Screenable Marker Genes Research Articles

In Planta Genetic Transformation of Mungbean (Vigna radiata (L.) Wilczek) with Marker Gene

In genetic improvement of mungbean much success has not been achieved due to its recalcitrant nature towards in vitro regeneration. An attempt was made to develop an Agrobacterium-mediated in planta genetic transformation protocol for a locally grown mungbean variety BARI Mung-3 using a screenable marker gene. Two minutes of vacuum infiltration followed by 60 minutes of incubation period in Agrobacterium suspension of Winans’ AB medium containing wounded tobacco leaf extract was found most suitable towards genetic transformation in pricked de-coated half seed explants. An optical density (OD600) of 0.7 was found most effective for transient gus gene expression. Chimeric GUS expression was observed in the root and leaf tissues from the successfully transformed plantlets obtained through in planta transformation. This methodology of genetic transformation was found more suitable, easier and less time consuming than tissue culture based genetic transformation, which may be used for the genetic improvement of mungbean.

Generation of white-eyed Daphnia magna mutants lacking scarlet function.

The crustacean Daphnia magna is an important model in multi-disciplinary scientific fields such as genetics, evolutionary developmental biology, toxicology, and ecology. Recently, the draft genome sequence and transcriptome data became publicly available for this species. Genetic transformation has also been achieved via the introduction of plasmid DNA into the genome. The identification of a screenable marker gene and generation of mutant strains are essential to further advance D. magna functional genomics. Because crustaceans are closely related to insects, we hypothesized that, similar to Drosophila genetic studies, eye color-related genes can function as marker genes in Daphnia. We searched orthologs of Drosophila eye pigment transporters White, Scarlet, and Brown in the genome of D. magna. Amino acid sequence alignment and phylogenetic analysis suggested that D. magna has six white and one scarlet orthologs, but lacks the brown ortholog. Due to the multiplicity of white orthologs, we analyzed the function of the scarlet ortholog, DapmaSt, using RNA interference. DapmaSt RNAi embryos showed disappearance of black pigments both in the compound eye and in the ocellus, suggesting that DapmaSt is necessary for black pigmentation in Daphnia eyes. To disrupt DapmaSt using the Crispr/Cas9 system, we co-injected DapmaSt-targeting gRNAs with Cas9 mRNAs into eggs and established white-eyed DapmaSt mutant lines that lack eye pigments throughout their lifespan. Our results suggest that DapmaSt can be used as a transformation marker in D. magna and the DapmaSt mutants would be an important resource for genetic transformation of this species in the future.

Generation of composite Persea americana (Mill.) (avocado) plants: A proof-of-concept-study.

Avocado (Persea americana (Mill.)), an important commercial fruit, is severely affected by Phytophthora Root Rot in areas where the pathogen is prevalent. However, advances in molecular research are hindered by the lack of a high-throughput transient transformation system in this non-model plant. In this study, a proof-of-concept is demonstrated by the successful application of Agrobacterium rhizogenes-mediated plant transformation to produce composite avocado plants. Two ex vitro strategies were assessed on two avocado genotypes (Itzamna and A0.74): In the first approach, 8-week-old etiolated seedlings were scarred with a sterile hacksaw blade at the base of the shoot, and in the second, inch-long incisions were made at the base of the shoot (20-week-old non-etiolated plants) with a sterile blade to remove the cortical tissue. The scarred/wounded shoot surfaces were treated with A. rhizogenes strains (K599 or ARqua1) transformed with or without binary plant transformation vectors pRedRootII (DsRed1 marker), pBYR2e1-GFP (GFP- green fluorescence protein marker) or pBINUbiGUSint (GUS- beta-glucuronidase marker) with and without rooting hormone (Dip 'N' Grow) application. The treated shoot regions were air-layered with sterile moist cocopeat to induce root formation. Results showed that hormone application significantly increased root induction, while Agrobacterium-only treatments resulted in very few roots. Combination treatments of hormone+Agrobacterium (-/+ plasmids) showed no significant difference. Only the ARqua1(+plasmid):A0.74 combination resulted in root transformants, with hormone+ARqua1(+pBINUbiGUSint) being the most effective treatment with ~17 and 25% composite plants resulting from strategy-1 and strategy-2, respectively. GUS- and GFP-expressing roots accounted for less than 4 and ~11%, respectively, of the total roots/treatment/avocado genotype. The average number of transgenic roots on the composite plants was less than one per plant in all treatments. PCR and Southern analysis further confirmed the transgenic nature of the roots expressing the screenable marker genes. Transgenic roots showed hyper-branching compared to the wild-type roots but this had no impact on Phytophthora cinnamomi infection. There was no difference in pathogen load 7-days-post inoculation between transformed and control roots. Strategy-2 involving A0.74:ARqua1 combination was the best ex vitro approach in producing composite avocado plants. The approach followed in this proof-of-concept study needs further optimisation involving multiple avocado genotypes and A. rhizogenes strains to achieve enhanced root transformation efficiencies, which would then serve as an effective high-throughput tool in the functional screening of host and pathogen genes to improve our understanding of the avocado-P. cinnamomi interaction.

English

Rubber tree belonging to the genus Hevea is an economically important crop of Thailand and South-east Asia . To optimize its agronomical trait for glyphosate-resistant, in vitro gene transformation through Agrobacterium tumefaciens was investigated. The bacteria carrying plasmid pCAMBIA 1304, harboring gus as screenable marker genes and EPSPs gene was used. The shoot tips were immersed in A. tumefaciens suspension at optical densities (OD 600 ) at 0.3, 0.6 and 0.9 for various times (15, 30 and 60 min). The results revealed that shoot explants immersed in A. tumefaciens suspension at OD 600 of 0.6 for 30 min gave the higher survival rate after being cultured on glyphosate containing MS medium for one and half months. Assessment of transformed shoots revealed positive results in GUS histochemical assay. The presence of the gus and EPSPs genes in transformed rubber tree were confirmed by polymerase chain reaction (PCR) technique, dot blot hybridization and Southern PCR hybridization. Specific primers for the gus and EPSPs genes were designed to amplify a 919 and 1,600 bps DNA fragment, respectively. Keywords: Transgenesis, glyphosate, inoculation time, Agrobacterium density, Hevea African Journal of Biotechnology , Vol 13(23) 2321-2329

Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat

Immature embryos have been used frequently as target tissues in the genetical transformation of wheat. However, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process, because of the need to cultivate the plants under controlled conditions. To circumvent this, we have employed mature embryos rather than immature ones as starter explants for Agrobacterium-mediated transformation of an elite wheat (Triticum aestivum L.) cultivar EM12. The neomycin phosphotransferase II, (npt II) and beta-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimize the performance of T-DNA delivery. With the aid of an orthogonal design, the effect of four factors in combination on transfer DNA (T-DNA) delivery was studied. These factors were preculture duration, different kinds of inoculation, length of inoculation and co-culture condition. Optimal conditions for T-DNA delivery were obtained for mature embryos precultured for 14 days, followed by immersing in inoculation suspension with full strength Murashige and Skoog (MS) salts in darkness at 23-25 degrees C for 3 h, and then co-culturing with Agrobacterium under desiccating condition in the dark at 23-24 degrees C for 2-3 days. Complete analysis of transgene insertion demonstrated that the optimized method for Agrobacterium-mediated transformation of mature embryos of wheat was efficient and practicable.

Calluses initiated from thin mature embryo fragments are suitable targets for wheat transformation as assessed by long-term GUS expression studies

Microprojectile- or Agrobacterium-mediated DNA delivery into calluses initiated from immature embryos has proven to be effective in transforming wheat. Yet, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process. To circumvent these limitations, we propose an alternative technique applying the particle bombardment technology to calluses derived from fragmented mature embryos rather than immature tissues. The phosphinothricin acetyl transferase (bar) and β-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimise the performance of the proposed technique. Primary requirement for genetic transformation method development, the regeneration capacity of bombarded calluses was established. A preculture duration of 6 days was identified as optimal for DNA uptake and β-glucuronidase (GUS) expression. The highest activity was recorded when calluses were selected. Long-term GUS expression studies (1–7 weeks subsequent to bombardment), showed differentiated behaviours for tissues obtained from mature versus immature embryos. Notably, mature embryos exhibited the greatest number of cells stably expressing the reporter gene, thus providing an excellent source material for developing a stable transformation procedure.

Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies

Genetic transformation has been achieved for several citrus genotypes. However, regeneration of escapes at high frequency is a major problem, making the available procedures rather inefficient. Attempts to improve selection by increasing the concentration of kanamycin, used as the selective agent, or substituting it by geneticin have been unsuccessful. Here, we have critically assessed the actual frequency and origin of escapes in citrus by using visual screening with β-glucuronidase (gusA) and green fluorescent protein (gfp) markers, by studying the persistence of engineered Agrobacterium in the explants, and by characterising through Southern blot analysis all the regenerants obtained under kanamycin selection. Our results show that inefficient selection could be attributed to the protection of the non-transformed cells from the selective agent by the surrounding transformed cells, and to the persistence of kanamycin-resistance Agrobacterium in explant tissues over long periods of time after co-cultivation. This also explained the high frequency (12%) of chimeric shoots that were commonly recovered. High frequency regeneration of chimeras that resulted from the fusion of different transformation events is reported for the first time. On the other hand, molecular analysis of all the regenerants reveals that transformation frequency is underestimated when based on the expression of a screenable marker gene, and that low expressors and silenced lines could account for at least 25% of those plants considered escapes based on selectable and screenable marker analysis. Consequences of these results at the practical level are also discussed.

Genetics of the DST-mediated mRNA decay pathway using a transgene-based selection.

mRNA sequences that control abundance, localization and translation initiation have been identified, yet the factors that recognize these sequences are largely unknown. In this report, a transgene-based strategy designed to isolate mutants of Arabidopsis thaliana that fail to recognize these sequences is described. In this strategy, a selectable gene and a screenable marker gene are put under the control of the sequence element being analysed and mutants are selected with altered abundance of the corresponding marker RNAs. The selection of mutants deficient in recognition of the DST (downstream) mRNA degradation signal is used as a test-case to illustrate some of the technical aspects that have facilitated success. Using this strategy, we report the isolation of a new mutant, dst3, deficient in the DST-mediated mRNA decay pathway. The targeted genetic strategy described circumvents certain technical limitations of biochemical approaches. Hence, it provides a means to investigate a variety of other mechanisms responsible for post-transcriptional regulation.

Genetic Transformation of Wheat (Triticum Aestivum L.) Anther Culture-Derived Embryos by Electroporation

ABSTRACTThis report describes the delivery of plasmid DNA into wheat anther culture derived embryos. Initial electroporation experiments were conducted using enzyme (NT-1) pretreatment, 500 μF capacitance, and plasmid pAM2100, which carries the herbicide resistance gene, bar, and the screenable marker gene, Gus. Electroporated embryos were allowed to recover 10 days on regeneration medium and then transferred to regeneration medium containing 0, 10, 50, and 100 ppm phoshinotricin (PPT). Twenty eight albino and 13 green plants were regenerated on control (0 ppm PPT) selection medium from 640 embryos treated with pAM2100. The regeneration frequency was similar to embryos electroporated without pAM2100 demonstrating that electroporation was not deleterious for regeneration. Four albino plants were regenerated on selection medium (10, 50, and 100 ppm PPT). One plant was screened and shown to be transgenic by Southern blot analysis of a bar gene.

Stable electrotransformation of symboint candidate diazotrophic bacterium with plasmids carrying selectable and screenable marker genes

Nitrogen-fixing symbioses had been established between the originally asymbiotic soil bacterium Azotobacter vinelandii CCM289 and different lower and higher plant species. Better characterization and further development of such artificial systems require a reliable genetic transformation method for the introduction of marker genes into symbiont candidates. The performance of electroporation was evaluated using pJB3 (4.8 kb), pBl121 (12.8 kb) and pFAJ31.2 (24 kb) plasmid DNAs containing selectable (Ap, Km, Tc) and screenable ( gusA, lacZ) marker genes. The adapted methods for the preparation of transformation-competent azotobacters and their electroporation (18 kV/cm electric field strength, 5 ms time constant, 0°C) provided up to 6.8 × 10 5 transformants per μg plasmid DNA, which is about 10 3 times the transformation efficiency achieved in control experiments. No electrotransformants were obtained with the 24-kb pFAJ31.2. The size of plasmid DNA did not significantly affect the efficiency of transformation. Transformants were able to grow at antibiotic concentrations that were 100–200 times greater than the lowest amounts that completely inhibited the growth of wild-type bacteria. A constitutive expression of gusA gene was observed in transformants with the CaMV 35S promoter- gusA fusion containing pBl121, while lacZ expression was not detected under the control of the lac promoter in pJB3 transformants. Electroporated plasmids were reisolated from transformants in their original form, while non-transformed bacteria did not contain indigenous plasmids. PCR amplification and Southern DNA blot hybridization showed the integration of plasmid DNA into the host genome as well. Transformants retained their nitrogen-fixing ability and had normal morphological and growth characteristics. Experimental findings proved the stable maintenance of plasmid DNA in azotobacters, making possible the routine transformation and detection of these symbiont candidates.

Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants

A set of plasmids has been constructed utilizing the promoter, 5' untranslated exon, and first intron of the maize ubiquitin (Ubi-1) gene to drive expression of protein coding sequences of choice. Plasmids containing chimaeric genes for ubiquitin-luciferase (Ubi-Luc), ubiquitin-beta-glucuronidase (Ubi-GUS), and ubiquitin-phosphinothricin acetyl transferase (Ubi-bar) have been generated, as well as a construct containing chimaeric genes for both Ubi-GUS and Ubi-bar in a single plasmid. Another construct was generated to allow cloning of protein coding sequences of choice on Bam HI and Bam HI-compatible restriction fragments downstream of the Ubi-1 gene fragment. Because the Ubi-1 promoter has been shown to be highly active in monocots, these constructs may be useful for generating high-level gene expression of selectable markers to facilitate efficient transformation of monocots, to drive expression of reference reporter genes in studies of gene expression, and to provide expression of biotechnologically important protein products in transgenic plants.

Stable transformation via electroporation into maize Type II callus and regeneration of fertile transgenic plants

Maize Type II callus tissue was used as the plant material for genetic transformation via electroporation. Plasmid DNA containing a selectable marker gene (either neomycin phosphotransferase (npt-II) or phosphinothricin acetyl transferase (bar)), and a screenable marker gene (gus A) was incubated with the tissue prior to electroporation. Electroporated callus tissue was placed on selection medium containing kanamycin sulfate or Bast(™). No kanamycin resistant colonies were recovered whereas four independent Basta(™) resistant callus isolates were recovered from a total of 544 cuvettes electroporated. After 8 to 16 weeks on the Basta(™) containing medium, selected calli were isolated and maintained in individual selection plates for 4 to 6 weeks until sufficient tissue accumulated. Enzyme assays and DNA analyses were performed to verify the transformation events. Several plants were regenerated from individual callus isolates. The plants derived from one callus isolate were male sterile while those derived from the other isolates were both male and female fertile. Most plants showed Basta(™) resistance. DNA analyses confirmed the presence of the introduced bar gene(s) in the primary regenerants and their progeny. The integration patterns of the inserted DNA appeared to be complex.

Molecular and genetic characterization of elite transgenic rice plants produced by electric-discharge particle acceleration

The recovery of transgenic rice plants expressing a number of exogenous genes was reported previously. Using immature embryo explants as the target tissue, plasmids containing both selectable and screenable marker genes were introduced into elite rice varieties via electric-discharge particle acceleration. Co-integration, copy number, expression, and inheritance of these genes were analyzed. A 100% co-integration frequency was confirmed by Southern-blot analyses of R0 plants. The majority of transgenic plants contained between one and ten copies of exogenous DNA and molecular and genetic analyses of progeny indicated that all copies in almost all R0 plants were inherited as a single dominant hemizygous locus. Co-expression of unselected genes ranged from 30-66% for gus/hmr constructs, depending on the promotor used, and up to 90% for bar/hmr constructs. The integrative structures of two unlinked transgenic loci of a rare R0 plant were analyzed in detail by Southern-blot analysis of its progeny.

A plant transformation vector with a minimal T-DNA.

Plant transformation, via Agrobacterium tumefaciens, is usually performed with binary vectors. Most of the available binary vectors contain within the T-DNA (which is transferred to the plant genome) components not required for the intended modification. These additional sequences may cause potential risks during field testing of the transgenic plants or even more in the case of commercialization. The aim of this study was to produce a plant transformation vector which only contains a selectable and screenable marker gene and a multiple cloning site for insertion of promoter::foreign gene::terminator cassettes from other plasmids.

Electroporation of rapeseed protoplasts - transient and stable transformation.

Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β-glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead-type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10-5 and a relative transformation frequency of 0.3% could be achieved. X-ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.