느티나무의 芽培養 Callus培養, 細胞培養, 原形質體培養 및 形質轉換에 關한 生物工學的 基礎硏究 = Biotechnological Studies on Bud Culture, Callus Culture, Cell Culture, Protoplast Culture and Gene Transformation in Zelkova serrata Makino

다국어 초록 (Multilingual Abstract)

In order to develop techniques for rapid propagation and early breeding test on Zelkova serrata through biotechnology, bud culture, callus culture, cell culture, protoplast culture and Agrobacterium tumefaciens inoculation were examined.
The obtained results were as follows :
1. In bud culture of 1-year-old seedling best shoot growth was observed in explants from middle part of the shoot, and better growth in the lower part than in the upper part. It is recommended that the explant sources are obtained from the middle part of shoots with healthy buds. When the middle part of flushing shoots was used for further culture on different media, shoot growth was better in the descending order of MS(36.4㎜), BTM(28.1㎜), WPM(22.1㎜), SH(20.7㎜), and GD(16.1㎜) media.
2. In multiple shoot induction from initial explants, WPM medium with 1.0㎎/ℓ BAP induced the largest(1.9 shoots/explant) number of shoots. When either BAP or thidiazuron was added to MS medium for multiplication of in vitro grown shoots the best result was at 0.5㎎/ℓ BAP (4.2 shoots/bud) in both quantity and quality of shoots. Multiple shoots were initiated from 0.002㎎/ℓ thidiazuron and the best result was obtained at 0.01㎎/ℓ thidiazuron (2.4 shoots/bud). However the concentration of thidiazuron is recommended to be used higher than 0.01㎎/ℓ to get a better result.
3. Bud culture of 20-year-old trees showed 83.3% in shooting when the buds were cultured on DCR medium supplemented with 10㎎/ℓ BAP, but the growth of new shoots was much inferior to that of 1-year-old seedlings.
4. When in vitro shoots were cultured for rooting on GD medium with different concentrations of IBA, the best result (56.7% in rooting) was obtained from the medium containing 0.05㎎/ℓ IBA. The initiated roots were the longest on the medium without IBA and the average number of initiated roots was one or two. When the rooted plantlets were transplanted in pots, they showed 75.7% in survival rate.
5. When leaf and hypocotyl were used for callus culture, induction of callus was the best on MS medium containing 200㎎/ℓ casein hydrolysate without 2,4-D and kinetin. Callus growth was the best on MS containing 200㎎/ℓ casein hydrolysate, 0.5㎎/ℓ 2,4-D and 0.2㎎/ℓ kindtin using leaf as explant sourse. When stem segments of in vitro grown shoots were used on MS, MMS, 1/2 MS, WPM containing 0.5㎎/ℓ NAA and 0.01㎎/ℓ BAP, all the media showed 100% in callus induction. Half-strength GD media added with 1.0㎎/ℓ IBA and 0.5㎎/ℓ NAA showed 100% in root formation, whereas WPM added with 0.01㎎/ℓ BAP and 0.5㎎/ℓ NAA did 30%
6. When leaf explants were cultured for cell suspension culture, large number of single cells and rapid cell proliferation were obtained from MS medium supplemented with 0.1㎎/ℓ BAP, 1.0㎎/ℓ NAA and 1.0㎎/ℓ 2,4-D. Stem segments also showed same response.
7. In protoplast culture, the best condition for protoplast isolation was the following combination of 0.5% Macerozyme, 3% cellulase and 0.6M mannitol at pH6.2. Budding were initiated at the cell density of 2.5×10^5 and 5×10^4/㎖ when protoplasts were cultured on WPM plating media containing 0.02㎎/ℓ BAP and 0.2㎎/ℓ NAA. Two or three times of binary cell divisions were also noticed, but further cell divisions were not observed.
8. When leaf and stem segment were inoculated with Agrobacterium tumefaciens wild types, pTiC58, pTiBo542, tumor-like tissue was formed by all the three wild types. However gene transformation was not confirmed in this study.

목차 (Table of Contents)

• Ⅰ. 緖 言
• Ⅱ. 硏 究 史
• Ⅲ. 材料 및 方法
• Ⅳ. 結果 및 考察
• 1. 芽 培 養
• 2. Callus 培養
• 3. 細胞懸獨培養
• 4. 原形質體 分離 및 培養
• 5. Agrobacterium tumefaciens의 器內 接種
• Ⅴ. 摘 要
• 參考文獻
• Summany