Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.     

Production of transgenic pea (Pisum sativum L.) plants resistant to the herbicide pursuit

Transgenic pea (Pisum sativum L.) plants containing mutant ahas/als gene were obtained using Agrobacterium-mediated genetic transformation. Transformation has been carried out using cocultivation of pea explants with Agrobacterium tumefaciens strain lBA4404 carrying genetic vectors pCB004, pCB006 and pCB007 containing ahas/als and nptII genes. The presence of transferred genes in the genomes of transgenic plants has been confirmed by PCR analysis.     

Transgenic indica rice resistant to sap-sucking insects

Agrobacterium-mediated genetic transformation has been optimized in indica rice susceptible to sap-sucking insects, viz., brown planthopper (BPH) and green leafhopper (GLH). Snowdrop lectin gene (gna) from Galanthus nivalis, driven by phloem-specific rice-sucrose-synthase promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was employed for genetic transformation. Embryogenic calli--after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-gna--were selected on the medium containing phosphinothricin. PCR and Southern blot analyses confirmed the stable integration of both the genes into genomes of transgenic (T0) rice plants. Northern and Western blot analyses revealed the expression of gna in the transgenic plants. In the T1 and T2 generations, the gna and bar transgenes showed co-segregation at a ratio of 3 : 1. Plant progenies expressing gna, in T1 and T2, exhibited substantial resistance against BPH and GLH pests. This is the first report dealing with transgenic indica rice exhibiting high resistance to both insects.     

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.)

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.     

Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.     

Effect of lox-sites of the Cre lox recombination system on promoterless bar gene expression in transgenic plants

We demonstrate that localization of lox site between the right border of T-DNA and promoterless bar gene (RB-lox-bar-) led to its highly efficient expression in transgenic plants of Nicotiana tabacum and N. africana. Plasmid vectors used in gene integration experiments contained neomycin phosphotransferase II (npt II) gene under nos promoter as well. Transgenic plants were selected according to their capacity to grow on the medium with kanamycin and then they were tested on the selective medium containing phosphinothricin. 80% of transgenic plants expressed bar gene at the level similar to that in plants transformed with the bar gene under widely used constitutive promoter. Transformation of plants with the plasmid vector containing only promoterless bar gene near T-DNA right border (RB-bar-) and with the vector containing lox site and promoterless bar gene in the middle of the construction (-lox-bar-) led to obtaining no more than 4.5% of transgenic plants resistant to phosphinothricin. PCR analyses confirmed both the absence of tandem repeats and of plasmid recombination resulting in transference of bar gene under promoter in plasmid vector. Nos-terminator situated between the lox site and the right border of T-DNA did not decrease bar gene expression.     

Transformation and Regeneration of Two Cultivars of Pea (Pisum sativum L.)

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.     

Comparing constitutive promoters using CAT activity in transgenic tobacco plants

The effectiveness of different promoters for use in transgenic tobacco was compared using a reporter gene expressing chloramphenicol acetyl transferase (CAT). Plasmids with CAT gene controlled by cauliflower mosaic virus 35S (CaMV 35S), rice actin1 (Ract1) and tobacco polyubiquitin (Tubi.u4) promoters were delivered into tobacco plants by Agrobacterium-mediated transformation. The Ract1 promoter, previously shown to be a strong promoter in rice and other monocots, failed to promote strong expression in tobacco. CAT expression was greatest from the vector carrying Tubi.u4 with a 5'UTR and leader intron without a ubiquitin monomer. In transgenic plants harboring the Tubi.u4 promoter, CAT expression was approximately twice that of the CaMV 35S promoter. Our results suggest that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco.     

高效烟草遗传转化体系的建立及甜蛋白基因的导入

以烟草无菌茁叶片为外植体,通过根癌农杆菌LBA4404介导法,将Thamnatin基因导人烟草中,经梯度卡那霉素(Kana-mycin,Km)筛选,获得可在含75mg／L、100mg／L Km选择生根培养基上再生的抗性植株,其中部分Km抗性植株经PCR检测为阳性,转化率为31．3％,初步鉴定已成功地建立了烟草遗传转化系统,为进一步探讨甜蛋白在植物中的转化和表达情况奠定基础。     

Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains

An Agrobacterium-mediated transformation method of pea has been developed for several edible and fodder cultivars of pea (Pisum sativum L.), characterized previously in their potential for regeneration via organogenesis. The most appropriate explant, which was susceptible to Agrobacterium infection and capable of regenerating transgenic plants, turned out to be a slice of an immature embryo, including the embryo axis and the basal part of a cotyledon. Three hypervirulent strains of A. tumefaciens were tested: AgL0, AgL1 and EHA105. Each carried the binary vector pP35SGIB containing the uid gene, with an intron under control of the 35S promoter, and the bar gene conferring resistance to phosphinotricin. Strain AgL0 was found to be efficient for the majority of cultivars, followed by AgL1 and EHA105. Transformation efficiency varied from 0.7 to 4.1%, depending on cultivar and Agrobacterium strain. The transformation efficiency of particular pea cultivars did not clearly correspond to their regeneration capacity, which--although indispensable--was not a critical parameter of successful transformation. The presence of integrated genes in pea genomic DNA was detected by the PCR. T-DNA was stably transmitted to the progeny, as it was confirmed by Southern hybridization. The activity of introduced genes was analysed by the histochemical GUS assay and by painting leaves or by spraying transgenic plants with the herbicide Basta.     

利用双T-DNA载体系统培育无选择标记转基因烟草

建立了一种利用双T-DNA载体培育无选择标记转基因植物的方法.通过体外重组构建了双T-DNA双元载体pDLBRBbarm.载体中,选择标记nptⅡ基因和另一代表外源基因的bar基因分别位于2个独立的T-DNA.利用农杆菌介导转化烟草(Nicotiana tabacum L.),在获得的转化植株中,同时整合有nptⅡ基因和bar基因的频率为59.2%.对4个同时整合有nptⅡ和bar基因植株自交获得的T1代株系进行检测分析,发现在3个T1代株系2个T-DNA可以发生分离,其中约19.5%的转基因T1代植株中只存在bar基因而不带选择标记nptⅡ.这一结果说明双T-DNA载体系统能有效地用于培育无选择标记的转基因植物.研究还利用位于2个不同载体上的nptⅡ基因与 bar基因通过农杆菌介导共转化烟草,获得共转化植株的频率为20.0%～47.4%,低于使用双T-DNA转化的共转化频率.     

农杆菌介导籼稻优良恢复系bar基因的遗传转化研究

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。     

Stable and specific expression of 4-coumarate:coenzyme A ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco

The ability of 4-coumarate:coenzyme A ligase promoter from Populus tomentosa (Pto4CL1p) to drive expression of the GUS reporter gene and 4-coumarate:coenzyme A ligase gene in tobacco has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Intense GUS histochemical staining was detected in the xylem of stem in transgenic tobacco plants carrying the 1140 bp Pto4CL1p promoter. To further investigate the regulation function of the tissue-specific expression promoter, Pto4CL1p, a binary vector containing Pto4CL1p promoter fused with 4CL1 gene was transferred into tobacco. The activity of the 4CL1 enzyme doubled in the stems of transgenic tobacco but did not increase in the leaves. The content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

Transformation and regeneration of carrot (Daucus carota L.)

A protocol is presented for the efficient transformation of carrot (Daucus carota L. cv. Nantaise) by Agrobacterium tumefaciens. The binary vector contained the marker gene -glucuronidase (GUS), driven by the 35S promoter of cauliflower mosaic virus, and the nptII gene, which confers kanamycin resistance. Highest T-DNA transfer rates were obtained by co-cultivating bacteria with hypocotyl segments of dark-grown seedlings on solidified B5 medium containing naphthaleneacetic acid and 6-benzylaminopurine. After 2 days, bacterial growth was stopped with antibiotics. Two weeks later, the explants were placed on agar containing the kanamycin derivate geneticin; antibiotic-resistant calli developed during the following 4 weeks. Suspension cultures were obtained from resistant calli and plants regenerated via somatic embryogenesis in liquid culture. The majority of plants were phenotypically normal and, depending on the Agrobacterium strain used, harbored single or multiple copies of the T-DNA. About equal levels of GUS activity were found in different organs of young plants up to 6 weeks after embryogenesis. In leaves of older plants, GUS activity was markedly reduced, whereas the activities in phloem and xylem parenchyma cells of developing tap roots were still high and fairly uniform. Thus, the 35S promoter may be a useful tool to drive the expression of transgenes in developing carrot storage roots.     

An efficient protocol for shoot regeneration and genetic transformation of pigeonpea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.] using leaf explants

A protocol for efficient plant regeneration from leaf explants of pigeonpea [ Cajanus cajan (L.) Millsp.] was developed for the production of transgenic plants. Leaf explants from 4- to 5-day-old in vitro raised seedlings were most efficient in producing multiple adventitious shoots in 90% of the explants on shoot induction medium [Murashige and Skoog (MS) medium +5.0 microM benzyladenine +5.0 microM kinetin]. Shoot buds originated from the petiolar cut end of the explants and elongated rapidly on medium containing 0.58 microM gibberellic acid. Over 80% of the elongated shoots rooted well on MS medium containing 11.42 microM indole-3-acetic acid and were transplanted with 100% success. The procedure reported here is very simple, efficient and reproducible, and is applicable across diverse genotypes of pigeonpea. The usefulness of this system for further studies on the genetic transformation of pigeonpea has been demonstrated in biolistics-mediated gene transfer by using nptII and uidA as marker genes, where 50% of the selected plants showed gene integration and expression.     

Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)

Highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.) was obtained by first wounding stem explants via microprojectile bombardment. When this was followed by cocultivation with disarmed Agrobacterium in the dark, the transformation frequency-based on transient GUS expression-increased to over 10-fold that of explants wounded by other means and cocultivated under constant light. Two cycles of regeneration/selection on kanamycin were employed to generate stably transformed carnation plants and eliminate chimeras: first, plantlets were regenerated from inoculated stem explants and then leaves from these plantlets were used to generate transgenes in a second selection cycle of adventitious shoot regeneration. Agrobacterium strain AGLO, carrying the binary vector pCGN7001 containing uidA and nptII genes, was used in the stable transformation experiments. The combination of wounding via bombardment, cocultivation in the dark and two cycles of kanamycin selection yielded an overall transformation efficiency of 1–2 transgenes per 10 stem explants for the three carnation varieties analyzed. Histochemical and molecular analyses of marker genes in T₀ and T₁ generations confirmed the transgenic nature of the selected plants.