【作者】 王燕；

【导师】 杨美纯；

【作者基本信息】 广西大学 ， 植物学， 2015， 硕士

【摘要】 广西是我国木薯种植第一大省,围绕木薯淀粉加工形成的一系列木薯加工产业链,是广西经济组成的重要部分。“西选03"是广西大学选育的木薯优良品种,耐旱、产量高,具有较高的经济和推广价值,采用转基因方法把抗逆基因导入优良木薯品种中可有效提高抗逆性,改良木薯品种。本试验主要研究以“西选03"幼嫩茎段腋芽为外植体建立快繁体系、以获得的无菌苗幼嫩叶片为材料建立离体叶片再生体系以及初步建立农杆菌介导的无菌叶片遗传转化体系,结果如下：1.外植体灭菌：经过0.1%KMnO4预处理10 mmin和75%酒精浸泡20s后,大田栽培、室内水培、大棚沙培来源的外植体用0.1%HgCl2分别灭菌10 min、5 min、 6 min效果最好,污染率分别为30.8%、11.1%、17.1%,成活率分别可达61.5%、80.0%、74.4%。2.初代培养：添加低浓度6-BA的培养基中外植体腋芽萌发优于KT和TDZ,最适宜初代诱导的培养基为MS+6-BA 0.05 mg/L,外植体在一周内萌发,萌芽率达90.0%,萌发芽长势良好。3.继代增殖：单芽增殖培养宜采用MS+6-BA 0.1 mg/L培养基,芽增殖倍数可达4.6；密集丛生芽培养宜采用MS+6-BA 0.3 mg/L+GA 30.5 mg/L培养基；无菌芽壮苗培养宜采用MS+PP333 3.0 mg/L培养基,无菌芽节间缩短、增粗。4.生根：单独添加NAA.PP333或者直接用MS培养基均可诱导无菌芽生根,其中以MS+PP333 0.3 mg/L诱导生根效果较好,根短而粗壮,生根率达100%,平均根数6.9。5.生根苗移栽：取生根3条左右,株高3～4 cm的健壮组培苗移栽至草炭：蛭石：菜园土=1：1：1的基质中,30 d成活率可达55.1%。6.愈伤组织诱导：MS+毒莠定12 mg/L+CuSO4 0.5 mg/L适合作为无菌叶片愈伤组织诱导培养基,出愈率达100%。形成愈伤组织呈淡黄色,部分愈伤组织块表面有颗粒状突起。7.愈伤组织继代：最佳的愈伤组织继代培养基为1/2 MS+毒莠定12 mg/L,增殖率达86.7%,用此培养基继代,可使硬化愈伤组织块变得疏松,表面颗粒状突起明显,为转化成胚性愈伤组织做准备。8.愈伤组织分化诱导：采用生长素类与细胞分裂素类植物生长调节剂配合使用诱导愈伤组织产生不定芽,分化率极低。采用MS+6-BA 1.0mg/L+IAA 0.5mg/L+ AgNO3 0.5 mg/L+CuSO4 0.5 mg/L培养基可一定程度上提高不定芽的分化率,但仍然不足15%。9.抗生素筛选：分别对带腋芽茎段、无菌叶片和愈伤组织块进行抗生素Km耐受压筛选,结果表明,三种材料的抗生素选择压培养基分别为MS+6-BA 0.05 mg、L +Km 400mg/L、Ms+毒莠定12 mg/L+Km 200 mg/L和MS+毒莠定12 mg/L+Km 150mg/L。10.农杆菌侵染无菌叶及GFP基因荧光检测：农杆菌侵染无菌叶以20min效果最好,GFP荧光表达率可达63.3%；农杆菌与无菌叶共培养4d效果较好,GFP荧光表达率为56.7%。更多还原

【Abstract】 Cassava is planted in Guangxi, the largest province in cassava planting in our county, and form a series of processing industry chain based on cassava starch processing, which is an important part of the economy of Guangxi. "Xixuan 03", a fine variety of cassava and can resist to drought, bred by Guangxi University, have the higher economic value and application prospects. It is the effective way to improve cassava quality to add the excellent resistance gene into cassava varieties through agrobacterium-mediated to obtain new varieties with higher resistance. This experiment focused on three aspects that establish rapid propagation system taken tender stem segments as materials, regeneration system based on young leaves of aseptic seedlings and genetic transformation system Agrobacterium-mediated, the results were as follows:1. Sterilization of explants:The best sterilization effect with 0.1% HgCl2 for explants taken from field garden, indoor and greenhouse respectively was 10 min,5 min,6 min after the same pretrement of 0.1% KMnO4 10 min and 75% alcohol 20 s. The contamination rate was 30.8%,11.1% and 17.1%, and the survival rate was up to 61.5%, 80.0% and 74.4%.2. Primary culture:The germination rate of explants with medium added low concentration of 6-BA was better than that in medium added KT and TDZ, and the most suitable medium for inducing buds was MS+6-BA 0.05 mg/L. The explants were induced to appear within a week, and the germination rate was 90.0%. The new buds were robust.3. Subculture:The suitable medium for single bud proliferation was MS+6-BA 0.1 mg/L, and the proliferation rate could reach 4.6. The suitable medium for the dense cluster buds was MS+6-BA 0.3 mg/L+GA3 0.5 mg/L. It was better to use MS+ PP333 3.0 mg/L as medium to make thin seedlings grow slower and stronger. The medium added with AC and low concentration of 6-BA could be used to make buds stronger in some extents.4. Root induced:The medium added NAA, PP333 alone or nothing added all could induce roots appear, of which MS+PP333 0.3 mg/L had the best roots induced effect. The rooting rate reached 100%, and the average root number was 6.9.The roots short and stout.5. Transplant:The robust seedlings that the number of roots was around 3 and the height were 3-4 cm should be choose for transplanting to the matrix of peat:vermiculite: garden soil=1:1:1, and the survival rate was up to 55.1% after 30 days later.6. Callus induction:MS+picloram 12 mg/L+CuSO4 0.5 mg/L suit as the medium of inducing sterile leaves to form callus, and the callus induction rate was up to 100%. The callus produced presented pale yellow and viscous soft, transparent, and part of the callus had the granular tower.7. Callus subculture:The best medium for callus subculture was 1/2 MS+ picloram 12 mg/L, and the proliferation rate was 86.7%. Took this medium to subculture callus could make the viscous soft to be hardened and prepare for transformated the normal callus into embryogenic callus.8. Differentiation induction of callus:The differentiation rate is very low if took the medium with the use of auxin and cytokinin plant growth regulators to induce callus to produce adventitious buds. Using MS+6-BA 1.0 mg/L+IAA 0.5 mg/L+AgNO3 0.5 mg/L+CuSO4 0.5 mg/L as differentiation induction medium could slightly improve the conversion rate of adventitious buds, but it was still less than 15%.9. Antibiotic selection pressure test:Stems with axillary buds, sterile leaves and callus were used to determine the minimum concentration of antibiotic caused all death. The results indicated that the medium for three explants respectively was MS+6-BA 0.05 mg/L+Km 400 mg/L, MS+Picloram 12 mg/L+Km 200 mg/L, MS+Picloram 12 mg/L+Km 150 mg/L.10. Infection and detection of GFP:Sterile leaves used as materials, were infected by agrobacterium tumefaciens-mediated which carried the target gene, and the suitable infection time was 20 min, the expression of GFP gene was 63.3%. And the test of different co-culture time showed that 4 d was the optimum time, up to 56.7%.更多还原