Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker

Beauveria bassiana has been investigated for use in the biological control of several insects in agricultural practice. To understand the molecular basis of virulence and host specificity and to improve the entomopathogenicity of B. bassiana, we have developed a simple, highly efficient and reliable Agrobacterium-mediated transformation method for B. bassiana using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. Most transformants contained single copies of T-DNA and the T-DNA inserts were stably inherited after five generations. With this highly efficient transformation method for B. bassiana, we also obtained two putative T-DNA-tagged mutants that may have altered growth habits or virulence. Thus, the described protocol could provide a useful tool to manipulate the genetic make-up and to tag genes that may be important for virulence or growth and development of B. bassiana.     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Green fluorescent protein as a visual marker for wheat transformation

 Wheat (Triticum aestivum L.) transformation via particle bombardment is now established in many laboratories, but transformation efficiencies are still largely low and the highest efficiencies can only be obtained with certain genotypes. For rapid optimization and improvement of wheat transformation protocols, a non-destructive marker which permits early detection of transformed cells is needed. We have assessed the ability of a modified version of the Aequorea victoria green fluorescent protein (GFP) to act as a marker for detecting transformed cells and tissues of wheat. Multicellular clusters emitting green fluorescence were observed 14 days after particle bombardment with a sGFPS65T gene construct, and gfp-expressing shoots (often with expressing roots) could be observed as early as 21 days after bombardment. These shoots can be removed from the callus and grown further until they are ready to transfer to soil. Transgenic wheat plants could be selected on the basis of gfp expression alone although the inclusion of antibiotic resistance as a selectable marker could improve the efficiency. Using sgfpS65T as a marker gene in an experiment comparing bombardment parameters allowed the rapid identification of variables that could be targeted for optimization. Received: 29 March 2000 / Accepted: 29 March 2000     

The green fluorescent protein (GFP) as a vital screenable marker in rice transformation

 An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluorescence easily detectable and screenable in rice tissue 12–22 days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection, drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP expression was observed in primary transformed rice plants (T₀) and their progeny (T₁). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice genetic engineering programmes are discussed. Received: 6 October 1997 / Accepted: 9 October 1997     

Green fluorescent protein as a vital marker for non-destructive detection of transformation events in transgenic plants

Transformation of plants is a popular tool for modifying various desirable traits. Marker genes, like those encoding for bacterial β-glucuronidase (GUS), firefly luciferase (LUC) or jellyfish green fluorescent protein (GFP) have been shown to be very useful for establishing of efficient transformation protocols. Due to favourable properties such as no need of exogenous substrates and easy visualization, GFP has been found to be superior in to other markers in many cases. However, the use of GFP fluorescence is associated with some obstacles, mostly related to the diminishing of green fluorescence in older tissues, variation in fluorescence levels among different tissues and organs, and occasional interference with other fluorescing compounds in plants. This paper briefly summarizes basic GFP properties and applications, and describes in more detail the contribution of GFP to the establishment, evaluation and improvement of transformation procedures for plants. Moreover, features and possible obstacles associated with monitoring GFP fluorescence are discussed.     

Transformation of Metarhizium anisopliae with benomyl resistance and green fluorescent protein genes provides a tag for genetically engineered strains

A plasmid, pBGFP, carrying green fluorescent protein (gfp) and benomyl-resistance genes was constructed and transformed into Metarhizium anisopliae. The transformants grew normally and GFP fluorescence was detected. No change was found in virulence for the transformants. Fluorescence was detected in hyphae from the haemolymph of the infected locust, and the benomyl-resistance was maintained. Results suggested that the two markers provided a useful tool for screening and monitoring the engineered strains even after infection.     

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.     

Use of the GUS gene as a selectable marker for Agrobacterium-mediated transformation of Rubus

A transformation system was established for red raspberry, blackberry and blackberry x raspberry hybrids, utilizing the binary vector system of Agrobacterium tumefaciens. Leaf discs or internodal stem segments were inoculated with Agrobacterium strain LBA4404 containing the binary vectors PBI121.X, which has the -glucuronidase (GUS) marker gene, or Bin 19, which has the neomycin phosphotransferase II (NPT II) gene. Regenerants were produced on media containing MS salts, 20 gl^-1 sucrose, 7 gl^-1 agar, 100 mgl^-1 inositol, 0.5 mgl^-1 nicotinic acid, 0.5 mgl^-1 pyridoxine-HCl, 0.1 mgl^-1 thiamine, and either 0.1 mgl^-1 IBA and 2 mgl^-1 BAP for leaf discs, or 0.2 mgl^-1 BAP and 0.2 mgl^-1 2,4-D for stem segments. Kanamycin sulphate, which was used as a selective agent for the NPT II gene, inhibited organogenesis at 50 mgl^-1 and was therefore unsuitable for use as a selectable marker gene in Rubus. All regenerants were assayed utilizing the fluorogenic assay procedure to determine if the GUS gene had been transferred into the material and could therefore cleave the substrate 4-methyl-umbelliferyl--D-glucuronide. Seven GUS-positive plantlets were obtained which confirmed that this marker gene had been transferred into Rubus. A dot blot assay was carried out on GUS-positive plant material to establish if the NPT II gene had also been transferred to the plant material.     

Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on GFP fluorescence with a very high accuracy. The method requires no selection agents like antibiotics or herbicides and enables a reduction of labour and time necessary for tissue culture. Moreover, individual transformants can be easily excised from the host tissue and cultured separately. All of the 12 used carrot cultivars produced transformed adventitious roots and the frequency of discs producing GFP expressing adventitious roots varied from 13 to 85%. The highest transformation rate was found for A4T and LBA1334 strains possessing chromosomal background of A. tumefaciens C58. The results encourage that visual selection of transformed, fluorescing adventitious roots can be highly effective and applied routinely for the production of carrot transgenic plants.     

Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungusUstilago maydis

Pathogenic development ofUstilago maydis, the causative agent of corn smut disease, is a multistep process. Compatible yeast-like cells fuse and this generates the infectious dikaryon which grows filamentously. Having entered the plant the dikaryon induces tumors in its host in which massive proliferation of fungal material, karyogamy and spore formation occur. In order to follow fungal development from the initial steps to the final stage we have expressed the green fluorescent protein (GFP) fromAequorea victoria as a vital marker inU. maydis and demonstrate that GFP-tagged strains can be used to study host-pathogen interactions in vivo.     

Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.     

Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation

Summary Development of new selectable markers is needed to increase the efficiency and flexibility of plant transformation, and to overcome drawbacks sometimes associated with use of existing markers. A useful alternative to chemical-based selection systems would be a system using visual screening to obtain transgenic lines. Investigations were carried out to determine if the green fluorescent protein (gfp) gene could be utilized alone as a visual screenable marker to produce stably transformed, fertile oat plants. Twelve experiments were conducted in which gfp-based selection was utilized to obtain routinely stable transgenic lines in oat. A synthetic gfp gene under the control of the maize ubiquitin promoter was delivered into embryogenic oat callus via microprojectile bombardment. Cell clusters (1–3 mm) expressing gfp were visually identified using epifluorescence microscopy and physically isolated approximately 3 wk post-bombardment. Fertile, gfp-expressing T0 plants were regenerated from 78% of the glowing cell sectors placed on regeneration medium. T0 plants from 55% of the events produced gfp-expressing progeny in a 3∶1 Mendelian ratio. Southern blot and PCR analysis confirmed transgene integration and transmission to progeny. Expression of gfp did not reduce plant growth or fertility. Transgene copy number and integration patterns were similar to those in transgenic plants derived from chemical-based selection systems. The mean transformation frequency, based on fertile events obtained per bombarded plate, was 1.8%. Over 180 independent transgenic oat lines were produced, to date, using only visual screening for expression of gfp, demonstrating efficiency and repeatability of the selection system. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the University of Wisconsin or the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

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.     

Arabitol dehydrogenase as a selectable marker for rice

Arabitol dehydrogenase has been adapted for use as a plant selectable marker. Arabitol is a five-carbon sugar alcohol that can be used by E. coli strain C, but not by the laboratory K12 strains. The enzyme converts the non-plant-metabolizable sugar arabitol into xylulose, which is metabolized by plant cells. Rice was transformed with a plant-expression-optimized synthetic gene using Biolistic-mediated transformation. Selection on 2.75% arabitol and 0.25% sucrose yielded a transformation efficiency (9.3%) equal to that obtained with hygromycin (9.2%). Molecular analyses showed that the atlD gene was integrated into the rice genome of selected plants and was inherited in a Mendelian manner. This study indicates that arabitol could serve as an effective means of plant selection.     

绿色荧光蛋白和潮霉素抗性双标记载体转化草酸青霉菌P8的研究

草酸青霉菌P8(Penicillium oxalicum)溶磷菌株具有较强的溶解多种无机难溶磷的能力和促进作物生长的作用。为研究P8在作物根际的定殖状况,用携带加强型绿色荧光蛋白和潮霉素抗性的双标记载体转化溶磷青霉菌P8的原生质体,筛选出稳定、高效表达GFP并对潮霉素产生抗性的转化菌株,Southern杂交分析结果显示,外源质粒DNA是以非同源重组方式整合到转化菌株基因组染色体上。试验证明转化菌株的形态学特征和溶磷能力与野生型菌株无明显差别。     

Identification of the molecular origin and development of a panzootic caused by Beauveria bassiana in praying mantis populations in eastern China

A panzootic in praying mantid species Tenodera sinensis and Statilia maculate, caused by Beauveria bassiana, occurred in north, southwest and southeast regions of Anhui Province, eastern China in Autumn, 2009. A 3-d principal component analysis (PCA) of 123 isolates from three sites revealed that the B. bassiana populations were heterogeneous with obvious dominance. Furthermore, the causal source of the panzootic in Anhui was shown to be polyphyletic. The populations were homogenized into homogenous subunits for investigation of genetic structure by inter-simple sequence repeat (ISSR) markers. Variance was greater than 70%, largely due to genetic differences within populations and subpopulations. Genetic distances and genetic differentiation were negatively associated with geographic distances and it was speculated that this was due to the effects of monsoons and topography. Mantid isolates were divided into five pathotypes based on a two-way cluster analysis of genetic distance. Pathotype I consisted of the predominant subpopulations of Huangcangyu and Chashui populations, with a genetic distance of 0.120 and gene flow up to 1.833. This pathotype caused a widespread epizootic in north and southwest Anhui, and Pathotype III caused enzootic at Site A in September and then epizootic in October, while the other three pathotypes caused enzootics at all three investigation sites. The widespread epizootics and isolated enzootics composed the polyphyletic panzootic in Anhui. A strong gene flow between isolates from the two mantid species was identified, resulting in negligible gene differentiation. This indicated a lack of host specificity in mantid isolates of B. bassiana.