根癌农杆菌介导的小麦成熟胚遗传转化研究

以小麦栽培品种郑麦9023成熟胚半胚愈伤组织作为受体材料,通过农杆菌菌株GV3101将目的基因导入受体材料并对转化过程进行了优化。考查了MS培养基中NH4NO3浓度对组织培养的影响,以及高渗处理对转化过程的作用。结果显示,2.5倍的NH4NO3浓度可明显提高愈伤组织再生率,高渗处理对愈伤组织再生率的提高也有帮助。经抗性筛选和PCR鉴定,共得到了4株转基因小麦植株,平均转化效率为0.18%。     

根癌农杆菌对中华猕猴桃的转化

猕猴桃含有丰富的维生素Ｃ，被誉为“水果之王”本实验用中华猕猴桃无菌苗的茎段和叶片被根癌农杆菌感染后，能够自主生根，但茎段的生根率与叶片的生根率有一定的差异。     

根癌农杆菌转基因的分子机制

根癌农杆菌将T-DNA转移并插入寄主细胞的基因组,需要趋化与附着、vir区活化、T-DNA切割及转运、T-DNA整合及表达等步骤。本文对这些步骤分子机制的研究进展进行了综述。     

用根癌农杆菌转化获得胡萝卜转化细胞

将悬浮培养的胡萝卜(Daucus carota Var.Sativa DC.)单细胞与野生的胭脂碱型和章鱼碱型根癌农杆菌共同培养,在含羧(艹卡)青霉素的无激素MS培养基上筛选,获得生长旺盛的转化细胞,转化频率为10~(-2)。经基因产物——冠瘿碱检测,这些细胞均有冠瘿碱合成酶活性。     

根癌农杆菌对结球白菜的转化

为了给外源有用基因导入结球白菜并高效稳定地表达提供依据，以结球白菜的子叶－子叶柄为外植体，采用农杆菌共培养法把外源基因C58／pV11导入结球白菜，在选择最优的转化条件－根癌农杆菌侵染外植体15min，农杆菌培养和共培养时在培养基中均加入AS，培养基pH为5．2，共培养3d的条件下，获得转基因植株，其中抗性植株的抗草丁膦试验，转基因植株DNA的PCR及PCR－Southern斑点杂交鉴定等结果表明，50％的抗性植株的基因组中有外源基因的整合。     

野生型根癌农杆菌对马铃薯的遗传转化研究

用不同的野生型根癌农杆菌(Agrobacterium tumefaciens)T37、B6、TFA16YET、FA16YK对马铃薯普通栽培种甘农薯1号的块茎圆片、茎段、叶切片、试管苗,美国引进品种褐皮.布尔班克的块茎圆片以及新型栽培种NW-174实生苗的子叶及下胚轴等组织进行了离体侵染和整株侵染实验,发现:不同的菌种对同一组织以及相同的菌种对不同组织的侵染效果均不同。在供试菌株中,以TFA16YE的转化效果最好。对NW-174新型栽培种实生苗的子叶、下胚轴进行的侵染实验表明,这两种组织的转化率较高。结果表明,在离体转化程序中,外植体的生理状态、发育阶段以及供试菌株的代谢活性及培养条件等均是影响转化效率的重要因素。     

冬小麦遗传再生体系及根癌农杆菌介导的转化研究

以临优145的幼胚为外植体诱导植株再生,并进行农杆菌转化研究.以冬小麦品种临优145的幼胚为外植体,消毒后用解剖刀挑取幼胚,盾片朝上接种于MS+2,4-D 2 mg/L+肌醇100 mg/L+MES 400 mg/L+CH 100 mg/L+40 g/L麦芽糖+8 g/L琼脂的培养基中诱导愈伤组织,每2周继代一次,将诱导出的淡黄色、颗粒状Ⅱ型胚性愈伤组织接种于分化培养基(MS+ZT 1 mg/L+IAA 1mg/L+MES 400 mg/L+CH 100 mg/L+麦芽糖40 g/L+gelrite 2.6 g/L,pH 5.8)上培养2-3周,然后转接到再生培养基(1/10 MS+NAA 0.5mg/L+KT0.5 mg/L+蔗糖30 g/L+gelrite 2.6 g/L,pH 5.8)中进行再生成苗.接种1229块幼胚,得到987块愈伤组织,Hyg抗性愈伤组织123块,抗性再生植株34株.在建立了再生体系的基础上,用根癌农杆菌介导法将GUS基因导入幼胚愈伤组织,抗性植株的PCR检测呈阳性.X-gluc染色表明,少部分愈伤组织出现肉眼可见的蓝色晕斑,说明GUS基因已经在小麦幼胚愈伤组织中表达.     

根癌农杆菌介导的植物遗传转化研究

介绍了农杆菌介导的植物原位转基因方法发展状况、技术环节以及在转化机制上的研究结果,并对存在的问题以及应用前景进行了讨论。     

根癌农杆菌介导FPF1基因转化菊花的研究

经过转化材料的筛选、KM筛选浓度、Cef抗菌浓度以及延迟筛选时间的测定,以根癌农杆菌（Agrobactriumtumefaciens）LBA4404菌株介导FPF1基因转化菊花,对转化材料进行连续KM筛选,获得105株抗性苗,部分植株经PCR检测呈阳性.田间观察部分抗性株与对照相比在株型、叶色及花期上表现出差异.     

The Efficient Transformation of Wheat in Planta by Agrobacterium tumefaciens

Transformation of wheat was performed by pipetting spikelets with Agrobacterium tumefaciens which contained expression vectors using Npt Ⅱ as reporter gene. Transformants were identified through kanamycin resistance, PCR and Southern blot. The results showed that transformation efficiency was within 2.0 to 3. 2 % in all tested varieties of wheat. Then the simple and efficient protocol of wheat transformation by Agrobacterium tumefaciens in planta was primarily established.     

农杆菌介导的小麦遗传转化研究进展

从转化的机理、应用现状、影响因素和应用前景4个方面对农杆菌介导的小麦遗传转化方法进行了综述。     

农杆菌介导的小麦活体转化

 将含有NptII基因表达载体的农杆菌滴入小麦小花中进行小麦活体转化 ,通过对幼胚和成熟种子进行卡那霉素抗性初步筛选 ,并通过PCR和Southern杂交进行分子水平鉴定。结果显示 ,在所试的 5个品种 (系 )中 ,获得转化植株效率为 2 .0 %～ 3 .2 % ,由此初步建立农杆菌介导的简便有效的小麦活体转化方法     

农杆菌介导小麦遗传转化的影响因素

[目的]对农杆菌菌株、小麦品种、培养基状态及报告基因选择等进行优化筛选,为进一步完善农杆菌介导小麦遗传转化方法提供参考。[方法]用携带质粒pCAMBIA1304的农杆菌菌株LBA4404、EHA105,对温麦19、郑离03和郑离06小麦的幼胚愈伤组织进行遗传转化,从农杆菌菌株、小麦品种、共培养基状态及报告基因选择等方面进行探讨。[结果]农杆菌菌株EHA105转化效率较高,最高达到22.58%;姊妹系郑离03与郑离06的转化率差别极大,郑离06转化率最高而郑离03最低;农杆菌与愈伤组织共培养时,使用MS半固体培养基的转化率高于MS固体培养基;GFP和GUS都可以用作判断转化率的报告基因,GFP优势更明显。[结论]高毒性农杆菌菌株EHA105转化效率明显高于菌株LBA4404;小麦基因型是影响转化效率的重要因素之一;农杆菌与愈伤组织共培养时使用半固体培养基可提高转化率;通过GFP瞬时表达,可有效进行小麦转化效率的研究。     

小麦农杆菌介导转基因植株的稳定获得和检测

 以预培养 4 d的小麦幼胚愈伤组织为受体 ,L B培养基上激活培养 3 d或 AB培养基上激活培养 4 d,并在WCC培养基中重悬的 C5 8c1农杆菌菌系为供体 ,将含有目的基因、标记基因的 p PTN2 4 9、p PTN 2 5 4、p PTN2 70、p SIS-GFP等重组 DNA质粒转入了小麦 ,经在含 10～ 5 0 mg/ L geneticin( G4 18)、5 0 mg/ L cefotaxime、5 0 m g/ L vancomycin、5 0 m g/ L ticillicin选择培养基上多次选择 ,移栽前利用 npt EL ISA检测 ,移栽后利用叶片离体退绿、PCR和 South-ern blot 4种方法综合检测 ,共获得了 2 9株转基因植株 ,转化频率平均为 0 .82 %。在所获得的转基因植株中 ,单拷贝整合植株占 65 .5 2 %。研究结果还表明 ,不同基因型的转化效率显著不同 ,除了 Bobwhite外 ,笔者也从扬麦 10号获得了转基因植株 ;EL ISA测定值的高低与外源 DNA整合的拷贝数有一定关系 ;初步建立了农杆菌介导稳定转化小麦的技术体系。     

农杆菌介导转化小麦的研究进展

在小麦转基因技术研究中,遗传转化效率一直不高。随着转基因技术的发展,人们对小麦转基因研究逐渐增多,目前使用的主要方法有基因枪法、农杆菌介导法和花粉管通道法等。文中对农杆菌介导转化小麦的研究进展进行综述,为小麦遗传转化研究奠定了基础。。。     

Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by Agrobacterium tumefaciens

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gone delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid(2,4-D) and 3,6-dichloro-o-anisic acid (dicamba) were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn't be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS (Murashige and Skoog) components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.     

农杆菌介导小麦HMW-Glu(5+10)基因转化研究

以含有HMW-Glu(5 10)基因的农杆菌(EHA 105)对豫麦18号幼胚愈伤组织进行了遗传转化,得到的6株转基因再生植株.经PCR初步检测,发现有2株的基因组中5亚基基因呈现阳性,其中1株的基因组5亚基和10亚基基因均呈现阳性.表明目的基因可能已经整合到了受体基因组中.同时对影响农杆菌介导遗传转化的几个重要因素进行了探讨,发现乙酰丁香酮(AS)浓度为200μmol.L-1、菌液OD600值为0.5、侵染时间为1 h、侵染后对愈伤组织适当干燥处理等可有效提高农杆菌的转化效率.     

利用农杆菌浸种法将叶片衰老抑制基因P_(SAG12)-IPT导入普通小麦的研究

 以普通小麦品种西农1376和西农2611为材料,利用农杆菌浸种法获得1株导入了叶片衰老抑制基因PSAG12-IPT转基因植株。经PCR、GUS组织化学染色和Southern杂交分析,证明目的基因已整合到小麦基因组中并在转基因植株中能够稳定遗传。转基因小麦在叶片细胞分裂素(异戊烯基腺嘌呤)含量、叶片衰老进程及农艺性状方面与对照无明显差异,初步表明叶片衰老抑制基因PSAG12-IPT,在转基因植株体内可能未表达或表达量不足。     

Regular Production of Transgenic Wheat Mediated by Agrobacterium tumefaciens

Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0-1.5mm in size, C58c1 of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production,shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptⅡ ELISA (nptⅡ enzyme assay), PCR, Southern blot and leaf bleach,29 positive plants were identified from two genotypes of Bobwhite and Yanglmai 10, with an average transformation efficiency of 0.82 %. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration.