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本研究以GUS基因在子叶节区的瞬时表达为依据,通过探讨影响农杆菌转化效率的因素,优化了大豆子叶节非组织培养遗传转化体系;利用该体系对冀豆16号进行Bar基因的遗传转化,并使用针刺法对转基因植株进行草铵膦筛选.结果表明,侵染液中附加3%蔗糖、OD600=0.6、以脱脂棉作为菌液附着介质同时不添加表面活性剂Silwet L-77、侵染1次的GUS阳性率最高达到62.13%.草铵膦抗性植株经PCR检测获得T0阳性植株10个,转化率为2.5%.经PCR和RT-PCR鉴定共获得3株T1阳性植株,初步证明目的基因已整合到大豆基因组中. 相似文献
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根癌农杆菌介导的大豆遗传转化 总被引:7,自引:1,他引:6
农杆菌介导法是大豆遗传转化的重要方法之一 ,许多实验室应用该方法得到了转基因大豆 ,但目前使用该方法进行转化的效率还比较低 ,尚需深入研究。农杆菌菌株、大豆基因型、组织培养条件、T-DNA的转移效率和转化后的筛选模式都会影响大豆转化的效率。概述了近年来根癌农杆菌介导的大豆遗传转化的一些重要成果 ,以及转化过程中大豆的易感性与农杆菌的转化能力、乙酰丁香酮促进vir基因活化、转化的受体系统和巯基混合物减轻受体材料的褐化、提高T DNA的转移效率等几个重要因素的研究进展 ,并介绍了转化中常用的几个筛选标记基因 (nptⅡ、hpt、bar基因和突变的ahas基因 )及通过共转化法去除标记基因的方法 ,同时对今后研究的重点进行了讨论. 相似文献
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农杆菌介导的玉米遗传转化研究进展 总被引:1,自引:0,他引:1
邓向阳 《基因组学与应用生物学》2010,29(4)
农杆菌介导的转基因法是目前玉米遗传转化的主流方法之一。目前,模式玉米种质幼胚的转化体系已程式化,且开发了新筛选基因和获得不含筛选基因转基因玉米的方法,但是大多数育种骨干自交系转化频率低和转化受体基本上是幼胚。从农杆菌、受体及培养条件多方面各种因素对问题进行分析,多数研究认为针对特定基因型和受体材料建立好的受体再生系统,结合高效率农杆菌转化体系,获得多目的基因聚合(无其它外源片段)的转基因玉米将是农杆菌介导玉米转化体系研究的最终目标。本文主要从农杆菌介导(转基因)法应用于玉米遗传转化的历史、现状、问题等方面进行综述,为同领域的研究者提供一定的参考。 相似文献
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影响农杆菌介导的大豆子叶节遗传转化的因素 总被引:1,自引:0,他引:1
利用携带pCAMBIA1301质粒(含hpt和gus基因)的超毒根癌农杆菌菌株EHA105对大豆子叶节外植体进行遗传转化,研究了影响农杆菌介导的大豆子叶节遗传转化的因素。研究结果表明.农杆菌侵染液和共培养培养基中添加200μmok/L乙酰丁香酮和50mg/L抗坏血酸可以有效促进农杆菌对大豆子叶节的转化。农杆菌与子叶节共培养后羧苄青霉素(250mr/L)和头孢霉素(100mg/L)结合使用能有效抑制农杆菌过度繁殖并提高转化芽诱导频率;在转化细胞的分化和转化芽伸长过程中,改进的筛选策略可以明显改善对转化芽的筛选效果,从而提高转化频率。应用优化后的转化体系.获得了3个国内大豆主栽品种的转基因植株,PCR阳性植株频率为3.8%~7.6%。转化植株叶片总DNA的PCR和Southern blot实验表明,T-DNA上的外源基因已经整合到大豆基因组中。 相似文献
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大豆转基因的研究进展 总被引:1,自引:0,他引:1
近年来利用基因工程技术,大豆的遗传转化取得了较大的进展。该文介绍了几种主要的大豆遗传转化系统的优点和缺点。探讨了影响农杆菌介导大豆遗传转化的因素。分析了大豆遗传转化中存在的一些问题及其解决办法,评价了转基因大豆的生物安全性,展望了未来大豆遗传转化的发展前景。 相似文献
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84K杨愈伤组织再生体系和直接分化再生体系遗传转化的比较性研究 总被引:1,自引:0,他引:1
《植物研究》2017,(4)
植物转基因必须以建立良好的植物转化受体系统为基础,而良好的受体系统必须以具有高频率的转化率及较强的再生能力为前提。实验以84K杨叶片为材料研究其在不同培养条件、不同种类及浓度的植物激素对其脱分化及再分化的影响,并建立了两种成熟的组织再生系统,得出组织培养体系与遗传转化体系的受体系统在激素配比方面存在一定差距,所以在此基础上进行了PCK1、PCK2两个基因的愈伤途径和不定芽途径途径的遗传转化,并对两种遗传转化体系进行了对比性分析并总结出两种体系的优缺点。 相似文献
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Advances in Pollen-Mediated Genetic Transformation 总被引:4,自引:0,他引:4
植物遗传转化技术是植物科学基础理论与应用研究的有力武器,已成为植物遗传改良的重要途径之一。但是、目前遗传转化所采用的受体系统,大都需要体外培养和植株再生过程,才能获得转基因植株。其中、基因型限制和遗传变异是该技术不可逾越的两大障碍。花粉管通道法可省去转化体的离体培养,不过、多数植物受花器结构的限制而难以经花柱注射DNA,只能向子房注射,并不是真正的“花粉管通道”。又由于此法外源基因的导入发生在授粉之后,因此该方法亦不属于花粉遗传转化。利用小孢子胚胎发生体系进行遗传转化与利用花粉作为外源DNA的媒介,继而、通过授粉受精获得转基因种子,是目前花粉遗传转化的两个重要方面和活跃的研究方向。前者仍需要离体再生系统,后者则可以利用植物自身的再生机制,本文称之为花粉介导法(polen-mediatedtransformation)。该方法通过自然的胚胎发育过程获得转基因子代,避免了组织培养过程中的遗传变异和转基因植株的嵌合现象。可望成为简便快速的植物遗传转化体系。目前对花粉介导的遗传转化进行专门评述的文献较少,本文对该领域的研究分三个层次进行了综述。一、外源基因转化方法小孢子或由小孢子形成的胚状体是很有潜力的遗传转化受体 相似文献
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Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants
of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental
phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set
of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection
steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars. 12 of 16 tested
cultivars were sufficiently amenable to existing routine tissue-culture conditions for practical genetic transformation. Three
cultivars required adjustments to 2,4-D levels during callus proliferation, geneticin concentration during selection, and/or
light intensity during regeneration. One cultivar gave an extreme necrotic response in leaf spindle explants and produced
no callus tissue under the tested culture conditions. It was helpful to obtain spindle explants for tissue culture from plants
with good water supply for growth, especially for genotypes that were harder to culture. It was generally possible to obtain
several independent transgenic plants per bombardment, with time in callus culture limited to 11–15 weeks. A caution with
this efficient transformation system is that separate shoots arose from different primary transformed cells in more than half
of tested calli after selection for geneticin resistance. The results across this diverse cultivar set are likely to be a
useful guide to key variables for rapid optimisation of tissue culture conditions for efficient genetic transformation of
other sugarcane cultivars. 相似文献
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采用GUS基因瞬时表达检测方法,通过正交试验以AS浓度、侵染菌液OD值、侵染时间、共培养时间和恢复培养时间5个因素在4个水平上进行分析,优化了农杆菌介导的大豆胚尖遗传转化体系,并在此基础上进行了抗逆基因GmPK的遗传转化。结果表明,采用共培养培养基中添加100μmol/L AS、侵染菌液OD600值0.9、侵染15h、共培养5d和恢复培养3d的转化条件最佳,GUS阳性率达74.59%,经PCR及RT-PCR进一步验证获得了转基因阳性植株。利用优化的最佳条件进行抗逆基因GmPK的转化,炼苗移栽成活的再生植株经PCR及PCR-Southern blotting验证,初步证明外源基因已经整合至大豆基因组,转化率为0.6%。 相似文献
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An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics. 相似文献
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为了研究甘露糖正向筛选体系在巨尾桉遗传转化过程中的有效性,构建了以6-磷酸甘露糖异构酶(6-phosphomannose isomerase,PMI)为筛选标记的pCAMBIA1301植物表达载体,并将该载体通过农杆菌介导的遗传转化转入木本植物巨尾桉中。将获得的阳性植株通过氯酚红(chlorophenol red,CPR)法及PCR检测,桉树遗传转化的阳性率达到26.09%。另外,通过正交试验优化法,对巨尾桉组培快繁体系建立过程中不同浓度激素配比进行了研究,建立起良好的巨尾桉组织培养再生体系,由甘露糖筛选敏感性测试,获得了巨尾桉筛选临界浓度,蔗糖与甘露糖比例为19∶11,优化了巨尾桉遗传转化体系,为今后巨尾桉组织培养与遗传转化研究提供了重要的参考依据。 相似文献
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Transformation of Soybean (Glycine max) by Infecting Germinating Seeds with Agrobacterium tumefaciens 总被引:9,自引:1,他引:8
The transfer of genetic material into soybean tissue was accomplished by using an avirulent strain of Agrobacterium tumefaciens which contained the binary vector pGA482. The method used for transformation requires no tissue culture steps as it involves the inoculation of the plumule, cotyledonary node, and adjacent cotyledon tissues of germinating seeds. The identification of neomycin phosphotransferase (NPT) II enzyme activity in the tissues of 16 (R0) soybean plants indicated that the plant expressible Nos-NPT II gene, contained within the T-DNA region from pGA482, had been transferred at least into somatic tissues. Putative transformed R0 soybean plants were advanced to produce R1 plants which were also assayed for the presence of the transferred Nos-NPT II gene. The combined results of these assays indicated that about 0.7% of the surviving inoculated seeds yielded transformed tissues in the R0 plant, and that about 1/10 of these plants yielded transformed R1 plants. The presence of the Nos-NPT II gene in DNAs isolated from both R0 and R1 plant was demonstrated by using genomic blot hybridization and polymerase chain reaction methods. Integration of this gene into the soybean genome was demonstrated for three R1 soybean plants. 相似文献
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Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system 总被引:16,自引:0,他引:16
Corneille S Lutz K Svab Z Maliga P 《The Plant journal : for cell and molecular biology》2001,27(2):171-178
Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants. 相似文献
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魔芋组织培养与细胞工程 总被引:3,自引:0,他引:3
近几年来,魔芋组织培养研究进展较快,其细胞工程领域也取得了一定的研究成果。现对魔芋组织培养过程中外植体取材、愈伤组织诱导与分化的激素应用进行了概述,重点介绍了魔芋离体形态建成的几种模式及其调控机制的研究新进展。有关魔芋种质资源离体保存和突变体的筛选的研究工作已经展开,其遗传转化体系也逐渐完善,外源基因如抗病基因、抗除草剂基因等现已转化成功。最后还对魔芋今后的研究方向进行了讨论,指出了目前存在的主要问题并提出了相应的对策。 相似文献
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大豆体细胞胚增殖保存与萌发植株体系的建立(英文) 总被引:1,自引:0,他引:1
以大豆(Glycine max(L.)Merr.)未成熟子叶为外植体,用高浓度生长素诱导东北地区主栽大豆的18个基因型体细胞胚胎发生,诱导率为0.29%~77.62%。在此基础上成功地诱导10个大豆基因型产生可继代增殖的体细胞胚,诱导率在5.2%~22.1%之间。经在固体培养基上多次继代增殖,首次建立了可在固体培养基上继代增殖的大豆体细胞胚萌发再生体系,继代一年以上的体细胞胚仍具有萌发能力和正常育性,得到了结荚植株。此体系的建立为大豆的遗传转化提供了新的、更为有效的受体体系。 相似文献
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Mature seed-derived callus of the model indica rice variety Kasalath is highly competent in Agrobacterium-mediated transformation 总被引:1,自引:0,他引:1
We previously established an efficient Agrobacterium-mediated transformation system using primary calli derived from mature seeds of the model japonica rice variety Nipponbare. We expected that the shortened tissue culture period would reduce callus browning—a common problem
with the indica transformation system during prolonged tissue culture in the undifferentiated state. In this study, we successfully applied
our efficient transformation system to Kasalath—a model variety of indica rice. The Luc reporter system is sensitive enough to allow quantitative analysis of the competency of rice callus for Agrobacterium-mediated transformation. We unexpectedly discovered that primary callus of Kasalath exhibits a remarkably high competency
for Agrobacterium-mediated transformation compared to Nipponbare. Southern blot analysis and Luc luminescence showed that independent transformation
events in primary callus of Kasalath occurred successfully at ca. tenfold higher frequency than in Nipponbare, and single
copy T-DNA integration was observed in ~40% of these events. We also compared the competency of secondary callus of Nipponbare
and Kasalath and again found superior competency in Kasalath, although the identification and subsequent observation of independent
transformation events in secondary callus is difficult due to the vigorous growth of both transformed and non-transformed
cells. An efficient transformation system in Kasalath could facilitate the identification of QTL genes, since many QTL genes
are analyzed in a Nipponbare × Kasalath genetic background. The higher transformation competency of Kasalath could be a useful
trait in the establishment of highly efficient systems involving new transformation technologies such as gene targeting. 相似文献
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优化子叶节转化法培育大豆MtDREB2A转基因植株 总被引:4,自引:0,他引:4
将正交因素试验与GUS基因组织化学染色等技术相结合,优化大豆(Glycine max)品种东农50遗传转化体系,导入抗旱关键基因Mt DREB2A。结果表明,大豆种子表面消毒,NaClO溶液法与Cl_2气熏蒸法的去污染率分别达到98.67%和93.33%。子叶节法转GUS基因组织化学染色率(68.33%)显著高于下胚轴法(14.00%)和胚尖法(0.67%)(P0.05)。种子萌发5天,农杆菌(Agrobacterium tumefaciens)培养温度25°C,OD600=0.9,共培养5天的转GUS基因子叶节最高达72.00%;恢复培养5天,草丁膦(3 mg·L~(–1))、头孢噻肟钠(200 mg·L~(–1))和羧苄青霉素(300 mg·L~(–1))筛选诱导分化的转GUS基因不定芽最多为3.33%;优化的大豆遗传转化体系转化效率为1.11%。转Mt DREB2A基因大豆东农50植株根系更加密集,主根长度和侧根数量均显著高于对照(P0.05),证实Mt DREB2A基因具有促进大豆根系生长的作用,为利用该基因进行大豆抗旱育种奠定了坚实的基础并提供了理论依据。 相似文献