Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes

Experiments were conducted to produce transgenic barley plants following infection of immature embryos with Agrobacterium tumefaciens. Transformed callus was obtained using hygromycin resistance as a selectable marker and either green fluorescent protein (GFP) or -glucuronidase (GUS) as a reporter. Significantly reduced plant transformation frequencies were obtained with the GFP gene compared to GUS. However, GFP proved to be an excellent reporter of early transformation events and was used to compare four barley cultivars for efficiency in two phases of transformation: the generation of stably transformed barley callus and the regeneration of plantlets from transformed callus. Transformed callus was generated at a high frequency (47–76%) in all four cultivars. Regeneration of transformed plantlets was also achieved for all four cultivars although the frequency was much higher for Golden Promise than for the other three genotypes, reiterating that genotype is an important determinant in the regenerative ability of barley. This study has demonstrated for the first time that Agrobacterium-mediated transformation can be used to transform the Australian cultivars Sloop and Chebec.Communicated by W. Harwood     

Efficient genetic transformation of Sorghum using a visual screening marker.

To transform grain sorghum (Sorghum bicolor (L.) Moench) with a visual reporter gene (gfp) and a target gene (tlp), three genotypes (two inbreds, Tx 430 and C401, and a commercial hybrid, Pioneer 8505) were used. We obtained a total of 1011 fertile transgenic plants from 61 independent callus lines, which were produced from 2463 zygotic immature embryos via Agrobacterium-mediated transformation. The reporter gene, gfp, encoding green fluorescent protein (GFP), was used as a visual screening marker, and the target gene, tlp, encoding thaumatin-like protein (TLP), was chosen for enhancing resistance to fungal diseases and drought. Both genes were under the control of the maize ubi 1 promoter in the binary vector pPZP201. A total of 320 plants showing GFP expression, derived from 45 calli, were selected and analyzed by Southern blot analysis. There was a 100% correlation between the GFP expression and the presence of the target gene, tlp, in these plants. Transgenic plants showing strong TLP expression were confirmed by Western blotting with antiserum specific for TLP. The transgene segregated in various ratios among progeny, which was confirmed by examining seedlings showing GFP fluorescence. The progeny also showed different copy numbers of transgenics. This report describes the successful use of GFP screening for efficient production of stably transformed sorghum plants without using antibiotics or herbicides as selection agents.     

Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.     

转基因大麦中gfp基因的染色体位置及其表达

通过对大麦小孢子进行基因枪轰击获得4株转绿色荧光蛋白基因(gfp)的植株(A、C、D、E),以gfp基因为探针进行荧光原位杂交(FISH)研究转化植株中转基因插入位置和基因表达。4个株系在染色体7L(5HL)的不同位置都有一个插入点,而E株系在染色体5S(7HS)还有第2个插入点。所有的转基因T0代植株都是半合子并在T1、T2代发生分离。D株系GFP未表达,但FISH和PCR分析表明gfp基因已成功插入其染色体。各株系在根尖和花粉中的GFP表达水平不同：C株系在花粉表达强而在根尖表达中等;A株系在花粉中等表达而在根尖表达较淡;E株系则在根尖高表达,花粉中等表达。A和C株系在根尖和花粉的GFP分离都表现单位点特性,而E株系的根尖分离表现重叠作用(15：1)特征,但在花粉中表达GFP的频率低。PCR结果和3个分离株系的根尖表达结果一致。D和E株系的GFP表达不正常可能和加基因插入位置或基因的结构有关。     

Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.)

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and green fluorescent protein (gfp) as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL (targeting site) probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.     

Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation

We report a large-scale study on the frequency of transgene and T-DNA backbone integration following Agrobacterium-mediated transformation of immature barley embryos. One hundred and ninety-one plant lines were regenerated after hygromycin selection and visual selection for GFP expression at the callus stage. Southern blotting performed on a subset of 53 lines that were PCR positive for the GFP gene documented the integration of the GFP gene in 27 of the lines. Twenty-three of these lines expressed GFP in T₁ plantlets. Southern blotting with a vector backbone probe revealed that 13 of the 27 lines possessed one or more vector backbone fragments illustrating the regular occurrence of vector backbone integration following Agrobacterium infection of barley immature embryos.     

Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers

Early and reliable detection of plant transformation events is essential for establishing efficient transformation protocols. We have compared the effectiveness of using the gene encoding a green fluorescent protein (GFP) and a beta-glucuronidase (gus) as reporter genes for early detection of transgene expression in explants subjected to biolistic bombardment and Agrobacterium-mediated transformation. The results indicate that gfp gene is superior to gus gene in following transgene expression in transiently transformed materials in both methods of transformation. Using GFP as the screenable marker, we have optimized sorghum transformation with respect to the conditions for transformation, type of explants, promoters, and inbreds. These optimized conditions have been used to obtain stably transformed explants for subsequent regeneration.     

茉莉茎段遗传转化体系初步研究

以双瓣茉莉(Jasminum sambac)一年生茎段为外植体,探索茉莉花遗传转化体系。结果显示,预培养7 d后进行农杆菌侵染,然后置于含卡那霉素(Kan) 80～100 mg·L-1的筛选培养基上培养可获得成活率较高的抗性茎段。GFP转化预培养7 d的茉莉茎段再培养3 d及30 d后,以及转化预培养20 d茉莉茎段愈伤再培养14 d后愈伤组织中均能检测到GFP荧光。说明利用该转化系统进行茉莉遗传转化是可行的。     

Evaluation of different promoters driving the GFP reporter gene and selected target tissues for particle bombardment of <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17

Three different morphological callus types, identified as type A, B and C, and tips of in vitro inflorescences were used as target tissues for genetic transformation. Five different DNA plasmids carrying a synthetic green fluorescent protein (gfp) gene driven by different promoters, CaMV 35S, HBT, and Ubi1 were tested for the genetic transformation of Dendrobium Sonia 17. 35S-sgfp-TYG-nos (p35S) with the CaMV 35S promoter showed the highest GFP transient expression rate, while the HBT and Ubi1 promoters showed a relatively lower expression rate in all of the target tissues tested. The highest number of GFP-expressing cells was observed on day 2 post-bombardment, and the number declined gradually over the course of the next 2 weeks. Type A and B callus were found to be the best potential target tissues for genetic transformation.     

Promoter activity of the pER341-borne ST(Phsp) in heterologous gene expression in Escherichia coli and Streptococcus thermophilus(1).

A 138-bp EcoO109/HinfI fragment of Streptococcus thermophilus plasmid pER341 (2798 bp) including the promoter sequence of the heat stress protein gene hsp16.4 was tested in vector constructs for ability to activate the promoterless green fluorescent protein gene (gfp) from a jelly fish in Escherichia coli, S. thermophilus, and lactococci. ST(Phsp) promoted gfp expression in transformed hosts as evidenced by the presence of green fluorescent (GFP(+)) colonies under UV illumination. The results confirmed the potential of ST(Phsp) as a functional promoter in heterologous gene expression in dairy fermentation bacteria.     

A set of useful monocotyledon transformation vectors

The construction of four plasmids optimized for transgenic studies in barley and other monocot plants is presented. All vectors contain the promoter and first intron of the rice actin 1 (Act-1) promoter to drive expression of the coding sequence of choice. Two of the vectors utilize the gene for green fluorescent protein (GFP) from Aequorea victoria as a screenable marker.     

潮霉素在大麦遗传转化中的应用研究

以‘B1202’、‘U202’和‘云引1号’3个大麦品种(系)为试材,研究了潮霉素对大麦愈伤组织生长、分化及成熟种子的影响。结果表明:潮霉素对抗性愈伤的适宜筛选浓度为60 mg/L,分化阶段的适宜筛选浓度为40 mg/L,对转基因后代种子筛选以80 mg/L较为适宜,而不同材料对潮霉素的敏感性存在一定程度的差异。通过PCR检测采用农杆菌介导法转化获得的T0和T1代潮霉素抗性苗,结果证实在大麦遗传转化中采用潮霉素进行筛选是可行的。     

Easy detection of green fluorescent protein multicopy transformants in Penicillium griseoroseum

Penicillium griseoroseum, a deuteromycete fungus producer of pectinolytic enzymes, was transformed with a gene encoding for green fluorescent protein (GFP). The selection of transformants was based on the homologous nitrate reductase gene (niaD). Protoplasts of a P. griseoroseum Nia mutant (PG63) were co-transformed with the plasmids pNPG1 and pAN52-1-GFP. The plasmid pNPG-1 carries the homologous niaD gene and pAN52-1-GFP carries the SGFP-TYG version of GFP. The highest transformation efficiency (102 transformants/mug of pNPG1) resulted from the utilization of equimolar amounts of transforming and co-transforming vectors. Analysis of pAN52-1-GFP insertions into the genomic DNA of the transformants revealed single and multiple copy integrations. The transformants possessing a single copy of the gfp gene showed a low level of fluorescence, whereas multicopy transformants displayed strong fluorescence under visualization with fluorescent light. The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.     

小鼠脂联素与绿色荧光蛋白融合基因表达载体的构建及在COS-7细胞中的表达

以绿色荧光蛋白(GFP)基因(gfp)为报告基因,构建小鼠脂联素(mADPN)基因(mAd)与gfp的融合基因mAd/gfp表达载体pCI-neo-apoEHCR-hAATp-mAd-gfp,脂质体法转染体外培养的COS-7细胞,荧光显微镜观察GFP在细胞中的表达可间接反映mADPN的表达,并通过RT-PCR在核酸水平进一步确证mAd的表达.荧光显微镜观察及RT-PCR结果均证明mADPN在COS-7细胞中获得了高效表达,表明mADPN重组表达载体pCI-neo-apoEHCR-hAATp-mAd可以在真核细胞COS-7中高效表达mADPN,为进一步探讨mAd在小鼠体内的表达提供了可行性依据.     

Agrobacterium tumefaciens-mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression

To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l^–1 -naphthaleneacetic acid (NAA) and 1.0 mg l^–1 thidiazuron (TDZ). Putative transformed shoots were selected on SR medium containing 75 g ml^–1 Km, 200 g ml^–1 Timentin, and transformation was monitored by observation of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants have set seed. GFP expression segregated in the T₁ seedlings of the two lines tested in a 3 – 1 ratio. In addition to the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern and Western blot analyses.