Isolation, Culture, and Fusion of Nicotiana Protoplasts

원형질체 분리, 배양 및 Nicotiana 종간 세포융합에 관한 연구

For the preliminary study on tobacco cell fusion as one of new breeding techniques, the conditions that would be most effective in isolation, fusion, and culture of tobacco protoplasts were examined ; 1. The enzyme solution of 0.5% macerozyme and 2% cellulase( or meicellase) was the most economic and efficient in isolating protoplasts from tobacco leaves. 2. The proper incubation period of tobacco leaves in cell wall digesting solution was 4 hours. 3. As an osmotic stabilizer, sorbitol or mannitol solutions were employed. The concentration of 0.5~0.7 M of either hexitol gave satisfying results as the osmotic stabilizer. 4. The calcium concentration appeared to be an important factor in protoplast fusion. The adhesion of protoplasts was enhanced by enrichment of calcium ion in PEG solution. The highest frequency of protoplast fusion was obtained when tobacco protoplasts were incubated in PEG solution. containing 9mM CaCl2. 5. Cell divisions of the isolated protoplasts were continued and have generated colonies when they were grown on B-5 medium at 28$^{\circ}C$.

본 시험은 담배 신품종 육종기술확립을 위하여 효율적으로 原形質體(protoplast)를 얻을수 있는 방법과 protoplast 배양조건을 조사하였다. 1. protoplast를 효율적이며 경제적으로 얻을 수 있는 세포붕괴, 細胞模解離 酵素의 농도는 0.5% macerozyme + 2% cellulase (또는 meicellase)였다. 2. 효소처리시간은 품종간에 약간의 차이는 있었으나 4시간 이상이 필요하였으며 1인 작업량으로 보아 4시간이 가장 적합하였다. 3. 等張液을 만들기 위하여는 0.5∼0.7M의 mannitol이나 sorbitol을 이용하는 것이 좋았다. 4. 세포융합시에 Ca++ 이온의 농도는 중요하며 9mM CaCl2를 포함한 PEG용액(0.5g/ml)을 쓰는 것이 가장 효과적이었다. 5. 분리된 protoplast는 B-5 培地에서 계속분열하여 colony를 형성하였다.