【作者】 刘志洋；

【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， 2005， 硕士

【摘要】 唐菖蒲(Gladiolus hybridus)是鸢尾科唐菖蒲属多年生单子叶球根花卉,是世界著名的四大切花之一。唐菖蒲切花生产是我国花卉产业的重要组成部分。唐菖蒲切花生产中遇到的一个主要问题是病虫害繁多,尤其是真菌性病害发病十分严重。应用抗病品种是病害防治最直接有效的方法,但唐菖蒲抗病种质资源匮乏加之常规育种周期长,使得抗病品种远远不能满足生产上的需求。随着生物技术的迅猛发展,运用基因工程手段将外源抗病基因导入植物中,为唐菖蒲抗病育种带来了新的希望。本研究将抗真菌病基因(几丁质酶基因)通过农杆菌介导转化唐菖蒲。建立了唐菖蒲高频再生系统和遗传转化系统,经PCR 检测得到了PCR 阳性植株。为培育唐菖蒲抗真菌病新品种奠定了基础,为单子叶花卉植物遗传转化提供了理论依据。研究研究的主要内容和结果如下: 1.建立了唐菖蒲直接分化再生系统和愈伤组织再生系统:以子球茎为外植体,在MS+NAA0.2mg/L+6BA2.0mg/L+KT0.8mg/L 培养基上直接分化不定芽,在MS+ IBA0.5mg/L培养基上生根;以幼芽为外植体,在MS+2,4-D4.0 mg/L +6BA0.5 mg/L 培养基上诱导愈伤组织,在MS+NAA0.2mg/L+6BA2.0mg/L+KT0.8mg/L 培养基上诱导芽分化,在MS 培养基上生根。2.确定了抗生素选择压力:载体上含有NPTⅡ选择标记基因,使转化植株带有卡那霉素抗性,研究得到的卡那霉素选择压力为110mg/L。3.研究了预培养时间、侵染时间、共培养时间、共培养pH 值四个因素对遗传转化的影响:对于直接分化再生系统,最佳组合为:预培养0d+侵染15min+共培养2d+共培养pH 值5.6;对于愈伤组织再生系统,最佳组合为:预培养2d+侵染15min+共培养3d+共培养pH5.2。4.转化植株的PCR 检测:直接分化再生系统得到44 株抗性植株,其中1 株为PCR 阳性植株,阳性率为2.3%,转化率为0.07%。愈伤组织再生系统得到31 株抗性植株,其中3株为PCR 阳性植株,阳性率为9.7%,转化率为0.20%。更多还原

【Abstract】 gladiolus(Gladiolus hybridus) is one of perennial and monocotyledonous bulbous flowers,which belongs to the gladiolus of iridaceous and is one of the four world-famous cut flowers.Cut flower production of gladiolus plays an important role in Chinese folwer industry.The main problem in the production of gladiolus is that diseases and insect pests are so much, especially,the fungi disease is very serious. Using the disease-resistant varieties is the best way for preventing and curing the plant diseases.But the disease-resistant resource of gladiolus is so little and the cycle of the general breeding is so long that the disease-resistant variety is far from meeting the demand of the gladiolus production.With the rapid development of biotechnology, transferring new disease-resistant gene of other sources into the gladiolus by using genetic engineering means brings new hope in the disease-resistant breeding of gladiolus. The disease-resistant gene (chitinase gene) has been transferred into gladiolus mediated by the agrobacterium.Seted up high-frequency regeneration system and genetic transformation system,gained positive plant by PCR.Stablished the foundation for cultivation of new disease-resistant gladiolus variety , Offered the theoretical foundation for genetic transformation of the monocotyledon.The main content and result of experiment are as follows: 1.Established direct differentiation and callus differertiation regeneration system.Taking bulbs as explants , direct differentiated adventitious bud on the culture medium of MS+NAA0.2mg/L+6BA2.0mg/L+KT0.8mg/L,induced roots on the culture medium of MS+ IBA0.5mg/L.Taking young buds as explants,induced callus on the culture medium of MS+2,4-D4.0mg/L+6BA0.5mg/L,differentiated on the culture medium of MS+NAA0.2mg/L +6BA2.0mg/L+KT0.8mg/L and induced roots on the culture medium of MS. 2.Determined the select pressure of antibiotics.The select and sign gene NPTⅡon the carrier,which can make the trangenic plants have the resistance to kanamycin. The select pressure of kanamycin was determined as 110mg/L. 3.Studied four factors effect on genetic transformation which included pre-culture time,infect time,co-culture time and co-culture PH.For direct differentiation regeneration system,the best combination is:pre-culture 0d + infect 15min + co-culture 2d + co-culture pH 5.6.For callus differertiation regeneration system,the best combination is:pre-culture 2 d + infect 15min + co-culture 3d + co-culture pH5.2. 4.Detection of the transformed plant:For direct differentiation regeneration system, we gained 44 resistant plants.One of them is PCR positive plant,positive percentage is 2.3% and transformation percentage is 0.07%.For callus differentiation regeneration system,we gained 31 resistant plants.Three of them are PCR positive plants,positive percentage is 9.7% and transformation percentage is 2.0%.更多还原