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.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

Transgenic peppers that are highly tolerant to a new CMV pathotype

The CMV (cucumber mosaic virus) is the most frequently occurring virus in chili pepper farms. A variety of peppers that are resistant to CMVP0 were developed in the middle of 1990s through a breeding program, and commercial cultivars have since been able to control the spread of CMVP0. However, a new pathotype (CMVP1) that breaks the resistance of CMVP0-resistant peppers has recently appeared and caused a heavy loss in productivity. Since no genetic source of this new pathotype was available, a traditional breeding method cannot be used to generate a CMVP1-resistant pepper variety. Therefore, we set up a transformation system of pepper using Agrobacterium that had been transfected with the coat protein gene, CMVP0-CP, with the aim of developing a new CMVP1-resistant pepper line. A large number of transgenic peppers (T₁, T₂ and T₃) were screened for CMVP1 tolerance using CMVP1 inoculation. Transgenic peppers tolerant to CMVP1 were selected in a plastic house as well as in the field. Three independent T₃ pepper lines highly tolerant to the CMVP1 pathogen were found to also be tolerant to the CMVP0 pathogen. These selected T₃ pepper lines were phenotypically identical or close to the non-transformed lines. However, after CMVP1 infection, the height and fruit size of the non-transformed lines became shorter and smaller, respectively, while the T₃ pepper lines maintained a normal phenotype.     

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) and Regeneration of Salt Tolerant Plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (km^r) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T₁, T₂, and T₃ progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T₁ AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.     

Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants

 The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus, most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic and escape shoots. The selection of transgenic shoots could be easily favored by eliminating the escapes and/or by performing shoot-tip grafting of the transgenic buds soon after their origin. GFP-expressing shoots have also been observed in citrus explants co-cultivated with Agrobacterium but cultured in a medium without the selective agent kanamycin. This opens the possibility to rescue the transgenic sectors and to regenerate transgenic plants without using selectable marker genes conferring antibiotic or herbicide resistance, which is currently a topic of much discussion for the commercialization of transgenic plants. Received: 28 October 1998 / Accepted: 28 November 1998     

Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression

The effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and analysis system developed for continual monitoring of GFP expression. Suppressors were introduced into lima bean cotyledonary tissues either as 3′-GFP translational fusions or as co-introductions with the GFP gene on a separate plasmid. The resultant transient expression profiles for each suppressor depended on whether the suppressor was introduced as a fusion or co-introduced on separate plasmids. As co-introductions, the silencing suppressors HCPro (from Tobacco etch virus), p19 (from Tomato bushy stunt virus), γb (from Barley stripe mosaic virus) and p21 (from Beet yellows virus) led to an almost twofold increase in initial GFP expression levels, followed by a rapid decline. In contrast, fusions of HCPro, p19, and γb to the 3′-end of GFP resulted in slightly lower but more prolonged GFP expression. Compared with the co-introductions, all GFP::Suppressor translational fusions gave reduced GFP fluorescence levels, suggesting interference of the fusion partner with GFP fluorescence. Regardless of the configuration, introductions of the silencing suppressors AL2 (from Tomato golden mosaic virus) and 126-kDa protein (from Tobacco mosaic virus) resulted in very low GFP fluorescence. This is the first report that directly compares the effects of a large number of viral suppressors of silencing on transient transgene expression using both translational fusions and co-introductions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Monitoring the expression of green fluorescent protein in carrot

Green fluorescent protein (GFP) was successfully used as a visual reporter at various stages of carrot (Daucus carota L.) transformation. GFP-fluorescence was non-invasively observed in protoplasts, callus and plants after the delivery of mgfp5-er gene using two transformation methods: direct DNA transfer into polyethylene glycol (PEG) -treated protoplasts and inoculation of root discs with Agrobacterium rhizogenes. Transient GFP-expression was detected in the treated protoplasts and monitored during the first week of the cell culture until the stable level of expression was observed. It was useful for the comparison of protoplast susceptibility to DNA uptake and the transgene expression as the fluorescence declined with various rates depending on the used carrot genotype and PEG-concentration. GFP-monitoring in callus enabled the selection of stably expressing lines. It also allowed verification of the homogeneous tissue composition with regard to the expression of the transgene. In plants, GFP-performance depended on the assayed tissue and organ despite of the constitutive 35S promoter. The expression was visually detected in both vegetative and generative parts, but particularly strong fluorescence was observed in leaf marginal meristems, petioles, stems, and styles. Those tissues can be convenient for examination of the transgenic plants during their growth. The results encourage that GFP is a valuable reporter and can be routinely used for optimization of transformation protocol, selection of transformants and monitoring transgenic carrot.     

Selection of transgenic <Emphasis Type="Italic">Petunia</Emphasis> plants using the green fluorescent protein (GFP)

Selectable marker genes are needed for efficient transformation of plants. The present study focused on testing the applicability of green fluorescent protein (GFP) for selecting transgenic Petunia hybrida plants without applying antibiotics or herbicides. Based on a transient gene expression assay, the efficiency of two gfp genes, mGFP-4 and smRS-GFP, was compared. Two days after infiltration of Agrobacterium tumefaciens, GFP expression was recorded in leaf epidermal cells. The intensity of smRS-GFP fluorescence was higher than that of mGFP-4 and easier to distinguish from other unspecific fluorescent signals in Petunia. Transformations using the pMen65smRS-GFP vector, which contained the neomycin phosphotransferase II (nptII) gene, resulted in callus and shoots that visually and clearly expressed detectable GFP levels; in addition, this vector made it possible to exclusively select transformed plants using GFP. The transformation efficiencies achieved by using GFP selection versus combined kanamycin and GFP selection (nptII+GFP) were compared in four Petunia genotypes with a transformation experiment with four replications. In three out of four Petunia cultivars a higher transformation frequency was achieved by using nptII+GFP selection. Southern blot hybridisation revealed single and multiple integrations of smRS-GFP in Petunia. Single copy plants showed intensive expression in all parts of the plants, whereas a higher copy number led to only weak or partial expression of smRS-GFP allowing the visual selection of single copy events. Thus, it is possible to select transgenic Petunia plants based on their GFP expressions without applying antibiotics or herbicides.     

Delayed and Restricted Expression of UAS-Regulated GFP Gene in Early Transgenic Zebrafish Embryos by Using the GAL4/UAS System

A stable Tg(UAS:GFP) zebrafish line was generated and crossed with Tg(hsp70:GAL4) line, in which the GAL4 gene is under the control of an inducible zebrafish promoter derived from the heat shock 70 protein gene (hsp70). The dynamic green fluorescent protein (GFP) expression in early zebrafish embryos in the GAL4/UAS binary system was then investigated. We found that, at early developmental stages, expression of GFP effector gene was restricted and required a long recovery time to reach a detectable level. At later developmental stage (after 2 days postfertilization), GFP could be activated in multiple tissues in a shorter time, apparently due to a higher level of GAL4 messenger RNA induction. It appears that the type of tissues expressing GFP was dependent on whether they had been developed at the time of heat shock. Therefore, the delayed and restricted transgene expression should be taken into consideration when GAL4/UAS system is used to study transgene expression in early developmental stages.     

Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants

The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.Abbreviations ELISA Enzyme-linked immunosorbent assay - HRP Horseradish peroxidase - GFP Green fluorescent protein Communicated by M.C. Jordan     

Development of a strategy for transgenic studies and monitoring of transgene expression in two closely related Moricandia species possessing a C3 or C3–C4 intermediate photosynthetic phenotype

In order to establish a model system for comparative studies of C₃ and C₃–C₄ intermediate photosynthesis, the development of efficient transformation systems and the monitoring of transgene behaviour and stability were carried out in two closely related Moricandia species (Brassicaceae): the C₃–C₄ photosynthetic intermediate species M. arvensis and the C₃ species M. moricandioides. In this study the green fluorescent protein (gfp) reporter gene was used as a vital marker gene while the use of the β‐glucuronidase (gusA) gene was based on the highly sensitive detection of its activity. For Agrobacterium‐mediated transformation of leaf explants, a cauliflower mosaic virus 35S promoter‐driven, modified version of gfp, the mgfp5‐ER gene and the gusA gene, respectively, were introduced into the new dual binary transformation vector system pGreen/pSoup (Hellens et al. 2000, Plant Mol Bio 42: 819–832). GFP5 produced bright‐green fluorescence in transformed tissues that was distinctly detected 5–12 days following transformation in developing calli of the two species. Visual screening, combined with antibiotic selection, enabled early and easy identification of transformation events and contributed to improvements in the transformation strategies. Transgene integration studies demonstrated that mgfp5‐ER was inserted with low copy number in the M. arvensis plant lines and the transgene was transmitted in a Mendelian fashion to T₁ and T₂ progenies. GFP5 expression levels in a population of 100 independent primary transformed M. arvensis plant lines (T₀) showed great variation between transformation events (coefficient of variation of 108%). The mgfp5‐ER or gusA reporter genes were expressed in 90–95% of the kanamycin‐resistant M. arvensis plant lines and in up to 98% of the independent M. moricandioides plant lines.     

RNA silencing in the life cycle of soybean: multiple restriction systems and spatiotemporal variation associated with plant architecture

The expression of transgenes introduced into a plant genome is sometimes suppressed by RNA silencing. Although local and systemic spread of RNA silencing have been studied, little is known about the mechanisms underlying spatial and temporal variation in transgene silencing between individual plants or between plants of different generations, which occurs seemingly stochastically. Here, we analyzed the occurrence, spread, and transmission of RNA silencing of the green fluorescent protein (GFP) gene over multiple generations of the progeny of a single soybean transformant. Observation of GFP fluorescence in entire plants of the T₃–T₅ generations indicated that the initiation and subsequent spread of GFP silencing varied between individuals, although this GFP silencing most frequently began in the primary leaves. In addition, GFP silencing could spread into the outer layer of seed coat tissues but was hardly detectable in the embryos. These results are consistent with the notion that transgene silencing involves its reset during reproductive phase, initiation after germination, and systemic spread in each generation. GFP silencing was absent in the pulvinus, suggesting that its cortical cells inhibit cell-to-cell spread or induction of RNA silencing. The extent of GFP silencing could differ between the stem and a petiole or between petiolules, which have limited vascular bundles connecting them and thus deter long-distant movement of silencing. Taken together, these observations indicate that the initiation and/or spread of RNA silencing depend on specific features of the architecture of the plant in addition to the mechanisms that can be conserved in higher plants.     

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize

A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo₃ in the infection medium and adding AgNo₃ in co-cultivation, resting and selection medium; Agrobacterium concentration at 0.5×10⁹ c.f.u. ml^–1 for bacterium inoculation; 100 mg l^–1 carbenicillin used in the medium to eliminate Agrobacterium after inoculation; and 3 days for co-cultivation and 4 days for resting. A combination of all of these conditions resulted in establishing a high throughput transformation system. Over 500 T₀ plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T₁ plants were analyzed and transmission of transgenes to the T₁ generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.     

High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD₆₀₀ 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T₀ transgenic plants were generated, representing 3.6% transformation frequency. T₀ transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T₁ progeny. PCR, RT-PCR, and Southern hybridization analysis of T₀ and T₁ transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T₀ and T₁ transgenic chickpea plants was achieved maximum up to 116 ng mg⁻¹ of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.     

Investigations of copy number of transgene,fertility and expression level of an introduced GUS gene in transgenic NERICA produced by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated methods

In the use of genetic transformation in breeding, there are several possible problems including multiple copy insertion of transgene, sterility caused by somaclonal variation and gene silencing. In this study, we characterized transgenic New Rice for Africa (NERICA) produced by Agrobacterium-mediated methods with respect to copy number of transgene, fertility, and expression level of an introduced GUS gene. Southern blot analysis of primary transformants demonstrated that about half of the events carried a single copy of the transgene regardless of the cell density of Agrobacerium for inoculation. We examined ten procedures, consisting of different time periods and times of subculture for callus formation and the starting times of hygromycin-based selection of transformed cells, for transformation of NERICA cultivars to produce transformants within a short culture period at high frequency. A new culture method developed in this study required only about 1.5 mo from the beginning of tissue culture to transformants, whereas a standard protocol we developed previously needed about 2 mo of culture; however, it did not significantly reduce percentages of sterile plants. Fertile T₀ plants produced fertile T₁ plants at higher frequency. However, fertility was not inherited in a simple fashion: both fertile and partially sterile T₀ plants produced fertile, partially sterile and sterile T₁ plants. Expression assay of an introduced GUS gene revealed position effects in seven independent homozygous transformed lines carrying one copy of the transgene. Points to pay attention to in the use of genetic transformation in breeding are discussed.     

Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny

To investigate the effect of matrix attachment regions (MARs) on transgene expression levels and stability in cereal crops, we generated 83 independent transgenic rice callus lines containing a gusA expression cassette either as a simple expression unit, or flanked with MARs from tobacco (Rb7) or yeast (ARS1). Transgenic rice plants were regenerated from these callus lines and analysed at the structural and expression levels over two generations. In the first generation (T₀), both Rb7 and ARS1 MARs significantly increased transgene expression levels. In the populations of plants containing MARs, we observed a significant reduction in the number of non-expressing lines compared to the population of plants without MARs. However, variation in β-glucuronidase (GUS) expression levels between independent lines was similar both in the presence and absence of flanking MARs. In the presence of MARs, GUS activity increased in proportion to transgene copy number up to 20 copies, but was generally reduced in lines carrying a higher copy number. In the population of plants without MARs, there was no correlation between expression level and transgene copy number. In the second generation (T₁), transgene expression levels were significantly correlated with those of the T₀ parents. The Rb7 MARs significantly improved the stability of transgene expression levels over two generations, and therefore appear to offer protection against transgene silencing. Our study shows that the exploitation of MARs may be an important strategy for stabilising transgene expression levels in genetically engineered cereals.     

The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants

Segregating T₁, T₂ and T₃ transgenic rice populations, derived from independent particle-bombardment-mediated transformation events were examined in order to assess the effect of gene dosage on transgene expression levels and stability. The expression level of the unselected β-glucuronidase (gusA) reporter gene was quantified in plants from these populations. The gusA gene dosage was determined by segregation analysis of progeny seedlings at the structural level (by PCR) and at the expression level. For some transformation events a gene dosage effect on transgene expression was observed, leading to higher transgene expression levels in homozygous progeny than in hemizygous progeny or primary transgenic plants. However, in many other transformation events, the homozygous state appears to be disadvantageous, being associated with lower transgene expression levels, gene silencing or counter-selection of homozygous plants across generations. Change of gene dosage is probably one of the key factors influencing transgene expression levels and stability in transgenic rice. This is particularly important when considering molecular genetic studies and crop improvement programmes. The possible influence of matrix attachment regions (MARs) in increasing the likelihood of an additive effect on transgene expression level is discussed. Received: 21 March 2001 / Accepted: 29 June 2001     

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.