have been used extensively as food and beverage additives. The main pigments that accumulate in the storage roots of purple sweet potato(Ipomoea batatas L.) are anthocyanins with aromatic acyl groups. These pigments have a high thermostability and contribute towards antioxidative activi

Anthocyanins have been used extensively as food and beverage additives. The main pigments that accumulate in the storage roots of purple sweet potato(Ipomoea batatas L.) are anthocyanins with aromatic acyl groups. These pigments have a high thermostability and contribute towards antioxidative activity. Therefore, the sweet potato pigment is expected to be a high quality natural food colorant which may aid in the prevention of lifestyle-related diseases.
Plant cell cultures have been suggested as a feasible technology for the production of plant-derived secondary metabolites. Significantly progress has been made in the enhancement of both productivity and yield of anthocyanins in a number of plant cell cultures. However, very few tissue culture techniques have been applied to introduce the biosynthesis of anthocyanins in vitro from sweet potato. In this study, we have tested the effects of optimization of media and culture conditions on the pigments accumulation in the tissue culture of purple sweet potato .
1. Development of tissue culture method and the selection of pigmented callus
This study was carried out to establish a regeneration system and selection of high anthocyanin producing cell line from leaf and storage rootexplant of purple sweet potato (Ipomoea batatas L.). Callus induction was initiated from explants derived leaf and root storage. Using leaf, optimal combination of the growth regulators for callus formation was 1 μM 2,4-D and 5 μM BA, and the highest yield of embryogenic calli were observed with a Murashige and Skoog (MS) basal medium containing 0.5 μM 2,4-D under light condition after 4 weeks of culture. Embryogenic calli were subcultured on a medium supplemented with 5 μM ABA for 4 days. Regeneration of shoots occurred when those embryogenic calli were transferred onto the hormone free MS medium complemented with 3∼6 μM gibberellic acid. Regenerated shoots were developed into normal plantlets.
The optimal concentration for callus formation derived storage root explants was the best in 10 μM 2,4-D and 0.5∼5 μM BA combination. The embryogenic calli were observed on MS basal medium containing 0.5 μM 2,4-D under the light condition after 5 weeks of culture. Embryogenic callus subcultured on medium supplemented with 5 μM ABA for 4 days. Subsequently, the regeneration of shoots occurred when these embryogenic calli were transferred onto the MS hormone free medium. The highest frequency(2 shoots/explant) of shoot regeneration occurred in the line Muan 5. Regenerated shoots rooted rapidly and the plantlets transferred to soil in the greenhouse appeared normal. This system of somatic embryogenesis described here will facilitate tissue culture and gene transfer research of purple sweet potato due to its rapidly(8 ∼ 10 weeks).
For the selection of highly anthocyanin synthesizing cell line were used the callus derived storage root and leaf explants. Light intensity was identified as important influence on anthocyanin synthesis in callus culture. Under 3000 lux light intensity was the best for pigment production in this study. In these condition brought about a 2-fold increase in anthocyanin content compared to the original leaf tissue. Dark condition was not suitable for anthocyanin production. The blue, red, far red light irradiation were influenced on the piment accumulation in callus. Under the blue light irradiation for 2 h was most effective in anthocyanin accumulation, but little anthocyanin under the other light condition. The average anthocyanin contents was enhanced 2.2∼2.4 fold by light-emitting diodes(LED) treatment for 2∼4 h.
2. Development of effective suspension culture and cell line selection
We attempted to select a high pigmented callus(PC) suitable for the development of an efficient technological procedure for anthocyanin mass production. Suspended cell cultures were initiated by transferring about 0.1g (fresh weight) of callus to 10 ml of liquid medium in 50 ml flask. The cultures were incubated on rotary shaker (120 rpm) at 27°C in the dark. The growth and the anthocyanin accumulation in suspension cultures were monitored periodically. The cell growth observed a typical curve with induction period at 12 days and exponential phase between 12 and 30 days after inoculation. The cell growth to next subculture ncreased about 19 times during the whole growth period.
To select the high anthocyanin synthesizing cell line, we tested culture condition including the composition of plant growth regulators, light irradiation, and shaking speed etc. The PC appeared in the suspension culture after 20 weeks subculturing in MS medium. However, the frequency was very low and their formation suppressed by 2,4-D or BA. The maximum pigment accumulation on the present culture observed during the first 6 days (0.99 mg/mL), which was closed that of a pigment extracted from storage roots, (which was 1.5). The highest increase in the anthocyanin contents in culture was associated with a low growth rate. On the contrary the pigment contents was constant at the exponential growth phase.
Light irradiation or addition of jasmonic acid (20μM) enhanced anthocyanin accumulation in suspension culture, but inhibited cell growth. This represented an 8.5-fold increase compared with the control culture in the dark. Following the continuous light irradiation of 8000-8300 lux, the maximum anthocyanin accumulation was 6.8 CV(color value)/gFCW(fresh callus weight) at 10 days after inoculation, which was 4.8-fold that the control. A process that combined jasmonic acid treatment and light irradiation resulted in a significant synergistic enhancement of anthocyanin accumulation up to 22.8 CV/g FCW on day 7. This value was 13.8-fold that of the control. One anthocyanin was isolated from the highly pigmented callus derived from the storage root of purple sweet potato cv. muan. It was identified as cianidin by HPLC analysis.
3. Selection of new breeding lines with high anthocyanin content by hybridization breeding
Selection of new breeding line having high anthocyanin content was tried by hybridization breeding method. Seeds for new lines were obtained from artificial cross, and cutted at field as seedlings next year. 8 new breeding lines having anthocyanin pigment were selected from total 1,603 seeds by artificial cross. And, 6 genetic resources were selected from seedling 5 ∼ 7 generations. These resources have high anthocyanin contents and yields and will be used for new variety development of high anthocyanin content.
Cultivation periods were experimented for high anthocyanin content using purple sweetpotato. Even if cultivation period was prolonged, anthocyanin content was not changed from short day cultivation as 80 days. But, total anthocyanin yield can obtained higher and higher by fleshiness of storage root. Therefore, cultivation over 120 days was recommended for high yield of anthocyanin.

목차 Contents

• 제출문 ... 1
• 요약문 ... 2
• SUMMARY ... 8
• CONTENTS ... 13
• 목차 ... 17
• 제1장 총론 ... 21
• 제1절 연구개발의 필요성 ... 21
• 1. 기술적 측면 ... 21
• 2. 경제, 산업적 측면 ... 22
• 3. 사회, 문화적 측면 ... 23
• 제2절 연구개발의 목적 및 범위 ... 23
• 1. 연구개발의 목적 ... 23
• 2. 연구개발의 범위 ... 25
• 제2장 조직배양 방법의 확립 ... 31
• 제1절 실험재료 및 방법 ... 31
• 제2절 잎 조직을 이용한 조직배양과 자색캘러스 선발 ... 32
• 1. 캘러스의 유도 ... 32
• 2. 배발생 캘러스 형성 ... 38
• 3. 식물체의 재분화 ... 41
• 4. 높은 안토시아닌 생합성을 위한 배양 조건 ... 45
• 가. 자색 캘러스 선발 ... 45
• 나. 생장조절제 처리 ... 51
• 1) 2,4-D 처리 ... 51
• 2) ABA 처리 ... 53
• 3) Jasmonic acid 처리 ... 55
• 다. 광조사 기구(LED)를 통한 색소 생합성 조사 ... 57
• 제3절 괴근 조직을 이용한 조직배양 및 자색캘러스 선발 ... 59
• 1. 캘러스의 유도 ... 59
• 2. 광조건과 배양기간에 따른 배발생 캘러스 형성률 ... 62
• 3. 식물체의 재분화 ... 65
• 4. 높은 생합성을 위한 배양조건 ... 69
• 가. 자색 캘러스 선발 ... 69
• 나. 생장조절제 처리 ... 71
• 1) 2,4-D와 BA 처리 ... 71
• 2) NAA와 BA 처리 ... 73
• 5. EMS (Ethyl methane sulfonate) 처리에 의한 돌연변이체 유도 ... 73
• 제4절 변이체 캘러스를 이용한 조직배양 ... 75
• 1. 변이체 캘러스 선발 ... 75
• 2. 유지·증식 ... 77
• 3. 생장조절제의 효과 ... 77
• 가. 2,4-D 와 BA 처리 ... 77
• 나. 광조사 기구(LED)를 통한 색소 생합성 조사 ... 80
• 제5절 결과 ... 80
• 제3장 현탁배양 기술의 확립 ... 83
• 제1절 잎조직 유래 캘러스의 현탁배양 ... 83
• 1. 진탕 속도가 안토시아닌 생합성에 미치는 영향 ... 83
• 2. 생장조절제 처리 ... 87
• 3. 잎조직 유래 캘러스의 생장곡선 ... 91
• 제2절 괴근조직 유래 캘러스의 현탁배양 ... 93
• 1. 2,4-D와 BA 영향 ... 93
• 제3절 변이체 캘러스의 현탁배양 ... 95
• 1. 유지 및 증식 ... 95
• 2. 2,4-D와 BA 영향 ... 95
• 3. 고생합성 변이체 캘러스 선발 ... 98
• 가. 색소 생합성 캘러스의 선발 ... 98
• 나. 생장곡선 ... 102
• 디. 자색 변이체 캘러스의 안토시아닌 분석 ... 104
• 라. 배양조건 화립 ... 107
• 1) 배지조건의 실험 ... 107
• 2) 당의 영향 ... 109
• 3) 캘러스 접종량 ... 112
• 4) 2,4-D와 BAP 혼합 처리의 영향 ... 114
• 5) ABA 영향 ... 116
• 6) Jasmonic acid 영향 ... 118
• 제4절 결과 및 고찰 ... 120
• 제4장 자색고구마 색소함량 제고를 위한 적정조건 구명 ... 121
• 제1절 교잡육종에 의한 안토시아닌 색소 고함량 계통 선발시험 ... 121
• 1. 수행방법 ... 121
• 가. 인위 개화유도 ... 121
• 나. 인공교배 및 결실, 채종 ... 123
• 다. 실생종자 파종 및 선발 (실생1년, 실생개체선발시험) ... 123
• 라. 계통선발 예비시험(실생 2년) 및 본시험(실생 3년) ... 124
• 마. 안토시아닌 색가(color value)의 측정 ... 124
• 2. 시험결과 ... 126
• 가. 안토시아닌 색소 고함유 계통육성 ... 126
• 1) 인공교배 현황 및 채종립수 ... 126
• 2) 유망계통 선발 ... 128
• 3) 색가 비교 ... 130
• 3. 결과요약 ... 132
• 제2절 재배방법에 의한 안토시아닌 색소함량 제고 적정조건 구명 시험 ... 133
• 1. 수행방법 ... 133
• 2. 시험결과 ... 133
• 3. 결과요약 ... 134
• 끝페이지 ... 135