uidA Reporter Gene Research Articles

Simplified Method for Agrobacterium-Mediated Genetic Transformation of Populus x berolinensis K. Koch.

The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid in vitro growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, Populus × berolinensis K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for in vitro manipulations. Various protocols for the Agrobacterium-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the Agrobacterium-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L-1), TDZ (0.02 mg·L-1), and NAA (0.01 mg·L-1). Acetosyringone was not used as an induction agent for vir genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the nptII selective and uidA reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%.

Identification of Stigma Specific Expression Fragment in the Promoter of a Soybean Chitinase Class I Gene

The expression level of heterologous genes in transgenic plants serves as an important indicator of gene efficiency. The small number of currently known effective promoters, limits the possibilities in fine-tuning the expression of transgenes. We cloned and characterized a tissue-specific promoter fragment of the soybean chitinase class I gene (GmChi1). The GmChi1 promoter (GmChi1P) was cloned from Jungery soybean. The promoter sequence contains a number of putative cis-acting elements, including tissue-specific and stress-regulated motifs. By histochemical analysis, the GmChi1P-controlled β-glucuronidase (GUS) reporter enzyme activity was shown to be highest in the roots of transgenic Nicotiana tabacum cv. NC89 at the four-leaf sprout formation stage. Interestingly, the high GUS activity in transgenic tobacco roots was effectively suppressed by salicylic acid (SA) treatment. Deletion analysis of GmChi1P revealed that the sequences located between positions -719 and -382 contain key cis-elements responsible for the reporter uidA gene expression (encoding GUS) in leaves, roots, and wounds of Nicotiana tabacum. In addition, fluorometric analysis showed that the activity of the shortened ChiP(-1292) to ChiP(-719) promoters in the roots of transgenic tobacco was significantly suppressed by abscisic acid and completely suppressed by SA. The ChiP(-382) promoter was also found to be expressed exclusively in the stigma of transgenic tobacco flowers. Using the GUS reporter enzyme, no staining was detected in other flower organs in transgenic Nicotiana tabacum, including sepals, petals, anthers, filaments, and ovaries, or in any vegetative tissues. The results indicate that the promoter fragment ChiP(-382) can be used in tissue-specific regulation of gene expression and plant genetic engineering.

Agrobacterium-mediated transformation of potato Solanum tuberosum L. with constructs carrying the strong plant-derived promoter pro-SmAMP1 from Stellaria media L.

The effectiveness of plant genetic transformation is determined by the choice of genetic structures and their regulatory sequences that cause a high and stable expression level of heterologous genes. In this regard, the actual task of biotechnology is the use of highly effective plant promoters. The choice of promoter determines not only the level of the expression gene, but also the effectiveness of genetic transformation. The purpose of our study was to evaluate the influence of explant type and 5-deletion variants of the plant strong pro-SmAMP1 promoter, on the Agrobacterium -mediated transformation efficiency of potato ( Solanum tuberosum L.) cv. Udacha. To analyze the regenerative capacity of potato stem and leaf explants, AGL0 strain carrying constructs containing the 5-deletion variants of the promoter fragment of gene encoding antimicrobial peptide from Stellaria media L. ( pro-SmAMP1 ) was carried out. Four genetic constructs based on the plant expression vector pCAMBIA1381Z were used in this work, containing the selectable gene hptII and reporter gene uidA under different 5-deletion variants of the pro-SmAMP1 promoter (-442, -675, -732 and -1196 bp relative to the transcription initiation site); as well as two binary vectors based on the expression vector pCAMBIA1302 with 5-deletion pro-SmAMP1 promoter variants (-442 and -1196 bp), controlling the expression of gfp reporter gene. It was found that the effectiveness of Agrobacterium -mediated transformation depended on the type of genetic construction used, but not on the type of explant being cultivated. The insertion of the promoter region pro-SmAMP1 gene, hptII , as well as the absence of the bacterial Vir E gene was confirmed by PCR. Depending on the type of genetic construct, the transformation efficiency for the reporter gene varied from 2.0 to 7.2 %. The results are compared with previously conducted few studies, according to which the choice of promoter determines not only the expression level of marker genes, but also has a significant influence on the genetic transformation efficiency.

Cotton phloem loads from the apoplast using a single member of its nine-member sucrose transporter gene family.

Phloem loading and transport are fundamental processes for allocating carbon from source organs to sink tissues. Cotton (Gossypium spp.) has a high sink demand for the cellulosic fibers that grow on the seed coat and for the storage reserves in the developing embryo, along with the demands of new growth in the shoots and roots. Addressing how cotton mobilizes resources from source leaves to sink organs provides insight into processes contributing to fiber and seed yield. Plasmodesmata frequencies between companion cells and flanking parenchyma in minor veins are higher than expected for an apoplastic loader, and cotton's close relatedness to Tilia spp. hints at passive loading. Suc was the only canonical transport sugar in leaves and constituted 87% of 14C-labeled photoassimilate being actively transported. [14C]Suc uptake coupled with autoradiography indicated active [14C]Suc accumulation in minor veins, suggesting Suc loading from the apoplast; esculin, a fluorescent Suc analog, did not accumulate in minor veins. Of the nine sucrose transporter (SUT) genes identified per diploid genome, only GhSUT1-L2 showed appreciable expression in mature leaves, and silencing GhSUT1-L2 yielded phenotypes characteristic of blocked phloem transport. Furthermore, only GhSUT1-L2 cDNA stimulated esculin and [14C]Suc uptake into yeast, and only the GhSUT1-L2 promoter caused uidA (β-glucuronidase) reporter gene expression in minor vein phloem of Arabidopsis thaliana. Collectively, these results argue that apoplastic phloem loading mediated by GhSUT1-L2 is the dominant mode of phloem loading in cotton.

Structural analysis of regulatory stress elements in OsCIPK14 the rice homologue and functional validation in wheat

A major mechanism of stress tolerance in plants is to increase the cytosolic concentration of Ca2+ ion, a secondary messenger that mediates cellular responsiveness to the extracellular environment. The involvement of different types of Ca2+-sensing protein kinases such as CIPK in stress responses has been elucidated in different plant models. In this study, the upstream regulatory sequence of a CIPK homologue from an Egyptian rice cultivar (Oryza sativa cv Giza182) was structurally and functionally characterized and tested for its ability to drive the expression of β- glucuronidase reporter gene (uidA) in wheat. To that end, a 1275-bp DNA fragment, 49 nucleotides upstream of the putative transcription start site (TSS) of the OsCIPK14 coding sequence, referred in this study as URS-CIPK14, was cloned, sequenced and analyzed for the presence of stress-responsiveness cis-acting elements. Subsequently, the URS-CIPK14 was fused to the uidA gene with and without the Act-1 intron, cloned in pAB6 and transformed into wheat mature embryo for transient expression. Our results showed that URS-OsCIPK14 fused with the Act-1 intron was induced by NaCl and ABA, whereas URS-OsCIPK14 drove expression of uidA constitutively in the absence of Act-1 intron. Furthermore, rice wide-genome screening using URS-CIPK14 sequence obtained in this study identified another homologue on chromosome 11. Unlike URS-CIPK14, which is associated with chromosome 12, this homologue located at about 1015 nucleotides upstream from the putative TSS of OsCIPK15 coding sequence. Interestingly, the 5’-UTS in CIPK14 is longer than CIPK15 and can code for a putative short ORF that contains two Casein kinase II phosphorylation motifs (SXXD). The significance of this ORF is not known at this time and needs to be elucidated in future studies. We conclude that data from this work suggest that URS-CIPK14 can be used as a promoter candidate for high expression level under both constitutive and inducible conditions.

A novel and rapid method for Agrobacterium-mediated production of stably transformed Cannabis sativa L. plants

The development of genetically transformed plants is an elusive landmark in Cannabis sativa L. breeding. Despite its economic interest, at present, protocols for producing transgenic C. sativa plants are scarce. We studied the ability of hypocotyl, cotyledon and meristem explants from six C. sativa hemp varieties for transgenic plant regeneration. For this, we firstly evaluated in vitro regeneration rates of hypocotyls cultured in medium without plant growth regulators, and cotyledons cultured in medium supplemented with 0.4 mg L−1 of thidiazuron (TDZ) and 0.2 mg L−1 of α-naphthaleneacetic (NAA). Subsequently, the effect of different kanamycin concentrations (50, 100, 200, 500 and 750 mg L−1) on hypocotyl regeneration rate was determined. Finally, we assessed transformation rates after hypocotyl, cotyledon and meristem co-culture with Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid pBIN19 containing the β-glucuronidase (uidA) reporter gene and the kanamycin resistance neomycin phosphotransferase (nptII) genes. Plant transformation was validated through in vitro culture of regenerating shoots in kanamycin-containing selective regeneration medium, by GUS histochemical assay for uidA expression, and by PCR amplification of uidA and nptII genes. Our results showed that hypocotyls reached a higher regeneration rate (53.3 %) than cotyledons (18.1 %) without Agrobacterium co-culture. On the other hand, 100 mg L−1 kanamycin proved to be the best concentration in terms of regeneration rate (63.3 %) and spontaneous rooting rate of hypocotyl regenerating shoots (12.2 %), which displayed a 7.1 % of albinism rate. After co-culture with A. tumefaciens and subsequent culture in antibiotic-containing selective regeneration medium, hypocotyl was the best explant type achieving 23.1 % of regeneration rate, which contrasts with the 1.0 % regeneration rate detected for cotyledons. Transgenic plants were obtained from all explant types evaluated. Although there were significant differences among varieties evaluated, hypocotyls proved to be superior to already-developed meristems, reaching a transformation rate of 5.0 % and 0.8 % respectively. Despite the extremely low regeneration rate of cotyledons after A. tumefaciens co-culture, all cotyledon-derived regenerating shoots analyzed were successfully transformed. Our hormone-free protocol doubles the transformation rate of regenerating shoots, also producing transgenic plants three times faster than other already published protocols. This has relevant implications for C. sativa breeding, enabling not only genetic transformation, but also the use of new plant breeding techniques such as targeted genome editing by using CRISPR/Cas systems. This may foster the development of C. sativa varieties with specific biochemical profiles, or tolerant to biotic and abiotic stresses among others.

Novel and efficient transformation of wild passion fruit (Passiflora cincinnata Mast.) using sonication-assisted Agrobacterium-mediated transformation

Passiflora cincinnata stands out among Passifloraceae because of its medicinal properties and its resistance to pathogens, which promotes its use as a rootstock for other Passiflora species. A valuable strategy for obtaining disease-resistant passion fruit plants is represented by genetic transformation; however, this requires efficient regeneration. Here, we aimed to establish an efficient protocol for generating transgenic passion fruit plants using sonication-assisted Agrobacterium-mediated transformation of somatic embryos. The latter were obtained from anthers, sonicated, and exposed to an Agrobacterium suspension for 15 or 30 s. As a comparison, non-sonicated embryos were also exposed to the same bacterial treatment, whereas non-infected embryos were used as controls. Plant identity was confirmed by PCR, qPCR, and histochemical assay. Transgenic plants were obtained at higher rates in the treatments applying sonication. Overall, 171 plantlets were regenerated, 38 of which showed stable uidA reporter gene expression. Of these 38 transgenic plants, 22 (57.89%) and 13 (34.21%) were obtained by sonication-assisted Agrobacterium-mediated transformation for 30 s and 15 s, respectively, whereas the remaining 3 (7.89%) were exposed for 30 s but without prior sonication. Our results indicate that sonication-assisted Agrobacterium-mediated transformation for 30 s enhanced transformation efficiency in P. cincinnata. We believe that this system will allow for more efficient production of transgenic passion fruit plants.

Overexpression of ICE1 gene in mungbean (Vigna radiata L.) for cold tolerance

The putative transgenic mungbean (Vigna radiata Var. OBGG-52) plants were regenerated on Murashige and Skoog (1962) medium supplemented with 1.0 mg/L 6-benzylaminopurine (BA). The transgenic status of transformants (T2 generation) was determined through PCR analysis using nptII and ICE1 gene-specific primers as well as uidA reporter gene expression. About 75% of the transformed plantlets were successfully established in the greenhouse. Overexpression of ICE1 gene in transgenic mungbean plants resulted in cold-tolerance at the seedling stage when compared to non-transformed plants. Seeds from T2 generation were further studied for cold tolerance through nutrient culture. Transgenic seeds had significantly increased germination and growth on a nutrient medium at 10–14 °C in growth chamber compared to the non-transgenic control. Moreover, the root and shoot length of transformed plants were significantly increased at 10–14 °C as compared with the non-transformed plants. Under cold stress, it was also observed that the transformed plantlets have significantly higher chlorophyll, proline, and lipid content as well as anti oxidative enzyme activity as compared to non-transformed plants. This study will help in mungbean improvement program for abiotic stress tolerance. Cold temperature damaged the crop productivity of mungbean (Vigna radiata L. Wilczek), a high protein rich legume crop. The overexpression of cold responsive transcription factor, INDUCER OF CBF EXPRESSION 1 (ICE1) in mungbean enhanced cold tolerance.

Overexpression of annexin gene in rice (Oryza sativa L.) for salinity and water stress

The aim of the study is to develop transgenic rice with annexin genes (AnnBj2) to determine its salinity and water stress tolerance through Agrobacterium-mediated genetic transformation. The transgenic somatic embryos were developed on Murashige and Skoog medium fortified with 0.5 mg L−1 kinetin and 3.0 mg L−1 2,4-D. About 80% of the transformed somatic embryos germinated and was successfully established in the transgenic greenhouse. The transformed plantlets (T0 and T1 generation) were identified through PCR using NPT-II and AnnBj2 gene–specific primers as well as positive uidA reporter gene expression. Compared with non-transformed plants, the transgenic rice plants overexpressing AnnBj2 gene exhibited salt tolerance at the seedling stage. Seeds generated at T0 and T1 generations were further studied to determine their salinity tolerance through nutrient culture and pot culture experiments for water stress. When compared with the non-transgenic control, transgenic seeds (T0 and T1) had increased germination on a nutrient medium having 200 mM NaCl. The data showed that the shoot and root lengths were longer at 200 mM NaCl when compared with those of the non-transformed plants. Further, the biochemical assessment (photosynthetic pigment analysis, protein, proline content, and oxidative enzyme activity) was performed between transformed (T0 and T1) and non-transformed plantlets under stress. It was noted that the transformed plantlets were higher in chlorophyll and proline content as well as oxidative enzyme activity as compared with the non-transformed plants.

Functional characterization of wheat myo-inositol oxygenase promoter under different abiotic stress conditions in Arabidopsis thaliana.

The production of wheat is severely affected by abiotic stresses such as cold, drought, salinity, and high temperature. Although constitutive promoters are frequently used to regulate the expression of alien genes, these may lead to undesirable side-effects in transgenic plants. Therefore, identification and characterization of an inducible promoter that can express transgene only when exposed to stresses are of great importance in the genetic engineering of crop plants. Previous studies have indicated the abiotic stress-responsive behavior of myo-inositol oxygenase (MIOX) gene in different plants. Here, we isolated the MIOX gene promoter from wheat (TaMIOX). The in-silico analysis revealed the presence of various abiotic stress-responsive cis-elements in the promoter region. The TaMIOX promoter was fused with the UidA reporter gene and transformed into Arabidopsis thaliana. The T3 single-copy homozygous lines were analyzed for GUS activity using histochemical and fluorometric assays. Transcript expression of TaMIOX::UidA was significantly up-regulated by heat (fivefold), cold (sevenfold), and drought (fivefold) stresses as compared to transgenic plants grown without stress-induced conditions. The CaMV35S::UidA plants showed very high GUS activity even in normal conditions. In contrast, the TaMIOX::UidA plants showed prominent GUS activity only in stress treatments (cold, heat, and drought), which suggests the inducible behavior of the TaMIOX promoter. The substrate myo-inositol feeding assay of TaMIOX::UidA plants showed lesser GUS activity as compared to plants treated in abiotic stress conditions. Results support that the TaMIOX promoter could be used as a potential candidate for conditional expression of the transgene in abiotic stress conditions.

Molecular Functional Characterisation of MechlPPDK Promoter in Transgenic Tobacco

Chloroplastic pyruvate phosphate dikinase (PPDK) (chlPPDK) is a key enzyme in the photosynthesis of C4 plants. PPDK is expressed in high abundance in C4 plants but only in trace amounts in C3 plants. The existing research reveals a higher expression of MechlPPDK in cultivated cassava varieties than that in wild cassava W14. However, knowledge about the transcriptional regulation of the MechlPPDK gene in cassava (Manihot esculenta Crantz) is insufficient. Therefore, we aim to identify the transcription profile of MechlPPDK and the core promoter region of MechlPPDK. We cloned the MechlPPDK coding sequence and its 5′-flanking sequence from the cassava variety Ku50. A series of deletion constructions fused to a uidA reporter gene were performed in the 5′-flanking sequence and stably transformed into tobacco. We found that P1 (from −590 bp to +114 bp) had the highest promoter activity among P1 to P3. The 5′-flanking sequence of MechlPPDK responded differently to varied abiotic stresses. Our results will further the understanding of the regulation of MechlPPDK expression.

Microbe-plant interactions between Streptomyces and model agricultural plants – Hordeum vulgare and Lycopersicon esculentum (Microtom)

Aim. Microbe-plant interactions (MPI) constitute an important aspect of ecology because of their significant influence on plant’s ability to withstand abiotic stress and infection. In comparison to proteobacteria and bacilli, the roles of streptomycetes in MPI remain poorly studied. Here, we elucidate some aspects of MPI between two model plant species, Hordeum vulgare and Lycopersicon esculentum, and several strains of Streptomyces lividans 1326 and S. ghanaensis ATCC14672. Methods. Microbiology, microscopy and molecular genetics were combined to reveal the MPI. Results. We demonstrate the colonization of H. vulgare and L. esculentum roots by different strains of S. ghanaensis deficient in production of either the antibiotic moenomycin or signaling molecule of the γ-butyrolactone type. The treatment of H. vulgare seeds with S. lividans spores increased the root biomass. Plants treated with 1,4-butyrolactone had no positive influence on plants, at milimolar concentrations this compound inhibited the root and shoot growth of L. esculentum. Conclusions. Roots of two mono- and dicot plants are colonized by Streptomyces; reporter gene uidA is useful to monitor the colonization. Under our experimental conditions the ability to colonize plants by streptomycetes was not affected by the deficiency in antibiotic or butenolide production.
 Keywords: Streptomyces ghanaensis, moenomycinA, low-molecular weight signal compounds, root colonization.

Engineered Male Sterility by Early Anther Ablation Using the Pea Anther-Specific Promoter PsEND1.

Genetic engineered male sterility has different applications, ranging from hybrid seed production to bioconfinement of transgenes in genetic modified crops. The impact of this technology is currently patent in a wide range of crops, including legumes, which has helped to deal with the challenges of global food security. Production of engineered male sterile plants by expression of a ribonuclease gene under the control of an anther- or pollen-specific promoter has proven to be an efficient way to generate pollen-free elite cultivars. In the last years, we have been studying the genetic control of flower development in legumes and several genes that are specifically expressed in a determinate floral organ were identified. Pisum sativum ENDOTHECIUM 1 (PsEND1) is a pea anther-specific gene displaying very early expression in the anther primordium cells. This expression pattern has been assessed in both model plants and crops (tomato, tobacco, oilseed rape, rice, wheat) using genetic constructs carrying the PsEND1 promoter fused to the uidA reporter gene. This promoter fused to the barnase gene produces full anther ablation at early developmental stages, preventing the production of mature pollen grains in all plant species tested. Additional effects produced by the early anther ablation in the PsEND1::barnase-barstar plants, with interesting biotechnological applications, have also been described, such as redirection of resources to increase vegetative growth, reduction of the need for deadheading to extend the flowering period, or elimination of pollen allergens in ornamental plants (Kalanchoe, Pelargonium). Moreover, early anther ablation in transgenic PsEND1::barnase-barstar tomato plants promotes the developing of the ovaries into parthenocarpic fruits due to the absence of signals generated during the fertilization process and can be considered an efficient tool to promote fruit set and to produce seedless fruits. In legumes, the production of new hybrid cultivars will contribute to enhance yield and productivity by exploiting the hybrid vigor generated. The PsEND1::barnase-barstar construct could be also useful to generate parental lines in hybrid breeding approaches to produce new cultivars in different legume species.

English

Horticulture is one of the fastest growing sectors in Uganda, exporting products worth US$100 million annually. Passion fruit (Passiflora edulis) growing and export is one of the critical contributors to this sector employing over a million farmers. However, a number of biotic and abiotic constraints have initiated widespread enterprise abandonment by farmers. Passiflora improvement efforts by conventional breeding has had limited success calling for research into alternative approaches such as genetic engineering. The study aimed at optimizing existing protocols to develop an efficient and reproducible Agrobacterium mediated transformation system to suit Uganda’s Passiflora cultivars. Agrobacterium tumefaciens strain AGL1 (OD600 of 0.5) harbouring pCAMBIA2301 containing the GUS (uidA) reporter gene was used to infect pre-cultured leaf discs. Leaf discs were then vacuum infiltrated for 1.5 min at 750 mmHg followed by a three day co-cultivation period on MS + acetosyringone (100 µml-1). Putatively transgenic yellow passion fruit shoots were induced on Murashige and Skoog (MS) selection media supplemented with benzylaminopurine (BAP) 8.9 μM, kanamycin (100 mgL-1mgl) and cefotaxime (500 mgL-1). Developed shoots were then transferred to elongation media (MS + 0.44 µM BAP) and later rooted on 5.37 µM naphthaleneacetic acid (NAA). Genetic transformation was monitored using GUS staining. A single independently transformed plant was confirmed by polymerase chain reaction (PCR), translating in a transformation efficiency of 0.456%. A viable in vitro transformation protocol for Uganda’s yellow passion fruit directly from leaf discs was developed using GUS reporter gene. Further investigations are required to improve the reported protocols transformation efficiency.   Key words: Passion fruit, Passiflora edulis, Passiflora improvement, genetic engineering, transformation system.

Transient gene expression in western white pine using agroinfiltration

A method for transient gene expression was developed for western white pine (WWP, Pinus monticola Dougl. ex D.Don) using reporter gene uidA encoding β-glucuronidase (GUS). GUS was transiently expressed in cross sections of primary and secondary needles, cotyledons, and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens. Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration (dpa). GUS activity expressed inside WWP cells was confirmed using light microscopy. In fluorometric assays, GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles, while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly. Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons, all tested WWP tissues using the protocol had high levels of transient GUS expression. Thus, heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach. The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.

Identification and validation of organ-preferential genes and analysis of corresponding upstream tissue-specific promoters in wheat

Tissue/organ-specific promoters are important tools in genetic engineering and crop molecular breeding. They are well characterized in dicots, such as Arabidopsis, tobacco, and tomato, but not sufficiently in monocots, especially in wheat. In this study, the genes specifically expressed in seven different tissues, including coleoptile, root, leaf, pistil, anther, embryo, and endosperm were identified through analyzing the public transcriptome data from a wheat microarray using the ROKU method. The expression patterns of selected genes were validated by reverse transcription polymerase chain reaction. The results showed that these selected genes were expressed specifically or preferentially in each representative tissue/organ. Moreover, the function of their promoters was verified by transient expression in wheat or stable transformation in Arabidopsis. The results showed that these promoters can efficiently and predominantly drive uidA (β-glucuronidase) reporter gene expression in different tissues. Due to their tissue-specific nature, these promoters can be used as potential candidates in plant genetic engineering.

Expression of uidA gene in tobacco transplastomic plants under the control of 5'-untranslated region of genomic RNA of potato virus Y

Regulation of the expression of chloroplast genes mainly occurs at the post-transcriptional level, in which 5'-untranslated regions (5'UTR) of plastid mRNA play a significant role. In transplastomic tobacco plants, we studied the effect of the 5’UTRs of the genomic RNA of potato Y-virus (5’PVY), as well as the 5’UTR of the plastid psbA gene (5’psbA) on the expression of the bacterial reporter uidA gene encoding β-glucuronidase (GUS). Genetic constructs containing the uidA gene were created under the control of identical transcriptional regulatory elements: the promoter and terminator of the psbA gene and two different 5’UTRs — 5’psbA and 5’PVY. Тobacco chloroplasts were transformed with these constructs by bioballistic method. Transplastomic lines showed approximately 24-fold differences in GUS activity. Replacing the natural 5’psbA with the heterologous 5’PVY resulted in a significant decrease in the synthesis of GUS protein. Key words: chloroplasts, protein synthesis, 5’UTR, transplastomic plants, reporter protein.

Evaluación del potencial biotecnológico de un promotor derivado del virus de la distorsión de la hoja de maracuyá (PLDV), un begomovirus que infecta maracuyá

Los avances biotecnológicos en plantas requieren la bioprospección de nuevos promotores para la expresión de genes de interésagronómico, en particular, es necesario caracterizar nuevos promotores con expresión tejido específica. El objetivo de esta investi-gación fue evaluar la actividad de expresión del promotor del gen AV1que codifica para la proteína de la cápside (CP) del virus de la distorsión de la hoja de maracuyá (Passion fruit leaf distortion virus,PLDV) mediante ensayos transitorios de biobalística de baja presión. Se realizó un análisis de la región promotora del gen AV1empleando herramientas bioinformáticas. Se construyó una fu-sión traduccional (CP-PLDV-GUS), que porta la región promotora del gen AV1de PLDV fusionada al gen reportero uidA(GUS). CP-PLDV-GUS fue bombardeado sobre hojas de plántulas de tabaco cultivadas in vitro empleando una pistola de genes. Como control positivo se utilizó el plásmido pBI121 que porta el gen GUS bajo el control del promotor 35S de CaMV. Se llevaron a cabo 11 re-peticiones, donde la unidad experimental fue la hoja y la variable de respuesta, la expresión transitoria del gen GUS representado por el número de puntos azules observados en las hojas bombardeadas. Como resultado, el análisis estadístico no paramétrico demostró que existe evidencia muestral suficiente para confirmar que, tanto el promotor AV1del PLDV y 35S de CaMV presentan una actividad de expresión semejante. Finalmente, el promotor del gen AV1de PLDV mostró una fuerte actividad de expresión del gen reportero en las células del mesófilo de las hojas, el cual podría ser usado para conferir expresión tejido específica enplantas transgénicas

Susceptibility of Maize Mesocotyl Culture to Agrobacterium Transformation and Its in vitro Regeneration

The capacity of mesocotyls to agrobacterially transform four maize lines 4766, RB-179, L-1, L-2 and the conditions for their in vitro regeneration have been studied. Regeneration of maize mesocotyls was observed in media containing various auxins and cytokinins combinations. The efficiency of the process depended on the qualitative and quantitative compositions of growth regulators. The frequency of regenerated explants and the average number of shoots that form on one explant increased with increased cytokinin 6‑BAP and defoliant Dropp (thidiazuron) concentrations in the medium. 6-BAP (3–5 mg/L) and Dropp (3 mg/L) in combination with auxins (IAA, 0.5 mg/L and 2,4-D, 2 mg/L) were shown to be most effective for the mesocotyl regeneration. The number of regenerated explants reached 59.0%, whereas the average number of shoots per explant was 7.5 after 6 weeks of cultivation. The rooting and growing of regenerated shoots was successfully carried out on MS medium supplemented with 0.2 mg/L IBA. The capacity of the mesocotyl was determined by Agrobacterium tumefaciens–mediated transformation (ATMT) with an AGL0 strain containing the pVecActI-GUS genetic construction encoding the reporter gene uidA under the control of the promoter of rice actin ActI gene and the selective hptII hygromycin resistance gene. PCR analysis demonstrated that few regenerants with resistance to the selective agent contained the insertion of the hptII antibiotic-resistance gene in their genomes. Thus, it was shown that maize mesocotyls can be used as explants for agrobacterial transformation.

Cloning of the Coffea canephora SERK1 promoter and its molecular analysis during the cell-to-embryo transition

Somatic embryogenesis receptor-like kinase 1 (SERK1) is a cell membrane receptor active in different plant tissues and involved in cell differentiation activities including somatic embryogenesis. The identification of promoter elements responsible for SERK1 expression during the onset of somatic embryogenesis can be useful to understand the molecular regulation of the cell-to embryo transition, and these promoter elements represent biotechnological tools in plant organ tissue culture. A − 1,620 bp DNA sequence located upstream of the Coffea canephora SERK1 gene homologue (CcSERK1) was isolated, and then, different segments containing key response elements (REs) for somatic embryogenesis onset and development were fused to the uidA (encoding a β-glucuronidase, GUS) reporter gene to evaluate its expression in transgenic leaf explants. DNA segments of − 1,620 and − 1048 bp in length directed uidA expression with patterns in leaf explants similar to those occurring during somatic embryogenesis. When a − 792-bp fragment was used, uidA expression disappeared only in leaf explants and pro-embryogenic mass but persisted in developing embryos. No uidA expression was detected in any embryogenic stage when a − 618-bp fragment was used. DNA deletions showed that a − 1048-bp sequence located upstream of the CcSERK1 gene is sufficient to direct gene expression during the onset and the development of C. canephora somatic embryogenesis. The DNA segment located between − 1048 and − 792 bp (containing BBM and WUS REs) is needed for gene expression before embryogenesis onset but not during embryo development. The promoter segment between − 792 and − 618 bp (including GATA, ARR1AT, and ANT REs) regulates gene expression in developing embryos. How to cite: Jiménez-Guillen D, Pérez-Pascual D, Souza-Perera R, et al. Cloning of the Coffea canephora SERK1 promoter and its molecular analysis during the cell-to-embryo transition. Electron J Biotechnol 2018;36.https://doi.org/10.1016/j.ejbt.2018.08.005.