The elimination of a selectable marker gene in the doubled haploid progeny of co-transformed barley plants

Following the production of transgenic plants, the selectable marker gene(s) used in the process are redundant, and their retention may be undesirable. They can be removed by exploiting segregation among the progeny of co-transformants carrying both the selectable marker gene and the effector transgene. Here we show that the doubled haploid technology widely used in conventional barley breeding programmes represents a useful means of fixing a transgene, while simultaneously removing the unwanted selectable marker gene. Primary barley co-transformants involving hpt::gfp (the selectable marker) and gus (a model transgene of interest) were produced via Agrobacterium-mediated gene transfer to immature embryos using two respective T-DNAs. These plants were then subjected to embryogenic pollen culture to separate independently integrated transgenes in doubled haploid progeny. A comparison between 14 combinations, involving two Agrobacterium strains carrying various plasmids, revealed that the highest rate of independent co-transformation was achieved when a single Agrobacterium clone carried two binary vectors. Using this principle along with Agrobacterium strain LBA4404, selectable marker-free, gus homozygous lines were eventually obtained from 1.5 per 100 immature embryos inoculated. Compared to the segregation of uncoupled T-DNAs in conventionally produced progeny, the incorporation of haploid technology improves the time and resource efficiency of producing true-breeding, selectable marker-free transgenic barley.     

Genetic transformation of the commercial barley (Hordeum vulgare L.) cultivar Conlon by particle bombardment of callus

Commercial barley cultivars are difficult to transform because of the lack of an efficient regeneration system. By modifying certain components in the standard culture medium, we have developed a reproducible and more efficient regeneration system. Herbicide-resistant transgenic plants from barley (Hordeum vulgare L. cv. Conlon) were obtained using this medium. Embryo-derived callus was bombarded with pAHC25, which contains the screenable marker gus (#-glucuronidase) and the selectable marker bar (bialaphos resistance gene), both driven by the maize ubiquitin promoter (Ubi1) and followed by the nos terminator. Following bombardment, callus was transferred to callus-induction medium supplemented with 5 mg/l bialaphos for selection. Resistant calli were subsequently transferred to maintenance medium containing 5 mg/l bialaphos for further selection and finally transferred to regeneration medium with 5 mg/l bialaphos. Green shoots that developed on the regeneration medium were transferred to rooting medium containing 3 mg/l bialaphos. Eighty-five transgenic plants were obtained from 13 independent transformation events. Progeny tests showed Mendelian inheritance for the transgenes. This is the first report of the production of large numbers of transgenic plants from a commercial cultivar adapted to Midwestern US barley production.     

Factors affecting the regeneration capacity of isolated barley microspores (Hordeum vulgare L.)

The culture of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite malting barley cultivar) was studied. A careful choice of culture steps resulted in an average regeneration frequency of 300 green plants per starting material spike. Strong seasonal variation in regeneration capacity was observed. The choice of a cold pretreatment method affected the viability of microspores. A cold pretreatment of the collected starting material at +4°C for 4 weeks was needed for the efficient regeneration of green plants from isolated microspore cultures. Glutamine omission from and copper additions to microspore culture were studied. The omission of glutamine did not affect the number of regenerated green plants but did result in an increase in the number of regenerated albino plants. The addition of copper did not improve the regeneration capacity of isolated barley microspores. Transformation by particle bombardment of isolated microspores did not result in the production of transgenic plants.     

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F₁, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.     

Development of analytical tools for evaluating the effect of T-DNA chimeric integration on transgene expression in vegetatively propagated plants

T-DNA chimeric integration and unexpected transgene expression are relevant constraints affecting transgenic plants. This study aims to properly investigate the occurrence of these events and to what extent they may be related. The final goal is to develop an effective screening tool for earlier selection of proper transgenic lines. A strategy based on qPCR and Southern blot was adopted for evaluating gus and Egfp chimerism degree in transgenic Vitis vinifera cv ‘Chardonnay’. Of nine transgenic lines, one had a very high chimerism value, which was shown to be associated with minimal transgene expression. The evaluation of the gus gene over time and space on a line selected as a model showed that transgene’s chimerism was stable and uniform throughout plant tissues whilst its expression was highly variable. Transgene chimerism issue was investigated in detail and useful hints were given for selecting the most favorable transgenic plants and for proper planning of in vitro and ex vitro experiments.     

Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.)

Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion.     

GFP accumulation controlled by an auxin-responsive promoter as a non-destructive assay to monitor early auxin response

We have constructed transgenic Arabidopsis lines that contain a gene for green fluorescent protein (GFP) under the control of auxin-responsive domains A and B of the promoter from the pea PS-IAA4/5 gene. The chimeric transgene was named BA-mgfp5-ER. GFP was detected after the application of indole-3-acetic acid at concentrations as low as 100 nM in epidermal cells in the root elongation zone. The induction of the reporter gene was highly specific to auxin and was correlated with the auxin-induced change in epidermal cell shape. No GFP accumulation was observed in the lateral root meristem that was formed as a result of exogenous auxin application. These results suggest that auxin signals were transmitted through several distinct pathways depending on the cell type. The intensity of the GFP signal was strong enough to be observed through the plastic lid of the culture dish using a dissecting microscope, thereby enabling GFP expression to be monitored in an aseptic environment. Thus, the BA-mgfp5-ER transgenic plant can be a powerful tool for screening mutants that are defective in auxin signaling and the expression of early auxin-response genes.     

Overexpressions of dihydroxyacetone synthase and dihydroxyacetone kinase in chloroplasts install a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco using modified Gateway entry vectors

Dihydroxyacetone synthase (DAS) and dihydroxyacetone kinase (DAK) are two key enzymes for formaldehyde assimilation in methylotrophic yeasts. In order to using a Gateway LR recombination reaction to construct a plant expression vector that contains the expression cassettes for the das and dak genes and allow the proteins encoded by the two target genes to be localized to the chloroplasts of transgenic plants, the entry vector pEN-L4*-PrbcS-*T-gfp-L3* contained the tomato rbcS 3C promoter (PrbcS) with its transit peptide sequence (*T) and a GFP reporter gene (gfp) was constructed in this study. To verify the applicability of pEN-L4*-PrbcS-*T-gfp-L3*, we generated an entry vector for the dak gene by replacing the gfp gene in this entry vector with the dak gene. We also generated an entry vector for the das gene by replacing the gus gene in another entry vector (pENTR*-PrbcS-*T-gus) with the das gene. Using these entry vectors and pK7m34GW2-8m21GW3, we successfully constructed the pKm-35S-PrbcS-*T-gfp-PROLD-PrbcS-*T-gus and the pKm-35S-PrbcS-*T-dak-PROLD-PrbcS-*T-das expression vectors. Our results showed that high expression of GUS was achieved in leaves, and the expressed GFP, DAS and DAK proteins could be targeted to the chloroplasts after the two expression vectors were used to transform tobacco. The overexpressions of DAS and DAK in the chloroplasts successfully created a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco. By utilizing these expression vectors, we not only successfully expressed two target genes with one transformation but also localized the expressed proteins to chloroplasts via the transit peptide sequence (*T). Therefore, the construction of pEN-L4*-PrbcS-*T-gfp-L3* establishes a technique platform that provides a convenient means for chloroplast genetic engineering.     

Characterization of directly transformed weedy <Emphasis Type="Italic">Brassica rapa</Emphasis> and introgressed <Emphasis Type="Italic">B. rapa</Emphasis> with Bt <Emphasis Type="Italic">cry1Ac</Emphasis> and <Emphasis Type="Italic">gfp</Emphasis> genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.     

Green fluorescent protein gene as a tool to examine the efficacy of Agrobacterium-delivered CRISPR/Cas9 reagents to generate targeted mutations in the potato genome

CRISPR/Cas9 has emerged as a simple, yet efficient gene editing tool to generate targeted mutations in desired genes in crops plants. Agrobacterium tumefaciens, a reliable and inexpensive DNA-delivery mechanism into plant cells, has been used for the generation of CRISPR/Cas9-mediated mutations in crop plants, including potato. However, little information is available as to the progression of gene knockout during various stages of culture following the introduction of CRISPR components in this species. In the current study, the green fluorescent protein (gfp) transgene was first introduced in the genome of a potato variety, Yukon Gold. Two GFP-expressing lines, one with a single gfp copy integrated and another with four gfp copies integrated, were subjected to CRISPR/Cas9-mediated mutations in the transgene(s) using three different gRNAs. Disappearance of GFP fluorescence was monitored during the entire culture/regeneration process. Although all three gRNAs successfully knocked out the transgene(s), their efficiencies differed greatly and did not completely match the predicted scores by some guide RNA prediction tools. The nature of mutations in various knockout events was analyzed. Several lines containing four gfp-copies showed four different types of mutations. These findings suggest that it is possible to target all four alleles of a desired native gene in the tetraploid potato.

     

Choosing a reporter for gene expression studies in transgenic actinorhizal plants of the Casuarinaceae family

Transgenic Casuarinaceae and reporter genes provide valuable tools to study gene expression in transgenic actinorhizal nodules. In this paper, we discuss the use of ß-glucuronidase for the histochemical localization and quantification of gene expression in transgenic plants of Allocasuarina verticillata and Casuarina glauca nodulated by the actinomycete Frankia. We also report on the genetic transformation of A. verticillata by the Agrobacterium tumefaciens strain C58C1(pGV2260) containing the 35S-mgfp5-ER construct encoding a modified green fluorescent protein of Aequorea victoria in a binary vector. The evolution of the GFP fluorescence was monitored through all stages of the regeneration process. The data indicate that GFP is not toxic in Casuarinaceae and that this reporter gene can be used for visual screening of transformed calli and transgenic plants. The fluorescence pattern of gfp provides a new tool for monitoring in vivo transgene expression in actinorhizal plants.     

Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model

To characterize the process of vertebral segmentation and disc formation in living animals, we analyzed tiggy-winkle hedgehog (twhh):green fluorescent protein (gfp) and sonic hedgehog (shh):gfp transgenic zebrafish models that display notochord-specific GFP expression. We found that they showed distinct patterns of expression in the intervertebral discs of late stage fish larvae and adult zebrafish. A segmented pattern of GFP expression was detected in the intervertebral disc of twhh:gfp transgenic fish. In contrast, little GFP expression was found in the intervertebral disc of shh:gfp transgenic fish. Treating twhh:gfp transgenic zebrafish larvae with exogenous retinoic acid (RA), a teratogenic factor on normal development, resulted in disruption of notochord segmentation and formation of oversized vertebrae. Histological analysis revealed that the oversized vertebrae are likely due to vertebral fusion. These studies demonstrate that the twhh:gfp transgenic zebrafish is a useful model for studying vertebral segmentation and disc formation, and moreover, that RA signaling may play a role in this process.     

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     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Avocado globular somatic embryos were transformed with three binary vectors, pK7FNF2, pK7RNR2 and pK7S*NF2, harboring the marker genes gfp, DsRed and a gfp-gus fusion gene, respectively. GFP and DsRed fluorescence was detected in embryogenic lines growing in selection medium 2 months after Agrobacterium inoculation. The fluorescence signal was maintained thereafter in transgenic calli, as well as in mature somatic embryos. Red fluorescence in pK7RNR2 transgenic lines was higher and more easily observable than GFP fluorescence. Furthermore, calli transformed with pK7S*NF2, harboring gfp-gus, showed higher level of fluorescence than those transformed with pK7FNF2, containing two gfp. To improve plant recovery, maturated transgenic embryos that failed to germinate or showed an underdeveloped shoot were cultured for 4 weeks in a medium with 1 mg l^?1 TDZ and 1 mg l^?1 BA after partial removal of cotyledons. A 50% of embryos developed one or several shoots on the cut surface. These embryos were cultured for 4 additional weeks in a medium with 1 mg l^?1 BA for shoot elongation and then, shoots were grafted in vitro onto seedling rootstocks. Culture of micrografts in solid MS medium supplemented with 1 mg l^?1 BA allowed a 60–80% success rate. Young leaves from transgenic plants showed GFP or DsRed fluorescence located in the nucleus. The results obtained indicate that fluorescent marker genes, especially DsRed, could be useful for early selection of transgenic material and optimization of the transformation parameters in avocado. Furthermore, the protocol established allowed the successful recovery of transgenic plants, one of the main limiting steps in avocado transformation.     

Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system

To test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp(green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system.     

Expression of a gene encoding trichosanthin in transgenic rice plants enhances resistance to fungus blast disease

. The gene encoding mature trichosanthin, a type I ribosome-inactivating protein isolated from the tuber of Trichosanthes kirilowii Maximowicz, was transformed into calli of rice (Oryza sativa L.) by bombardment. Transgenic rice plants were obtained and confirmed by Southern and Western blot analysis. When transgenic rice plants expressing trichosanthin were inoculated with the spores of Pyricularia oryzae, a major rice fungus blast pathogen, the lesions on leaves were much less severe, and the seedling survival rate and whole plant weight were higher than those of control plants with the gus gene. The presented data demonstrate a novel, potential role of trichosanthin in antifungal protection in transgenic plants.     

Use of doubled haploid technology for development of stable drought tolerant bread wheat (Triticum aestivum L.) transgenics

Anther culture–derived haploid embryos were used as explants for Agrobacterium‐mediated genetic transformation of bread wheat (Triticum aestivum L. cv CPAN1676) using barley HVA1 gene for drought tolerance. Regenerated plantlets were checked for transgene integration in T₀ generation, and positive transgenic haploid plants were doubled by colchicine treatment. Stable transgenic doubled haploid plants were obtained, and transgene expression was monitored till T₄ generation, and no transgene silencing was observed over the generations. Doubled haploid transgenic plants have faster seed germination and seedling establishment and show better drought tolerance in comparison with nontransgenic, doubled haploid plants, as measured by per cent germination, seedling growth and biomass accumulation. Physiological evaluation for abiotic stress by assessing nitrate reductase enzyme activity and plant yield under post‐anthesis water limitation revealed a better tolerance of the transgenics over the wild type. This is the first report on the production of double haploid transgenic wheat through anther culture technique in a commercial cultivar for a desirable trait. This method would also be useful in functional genomics of wheat and other allopolyploids of agronomic importance.     

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.