根癌农杆菌介导的水稻遗传转化

就根癌农杆菌介导水稻遗传转化的研究历程和影响农杆菌转化水稻的几个关键因素以及这一问题的前景作了评述和展望     

根癌农杆菌介导的大豆遗传转化

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.     

根癌农杆菌介导的巨大口蘑遗传转化体系的构建

以巨大口蘑菌丝为受体材料,利用含有双元质粒plasmid4的根癌农杆菌EHA105介导,首次成功建立了巨大口蘑的遗传转化体系。通过潮霉素抗性筛选、PCR鉴定和绿色荧光蛋白的检测,表明潮霉素抗性基因（Hyg）已经整合到巨大口蘑基因组中,增强型绿色荧光蛋白基因（eGFP）在巨大口蘑菌丝中获得表达,并能够稳定遗传。本研究建立了农杆菌介导的巨大口蘑遗传转化体系,为今后巨大口蘑的基因功能研究奠定了基础。     

根癌农杆菌介导丝状真菌遗传转化的研究进展

根癌农杆菌介导的丝状真菌遗传转化是近年建立起来的一种新方法,该方法和以往的真菌转化体系相比具有转化方法简单、材料易得、效率高以及转化子中T-DNA单拷贝插入比例高等特点。就根癌农杆菌转化的丝状真菌种类、转化的具体过程以及影响转化效率的因素等方面进行了综述,并展望了该方法的应用前景。     

根癌农杆菌介导真菌遗传转化的研究进展

根癌农杆菌介导的真菌遗传转化是近年来发展的一种新方法 ,与其它方法相比 ,该方法具有操作简便、转化效率高和易得到稳定转化子等特点。目前 ,在根癌农杆菌介导下已实现了多个属种真菌的遗传转化 ,显示出良好的应用前景。综述了根癌农杆菌介导真菌遗传转化的转化机理和T DNA在真菌细胞中的存在方式等方面的研究结果 ,并展望这一方法的应用前景。     

根癌农杆菌介导的香蕉遗传转化研究进展

香蕉的栽培品种绝大多数是三倍体, 常规育种难度大, 工作周期长, 难以采用传统的育种方法进行遗传改良, 因此转基因技术成为改良香蕉种质的有效方法。根癌农杆菌介导的香蕉的遗传转化从20世纪90年代起取得的了较大的进步, 但仍然存在着很多问题。文章分析了影响农杆菌介导香蕉转化的几个重要环节, 并对其研究现状、存在的问题及应用前景作简要概述。     

根癌农杆菌介导真菌遗传转化的研究及应用

根癌农杆菌介导转化法（Agrobacterium tumefaciens-mediated transformation,ATMT）具有转化效率高、遗传稳定、适用范围广等诸多优点,已成为真菌遗传转化研究中的强有力手段,在真菌基因资源开发、真菌性疾病研究和外源蛋白表达研究中发挥巨大作用。本文概述了根癌农杆菌转化法在真菌转化中的研究进展、技术优缺点、转化机制、实验方法和应用现状,着重介绍影响其转化效率的因素并对优化方法进行探讨,展望了该技术在真菌基因资源发掘、基因编辑等方面的应用前景,为今后真菌的遗传转化研究提供参考。     

根癌农杆菌介导灭蚊卵菌贵阳腐霉的遗传转化

Pythium guiyangense is a mosquito pathogen, and has been proved to be a promising agent for biological control of mosquitoes. In order to develop the strains adaptable to different ecological environment having stable virulence to mosquito larvae, and being able to prolong the shelf life, an effort was made on transforming the fungus by using homologous or heterologous virulence genes. In this paper, a genetic transformation experiment of P. guiyangense mediated by Agrobacterium tumefaciens is reported. As a result, an A. tumefaciens mediated genetic transformation system was established successfully.     

根癌农杆菌介导的草莓遗传转化研究进展

随着植物离体再生技术的日益成熟和多种外源目的基因的分离与克隆,转基因技术以目的性强、周期短等优点成为草莓品种改良的重要途径。在草莓上采用的遗传转化方法主要是农杆菌介导法。概述了农杆菌介导法的转化机理以及基因型、农杆菌菌株、侵染时间和菌液浓度、共培养时间、酚类物质等对草莓遗传转化的影响,并从草莓果实贮藏保鲜、抗除草剂、抗病毒、抗虫、抗真菌、转基因疫苗等方面对近年来草莓的转基因研究进展进行了总结。     

根癌农杆菌介导的高羊茅遗传转化研究

采用携带卡那霉素抗性基因nptⅡ和Na^ ／H^ 反向运输AtNHX1基因的表达载体pROK2／AtNHX1(带有35S启动子)和pROK2U／AtNHX1(带有ubi1启动子)的根癌农杆菌AGL1和GV3101,对4个品种高羊茅下胚轴来源的胚性愈伤组织进行了遗传转化。胚性愈伤组织经根癌农杆菌感染和共培养后,用50～150mg／L巴龙霉素筛选抗性愈伤组织,获得1126棵再生植株,用10～20mg／L卡那霉素进一步筛选再生植株,总共得到525棵绿色抗性植株。抗性植株的总DNA用AtNHX1基因的特异引物进行PCR检测,其中21棵为PCR阳性,最高转化频率为1．77％。Southern杂交结果证实,外源基因以低拷贝整合到高羊茅的基因组中,实验发现,在不同品种之间转化效率有所差异。     

Agrobacterium tumefaciens-mediated transformation of the soybean pathogen Phomopsis longicolla

To facilitate functional genomics in the soybean pathogen Phomopsis longicolla, we developed a robust Agrobacterium tumefaciens-mediated transformation system that yielded 150–250 transformants per 1 × 10⁶ conidia of P. longicolla. This first report of P. longicolla transformation provides a useful tool for insertional mutagenesis in an increasingly important pathogen of soybean.     

Agrobacterium tumefaciens-mediated barley transformation

Genetically transformed barley was produced by eco-cultivating immature embryo explants with Agrobacterium tumefaciens carrying a binary vector coding for chimaeric bacterial genes, bar and gus , and selecting for bialaphos-resistant cultures from which plants were regenerated. Integration of both genes was confirmed by gel blot hybridization analysis of DNA from the transformed plants and their progenies. From 1282 embryos, plants were recovered for 54 independently transformed lines, giving a transformation efficiency of 4.2%. Transgene numbers in the different lines ranged from single copy insertion to at least ten copies. Sixteen out of 18 plants grown to maturity were fully fertile. Both marker genes, bar and gus , were expressed and co-segregated in the T₁ progeny plants. In the majority of cases, the genes showed Mendelian segregation predicted for transgene insertion at a single locus. In one family with multiple transgene insertions, molecular analysis of T₁ and T₂ plants suggested that the T-DNA had inserted at two unlinked loci.     

Agrobacterium tumefaciens-mediated transformation ofMucor circinelloides

The Agrobacterium tumefaciens-mediated transformation of the zygomycetous fungus Mucor circinelloides is described. A method was also developed for the hygromycin B-based selection of Mucor transformants. Transformation with the hygromycin B phosphotransferase gene of Escherichia coli controlled by the heterologous Aspergillus nidulans trpC promoter resulted in hygromycin B-resistant clones. The presence of the hygromycin resistance gene in the genome of the transformants was verified by polymerase chain reaction and Southern hybridization: the latter analyses revealed integrations in the host genome at different sites in different transformants. The stability of transformants remained questionable during the latter analyses.     

Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation

A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.     

Agrobacterium tumefaciens-mediated transformation of Atropa belladonna

Belladonna or deadly nightshade (Atropa belladonna L.) is an important medicinal plant in the family Solanaceae. It is a model plant for studying plant alkaloid biosynthesis. In this study, a reliable protocol for efficient transformation of A. belladonna using Agrobacterium tumefaciens was developed. Hypocotyl and cotyledon explants were co-cultivated with three opine-type Agrobacterium strains (LBA4404: pBISN1, GV3101: pBISN1, and EHA105: pBISN1). Selection and regeneration of transformed cells were conducted on two regeneration media; RM1 (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium (MS) salts, Gamborg B5 vitamins (Gamborg et al. in Exp Cell Res 50:151–158, 1968), 4.56 μM zeatin, and 2.9 μM indole-3-acetic acid (IAA)] and RM2 (MS salts, B5 vitamins, 4.65 μM kinetin, and 1.14 μM IAA), each containing 100 mg l^?1 kanamycin and 250 mg l^?1 timentin. Both regeneration media and type of explant had significant effects on frequencies of transformation. Using an optimal regeneration medium and regardless of the strain of Agrobacterium used, over 80 % of hypocotyl explants and 60 % of cotyledons developed at least one transformed shoot after 2–3 months of selection. Most transformants exhibited a normal phenotype while growing in the greenhouse. Southern blot analysis confirmed the stable integration of the nptII transgene in T₁ plants.     

Agrobacterium tumefaciens-mediated transformation of Robinia pseudoacacia

Robinia pseudoacacia (black locust) plants were regenerated after co-cultivation of stem and leaf segments with Agrobacterium tumefaciens strain GV3101 (pMP90) that harbored a binary vector that included genes for β-glucuronidase (GUS) and hygromycin phosphotransferase. Successful transformation was confirmed by the ability of stem and leaf segments to produce calli in the presence of hygromycin, by histochemical and fluorometric assays of GUS activity in plant tissues, and by Southern blotting analysis. In this transformation system, about 2 months were required for regeneration of transgenic plants from stem and leaf segments. The frequency of transformation from stem segments was approximately 24%, and the morphology of regenerated plants resembled that of the original parental strain. Received: 2 September 1999 / Revision received: 30 November 1999 / Accepted: 4 December 1999     

Agrobacterium tumefaciens-mediated transformation of Rhipsalidopsis gaertneri

A protocol for Agrobacterium tumefaciens-mediated genetic transformation of Rhipsalidopsis cv. CB5 was developed. Calluses derived from phylloclade explants and sub-cultured onto fresh callus induction medium over a period of 9–12 months were co-cultivated with A. tumefaciens LBA4404. Plasmid constructs carrying the nptII gene, as a selectable marker, and the reporter uidA gene were used. Transformed Rhipsalidopsis calluses with a vigorous growth phenotype were obtained by extended culture on media containing 600 mg l⁻¹ kanamycin. After 9 months of a stringent selection pressure, the removal of kanamycin from the final medium together with the culture of the transformed calluses under nutritional stress led to the formation of several transgenic adventitious shoots. Transformation was confirmed by GUS staining (for uidA gene), ELISA analysis and Southern blot hybridization (for the nptII gene). With this approach, a transformation efficiency of 22.7% was achieved. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for this cactus species.     

Agrobacterium tumefaciens-mediated transformation of recalcitrant crops

The most widely used technique for the introduction of new genetic information into plant cells is based on the natural gene transfer capacity ofAgrobacterium tumefaciens. Currently, this technique is routinely applicable in just a few model species, like tobacco and petunia. Thus far, the numerous efforts to apply the technique to crop species have had limited success. In this review, an attempt is made to survey all the research experience onAgrobacterium tumefaciens-mediated transformation of recalcitrant crops and to highlight the problems generally encountered. The main difficulty appears to be directing the gene transfer towards those plant cells that are amenable to regeneration. The various ways to reduce stress during the transformation and regeneration process are often beneficial. The influence of the developmental stage of the plant material and the host range of theAgrobacterium strain depends largely on the plant species used, which hampers the formulation of common procedures. However, some general guidelines for the development of a transformation protocol are discussed.     

Factors affecting Agrobacterium tumefaciens-mediated transformation of peppermint

 Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic evaluation of co-cultivation parameters established that deletion of coconut water from the co-cultivation medium resulted in substantially increased transient β-Glucuronidase (GUS) activity, in both the frequency of explants expressing gusA and the number of GUS foci per explant (>700 explants). Co-cultivation on a tobacco cell feeder layer also enhanced A. tumefaciens infection. Enhanced transformation efficiencies were further facilitated by increased selection pressure mediated by higher concentrations of kanamycin in the medium during shoot induction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/regeneration medium and from 15 to 30 mg/l in rooting medium. Raising the concentration of kanamycin in media substantially lowered the number of "escapes" without significant reduction in plant regeneration. These modifications to the protocol yielded an average transformation frequency of about 20% (>2000 explants) based on expression of GUS activity or the tobacco antifungal protein, osmotin, in transgenic plants. Genetic transformation of peppermint has been enhanced to the extent that biotechnology is a viable alternative to plant breeding and clonal selection for improvement of this crop. Received: 7 December 1998 / Revision received: 27 April 1999 / Accepted: 14 May 1999