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1.
农杆菌介导籼稻优良恢复系bar基因的遗传转化研究   总被引:2,自引:0,他引:2  
应用农杆菌介导转化体系,成功地将含有CaMv35s启动子启动的bar基因导入籼稻幼胚来源的愈伤组织,获得籼稻优良恢复系T461、R402和752三个品种(系)共47个抗除草剂Basta的转基因株系,Southem分析结果表明,转基因植株基因组中检测到bar基因的整合,转基因植株自交后代Basta除草剂抗性鉴定表现出分离,且大多数为1-2个整合位点的孟德尔方式遗传。结果表明,根癌农杆菌介导法可以有效且可靠地转化籼稻。  相似文献   

2.
农杆菌介导转化小麦幼胚获得抗除草剂再生植株   总被引:9,自引:0,他引:9  
采用农杆菌介导法转化小麦品种G54授粉10 d后的幼胚,经5‰和2‰ PPT筛选获得83株正常再生植株.PCR及Southern杂交检测证明其中8株再生苗为转bar基因植株,这些植株对除草剂Basta的抗性明显提高.实验结果还证明,高糖浓度的培养基对愈伤组织诱导、植株再生及生根都有显著的促进作用;在感染液和共培养基中添加乙酰丁香酮有利于转化株的筛选.  相似文献   

3.
该实验构建了含甘蓝型油菜黄化相关基因BnCr4特异片段反向重复结构的RNA干扰(RNAi)载体pFGC5941-Cr4,通过根癌农杆菌介导转化油菜,获得47株抗Basta的抗性再生油菜植株,其中10株经PCR鉴定为阳性转基因植株.随机选取3株经鉴定的转基因阳性油菜植株进行半定量RT-PCR分析,结果显示,相对于非转基因的野生型油菜,3株转基因植株中BnCr4基因的表达量分别降低了78.5%、8.5%、11.8%,表明该干扰载体转入油菜能特异引起植株BnCr4基因表达量下降.  相似文献   

4.
以马铃薯脱毒试管苗茎段为转化受体材料,建立并优化了农杆菌介导的马铃薯遗传转化体系.通过农杆菌介导法将玉米淀粉分支酶基因(Starch branching enzyme b,SBEⅡb)的过表达载体转化马铃薯,接种762个茎段,共获得35株抗性植株.经PCR检测获得了4株转基因阳性植株;对转基因植株进一步进行GUS活性组织化学染色,发现转基因植株的茎段与试管薯均被染上蓝色,表明外源SBEⅡb基因已整合到马铃薯基因组,且正常表达.  相似文献   

5.
利用根癌农杆菌法获得转基因水稻植株及其后代   总被引:14,自引:1,他引:13  
尹中朝  杨凡 《遗传学报》1998,25(6):517-524
在100μmol/L乙酰丁香酮(AS)等vir基因诱导分子存在的情况下,用含双元载体pBYT2的根癌农杆菌菌株EHA101同水稻(OrizasativaL.)台北309悬浮培养细胞共培养3天。经过2个月的连续筛选,共从364颗同根癌农杆菌共培养的悬浮培养细胞团中得到17个具有稳定潮霉素抗性和GUS表达的愈伤组织。对从8个转化组织中得到的10株可能的R0代转基因植株及其后代进行外源基因的整合和表达分析,Southern分析表明外源基因已稳定地整合进水稻基因组中并实现了有性遗传传递。杂交结果显示在其中一个转化系的植株中有5个拷贝的T-DNA整合,而其余的转化系则只整合了1个拷贝。转基因水稻细胞及植株中GUS活性的组织化学染色观察和荧光分析表明玉米ubiquitin基因启动子在水稻细胞中能高效启动gus报告基因的表达。ndPAGE-X-Gluc法检测表明转基因水稻细胞中表达的GUS蛋白比Sigma公司的标准GUS蛋白(SigmaCo.G0786)要小,而与来自大肠杆菌HB101(pBI1121)中的GUS蛋白大小相同。结果表明,根癌农杆菌可有效且可靠地介导外源基因转化水稻。  相似文献   

6.
以马铃薯脱毒试管苗茎段为转化受体材料,建立并优化了农杆菌介导的马铃薯遗传转化体系。通过农杆菌介导法将玉米淀粉分支酶基因(Starch branching enzyme b,SBEⅡb)的过表达载体转化马铃薯,接种762个茎段,共获得35株抗性植株。经PCR检测获得了4株转基因阳性植株;对转基因植株进一步进行GUS活性组织化学染色,发现转基因植株的茎段与试管薯均被染上蓝色,表明外源SBEⅡb基因已整合到马铃薯基因组,且正常表达。  相似文献   

7.
CHI-PAT双价基因遗传转化贵州禾来拢   总被引:1,自引:0,他引:1  
以贵州禾来拢幼胚为转化受体,用农杆菌介导法将几丁质酶和抗除草剂抗性双价基因(CHI-PAT)导入来拢幼胚,筛选出抗性愈伤组织并获得抗性植株.抗性植株经GUS组织化学及PCR检测呈阳性,转基因植株对50 mg/L的Basta溶液有抗性.初步证明CHI和PAT基因已整合进了水稻基因组中.  相似文献   

8.
利用生物技术方法对棉花进行遗传改良主要限于有效的遗传转化系统。以新疆主栽优良陆地棉品种‘新陆早33号’为材料,利用下胚轴作为外植体对影响农杆菌介导的棉花遗传转化及体细胞胚胎发生的因素进行研究,成功建立了除草剂Basta筛选的棉花遗传转化技术体系。同时将植物抗病相关基因多聚半乳糖醛酸酶抑制蛋白基因AtPGIP1导入棉花,经过对再生转化植株的PCR鉴定,初步证明外源基因已经整合到棉花基因组。研究发现:Basta是棉花遗传转化中很有效的筛选剂,低浓度Basta(2.5mg/L)就能够获得很好的筛选效果;较低的共培养温度(20℃)及合适的农杆菌浓度(OD600=0.5)有助于提高转化效率。该研究结果表明,‘新陆早33号’具备作为棉花优良遗传转化受体的基本特征,研究中获得的15株AtPGIP1转基因植株经PCR分子检测均为阳性植株。该研究为新疆棉区棉花分子生物学研究及转基因育种研究奠定了重要基础。  相似文献   

9.
已经成功报道的农杆菌介导的水稻遗传转化多以活力较高的胚性愈伤为材料,很少以水稻悬浮细胞作为受体.另外,利用农杆菌转化多数都是通过浸泡的方式进行侵染.本实验利用滴加浸染法进行农杆菌介导转化水稻悬浮细胞,探讨影响 DNA 转化效率的因素.研究显示,在转化前,将水稻悬浮细胞在愈伤诱导培养基上培养1~2周,诱导产生直径为2~3 mm的微小愈伤组织对转化非常重要.微小愈伤组织大小不应小于 2 mm;对悬浮细胞短时间培养不但会缩短植株再生时间,而且会提高转化效率.此外,侵染农杆菌的浓度、侵染时间和不同侵染方法也影响 T-DNA 插入基因组的效率.用 1 ml A600值为 0.5 浓度的农杆菌悬液滴加在水稻悬浮细胞诱导的愈伤,培养3 d或直到可见农杆菌菌落,此方法可以得到较高转化效率.将再生的潮霉素抗性的转化植株在含有 50 mg/L 潮霉素的分化和生根培养基中筛选得到,并对转化植株 gus 基因的表达进行 PCR 检测.结果显示,用 A600值为 0.5 浓度的农杆菌浸泡侵染 20 min和滴加浸染法,分别得到PCR阳性植株率为 70% 和92%.  相似文献   

10.
目的:通过农杆菌介导法遗传转化大豆。方法:通过热激法将质粒pCAAFP66导入根癌农杆菌菌株EHA105中获得含有抗冷冻蛋白基因(afp)及除草剂抗性筛选标记基因(bar)的农杆菌工程菌株;以大豆品种华春6号和马祖1号种子的下胚轴为外植体,经过农杆菌介导将抗冷冻蛋白基因导入大豆基因组中,在含有除草剂草丁膦(PPT)的培养基中筛选、并经过PCR鉴定获得大豆转化植株。结果:PPT的最佳筛选浓度为1.0mg/L,华春6号和马祖1号的阳性植株数分别为6株和2株,转化效率分别为3.70%和0.94%。结论:不同基因型大豆的转化率存在差异,抗冷冻蛋白基因成功遗传转化进大豆细胞中。  相似文献   

11.
 Transgenic plants of cassava (Manihot esculenta) resistant to the herbicide Basta were obtained through Agrobacterium-mediated transformation. The plants also expressed the uidA gene and two were positive for PCR- and/or Southern-based detection of the nptII gene. Somatic-embryo-derived cotyledons were used as source of explants. A non-disarmed Agrobacterium strain (CIAT 1182) was used to transfer the genes of interest into cassava cultivar MPer183. Greenhouse tests of resistance to Basta (Hoechst) showed three plant lines with different levels of tolerance to the herbicide. Based on Southern tests of transgenesis, the transformation efficiency was 1%. The results constitute the first report of the bar gene conferring herbicide resistance to cassava plants. Received: 9 January 1999 / Revision received: 10 May 1999 / Accepted: 15 June 1999  相似文献   

12.
Transgenic radish (Raphanus sativus L. longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the -glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences. The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production. Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds. The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant. Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically. Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits. These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop.  相似文献   

13.
A reproducible transformation system was developed for pea (Pisum sativum L.) using as explants sections from the embryonic axis of immature seeds. A construct containing two chimeric genes, nopaline synthase-phosphinothricin acetyl transferase (bar) and cauliflower mosaic virus 35S-neomycin phosphotransferase (nptII), was introduced into two pea cultivars using Agrobacterium tumefaciens-mediated transformation procedures. Regeneration was via organogenesis, and transformed plants were selected on medium containing 15 mg/L of phosphinothricin. Transgenic peas were raised in the glasshouse to produce flowers and viable seeds. The bar and nptII genes were expressed in both the primary transgenic pea plants and in the next generation progeny, in which they showed a typical 3:1 Mendelian inheritance pattern. Transformation of regenerated plants was confirmed by assays for neomycin phosphotransferase and phosphinothricin acetyl transferase activity and by northern blot analyses. Transformed plants were resistant to the herbicide Basta when sprayed at rates used in field practice.  相似文献   

14.
We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The To plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T1 and T2 progenies. Enzyme analyses on T1 progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of To plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.  相似文献   

15.
Indica rice cultivar IR64 is most recalcitrant to regenerate, which affects the transformation efficiency especially when mature seed-derived callus tissues are used as explants. Therefore, a simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation. With different hormonal combination tested, the maximum callus induction was observed on MS medium supplemented with 2.5 mg/l 2,4-D and 0.15 mg/l BAP from the scutellum explants. Three weeks old scutellum derived callus explants were immersed in Agrobacterium suspension (strain LBA4404, OD600=1.0) and co-cultured at 26±2°C in dark for 2 d. The maximum transformation efficiency (12%) was achieved with infection of callus explants for 20 min along with use of 150 μm acetosyringone. The maximum plant regeneration was observed on MS medium supplemented with 3 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA. The maximum root induction was observed on MS medium along with 10 g/l glucose and 20 g/l sucrose. The integration of the transgene in T1 transgenic plants was confirmed by polymerase chain reaction and Southern blot analyses. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants. By using this improved method we have successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration.  相似文献   

16.
Summary Tall fescue (Festuca arundinacea Schreb.) is the most important forage species worldwide of the Festuca genus. Single genotype-derived embryogenic suspension cultures were established from tall fescue cultivar Kentucky-31, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric β-glucuronidase (gusA) gene was co-transformed with hph. Transgenic plants were recovered after microprojectile bombardment of suspension cells and subsequent selection in the presence of a high concentration of hygromycin. Fertile transgenic plants were obtained after vernalization under field conditions. T1 and T2 progenies were obtained after reciprocal crosses between transgenic and untransformed control plants. PCR and Southern hybridization analyses revealed a 1∶1 segregation ratio for both transgenes in the T1 and T2 generations. Southern hybridization patterns were identical for T0, T1, and T2 plants. The results demonstrated for the first time the stable meiotic transmission of transgenes following Mendelian rules in transgenic tall fescue.  相似文献   

17.

Key message

An efficient protocol of transformation and selection of transgenic lines of Micro-tom, a widespread model cultivar for tomato, is reported. RNA interference silencing efficiency and stability have been investigated and correlated with the number of insertions.

Abstract

Given its small size and ease of cultivation, the tomato (Solanum lycopersicon) cultivar Micro-tom is of widespread use as a model tomato plant. To create and screen transgenic plants, different selectable markers are commonly used. The bar marker carrying the resistance to the herbicide glufosinate/Basta, has many advantages, but it has been little utilised and with low efficiency for identification of tomato transgenic plants. Here we describe a procedure for accurate selection of transgenic Micro-tom both in vitro and in soil. Immunoblot, Southern blot and phenotypic analyses showed that 100 % of herbicide-resistant plants were transgenic. In addition, regeneration improvement has been obtained by using 2 mg/l Gibberellic acid in the shoot elongation medium; rooting optimisation on medium containing 1 mg/l IAA allowed up to 97 % of shoots developing strong and very healthy roots after only 10 days. Stable transformation frequency by infection of leaf explants with Agrobacterium reached 12 %. Shoots have been induced by combination of 1 mg/l zeatin-trans and 0.1 mg/l IAA. Somatic embryogenesis of cotyledon on medium containing 1 mg/l zeatin + 2 mg/l IAA is described in Micro-tom. The photosynthetic psbS gene has been used as reporter gene for RNA silencing studies. The efficiency of gene silencing has been found equivalent using three different target gene fragments of 519, 398 and 328 bp. Interestingly, silencing efficiency decreased from T0 to the T3 generation in plants containing multiple copies of the inserted T-DNA, while it was stable in plants containing a single insertion.  相似文献   

18.
Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 μM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 μM benzylaminopurine, 2.6 μM indole-3-acetic acid, 1 g l−1 activated charcoal, 1.5 mg l−1 phosphinothricin, and 300 mg l−1 cefotaxime. Phosphinothricin at 1.5 mg l−1 was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 μM 6-benzylaminopurine and 1.5 mg l−1 phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l−1 phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta.  相似文献   

19.
Herbicide-resistant transgenic Panax ginseng plants were produced by introducing the phosphinothricin acetyl transferase (PAT) gene that confers resistance to the herbicide Basta (bialaphos) through Agrobacterium tumefaciens co-cultivation. Embryogenic callus gathered from cotyledon explants of P. ginseng were pre-treated with 0.5 M sucrose or 0.05 M MgSO(4 )before Agrobacterium infection. This pre-treatment process markedly enhanced the transient expression of the beta-glucuronidase (GUS) gene. Embryogenic callus was initially cultured on MS medium supplemented with 400 mg/l cefotaxime for 3 weeks and subsequently subcultured five times to a medium containing 25 mg/l kanamycin and 300 mg/l cefotaxime. Somatic embryos formed on the surfaces of kanamycin-resistant callus. Upon development into the cotyledonary stage, these somatic embryos were transferred to a medium containing 50 mg/l kanamycin and 5 mg/l gibberellic acid to induce germination and strong selection. Integration of the transgene into the plants was confirmed by polymerase chain reaction and Southern analyses. Transfer of the transgenic ginseng plantlets to soil was successfully accomplished via acclimatization in autoclaved perlite. Not all of the plantlets survived in soil that had not been autoclaved because of fungal infection, particularly in the region between the roots and leaves. Transgenic plants growing in soil were observed to be strongly resistant to Basta application.  相似文献   

20.
根癌农杆菌介导的高效大豆遗传转化体系的建立   总被引:7,自引:1,他引:6  
利用根癌农杆菌对来自大豆成熟种子的胚尖进行遗传转化,研究了影响农杆菌介导大豆转化的各种因素,建立了一套优化的大豆遗传转化体系。研究结果表明:菌株KYRT1比EHA105和LBA4404具有更强的侵染能力;较酸的共培养基(pH5.4)、较低的培养温度(22℃)均有利于提高转化效率;恢复培养和分步抗性筛选方式有利于提高抗性组织的存活率和分化率。同时应用这种优化的遗传转化体系,获得了7个大豆品系的转基因植株,转化频率为4.29%-18%。经过PCR和Southern分析证明外源的双价抗虫基因cryIA(c)和pta已经整合到大豆的基因组中。  相似文献   

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