【作者】 王莎莎；

【导师】 车代弟；

【作者基本信息】 东北农业大学 ， 园林植物与观赏园艺， 2008， 硕士

【摘要】 百合(Lilium spp.)不但花姿独特,同时还具备药用和食用价值,深受国内外市场的喜爱。随着百合栽培面积的不断扩大,这种易感病的植物常常引起大面积的病害,造成巨大的经济损失。然而传统的药剂防治不但增加人工成本,还会对环境造成污染,应用抗病品种是病害防治的最直接有效的方法。但采用传统的育种方法不但周期较长,受亲本影响较大,因此这种育种方法不能满足生产的需要。随着植物生物技术的不断发展,直接将外源目的基因导入植物材料中,这为百合培育新品种开辟了新的道路。本研究以抗病能力差的东方百合“Sorbonne”为试材,对东方百合鳞片进行了直接再生不定芽系统的建立,同时以鳞片为外植体进行了愈伤组织的诱导,进行了愈伤组织经体细胞胚发生途径再生的研究。并对直接再生过程中的生根情况进行了比较,对鳞片愈伤的诱导体胚发育进行了较为深入的研究。在此基础上以百合鳞片为遗传转化受体,以鳞片直接再生芽方式为再生途径,将植物广谱抗病基因(NPR1基因)通过农杆菌介导转化百合,建立了稳定的遗传转化体系,得到了经PCR检测为阳性的植株。本研究的主要结果如下:1.百合鳞片直接再生不定芽体系:以百合鳞片为外植体,最佳芽诱导培养基为MS+6-BA0.4mg/L+NAA1.0mg/L,芽诱导率达到96.7%;最佳生根培养基为MS+IBA0.5mg/L,生根率为95.2%。2.百合愈伤组织体细胞胚发生途径的再生体系:以百合鳞片作为外植体,最佳愈伤诱导培养基为MS+2,4-D 2.0mg/L +6-BA 0.5mg/L+水解酪蛋白300mg/L,诱导率为68.3%;最佳胚性愈伤诱导培养基为MS+2,4-D2.0mg/L +6-BA0.5mg/L+水解酪蛋白150mg/L+谷氨酰胺150mg/L,诱导率为75.0%;将其放入MS+2,4-D 0.5 mg/L +KT 0.3 mg/L+蔗糖40g/L中培养,体胚形成率为60.0%,最佳体细胞胚萌发培养基为MS+6-BA0.5 mg/L。通过石蜡切片法对鳞片愈伤的发育过程观察发现,首先外植体表层细胞形成愈伤组织,随后在愈伤组织表面形成许多瘤状凸起,这些凸起继续发育成球形胚、心形胚,最后发育成熟形成完整植株。3.百合遗传转化体系:以百合鳞片为转化受体,在用农杆菌侵染前预培养2天,侵染时菌液浓度为OD600=0.5,侵染时间为20min,20℃下共培养时间2天,共培养之后进行Km浓度为30―80―130mg/L的依次筛选,头孢霉素在除菌过程中使用浓度为400mg/L,直至抗性芽的形成。通过实验发现在共培养时,去除培养基中NH4NO3,并向其中加入100mg/L的乙酰丁香酮可提高转化率。4.抗性植株的PCR检测:由直接芽再生途径得到的56株抗性植株中有3株呈阳性,阳性率为5.4%,占该再生系统总转化外植体900块的0.33%。更多还原

【Abstract】 Lilies(Lilium spp.)not only have special beautiful flowers, but also have officinal and edible value. They are very popular for many people in the world. With the planting areas expanded gradually, the flowers often bring lots of disease on the field which are sensitive for illness. So they produced hugeness lost. But tradition preventions increase labour cost,meanwhile they could effect the environment. It is more effective and direct method for preventing the plant diseases to use using the disease-resistant varieties. Because the tradition breeding method need a long time for breeding and is effected by F1 generation greatly, the demand of the Lilies production couldn’t be meted. On the background of biology technique fast development, we could transfer new disease-resistant gene of other sources into the Lilies by using genetic engineering means .It will break a new path in the new disease-resistant breeding of Lilies.This study took the less-disease resistant oriental lily‘Sorbonne’as material. We establish regeneration system of direct shoot method, introduce callus from Lilies bulbs。We observe the transformation from callus to somatic embryo. We compare the cases of shoot on different medias in the process of direct regeneration. We use the bulbs of Lilies as the explants of genetic transformation, The broad-spectrum disease resistance gene (NPR1 gene) transferred into Lilies mediated by the agrobacterium. we establish genetic transformation system of Lilies. Finally, we get positive plants after PCR. The main content and result of experiment are as follows:1.The regeneration system though bulbs: we use the slice of Lilies bulbs as explants, MS basal medium supplemented with 6-BA0.4mg/L and NAA1.0mg/L was advantageous to shoot induction, the frequency of shoot induction is 96.7%; MS basal medium supplemented with IBA0.5 mg/L could develop their roots, the rate of robust plantlets is 95.2%.2.The callus regeneration of Lilies by somatic embryogenesis: we use the bulbs of Lilies as explants, the good method for induce callus is MS basal medium supplemented with 2,4-D2.0mg/L and 6-BA0.5 mg/L and LH 300mg/L, the rate of produce callus is 68.3%. MS+2,4-D 2.0 mg/L +6-BA 0.5mg/L+LH150mg/L+Gln150mg/L is the best culture medium that the induction of embryogenesis callus ,and the rate of produce embryogenesis callus is 75.0% .Then transfer the embryogenesis callus into the culture media MS+2,4-D 0.5 mg/L +KT 0.3 mg/L + Sucrose 40g/L, which rate is 60.0%. Somatic embryo could germinate on the MS basal medium supplemented with 6-BA 0.5mg/L. The appearance and growth of somatic embryo were observed through paraffin slices. Histological observations showed that surface cells first experienced dedifferentiation and followed with callus-forming, then the top of callus appear a tubercular structure. These embryogenic cell masses experienced globular, heart stages and developed into whole plantlets .3.The establishment of genetic transformation system: Taking the slice of Lilies bulbs as explants, pre-culture of callus for 2d;the agrobacterium tumefaciens solution was OD600=0.5,the time of infect was 20min,the time of co-culture was 2d at 20℃,the step up the concentration of Km 30mg/L from the first day,80mg/L from the third day,130mg/L from the sixth day. Concentration of Cef was 400mg/L in the process of sterilization, until the plantlet formed. MS added 100mg/L trans-Rerulic acid and subtracted NH4NO3 can improve the transformation ratio.4.Detection of the transformed plant:For shoot regeneration,we gained 56 resistant plants.Three of them is PCR positive plant, positive percentage is 5.4% and transformation percentage is 0.33% in 900 explants.更多还原