【作者】 何晔；

【导师】 窦秉德； 曲延英；

【作者基本信息】 新疆农业大学 ， 作物遗传育种， 2007， 硕士

【摘要】 花生和甜玉米都是重要经济作物。它们自身营养价值丰富,口感好,深受广大人民喜爱,所以其研发价值受到科研界的高度关注,并成为生物反应器技术领域开发的重要功能食品。近些年来,花生和玉米在组织培养及遗传转化方面已经有了较快发展,但仍然存在许多尚未解决的问题,如植株再生品种少、不同基因型间再生差异大、畸形苗多、转化率低等问题。所以,不断发展新的再生体系和摸索有利的转化条件是当前两大作物的新趋势。针对上述问题,本研究分别以花生成熟胚各外植体及玉米幼胚为研究对象,对不同基因型的取材背景、培养条件进行实验,考察其体细胞植株再生能力的差异,优化植株再生条件,通过对影响花生成熟胚各外植体再生能力差异的各要素进行对比分析,综合考虑各分析结果,发现均以胚小叶外植体为优,最后以花生胚小叶为外植体,通过农杆菌介导法对其进行遗传转化,并对影响花生农杆菌遗传转化的各因素进行详细分析并探讨,主要研究结果如下:1.以玉米幼胚为外植体,以复合型培养基为基本培养条件,应用正交试验设计方法对影响玉米幼胚再生的4个生长调节物质的作用进行探讨,并找出适合本实验所用基因型的最佳培养基。结果发现AgNO3是诱导Ⅱ型愈伤的关键因素,其次是2,4-D和L-pro,而CH影响最弱。本实验的优化培养基成分为N6大量+ B5微量+肌醇(0.1g/l)+ Pro(500mg/l)+ 2,4-D(2mg/l)+ AgNO3(10mg/l)。2.本试验以预培养的花生种子的上、下胚轴和叶片等外植体为对象,在离体培养下,考察上述外植体再生能力的差异并对其作出对比分析。结果表明:上述外植体均能诱导出愈伤组织并能够进一步再生出幼苗,但不同外植体的再生能力有所差异,其再生能力大小依次为:上胚轴>叶片>下胚轴,培养基的差异对外植体愈伤组织诱导及再生起决定性作用,不同预培养时间对愈伤诱导及再生也有影响,总的来看是预培养时间短的外植体要好于预培养时间长的外植体。3.以花生胚小叶两种不同外植体取材方式为培养背景,系统地探讨了两种取材方式下各个培养要素(基因型、培养基等)与脱分化、再分化之间的关系。在预培养取材条件下,品种四粒红预培养6天的外植体在2号培养基上分化最好,每愈伤再生芽2.34个,品种改良海花预培养5天的外植体在1号培养基上分化最佳,每愈伤再生芽2.08个。直接取材条件下,改良海花在1号培养基上每愈伤出芽数为6个,而四粒红为3个。直接取材在诱导愈伤及器官分化方面都好于预培养取材。方差分析认为,直接取材条件下,不同培养基对愈伤组织的诱导及芽分化的作用差异较大,而基因型间在诱导愈伤的作用上有显著差异,在出芽率上的作用差异并不显著。4.以花生胚小叶为外植体,利用不同液体培养基对四粒红,改良海花两个品种进行悬浮培养,发现3种基本培养基附加多种植物激素组合均能诱导出愈伤组织并进一步分化出芽,通过对愈伤率与芽点数的比较分析,得出①号培养基最好,在BA激素浓度相同条件下3mg/lNAA要好于1mg/lNAA。进一步的统计分析发现,不同培养基和基因型对愈伤组织和芽的诱导均有显著差异。5.应用花生胚小叶再生体系,以农杆菌介导法对质粒上的基因转化进行初步探讨。通过对影响基因转化的各因素,如抗生素的筛选压、共培养时间、AS的影响、及取材方式的不同等研究,结果表明:外植体在与加有AS(100umol/l)的改良MS液体培养基中共培养4d后,先进行10d的恢复培养,继而转入加有Hy(25mg/l)和Cef(200mg/l)的抗性筛选培养基中进行筛选培养40-50d,后转入含Hy(20mg/l)的分化培养基中分化,3个月后在首批材料中得到6个再生株,经PCR检测,有1株显示了800bp的GUS基因核酸片段,初步证实了外源基因的整合。更多还原

【Abstract】 Peanut and sweet corn are both important and economic crops. They have abundant nourish value, good taste, most peoples are extremely like them, so their search value had been interested by scientific researchers and have become important food in biosphere. In recent years, a great deal of progress has been made in tissue culture and genetic transformation for peanut and maize. However, some problems have still been not solved such as low germination rates of somatic embryos, significant differences between geneotypes, abnormal embryos proportion and low transformation efficiency etc. So developing new regenerative system constantly, broadening the number of varieties of peanut and maize and trying to find out favorable transform conditions are a new trend presently in these two crops.The paper deals with the culture effect of the different explant from peanut seed and immature embryo of maize by organogenesis, et al. To test the regeneration efficiency of the explant getting ways of different genotype and the culture elements in peanut. Thinking that every analyses result systematical ,find it was found that leaflet is the excellent explant .So, the leaflets of peanut was genetically transformated through Agrobacterium-mediated method. A transformed plant was confirmed and the experiment factors influencing transformation efficiency were discussed in detail. All the results were summarized as follows:1. Immature embryos of sweet corn was used as explant on compound culture medium, 4 culture factors that affecting the maize regenerateds were investigated by experiment design and the best culture medium was found out . The results showed that AgNO3 was the key factor of typeⅡcallus induction. 2, 4-D and L-pro take the second place, CH was the last. The optimal medium were N6(major)+B5(minor)+ Inositol (0.1g/l)+pro(500mg/l)+ 2,4-D(2mg/l) +AgNO3(10mg/l).2. Different explant from mature seed of peanut was used to test the plant regenerateion character esp.its’bud formation rate in vitro on different medium. Result indicated: all explant can induced callus and sprouting shoot ,but they had difference rate between explant: Epicoty>Blade>Hypocotyl. Medium’s differences play a decisive role in induced callus and regeneration for different explant, and the same medium had different effect to different explant. It was found that in the experiment, pre-culture time had influence in buds induced and regeneration. Generally, in my result, short time pre-culture was better than its long time.3. The cultured effects of two kinds of leaflet from peanut were compared.The relationship was also discussed between the cultured element (genotype, medium, etc.)and cultured effects (dedifferentiation and redifferentiation).In the Pre-culture explant condition ,Variety SiLiHong six days explants on the 2nd medium differentiated 2.34 buds per callus, Variety GaiLiangHaiHua five days explants on the 1st medium regenerated 2.08 buds per callus.In the direct explants conditions, Variety GaiLiangHaiHua callus sprouting six buds and three from SiLiHong on the 1st medium . The cultured effect of direct explants was better than that of pre-cultured explants based on callus induction and differentiation. Variance analysis showed media had signioficant influence on the callus induction and differentiation, and genotypes were significant influence on callus induction, but not on sprouting rate.4. Leaflet from peanut varaiety SiLiHong and GaiLiangHaiHua was used as explant to culture on the different liquid culture medium. It was found that three basic mediums with various plant hormone combination can induce callus and sproute further. But by comparison the bud number on callus, the medium No.1 was better than the others. 3mg/l NAA was better than 1mg/lNAA while other hormone was same, Statistic analysis indicated that the genotype and medium had notable effects on calli induction and plant regeneration.5. The peanut genetic transformation was carried out by Agrobacterium-mediated approach on leaflet regeneration system. The factors affecting the transformation, such as the selective pressure of antibiotic, co-cultivation time, AS effect and the cultured effects of two kinds of leaflet were examined. The optimized procedure was as following: leaflet was co-cultivation with a vector containing Agrobacterium for 4 days on modified MS liquid medium (100 umol/l AS), 10 day’s recovering cultivates, the explants were transferred to above selective media with 20mg/l Hy and 200mg/l Cef, 40-50 days later, the explants were transferred to above development media with 20mg/l Hy. 6 regeneration shoots were gained in first batch of material after 3 months culture. Out of these shoots, one was confirmed containing a characteristic 800bp DNA segment of the GUS by PCR analysis. The integration of foreign DNA into the peanut genome was confirmed in first step.更多还原