苦皮藤再生体系的建立及超声生物学效应的研究
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摘要
苦皮藤(Celastrus anyulatus Maxim)属卫矛科(Celastraceaes)南蛇藤属(Celastrus．L)多年生落叶藤状灌木，是我国特有的最具潜力的杀虫植物之一，是生产无公害农药的宝贵资源。目前已经从其根皮、种子中分离出20多种天然化合物，这些物质均具杀虫活性，近年研究表明该类化合物还具有抗肿瘤和杀菌效果。因此，开展对苦皮藤的研究工作具有重要意义。
植物组织培养在生物技术领域中占有不可替代的地位。对苦皮藤进行组织培养并建立其再生体系具有一定的理论意义和应用价值。主要结果如下：
1．建立了苦皮藤再生体系：MS+2，4-D 2mg／L培养基适合愈伤组织诱导；MS+6-BA 2mg／L+NAA 0.2mg／L培养基适合芽的分化；MS+6-BA 1mg／L+NAA 0.2mg／L培养基适合芽的增殖；多效唑、暗培养对根的分化是必需的，将芽转至MS+MET0.7mg／L+IBA 0.5mg／L培养基暗培养10d后，转入1／2MS培养基光下培养可形成根。
2．观察了组织培养条件下出现的苦皮藤白化苗及黄化苗的超微结构：白化苗、黄化苗的细胞器与正常苗叶片具有明显差异。多数黄化苗细胞中叶绿体数量较少，叶绿体内含淀粉粒较少，基粒和基质片层的形态变化较大；白化苗叶绿体多呈解体状态，部分细胞线粒体解体，少数细胞核解体。
3．研究了苦皮藤组培苗不定根的形成过程：不定根的根原基发生于维管形成层及其附近的薄壁细胞。苦皮藤茎中未发现有潜伏根原基存在，属诱发根原基类型。不定根的木质部及韧皮部与母体的木质部及韧皮部互相联结形成了连续的维管系统。
环境应力对植物影响已成为生物力学研究领域的一个热点。超声作为一种环境应力，对植物的生长发育有重要影响。但有关超声刺激对植物组培苗影响还少有报道。本文初步研究了超声对苦皮藤组培苗的生物学效应。主要结果如下：
1．不同频率和强度的超声刺激对苦皮藤组培苗的生长有一定影响：27KHz(250W)超声刺激3min，5min，7min可以促进苦皮藤不定芽的萌发；而1.1MHz(500W)超声刺激对苦皮藤组培苗生长有抑制作用。
2．27KHz(250W)超声刺激影响苦皮藤组培苗的超微结构：扫描电镜下，超声处理1min～5min，气孔开张程度与时间呈正相关：处理7min～11min，气孔开张程度下降，但比对照组高。透射电镜下：短时间的超声处理，细胞膜、细胞
核、叶绿体等细胞器有轻微损伤，随着处理时间的延长，细胞质与核内出现空区，
质膜有剥离现象，说明超声对细胞的损伤随着处理时间的延长而增大。
3.超声刺激增强了植物体内保护酶活性:27KHz超声波刺激苦皮藤组培苗
smin和7min，处理后继续培养，并分别于od，3d，6d，gd，12d，15d，18d，21d
测定其超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活性。
结果表明，超声处理不同程度地增强了这些保护酶的活性。
Celastrus angulatus M. is a defoliated vine and used to be as traditional Chinese herbs. C. angulatus is also the valuable resources to be made of biorational pesticide. More than twenty secondary metabolites have being isolated from its bark and seed, and the most of it is the active substance. C. angulatus has a significant and bright prospects of development, so making efforts on this field is very important.
The technologies of plant tissue culture has its unique place in biotechnology, and study of tissue culture and plant regeneration in C. anyulatus M. had significant theoretical meaning and applied value. The main contents and results of research in this thesis are as follows:
Firstly, Study of tissue culture and plant regeneration in C. anyulatus: The MS medium containing 2,4-D2mg/L is suitable for the induction of callus. The MS medium, which contained 6-BA2mg/L and NAA0.2mg/L, is suitable for shoots differentiating from the callus. The MS medium, which contained 6-BA2mg/L and NAA0.2mg/L, is suitable for shoots growing. The MS medium with MET0.7mg/Land IBA0.5mg/L , darkness and 1/2MS medium were advantageous for the roots occurred. The survival rate of plantlet was 85% after transplanting under suitable condition.
Secondly, the ultrastructure of leaf cells in albino seedlings, etionlated seedlings and normal seedlings of C. anyulatus was observed by ultrathin-section and electron microscopy techniques. The results showed that there were distinct differences among ultrastructure of the leaf cells in albino seedlings, etionlated seedlings and normal seedlings of C. anyulatus. The cytoplasma amount in the most cells of etionlated seedlings was few. There were few chloroplasts and the conformation of chloroplast varied from each other in the albino seedlings leaf cells. Few chloroplasts were normal and the most presented part degeneration. Some chloroplast in
albino seedlings took on the state that part membranes were fusing. There were little difference on cell wall, and cell form among albino seedlings, etionlated seedlings and normal ones.
Thirdly, the anatomical observation on adventitious root of C. anyulatus vitro was carried by means of paraffin section method. It is significantly clear that adventitious root primordium has not been found in the stem. The adventitious root primordium of stem initialed from the vascular cambium or phloem parenchyma cell.
The effect of environmental stresses on the growth and development of plant cells and tissues is one of important fields in plant physiology. Ultrasound as one kind of environmental stresses, it has been found that can greatly influence the growth and development of plant. Where as, the studies about the effect of ultrasound stimulation on vitro is rare so far. In this thesis, the biology effect of ultrasound on C. anyulatus vitro was primarily explored. The results of research in this thesis are as follows:
Firstly, when C. angulatus vitro were treated by Ultrasound with a certain intensity of 250w and frequency of 27KHz froml minute to7 minutes, the adventitious shoots from C. angulatus vitro can grow effectively. But treated by Ultrasound with a certain intensity of 500W and frequency of 1.1 MHz , the growth of the adventitious shoots were inhibited in initial stage.
Secondly, ultrastructure of the leaf cells of C. angulatus stimulated with 27KHz ultrasonic in different time was observed by scanning electron microscope and transmission electron microscope. The result showed that the stomas in the affected surface were more opened than in the surface of normal leaves. From 1 minute to 5 minutes, the stimulated time more long, the stomas were more opened. But when stimulated from 7 minutes to llminutes, the degree of stomas opened was minish. The ultrasonic also scathed cell membrance, cytoplasm and nucleus. With stimulated longer, the cells were injured greatly.
Thirdly, the effect of ultrasonic stimulation on protective enzymes of
Celastrus angulatus vitro was explored. When Celastrus angulatus vitro were treated by Ultrasound with a certai
植物组织培养在生物技术领域中占有不可替代的地位。对苦皮藤进行组织培养并建立其再生体系具有一定的理论意义和应用价值。主要结果如下：
1．建立了苦皮藤再生体系：MS+2，4-D 2mg／L培养基适合愈伤组织诱导；MS+6-BA 2mg／L+NAA 0.2mg／L培养基适合芽的分化；MS+6-BA 1mg／L+NAA 0.2mg／L培养基适合芽的增殖；多效唑、暗培养对根的分化是必需的，将芽转至MS+MET0.7mg／L+IBA 0.5mg／L培养基暗培养10d后，转入1／2MS培养基光下培养可形成根。
2．观察了组织培养条件下出现的苦皮藤白化苗及黄化苗的超微结构：白化苗、黄化苗的细胞器与正常苗叶片具有明显差异。多数黄化苗细胞中叶绿体数量较少，叶绿体内含淀粉粒较少，基粒和基质片层的形态变化较大；白化苗叶绿体多呈解体状态，部分细胞线粒体解体，少数细胞核解体。
3．研究了苦皮藤组培苗不定根的形成过程：不定根的根原基发生于维管形成层及其附近的薄壁细胞。苦皮藤茎中未发现有潜伏根原基存在，属诱发根原基类型。不定根的木质部及韧皮部与母体的木质部及韧皮部互相联结形成了连续的维管系统。
环境应力对植物影响已成为生物力学研究领域的一个热点。超声作为一种环境应力，对植物的生长发育有重要影响。但有关超声刺激对植物组培苗影响还少有报道。本文初步研究了超声对苦皮藤组培苗的生物学效应。主要结果如下：
1．不同频率和强度的超声刺激对苦皮藤组培苗的生长有一定影响：27KHz(250W)超声刺激3min，5min，7min可以促进苦皮藤不定芽的萌发；而1.1MHz(500W)超声刺激对苦皮藤组培苗生长有抑制作用。
2．27KHz(250W)超声刺激影响苦皮藤组培苗的超微结构：扫描电镜下，超声处理1min～5min，气孔开张程度与时间呈正相关：处理7min～11min，气孔开张程度下降，但比对照组高。透射电镜下：短时间的超声处理，细胞膜、细胞
核、叶绿体等细胞器有轻微损伤，随着处理时间的延长，细胞质与核内出现空区，
质膜有剥离现象，说明超声对细胞的损伤随着处理时间的延长而增大。
3.超声刺激增强了植物体内保护酶活性:27KHz超声波刺激苦皮藤组培苗
smin和7min，处理后继续培养，并分别于od，3d，6d，gd，12d，15d，18d，21d
测定其超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活性。
结果表明，超声处理不同程度地增强了这些保护酶的活性。
Celastrus angulatus M. is a defoliated vine and used to be as traditional Chinese herbs. C. angulatus is also the valuable resources to be made of biorational pesticide. More than twenty secondary metabolites have being isolated from its bark and seed, and the most of it is the active substance. C. angulatus has a significant and bright prospects of development, so making efforts on this field is very important.
The technologies of plant tissue culture has its unique place in biotechnology, and study of tissue culture and plant regeneration in C. anyulatus M. had significant theoretical meaning and applied value. The main contents and results of research in this thesis are as follows:
Firstly, Study of tissue culture and plant regeneration in C. anyulatus: The MS medium containing 2,4-D2mg/L is suitable for the induction of callus. The MS medium, which contained 6-BA2mg/L and NAA0.2mg/L, is suitable for shoots differentiating from the callus. The MS medium, which contained 6-BA2mg/L and NAA0.2mg/L, is suitable for shoots growing. The MS medium with MET0.7mg/Land IBA0.5mg/L , darkness and 1/2MS medium were advantageous for the roots occurred. The survival rate of plantlet was 85% after transplanting under suitable condition.
Secondly, the ultrastructure of leaf cells in albino seedlings, etionlated seedlings and normal seedlings of C. anyulatus was observed by ultrathin-section and electron microscopy techniques. The results showed that there were distinct differences among ultrastructure of the leaf cells in albino seedlings, etionlated seedlings and normal seedlings of C. anyulatus. The cytoplasma amount in the most cells of etionlated seedlings was few. There were few chloroplasts and the conformation of chloroplast varied from each other in the albino seedlings leaf cells. Few chloroplasts were normal and the most presented part degeneration. Some chloroplast in
albino seedlings took on the state that part membranes were fusing. There were little difference on cell wall, and cell form among albino seedlings, etionlated seedlings and normal ones.
Thirdly, the anatomical observation on adventitious root of C. anyulatus vitro was carried by means of paraffin section method. It is significantly clear that adventitious root primordium has not been found in the stem. The adventitious root primordium of stem initialed from the vascular cambium or phloem parenchyma cell.
The effect of environmental stresses on the growth and development of plant cells and tissues is one of important fields in plant physiology. Ultrasound as one kind of environmental stresses, it has been found that can greatly influence the growth and development of plant. Where as, the studies about the effect of ultrasound stimulation on vitro is rare so far. In this thesis, the biology effect of ultrasound on C. anyulatus vitro was primarily explored. The results of research in this thesis are as follows:
Firstly, when C. angulatus vitro were treated by Ultrasound with a certain intensity of 250w and frequency of 27KHz froml minute to7 minutes, the adventitious shoots from C. angulatus vitro can grow effectively. But treated by Ultrasound with a certain intensity of 500W and frequency of 1.1 MHz , the growth of the adventitious shoots were inhibited in initial stage.
Secondly, ultrastructure of the leaf cells of C. angulatus stimulated with 27KHz ultrasonic in different time was observed by scanning electron microscope and transmission electron microscope. The result showed that the stomas in the affected surface were more opened than in the surface of normal leaves. From 1 minute to 5 minutes, the stimulated time more long, the stomas were more opened. But when stimulated from 7 minutes to llminutes, the degree of stomas opened was minish. The ultrasonic also scathed cell membrance, cytoplasm and nucleus. With stimulated longer, the cells were injured greatly.
Thirdly, the effect of ultrasonic stimulation on protective enzymes of
Celastrus angulatus vitro was explored. When Celastrus angulatus vitro were treated by Ultrasound with a certai
引文
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