罗汉果的组织培养与农杆菌介导遗传转化体系的建立研究
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摘要
本论文以青皮果和圆形红毛子两个罗汉果品种为材料，系统地研究了罗汉果的组织培养，建立了农杆菌介导的罗汉果遗传转化体系，成功地获得了罗汉果GUS基因转化植株，并进行了检测。研究结果包括：
1、不定芽的直接分化途径可作为罗汉果组织培养的主要途径；脱分化途径产生的罗汉果愈伤组织分化频率低，不利于罗汉果组织培养。
2、叶片、腋芽是罗汉果组织培养的适宜材料，直接分化不定芽的频率高；愈伤组织的诱导分化以腋芽为外植体的芽分化率相对较高。
3、罗汉果叶片的分化培养基以MS+6-BA_(2.0mg／L)+IBA_(0.5mg／L)为最佳，分化率为61.9％，平均芽数为5.48个／块；腋芽的分化培养基以MS+6-BA_(1.0mg／L)+IBA_(0.5mg／L)最佳，芽分化率达98.9％，平均芽数5.27个／块。
4、在2～5代继代培养中，随继代增殖代数增加，罗汉果叶、芽直接分化芽的能力不断下降。
5、罗汉果愈伤组织的芽分化率随愈龄的增加而下降，以5天愈龄的愈伤组织芽分化率最高，分化率为8％。
6、罗汉果不同部位叶片、腋芽的芽分化效果存在差异，以发育较成熟的3～4节间的叶片、腋芽的芽分化率最高，分别达到48.7％和96.9％。
7、罗汉果二次培养苗叶片仍具有较高的分化能力，青皮果芽分化率可达65％，圆形红毛子芽分化率可达50％。
8、罗汉果组培苗的生根壮苗培养以1／2MS+IBA_(0.8～1.0mg／L)+MET_(0.4～0.6mg／L)
厂西大学硕士学位论文
罗汉果的组织培养与农杆菌介导遗传转化体系的建立研究
为最佳培养基，生根率达100%，根数约为5.5一6.03条/苗。
9、罗汉果叶盘的幻n基础抗性浓度为6m叭，罗汉果非转化体在含
Km的培养基中难以分化出根。
10、罗汉果预培养4天后进行转化，GUS基因的瞬时表达效果最好。
瞬时表达检测发现:21.7%叶盘被全面染成较深蓝色，69.6%叶盘边缘和
内部均有较深蓝斑，8.7%叶盘边缘被染成蓝色。瞬时表达率达100%。
11、罗汉果叶盘在菌液浓度OD60。七0 .1一0 .5的农杆菌中浸泡5一60
秒后，共培养48小时，GUS瞬时表达效果最佳，瞬时表达率为100%。
12、先再生后选择的转化体筛选方法导致大量假转化体形成，而先
选择后再生，结合生根筛选，能有效去除假转化体。
13、GUS基因能在罗汉果完整植株中稳定表达，已获得罗汉果GUS
基因转化植株。
In this experiment, two varieties of Siraitia grosvenorii (Swingle) C. Jeffery that named Qingpiguo and Yuanxinghongmaozi were used as the materials. The system of tissue culture and agrobacterium-mediated transformation of Siraitia grosvenorii was established and some GUS transgenic plants of Siraitia grosvenorii had been obtained successfully. The results of study were included in the following.
t
1. The pathway of adventitious bud differentiation may become the main pathway on tissue culture of the Siraitia grosvenorii. The differentiation rate of callus produced by the pathway of des-differentiation was low, which was disadvantage to tissue culture of Siraitia grosvenorii.
2. The leaves and buds were the suitable material for tissue culture of Siraitia grosvenorii, which showed the high differentiation rate of adventitious buds. The buds differentiation rate of callus, which induced by the axillary buds, was higher comparatively.
3. On the leaves culture, the optimal medium of the buds differentiation was MS medium supplemented with 2.0mg-L-1 BA and 0.5mg-L-1 IBA, the buds differentiation rate was 61.9%, and average number of buds was 5.48. On the axillary buds culture, the optimal medium of the buds differentiation was MS medium supplemented with 1.0mg-L-1 BA and 0.5mg-L-1 IBA, the bud differentiation rate was 98.9%, and average number of buds was 5.27.
4. In 2-5 generation subculture, the capability of buds differentiation was decline with the leaves subculture time increasing.
5. The differentiation rate of the callus was drop with the callus age increasing. When the callus age was five days, the differentiation rate was the highest, and the rate was 14%.
6. The effects of the bud differentiation were difference in the different position of the leaves and axillary buds, which the leaves and axillary buds lied in 3-4 node showed the higher differentiation rate of buds. The rate was 96.9% and 48.7%.
7. The leaves of subculture plant still had higher differentiation abilities. The bud differentiation rate of Qingpiguo and Yuanxinghongmaozi was 65% and 50%.
8. In the culture of root inducement and sound seedling, the optimal medium was 1/2MS medium supplemented with 0.8-1.0mg-L-1 IBA and 0.4-0.6mg-L-1 MET. The root induce rate was 100%, the number of root was 5.5-6.03.
9. The sensitive concentration of Km was 6mg/L on the leaf-fragment of Siraitia grosvenorii. The non-transgenic seedling couldn't induce roots on the medium contained Km.
10. When the leaf-fragment of Siraitia grosvenorii pre-cultured for 4 days before being transformed, GUS gene expressed perfectly. The test of instantaneous expression indicated: the leaf-fragment that the whole dyed to navy blue was 21.7%, the leaf-fragment that the edge and inner dyed to deeper blue-spot was 69.6%, and the leaf-fragment that the edge dyed to blue was 8.7%. The rate of instantaneous expression was 100%.
11. Leaf-fragment of Siraitia grosvenorii were dipped in agrobacterium
liquid for 5~60 seconds that concentration was OD600 0.1 ~0.5 and co-cultivated in 48 hours, then GUS gene expressed optimal, the expressed rate was 100%.
12. A lot of fake-transgenic seedling was generated because of the selection after regeneration. The method, which the selection before regeneration combines with the filtration of root generation, could wipe off the fake-transgenic seedling efficiency.
13. GUS gene had been expressed steadily in the Siraitia grosvenorii plants. The transgenic plants of Siraitia grosvenorii were gained in the experiment.
1、不定芽的直接分化途径可作为罗汉果组织培养的主要途径；脱分化途径产生的罗汉果愈伤组织分化频率低，不利于罗汉果组织培养。
2、叶片、腋芽是罗汉果组织培养的适宜材料，直接分化不定芽的频率高；愈伤组织的诱导分化以腋芽为外植体的芽分化率相对较高。
3、罗汉果叶片的分化培养基以MS+6-BA_(2.0mg／L)+IBA_(0.5mg／L)为最佳，分化率为61.9％，平均芽数为5.48个／块；腋芽的分化培养基以MS+6-BA_(1.0mg／L)+IBA_(0.5mg／L)最佳，芽分化率达98.9％，平均芽数5.27个／块。
4、在2～5代继代培养中，随继代增殖代数增加，罗汉果叶、芽直接分化芽的能力不断下降。
5、罗汉果愈伤组织的芽分化率随愈龄的增加而下降，以5天愈龄的愈伤组织芽分化率最高，分化率为8％。
6、罗汉果不同部位叶片、腋芽的芽分化效果存在差异，以发育较成熟的3～4节间的叶片、腋芽的芽分化率最高，分别达到48.7％和96.9％。
7、罗汉果二次培养苗叶片仍具有较高的分化能力，青皮果芽分化率可达65％，圆形红毛子芽分化率可达50％。
8、罗汉果组培苗的生根壮苗培养以1／2MS+IBA_(0.8～1.0mg／L)+MET_(0.4～0.6mg／L)
厂西大学硕士学位论文
罗汉果的组织培养与农杆菌介导遗传转化体系的建立研究
为最佳培养基，生根率达100%，根数约为5.5一6.03条/苗。
9、罗汉果叶盘的幻n基础抗性浓度为6m叭，罗汉果非转化体在含
Km的培养基中难以分化出根。
10、罗汉果预培养4天后进行转化，GUS基因的瞬时表达效果最好。
瞬时表达检测发现:21.7%叶盘被全面染成较深蓝色，69.6%叶盘边缘和
内部均有较深蓝斑，8.7%叶盘边缘被染成蓝色。瞬时表达率达100%。
11、罗汉果叶盘在菌液浓度OD60。七0 .1一0 .5的农杆菌中浸泡5一60
秒后，共培养48小时，GUS瞬时表达效果最佳，瞬时表达率为100%。
12、先再生后选择的转化体筛选方法导致大量假转化体形成，而先
选择后再生，结合生根筛选，能有效去除假转化体。
13、GUS基因能在罗汉果完整植株中稳定表达，已获得罗汉果GUS
基因转化植株。
In this experiment, two varieties of Siraitia grosvenorii (Swingle) C. Jeffery that named Qingpiguo and Yuanxinghongmaozi were used as the materials. The system of tissue culture and agrobacterium-mediated transformation of Siraitia grosvenorii was established and some GUS transgenic plants of Siraitia grosvenorii had been obtained successfully. The results of study were included in the following.
t
1. The pathway of adventitious bud differentiation may become the main pathway on tissue culture of the Siraitia grosvenorii. The differentiation rate of callus produced by the pathway of des-differentiation was low, which was disadvantage to tissue culture of Siraitia grosvenorii.
2. The leaves and buds were the suitable material for tissue culture of Siraitia grosvenorii, which showed the high differentiation rate of adventitious buds. The buds differentiation rate of callus, which induced by the axillary buds, was higher comparatively.
3. On the leaves culture, the optimal medium of the buds differentiation was MS medium supplemented with 2.0mg-L-1 BA and 0.5mg-L-1 IBA, the buds differentiation rate was 61.9%, and average number of buds was 5.48. On the axillary buds culture, the optimal medium of the buds differentiation was MS medium supplemented with 1.0mg-L-1 BA and 0.5mg-L-1 IBA, the bud differentiation rate was 98.9%, and average number of buds was 5.27.
4. In 2-5 generation subculture, the capability of buds differentiation was decline with the leaves subculture time increasing.
5. The differentiation rate of the callus was drop with the callus age increasing. When the callus age was five days, the differentiation rate was the highest, and the rate was 14%.
6. The effects of the bud differentiation were difference in the different position of the leaves and axillary buds, which the leaves and axillary buds lied in 3-4 node showed the higher differentiation rate of buds. The rate was 96.9% and 48.7%.
7. The leaves of subculture plant still had higher differentiation abilities. The bud differentiation rate of Qingpiguo and Yuanxinghongmaozi was 65% and 50%.
8. In the culture of root inducement and sound seedling, the optimal medium was 1/2MS medium supplemented with 0.8-1.0mg-L-1 IBA and 0.4-0.6mg-L-1 MET. The root induce rate was 100%, the number of root was 5.5-6.03.
9. The sensitive concentration of Km was 6mg/L on the leaf-fragment of Siraitia grosvenorii. The non-transgenic seedling couldn't induce roots on the medium contained Km.
10. When the leaf-fragment of Siraitia grosvenorii pre-cultured for 4 days before being transformed, GUS gene expressed perfectly. The test of instantaneous expression indicated: the leaf-fragment that the whole dyed to navy blue was 21.7%, the leaf-fragment that the edge and inner dyed to deeper blue-spot was 69.6%, and the leaf-fragment that the edge dyed to blue was 8.7%. The rate of instantaneous expression was 100%.
11. Leaf-fragment of Siraitia grosvenorii were dipped in agrobacterium
liquid for 5~60 seconds that concentration was OD600 0.1 ~0.5 and co-cultivated in 48 hours, then GUS gene expressed optimal, the expressed rate was 100%.
12. A lot of fake-transgenic seedling was generated because of the selection after regeneration. The method, which the selection before regeneration combines with the filtration of root generation, could wipe off the fake-transgenic seedling efficiency.
13. GUS gene had been expressed steadily in the Siraitia grosvenorii plants. The transgenic plants of Siraitia grosvenorii were gained in the experiment.
引文
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