Cauliflower Mosaic Virus Promoter Research Articles

A bidirectional promoter from Papaya leaf crumple virus functions in both monocot and dicot plants

The intergenic region of Papaya leaf crumple virus (PaLCrV) transcribes the complementary (C) and virion (V) strand promoters, expressing the C1-C4 and V1-V2 genes respectively in opposite directions. Activities of the bidirectional promoter of PaLCrV in the C and V directions were compared with the 35S promoter of Cauliflower mosaic virus (CaMV35S), using β-glucuronidase (uidA) as the reporter gene. The resulting promoter constructs were named as pC, pV, and p35S respectively, and transformed into Arabidopsis thaliana, wheat and rice. GUS activity in leaf, stem, root, and flower of Arabidopsis, and the calli of wheat and rice was estimated using histochemical staining, fluorometric assay, and real-time PCR. The expression study showed that the transcription from pC was 12–13 folds higher than that from p35S in root and leaf of Arabidopsis. It expressed at a higher level in root, stem, leaf, flower, and silique of Arabidopsis, and in the callus of wheat and rice, as compared to p35S. The pV also expressed in both monocots and dicots, though the activity was lower as compared to p35S in all the cases. Hence, the C promoter of PaLCrV is a good candidate for high-level expression of transgenes in a wide variety of tissues in dicot and monocot plants.

Construction of a novel vector for the nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii and its stable expression

Plasmid pBI221aadAGUS which carried both of GUS (β-glucuronidase) and aadA (aminoglyoside transferase) genes besides of the 35S cauliflower mosaic virus promoter was constructed and used for stable nuclear transformation of Chlamydomonas reinhardtii. The vector was transformed into the alga by particle gun bombardment and two positive colonies were selected on spectinomycin–containing medium. The restriction analysis of the DNA of the positive colonies showed that aadA was inserted in two orientations. The presence of introduced genes in the transformed colonies was confirmed by (PCR) using primers specific to GUS and aadA genes. The expression of aadA and GUS genes was revealed in all colonies that were grown on spectinomycin in liquid culture for 3–4 generations. The usefulness of this vector, differing in the orientation of the aadA cassette, was manisfested by transforming C. reinhardtii to spectinomycin resistance in the stable expression. This constructed plasmid-based expression vector system would help to unravel the functions of various genes in the green alga.

Functional characterization of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Arabidopsis thaliana and its potential in providing flood tolerance

Ethylene is a phytohormone that has gained importance through its role in stress tolerance and fruit ripening. In our study we evaluated the functional potential of the enzyme involved in ethylene biosynthesis of plants called ACC (aminocyclopropane-1-carboxylic acid) oxidase which converts precursor ACC to ethylene. Studies on ethylene have proven that it is effective in improving the flood tolerance in plants. Thus our goal was to understand the potential of ACC oxidase gene overexpression in providing flood tolerance in transgenic plants. ACC oxidase gene was PCR amplified and inserted into the pBINmgfp5-er vector, under the control of a constitutive Cauliflower Mosaic Virus promoter. GV101 strain of Agrobacterium tumefaciens containing recombinant pBINmgfp5-er vector (referred herein as pBIN-ACC) was used for plant transformation by the ‘floral dip’ method. The transformants were identified through kanamycin selection and grown till T3 (third transgenic) generation. The flood tolerance was assessed by placing both control and transgenic plants on deep plastic trays filled with tap water that covered the soil surface. Our result shows that wild-type Arabidopsis could not survive more than 20 days under flooding while the transgenic lines survived 35 days, suggesting development of flood tolerance with overexpression of ACC oxidase. Further molecular studies should be done to elucidate the role and pathways of ACC oxidase and other phytohormones involved in the development of flood adaptation.

Development of a Sri Lankan rice variety Bg 94-1 harbouring cry2A gene of Bacillus thuringiensis resistant to rice leaffolder [Cnaphalocrocis medinalis (Guenée)

The rice leaffolder (RLF) [ Cnaphalocrocis medinalis (Guenee)] is a major lepidopteran insect pest of rice in Sri Lanka and the larvae cause significant yield losses annually. Insecticidal crystal protein gene, cry2A driven by the 35S cauliflower mosaic virus (CaMV) promoter with a double enhancer sequence was transformed into scutellum derived embryogenic calli of Bg 94-1 via Agrobacterium tumefaciens mediated transformation procedure. Transformation vector pCAMBIA 1305.1 containing the transgene construct CaMV 35S d-cry2A-NOS, hygromycin phosphotransferase and β-glucuronidase (GUS)-Plus was employed in this study. Transformed calli were subjected to 4 ‒ 5 rounds of hygromycin selection. Molecular analysis of the generated plants together with an insect bioassay using RLF larvae confirmed successful transformation and expression of this transgene in T0 generation of rice plants. A total of 19 putative transgenic plants could be confirmed from a total of 60 T0 generation plants examined. Insect feeding bioassay demonstrated that T0 transgenic rice plants are effective against RLF larvae, and showed approximately 89 % larval mortality after 3 days and 100 % mortality after 5 days of feeding. Further molecular analysis of T1 generation of the transgenic lines confirmed the stable integration of cry2A transgene into the Bg 94-1 genome. Findings of this study demonstrated that the cry2A gene of Bacillus thuringiensis ( Bt ) was successfully transformed into Bg 94-1 via Agrobacterium mediated transformation and have developed resistance against the RLF larvae. Thus, this study highlights the importance of developing transgenic rice, which can provide insect resistance against a major lepidopteran insect pest of rice in Sri Lanka.

English

The present study was carried out to ascertain the genetic status of soybean and maize crops introduced in Algeria and to test local and imported products containing maize and soybean for the presence of genetically modified organisms (GMO into the food and feed chain). Samples from crops and processed food products were selected, randomly sampled and screened by polymerase chain reaction (PCR) for the presence of the 35S cauliflower mosaic virus promoter (P35S CaMV) and the Agrobacterium tumefaciens nopaline synthase (nos) terminator, commonly found in transgene cassettes. Results showed that GMOs were absent in all crop samples, but present in 29 food and feed samples. These analyses are discussed for the efficiency of the Algerian regulation and the absence of labelling and traceability mechanisms within the country. Key words: Genetically modified organisms (GMOs), detection, maize, soybean, seeds, food and feed products.

Construction of a full-length infectious cDNA clone of Cowpea mild mottle virus.

Infectious cDNA clones are an important tool to study the molecular and cellular process of RNA virus infection. In vitro and in vivo transcription systems are the two main strategies used in the generation of infectious cDNA clones for RNA viruses. This study describes the first generation of a full-length infectious cDNA clone of Cowpea mild mottle virus (CPMMV), a Carlavirus. The full-length genome was synthesized by Overlap Extension PCR of two overlapping fragments and cloned in a pUC-based vector under control of the SP6 RNA polymerase promoter. After in vitro run-off transcription, the produced RNA was mechanically inoculated into soybean plants cv. CD206. The systemic infection was confirmed by RT-PCR and further sequencing of amplified cDNA fragments. To simplify the transfection process, the complete genome was subcloned into a binary vector under control of the 35S promoter of cauliflower mosaic virus by the Gibson Assembly protocol. The resulting clones were inoculated by particle bombardment onto soybean seedlings and the recovery of the virus was confirmed 2 weeks later by RT-PCR. Our results indicate the constructs of the full-length cDNA of CPMMV are fully infectious in both in vitro and in vivo transcription strategies.

Cloning of the Self-incompatibility SFB Gene from Chinese Apricot ‘Xiaobaixing’ and Construction of the SFB Expression Vectors

Three kinds of expression vectors of a pollen-S determinant were constructed to provide a reference for molecular breeding of self-compatible (SC) Prunus species. An S-haplotype-specific F-box (SFB) protein gene from the ‘Xiaobaixing’ apricot (Prunus armeniaca) was cloned by reverse transcription polymerase chain reaction (RT-PCR) and 3′-rapid-amplification of cDNA ends (3′-RACE). A 1136-bp sequence complementary to the 3′-end of the cDNA (GenBank accession number KP938528.2) with a 912-bp complete open reading frame (ORF) was obtained. The deduced amino acid sequence contained an F-box domain, two variable regions, and two hypervariable regions with structural characteristics similar to SFB in other Rosaceae plants. Sense, antisense, and RNA interference (RNAi) vectors for SFB were constructed by enzyme restriction. The target fragment was restricted using the corresponding restriction enzyme and then directionally inserted between the 35S cauliflower mosaic virus promoter and the nopaline synthase terminator (NOS-ter) of the expression vector pCAMBIA-35S-MCS-NOS-NPTII. The intron-containing hairpin RNA (ihpRNA) was obtained by fusion PCR. The constructed vectors were transferred into Agrobacterium tumefaciens strain LBA4404 by freezing/thawing. The RNAi vector of SFB was also transformed in tobacco (Nicotiana tabacum). The successful construction of these three expression vectors provides a basis for transforming ‘Xiaobaixing’ apricot and the breeding of SC Prunus cultivars.

Novel antibiotics regeneration and genetic transformation with RsMYB1 gene of recalcitrant chrysanthemum cv. Shinma

This research was conducted to develop genetic transformation of the recalcitrant chrysanthemum cv. Shinma by application of appropriate antibiotics and selective agents. Clavamox had the least inhibitory effect on shoot regeneration compared to timentin, carbenicillin, and cefotaxime. Clavamox, at a concentration of 125 mg L−1, was found to be the most suitable for shoot regeneration and production of quality shoots, suppressing the growth of Agrobacterium in explants infected with strains GV3101 or C58C1 for 3 and 4 weeks, respectively. The concentration of phosphinothricin (PPT) was found to be 1.0 mg L−1 for screening of putative transgenic shoots. Moreover, transgenic chrysanthemums were obtained by culturing explants co-cultivated with A. tumefaciens strain GV3101 harboring an anthocyanin regulatory gene RsMYB1 isolated from radish (Raphanus sativus), which was placed under the control of cauliflower mosaic virus promoter (CaMV) 35S and petal-specific promoter InMYB1 isolated from the morning glory (Ipomoea nil), on shoot regeneration medium supplemented with recommended concentration of antibiotic and selective agent. Flow cytometry analysis revealed that there was no variation in ploidy level between transgenic plants and donor plants (non-transformants). To our knowledge, this is the first report of the use of Clavamox and MYB transcription factor for genetic transformation of this chrysanthemum.

Functional Analysis of Cotton Leaf Curl Kokhran Virus/Cotton Leaf Curl Multan Betasatellite RNA Silencing Suppressors.

In South Asia, Cotton leaf curl disease (CLCuD) is caused by a complex of phylogenetically-related begomovirus species and a specific betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB). The post-transcriptional gene silencing (PTGS) suppression activities of the transcriptional activator protein (TrAP), C4, V2 and βC1 proteins encoded by Cotton leaf curl Kokhran virus (CLCuKoV)/CLCuMuB were assessed in Nicotiana benthamiana. A variable degree of local silencing suppression was observed for each viral protein tested, with V2 protein exhibiting the strongest suppression activity and only the C4 protein preventing the spread of systemic silencing. The CLCuKoV-encoded TrAP, C4, V2 and CLCuMuB-encoded βC1 proteins were expressed in Escherichia coli and purified. TrAP was shown to bind various small and long nucleic acids including single-stranded (ss) and double-stranded (ds) RNA and DNA molecules. C4, V2, and βC1 bound ssDNA and dsDNA with varying affinities. Transgenic expression of C4 under the constitutive 35S Cauliflower mosaic virus promoter and βC1 under a dexamethasone inducible promoter induced severe developmental abnormalities in N. benthamiana. The results indicate that homologous proteins from even quite closely related begomoviruses may differ in their suppressor activity and mechanism of action. The significance of these findings is discussed.

CLONING AND EXPRESSING TRYPSIN MODULATING OOSTATIC FACTOR IN Chlorella desiccata TO CONTROL MOSQUITO LARVAE.

The insect peptide hormone trypsin modulating oostatic factor (TMOF), a decapeptide that is synthesized by the mosquito ovary and controls the translation of the gut's trypsin mRNA was cloned and expressed in the marine alga Chlorella desiccata. To express Aedes aegypti TMOF gene (tmfA) in C. desiccata cells, two plasmids (pYES2/TMOF and pYDB4-tmfA) were engineered with pKYLX71 DNA (5 Kb) carrying the cauliflower mosaic virus (CaMV) promoter 35S(2) and the kanamycin resistant gene (neo), as well as, a 8 Kb nitrate reductase gene (nit) from Chlorella vulgaris. Transforming C. desiccata with pYES2/TMOF and pYDB4-tmfA show that the engineered algal cells express TMOF (20 ± 4 μg ± SEM and 17 ± 3 μg ± SEM, respectively in 3 × 10(8) cells) and feeding the cells to mosquito larvae kill 75 and 60% of Ae. aegypti larvae in 4 days, respectively. Southern and Northern blots analyses show that tmfA integrated into the genome of C. desiccata by homologous recombination using the yeast 2 μ circle of replication and the nit in pYES2/TMOF and pYDB4-tmfA, respectively, and the transformed algal cells express tmfA transcript. Using these algal cells it will be possible in the future to control mosquito larvae in the marsh.

Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.

Evaluation of red fluorescent protein (DsRed) as alternative visual marker of genetic transformation in cassava (Manihot esculenta Crantz)

Red fluorescent protein (DsRed) from reef coral was evaluated in comparison with green fluorescent protein (GFP) as a reporter gene for cassava transformation. Cassava friable embryogenic callus (FEC) was transformed with ER-targeted versions of DsRed and GFP constructs driven by the 35S cauliflower mosaic virus promoter. Efficiency of transformation was comparable for both visual marker genes at averages of 119 and 163 expressing plants recovered per cc of settled cell volume FEC for GFP and DsRed, respectively. High and uniform DsRed expression was observed at the single cell and proliferating callus stages, in somatic embryos and within organs of whole in vitro and greenhouse-grown plants in a manner similar to GFP. Plants expressing GFP and DsRed were robust and phenotypically normal with regard to growth, vigor, and formation of storage roots when grown in the greenhouse. Expression of marker genes within cross sections of petiole, woody stem, and storage roots from greenhouse-grown plants was determined. The interference of phenolic compounds and chlorotic tissues characteristic of the signal from GFP-expressing tissues was not observed within tissues transgenic for DsRed. Tissues and plants co-expressing DsRed and GFP were produced by co-culturing FEC with a mixed Agrobacterium suspension carrying GFP and DsRed gene constructs or by re-transformation of an existing GFP transgenic line with DsRed. Re-transformation of GFP-expressing tissues was the more efficient method for production of GFP/DsRed stacked plants. Co-expression of both marker genes within the same transformation unit was easily visualized at their respective wavelength with the aid of appropriate filters thus validating their potential for co-expression studies.

Transgene expression in tick cells using Agrobacterium tumefaciens.

Ticks transmit infectious agents to humans and other animals. Genetic manipulation of vectors like ticks could enhance the development of alternative disease control strategies. Transgene expression using the phytopathogen Agrobacterium tumefaciens has been shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for A. tumefaciens to mediate transgene expression in HeLa cells as well as Rhipicephalus microplus tick cells. Translational fusions eGfp:eGfp or Salp15:eGfp, including the enhanced-green fluorescent protein and the Ixodes scapularis salivary factor SALP15 genes, were constructed using the CaMV 35S (cauliflower mosaic virus) promoter, "PBm" tick promoter (R. microplus pyrethroid metabolizing esterase gene) or the Simian Virus SV40 promoter. Confocal microscopy, RT-PCR and Western-blot assays demonstrated transgene(s) expression in both cell lines. Transgene expression was also achieved in vivo, in both R. microplus and I. scapularis larvae utilizing a soaking method including the A. tumefaciens donor cells and confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNA(s). This strategy opens up a new avenue to express exogenous genes in ticks and represents a potential breakthrough for the study of tick-host pathophysiology.

RNA based viral silencing suppression in plant pararetroviruses.

The 35S promoter of cauliflower mosaic virus and that of other plant pararetroviruses gives rise to an RNA, which is both a pre-genome and a polycistronic mRNA. The 600 nucleotide long very structured leader of this RNA is also transcribed separately. The resulting 8S RNA is then converted to a double strand giving rise to a huge set of siRNAs, which suppress silencing. In this Mini-Review I discuss how this versatile stretch of 600 nts constitutes a masterpiece of evolution.

Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

A Cassava vein mosaic virus promoter cassette induces high and stable gene expression in clonally propagated transgenic cassava (Manihot esculenta Crantz)

The study described a T-DNA vector with a Cassava vein mosaic virus promoter cassette (pCsVMV) and a kanamycin selectable marker gene driven by the 35S Cauliflower mosaic virus promoter with a view to stably express transgenes over repeated cycles of clonal propagation. A β-glucuronidase reporter gene under control of pCsVMV (pCsVMV-GUS) was introduced into the cassava landrace ‘Tokunbo’ via Agrobacterium-mediated genetic transformation. Transgenic tobacco plants (Nicotiana tabacum SR1) with the same gene construct were also produced. In tobacco, the pCsVMV-GUS was highly expressed in all tissues tested such as leaf, stem, petiole, and roots. In transgenic cassava, the pCsVMV-GUS gene was highly expressed in all tissues and most cell types of in vitro plants including leaf, stem, petiole, and fibrous roots. The pCsVMV-GUS gene was also highly expressed in these tissues as well as in tubers of greenhouse grown cassava. High and stable pCsVMV-GUS gene expression was maintained over 3 cycles of ratooning under greenhouse conditions, thus showing the absence of undesired gene silencing effects after repeated in vitro subculturing and vegetative propagation. From the high constitutive levels of GUS activity observed, the study concluded that the CsVMV promoter cassette was useful for high-level expression in cassava over repeated cycles of clonal propagation.

Tissue- and Organ-Specific Promoters for Expression of Heterologous Genes in Transgenic Cassava (Manihot Esculenta Crantz) Plants

Promoters are regions of DNA that initiates transcription of genes. A number of promoters have been identified that confer high level of expression of heterologous genes in transgenic plants. Some promoters have constitutive expression as they are active in all circumstances in the cell, while others are regulated, becoming active in certain cell or in response to specific stimuli. Despite the availability of tissue- and organ-specific promoters, most transgene expressions in Cassava are driven by constitutive cauliflower mosaic virus promoter. This paper examines the availability of promoters for transgene expression in plants, assesses the use of promoters for transgene expression in Cassava and establishes the need for tissue- and organ-specific promoters for expression of heterologous genes in Cassava.

Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem-associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression.

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23-kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N-terminal 157-amino-acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV-like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23-derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem-specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV-specific symptoms (vein clearing and necrosis, and stem pitting), but not the non-specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem-specific accumulation of the p23Δ158-209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N-terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.

A Comparison of Protein and Phenolic Compounds in Seed from GMO and Non-GMO Soybean

Soybean protein is a valuable and important component in human and animal diets. Approximately 94% of the soybean planted in the US is genetically modified (GM) to enhance quality and productivity. Since value-added traits are continuously being developed by genetic modification, it is important to determine if any unintended changes occur in GM soybean seeds. In this investigation, we have selected three different transgenic lines, denoted event 1,2 and 3 with a single Agrobacterium tumefaciens T-DNA insert that included genes for a herbicide-resistance selectable gene (bar) and a β-glucuronidase (GUS) reporter gene expressed using a double 35S Cauliflower Mosaic Virus (CaMV) promoter and a soybean polygalacturonase (Glyma12g01480) promoter, respectively. The transgenic lines and nontransgenic progenitor isoline (control) were used for both proteomic and phenolic compound analysis. Seed proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Out of approximately 1300 protein spots detected per protein extract, 30 spots were selected for further analysis based on software-determined differences (ANOVA) in their relative abundance in the protein gels for the control and three events. Subsequent statistical analysis after Bonferroni correction indicated that the abundance of only two of the thirty protein spots were significantly different at the 1% probability level. Two protein spots, an isoflavone reductase and a quinine oxidoreductase-like protein, in event 2 were significantly different from the control and the other two transgenic events. All thirty protein spots were analyzed and identified by mass spectrometry (MS) followed by a search of the NCBI databases using the Mascot search engine. In addition to protein, two classes of phenolic compounds, isoflavonoids and phenolic acids, were analyzed by LC-MS. The results indicated no systematic differences in the amount or profile for either class of phenolic compounds in the control or three transgenic events.

Comparison of the MpEF1α and CaMV35 promoters for application in Marchantia polymorpha overexpression studies

Constitutive promoters are essential tools for analyses of gene functions by transgenic approaches. For overexpression and silencing studies of genes, a ubiquitous and strong expression of genes under investigation as well as selection markers is preferred. For future applications in the emerging basal plant model system Marchantia polymorpha, a liverwort, activities of the viral 35S cauliflower mosaic virus promoter and the endogenous elongation factor 1α (MpEF1α) promoter were analyzed. Expression of the reporter gene β-glucuronidase (GUS), driven by the CaMV35 and MpEF1α promoters, was compared throughout plant development. Significant differences were observed between the two promoter activities. The CaMV35 promoter yields a weak reporter gene expression in the meristematic zones but drives a strong expression in the thallus. The MpEF1α promoter causes a strong meristematic GUS expression and is more active in female sexual tissues. Overall, the MpEF1α promoter seems to be the better option for obtaining a strong and ubiquitous transgene expression. Furthermore, a whole mount in situ hybridization protocol for Marchantia was established. Analysis of MpEF1α mRNA transcript in intact, whole tissues showed an expression pattern that is overall similar to the pattern of the GUS reporter gene expression driven by the MpEF1α promoter, including strong expression in meristematic zones. The whole mount technique reported here can be used to determine the mRNA expression in intact gemmae and archegonia, and has the potential to be applied for screening large numbers of transgenic plants, for instance to identify knock-down mutants.