【作者】 赵瑜；

【导师】 肖娅萍；

【作者基本信息】 陕西师范大学 ， 植物学， 2007， 硕士

【摘要】 绞股蓝（Gynostemma pentaphyllum（Thunb．）Makino）为葫芦科Cucurbitaceae绞股蓝属Gynostemma多年生草质藤本植物，是一种著名的中药。它在我国分布广泛，野生资源丰富。我国各地如广东、福建、陕西、浙江、湖北、江西、安徽、四川、山东、河北等省已引种栽培成功。超声辐照属于物理诱变源，与其他诱变源相比，具有方便廉价，绿色安全等优点。它对植物的生长发育有重要的影响。超声辐照对染色体的致畸作用，可以被利用，来进行遗传、育种方面的研究。目前，对超声辐照诱变效应的研究比较多，但其系统性、综合性研究还不够深入。因而，超声辐照诱变与分子生物学技术、生物工程等其他育种途径相互结合，进一步加强机理和效应等方面的研究，提高诱变效率是非常必要的。本试验选取绞股蓝作为材料，进行了悬浮细胞培养研究。并应用悬浮细胞系作为诱变材料，引入了超声辐照对目标植物进行诱变处理，综合细胞工程与超声辐照诱变方便快捷等特点，研究不同时间的超声辐照对绞股蓝的影响，尝试从形态特征、细胞和分子水平上揭示超声辐照对绞股蓝种子及悬浮细胞系的损伤效应。主要的研究结果如下：1．绞股蓝种子萌发对温度的适宜性较强，从4℃到35℃均可以发芽，在60℃和80℃的高温处理中，有个别种子仍然能够保持生活力。对不同级别种子发芽率的试验说明，大田收获的绞股蓝种子饱满度不一，通过种子精选，可以增加种子萌发率，提高其使用价值。同时超声处理绞股蓝种子相对于6-BA、GA₃和2，4-D等3种处理方法而言，可以有效提高发芽率，而且只有高剂量的超声处理才会对绞股蓝幼苗产生影响。处理后的植株没有发生形态学方面的变异，对各个时期的生长进程也未见明显变化。因此，可采用10min超声处理，作为大规模播种的前处理，应用于大田生产的前处理。2．筛选出了适宜进行悬浮培养，质地疏松且生长旺盛的淡黄色愈伤组织，确定最佳悬浮培养基配比为：MS+2.4-D1.0mg╱L+6-BA0.5mg／L。置于25℃散射光或黑暗条件，100—120r／min旋转式摇床上震荡培养，两周后，在无菌条件下先用80目筛网滤去较大颗粒，再用100目滤取较小细胞团。继代培养获得绞股蓝细胞悬浮液。3．使用不同的超声时间梯度处理绞股蓝根尖细胞，观察染色体的变化。发现超声辐照能够引起绞股蓝染色体畸变。产生的类型主要包括：微核、染色体断片、染色体桥、落后染色体等。这种影响随着超声时间的增加呈现上升的趋势，当超声时间超过30min，染色体畸变情况明显加剧，在一个细胞里可能出现多种畸变类型。4．使用不同的超声时间梯度处理绞股蓝悬浮细胞，悬浮细胞系生长进程变缓，需要经过一个相对较长的适应期之后才能进入到细胞的对数生长期。同时，细胞生长随着超声辐照时间的不同呈现规律性的变化，而长时间的超声辐照处理则会造成细胞膜受损破裂，胞内物质外溢，导致细胞死亡。5．经过超声辐照的愈伤组织都多少能够分化，说明绞股蓝对超声辐照是有一定程度的耐受性。而超声辐照后的愈伤组织分化率则受到了不同程度的影响，这种影响和超声辐照的时间呈正相关。短时间的超声辐照对愈伤组织的分化和再生植株的形态影响不大。而从8min超声辐照开始，再生率明显下降，部分愈伤组织出现褐化。同时使用超声辐照和细胞悬浮培养及组织培养的方式，可以切实产生畸变，经过超声辐照过后的再生植株产生了一系列的变化，表现为分枝增多、叶片畸形、叶片变小、节间变短等。6．我们利用RAPD（Random Amplified Polymorphic DNAs）方法，探讨了超声辐照对绞股蓝再生植株DNA多态性的影响，电泳的谱带光滑清晰，背景浅而且重复性比较好。在一定辐照时间范围内，谱带基本相似，超声辐照2min和对照基本保持一致，差异并不明显。而超声辐照时间超过10min，则表现出的遗传差异较大。不过，从总体上观察，经过超声辐照过后，再生植株所产生的差异，要小于系统进化的品种间差异。更多还原

【Abstract】 Abstract: The plant Gynostemma pentaphyllum(Thunb.)Makino is a kind of perennial herb liane, which belongs to Cucurbitaceae. it distributing widely in China, and the wild resource is also very abundance. Across the country, a lot of provinces, such as Shaanxi, Zhejiang, Hebei have successfully cultivation.Ultrasonic irradiation is belonging to physical revulsive factor. Compare to other revulsive factors it has the advantages of cheap, convenient, natural and safe. Ultrasonic irradiation has greatly impact to plants’ growth and development. Ultrasound treatment can cause chromosome damage, so it can be used in genetic and breeding research. Currently, systematic and comprehensive research on the effects of ultrasound is not deeply enough. So, it is a necessary way in the study to combining the ultrasonic irradiation mutagenesis with molecular biology, bio-engineering and other breeding approaches, in order to strengthen the mechanism and effects of Ultrasonic irradiation, and to improve the efficiency of mutation.Gynostemma pentaphyllum(Thunb.)Makino is chosen as materials in this study, we establish it’s cell suspension system, then, we treat cell suspension system by the Ultrasonic irradiation in order to study the biologic effect and the impact with different treatment time to find out the changes in morphological characteristics, cellular and molecular levels. The main findings are as follows:1. the seeds of Gynostemma pentaphyllum(Thunb.)Makino can be bourgeon in biggish range from 4℃to 35℃, even in high temperature of 60℃and 80℃several seeds can still keep vim. Compare to disposes of 6-BA、GA3 and 2,4-D, Ultrasound effective enhance bourgeon rate, and the plant after treatment have no morphological variation among the periods of growth process unless under high-dose ultrasound treatment. So it is helpful to be disposed 10min use Ultrasound first before semination to field production.2. The yellow, loose, fresh callus was chosen for cell suspension culture, the optimal suspension culture system containing MS+2.4-D1.0mg/L+ 6-BA0.5mg/L. After cultivated for two weeks we got a eugenic suspension cell culture system.3. Use different ultrasonic time to treat with root tip, observe the change of chromosomes. The results show that ultrasonic can cause aberrance of chromosomes. The main aberrance types such as: micronucleus, nuclear buds, chromosomal bridges and so on can be observed. The impact and ultrasonic processing time is positively correlated. As more than 30 minutes ultrasound, aberrance is obviously strengthened, different aberrances may be found in one abnormal cell.4. Use different ultrasonic time to treat with suspension cells, the growth of suspension cells is slow down. Require a long period of adjustment to be able to enter the log phase. Meanwhile, the ultrasound treatment of long time destroyed the cell membrane cause the intracellular material spills and cell death.5. Callus tissue of Gynostemma has a certain degree of tolerance to ultrasonic treatment, low intensity ultrasonic has only slight impact to of callus tissue’s differentiation rate, regeneration and modality. From 3min ultrasonic irradiation, the regeneration rate has declined, and partly callus appears browning phenomenon, simultaneity plants occur a series transformation, such as: increase of divarication, leave’s abnormality, diminish of leaf and shorten of internodes.6. We use RAPD marker to study the influence of ultrasonic to regenerative plant’s DNA polymorphism of Gynostemma pentaphyllum(Thunb.)Makino. In definite intensity radialization range, strips of PCR result are basically the same, however when the radialization time pass 10min, it appear relatively difference. But in general this difference can be omitted relative to the difference of different varieties.更多还原