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Article in English | WPRIM | ID: wpr-929063


Cucurbitaceae is an important family of flowering plants containing multiple species of important food plants, such as melons, cucumbers, squashes, and pumpkins. However, a highly efficient genetic transformation system has not been established for most of these species (Nanasato and Tabei, 2020). Watermelon (Citrullus lanatus), an economically important and globally cultivated fruit crop, is a model species for fruit quality research due to its rich diversity of fruit size, shape, flavor, aroma, texture, peel and flesh color, and nutritional composition (Guo et al., 2019). Through pan-genome sequencing, many candidate loci associated with fruit quality traits have been identified (Guo et al., 2019). However, few of these loci have been validated. The major barrier is the low transformation efficiency of the species, with only few successful cases of genetic transformation reported so far (Tian et al., 2017; Feng et al., 2021; Wang JF et al., 2021; Wang YP et al., 2021). For example, Tian et al. (2017) obtained only 16 transgenic lines from about 960 cotyledon fragments, yielding a transformation efficiency of 1.67%. Therefore, efficient genetic transformation could not only facilitate the functional genomic studies in watermelon as well as other horticultural species, but also speed up the transgenic and genome-editing breeding.

CRISPR-Cas Systems , Citrullus/genetics , Cucurbitaceae/genetics , Gene Editing , Plant Breeding , Transformation, Genetic
Article in Chinese | WPRIM | ID: wpr-928108


Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.

Agaricales , Gastrodia/genetics , Protoplasts , Symbiosis/genetics , Transformation, Genetic
Electron. j. biotechnol ; 51: 95-109, May. 2021. tab, ilus
Article in English | LILACS | ID: biblio-1343466


Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology's high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots.

Transformation, Genetic , Biological Products , Chloroplasts , Crops, Agricultural , Biotechnology , Recombinant Proteins/biosynthesis , Plants, Genetically Modified
Electron J Biotechnol ; 49: 42-49, Jan. 2021. tab, graf, ilus
Article in English | LILACS | ID: biblio-1291646


BACKGROUND: Late embryogenesis abundant (LEA) proteins were reported to be related to adversity stress and drought tolerance. Lea-3 from Arachis hypogaea L. (AhLea-3) was previously found to be related to salt tolerance according to the result of transcriptome profiling and digital gene expression analysis. So, AhLea-3 was cloned and the salt tolerance was validated by transgenic peanut plants. RESULTS: AhLea-3 was isolated from M34, a salt-resistant mutant of peanut, with its cDNA as the template. AhLea-3 contains one intron and two extrons, and the full-length cDNA sequence contains 303 bp. AhLea3 was ligated to pCAMBIA1301 to obtain the overexpression vector pCAMBIA1301-AhLea-3, which was then transferred into peanut variety Huayu23. The expression level of AhLea-3, as determined by qRTPCR analysis, was >10 times higher in transgenic than in non-transgenic plants. Five days after they were irrigated with 250 mM NaCl, the transgenic plants showed less severe leaf wilting, higher activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase), and lower malonic dialdehyde content than non-transgenic plants. Relative to non-transgenic plants, the transgenic plants had a higher photosynthetic net rate, stomatal conductance, and transpiration rate, and a lower intercellular CO2 concentration after salt stress treatment (250 mM NaCl). CONCLUSIONS: These results indicate that overexpression of AhLea-3 increased the salt tolerance of transgenic peanut plants. AhLea-3 might become a useful gene resource for the variety breeding of salinity tolerance in peanut.

Arachis/metabolism , Plant Proteins/metabolism , Plants, Genetically Modified/metabolism , Salt Tolerance , Arachis/genetics , Plant Proteins/isolation & purification , Transformation, Genetic
Electron. j. biotechnol ; 45: 1-9, May 15, 2020. ilus
Article in English | LILACS | ID: biblio-1177370


BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

Tobacco/metabolism , Chloroplasts/genetics , Chloroplasts/metabolism , Zea mays/genetics , Green Fluorescent Proteins/metabolism , Transformation, Genetic , Biotechnology , Polymerase Chain Reaction , Plants, Genetically Modified , Plastids/genetics , Green Fluorescent Proteins/genetics , Escherichia coli , Genome, Chloroplast
Braz. arch. biol. technol ; 63: e20180679, 2020. tab, graf
Article in English | LILACS | ID: biblio-1132162


Abstract we report A. rhizogenes-induced hairy root formation in S. bryopteris, a medicinally and commercially important plant. A. rhizogenes strain LBA1334 co-cultivated with explants (root, rhizophore, stem portion near the root, and stem with intact fronds) for 24 and 48 h after transformation for induction of hairy roots. The induction of hairy root was observed after 6 days of infection in case of 48 h co-cultivation only. PCR with rolA and virC gene specific primers confirmed the induced hairy roots were due to Ri T-DNA integration and not due to contaminating A. rhizogenes. The root network as explants showed the maximum transformation efficiency. We tested different media like MS, SHFR (Stage Hog Fern Root) and KNOP's during transformation for hairy root induction. The SHFR based media showed good response in transformation as well as propagation. Further, transformation efficiency was enhanced by addition of TDZ (2 mg/L) and Bevistin (0.1%) in SHFR media. The present work would be helpful in hairy roots-based in vitro production of secondary metabolites and on aspect of functional genomics of S. bryopteris.

Transformation, Genetic/genetics , Polymerase Chain Reaction , Selaginellaceae/microbiology , Agrobacterium/genetics , Genomics
Article in Chinese | WPRIM | ID: wpr-828008


The WRKY family genes, which play an important role in plant morphogenesis and stress response, were selected based on the data of the full-length transcriptome of Asarum heterotropoides. Using AtWRKY33, which regulates the synthesis of the camalexin in the model plant Arabidopsis to compare homologous genes in A. heterotropoides, primers were designed to amplify the open reading frame(ORF) fragment of AhWRKY33 gene by RT-PCR using total RNA of A. heterotropoides leaves as template. Real-time PCR results showed that there was a significant difference between the aerial part and the underground part of A. heterotropoides, the toxic aristolochic acid content is highly expressed in the leaves higher than the root. After verification, the WRKY33 gene of A. heterotropoides is ORF long 1 686 bp, encoding 561 amino acids.AhWRKY33 had two conserved WRKYGQK domains. According to the classical classification, it belongs to group Ⅰ WRKY transcription factor. A. heterotropoides WRKY33 had some homology with amino acids of other species. The study successfully constructed the plant eukaryotic expression vector PHG-AhWRKY33 and transformed Arabidopsis thaliana, the transgenic Arabidopsis was obtained by PCR detection and hygromycin resistant plate screening. It found that the germination of transgenic Arabidopsis seeds was accelerated and the stress resistance was increased. It laid a foundation for further analysis of WRKY transcription factor in the growth and development of A. heterotropoides and the synthesis of secondary metabolites.

Arabidopsis , Genetics , Arabidopsis Proteins , Genetics , Asarum , Cloning, Molecular , Gene Expression Regulation, Plant , Plant Leaves , Plant Proteins , Genetics , Transcription Factors , Transformation, Genetic
Chinese Journal of Biotechnology ; (12): 643-651, 2020.
Article in Chinese | WPRIM | ID: wpr-827004


Cucumber (Cucumis sativus) is an important vegetable crop in the world. Agrobacterium-mediated transgenic technology is an important way to study plant gene functions and improve varieties. In order to further accelerate the transgenic research and breeding process of cucumber, we described the progress and problems of Agrobacterium tumefaciens-mediated transgenic cucumber, from the influencing factors of cucumber regeneration ability, genetic transformation conditions and various additives in the process. We prospected for improving the genetic transformation efficiency and safety selection markers of cucumber, and hoped to provide reference for the research of cucumber resistance breeding and quality improvement.

Agrobacterium tumefaciens , Metabolism , Breeding , Cucumis sativus , Genetics , Microbiology , Plants, Genetically Modified , Microbiology , Research , Transformation, Genetic
Chinese Journal of Biotechnology ; (12): 700-706, 2020.
Article in Chinese | WPRIM | ID: wpr-826906


The responsibility of root is absorbing water and nutrients, it is an important plant tissue, but easily to be affected by biotic and abiotic stresses, affecting crop growth and yield. The design of a synthetic root-specific promoter provides candidate promoters for the functional analysis and efficient expression of stress-related genes in crop roots. In this study, a synthetic root-specific module (pro-SRS) was designed using tandem four-copies of root specific cis-acting elements (OSE1ROOTNODULE, OSE2ROOTNODULE, SP8BFIBSP8AIB, and ROOTMOTIFAPOX1), and fused with minimal promoter from the CaMV 35S promoter to synthesize an artificially synthetic SRSP promoter. The SRSP promoter was cloned in pCAMBIA2300.1 by replacing CaMV 35S promoter so as to drive GUS expression. The constructs with SRSP promoter were transformed in tobacco by Agrobacterium-mediated method. SRSP promoter conferred root-specific expression in transgenic tobacco plants through Real-time PCR (RT-PCR) analysis and GUS histochemical staining analysis. It is indicated that the repeated arrangement of cis-acting elements can realize the expected function of the promoter. This study laid a theoretical foundation for the rational design of tissue-specific promoters.

Agrobacterium , Genetics , Cloning, Molecular , Gene Expression Regulation, Plant , Plant Roots , Genetics , Plants, Genetically Modified , Promoter Regions, Genetic , Genetics , Stress, Physiological , Tobacco , Genetics , Transformation, Genetic
Electron. j. biotechnol ; 41: 22-29, sept. 2019. ilus
Article in English | LILACS | ID: biblio-1087255


We highlight the importance of the mixed genetic approaches (classical and currents) to improve the social perception related to the GMOs acceptance. We pointed out that CRISPR/Cas9 events could carry DNA variability/rearrangements related to somaclonal variations or epigenetic changes that are independent from the editing per se. The transformation of single cells, followed by plant regeneration, is used to generate modified plants, transgenic or genome editing (CRISPR/Cas9). The incidence of undesirable somaclonal variations and/or epigenetic changes that might have occurred during in vitro multiplication and regeneration processes, must be carefully analyzed in replicates in field trials. One remarkable challenge is related to the time lapse that selects the modified elite genotypes, because these strategies may spend a variable amount of time before the results are commercialized, where in all the cases it should be take into account the genotype × environment interactions. Furthermore, this combination of techniques can create an encouraging bridge between the public opinion and the community of geneticists who are concerned with plant genetic improvement. In this context, either transgenesis or genomic editing strategies become complementary modern tools to facing the challenges of plant genetic improvement. Their applications will depend on case-by-case analysis, and when possible will necessary associate them to the schemes and bases of classic plant genetic improvement.

Plants, Genetically Modified , Gene Transfer Techniques , CRISPR-Cas Systems , Gene Editing , Transformation, Genetic , Mutagenesis , DNA Methylation , Genetic Enhancement , Epigenesis, Genetic
Electron. j. biotechnol ; 40: 45-51, July. 2019. ilus, tab
Article in English | LILACS | ID: biblio-1053457


Background: This research is intended to determine suitable types and concentrations of plant growth regulators (PGRs) to induce callus on stem and leaf sections of 4 species of the genus Garcinia, namely, Garcinia mangostana, Garcinia schomburgkiana, Garcinia cowa, and Garcinia celebica. The base medium was MS medium containing 30 g l -1 sucrose, 0.5 g l-1 polyvinylpyrrolidone (PVP), and 7 g l-1 agar, and for the different treatments, PGRs were added to the medium as follows: thidiazuron (TDZ) at concentrations of 0, 0.1, 0.5, 1, and 2 mg l-1; 6-(3- hydroxybenzylamino) purine (meta-topolin) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; 4-amino-3,5,6- trichloro-2-pyridinecarboxylic acid (picloram) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; and 2,4- dichlorophenoxyacetic acid (2,4-D) at concentrations of 0, 0.5, 1, 2, and 4 mg l-1. The occurrence of callus was observed after 4 weeks. Results: A maximum of 100% and 93% of G. mangostana leaf explants formed callus in the 0.5 mg l-1 and 1 mg l-1 TDZ treatments, respectively, while 100% of G. schomburgkiana stem explants formed callus in the 1 mg l-1 TDZ treatment and 89% of G. schomburgkiana leaf explants formed callus in the 0.5 mg l-1 picloram treatment. The highest callus induction rate for G. cowa was 62% in the 1 mg l-1 TDZ treatment and for G. celebica was 56% in the 0.5 mg l-1•mT-1 treatment. Conclusions: For all 4 species, the greatest amount of large nodular callus was observed in the TDZ treatments. White, friable callus was observed on most of the 2,4-D and picloram treatment groups. Most meta-topolin treatments resulted in minimal callus formation.

Plant Growth Regulators/metabolism , Garcinia/growth & development , Phytochemicals/metabolism , Phenylurea Compounds , Thiadiazoles , Time Factors , Transformation, Genetic , Clusiaceae/growth & development , Garcinia/physiology , Tissue Culture Techniques
Electron. j. biotechnol ; 34: 51-58, july. 2018. ilus, tab, graf
Article in English | LILACS | ID: biblio-1046711


Background: Capsicum is a genus of an important spice crop that belongs to the chili lineage. However, many Capsicum species (family Solanaceae) are known to be recalcitrant to genetic transformation and in vitro regeneration, thus hampering the effort in using Capsicum species for detailed biological investigation. In this study, we have developed an optimized protocol for the direct transformation of Capsicum frutescens L. cv. Hot Lava using a biolistic particle delivery system. In addition, a procedure for in vitro whole plant regeneration from the hypocotyl explants of C. frutescens was established. Results: In this study on the biolistic system, explant target distance, bombardment helium (He) pressure, and the size of the microcarrier were the key parameters to be investigated. The optimized parameters based on the screening of GFP expression were determined to have a target distance of 6 cm, helium pressure of 1350 psi, and gold particle (microcarrier) size of 1.6 µm. The greatest number of shoots was obtained from hypocotyls as explants using Murashige and Skoog medium supplemented with 5.0-mg/L 6-benzylaminopurine and 0.1-mg/L 1-naphthaleneacetic acid. On an average, five shoots per explant were formed, and of them, one shoot managed to form the root and developed into a whole plant. Conclusions: We obtained an optimized protocol for the biolistic transformation of chili and in vitro regeneration of chili plantlets. The establishment of the protocols will provide a platform for molecular breeding and biological studies of chili plants.

Capsicum/growth & development , Regeneration , Transformation, Genetic , In Vitro Techniques , Capsicum/genetics , Polymerase Chain Reaction , Biolistics , Green Fluorescent Proteins , Tissue Culture Techniques , Metabolic Engineering
Electron. j. biotechnol ; 32: 6-12, Mar. 2018. tab, graf, ilus
Article in English | LILACS | ID: biblio-1022493


Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier.

Basidiomycota/metabolism , Fungal Proteins/genetics , Lentinula/genetics , Lentinula/metabolism , Transformation, Genetic , Basidiomycota/enzymology , Yeasts , Fungal Proteins/metabolism , Blotting, Southern , Cloning, Molecular , Agrobacterium tumefaciens/metabolism , Sequence Analysis , Emulsifying Agents , Electrophoresis, Polyacrylamide Gel , Real-Time Polymerase Chain Reaction , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Microscopy, Fluorescence
Electron. j. biotechnol ; 30: 1-5, nov. 2017. ilus, tab, graf
Article in English | LILACS | ID: biblio-1021034


Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Seeds/enzymology , Hordeum/enzymology , Thermococcus/metabolism , alpha-Amylases/metabolism , Seeds/genetics , Seeds/microbiology , Transformation, Genetic , Hordeum/genetics , Hordeum/microbiology , Beer , Enzyme Stability , Plants, Genetically Modified/enzymology , Cloning, Molecular , Gene Transfer Techniques , alpha-Amylases/genetics , Fermentation , Thermotolerance , Hot Temperature , Hydrogen-Ion Concentration
Electron. j. biotechnol ; 29: 32-38, sept. 2017. tab, ilus, graf
Article in English | LILACS | ID: biblio-1017075


Background: We aimed to test the possibility of improving polypeptide production from soybean meal fermentation by engineered Aspergillus oryzae strains. Four different protease genes were cloned and transformed into wild-type A. oryzae, and the engineered A. oryzae strains were then used for soybean meal fermentation. Results: The results showed different degrees of improvement in the protease activity of the four transformants when compared with wild-type A. oryzae. A major improvement in the polypeptide yield was achieved when these strains were used in soybean meal fermentation. The polypeptide conversion rate of one of the four transformants, A. oryzae pep, reached 35.9%, which was approximately twofold higher than that exhibited by wild-type A. oryzae. Amino acid content analysis showed that the essential amino acid content and amino acid composition of the fermentation product significantly improved when engineered A. oryzae strains were used for soybean meal fermentation. Conclusions: These findings suggest that cloning of microbial protease genes with good physicochemical properties and expressing them in an ideal host such as A. oryzae is a novel strategy to enhance the value of soybean meal.

Peptide Hydrolases/metabolism , Aspergillus oryzae/enzymology , Aspergillus oryzae/genetics , Peptide Hydrolases/genetics , Soybeans , Transformation, Genetic , Genetic Engineering , Cloning, Molecular , Fermentation , Flour , Amino Acids/analysis
Electron. j. biotechnol ; 29: 63-67, sept. 2017. ilus, tab, graf
Article in English | LILACS | ID: biblio-1017249


Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5' or 3' untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression. Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5' SD sequence at the 5' UTR of pulA. pulA-3t was constructed by the addition of a 3' stem-loop structure at the 3' UTR of pulA. SD-pulA-3t was constructed by the addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively. Conclusion: The addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.

Escherichia coli/enzymology , Escherichia coli/genetics , Glycoside Hydrolases/metabolism , Transformation, Genetic , Gene Expression , Reverse Transcriptase Polymerase Chain Reaction , RNA Stability , Fermentation , Genetic Vectors , Glycoside Hydrolases/genetics
Electron. j. biotechnol ; 26: 40-45, Mar. 2017. ilus, graf
Article in English | LILACS | ID: biblio-1009000


Background: Ethylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results: In total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion: These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.

Soybeans/enzymology , Soybeans/genetics , RNA Interference , Lyases/metabolism , Soybeans/growth & development , Transformation, Genetic , Gene Expression , Cell Differentiation , Polymerase Chain Reaction , Gene Expression Regulation, Plant , Ethylenes/biosynthesis , Herbicide Resistance , Genetic Vectors , Glucuronidase
Electron. j. biotechnol ; 25: 33-38, ene. 2017. tab, ilus
Article in English | LILACS | ID: biblio-1008414


Background: Banana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics. Results: In this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 µM benzylaminopurine and 9.1 µM thidiazuron. One immature male flower could regenerate 380­456, 310­372, 200­240, 130­156, and 100­130 well-developed shoots in only 240­270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless ß-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot. Conclusions: Our robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.

Musa/growth & development , Musa/genetics , Regeneration , Transformation, Genetic , Immunohistochemistry , Blotting, Southern , Polymerase Chain Reaction , Plants, Genetically Modified , Agrobacterium tumefaciens/physiology , Musa/microbiology , Organogenesis, Plant , Glucuronidase
Journal of Medicinal Plants. 2017; 16 (Supp. 10): 102-109
in Persian | IMEMR | ID: emr-185699


Background: Licorice, Glycyrrhiza glabra [belong to Leguminosae family] is one of the most popular medicinal plants in the world and it is widely used in many fields such as medical, pharmaceutical, confectionery and health industries. Different parts of licorice [shoots, leaves and roots] were had various components such as Glycyrrhzin that was used for some proposes

Objective: The current study was done with the aim of gene transfer via Agrobacterium rhizogenes by ex vitro method for hairy root production in licorice

Methods: The experiment was laid out as a completely randomized design [CRD] with five treatments in three replications. At first, root of young plantlets was eliminated and excited plantlets were putted in the glass wool contain suspension of bacteria. After 10 to 14 days of inoculation with Agrobacterium rhizogenes, the roots were appeared. The percentage of root induction by four strains of Agrobacterium [ATCC 15834, GMI 9534, A4 and A13] with check [without bacteria] was investigated

Results: The results of PCR analysis with specific primers for roots of composite plants [putative transgenic] was shown that three strains of bacteria [A4, A13 and GMI 9534] and strain ATCC 15834, were produced 100% and 66.66% transgenic roots respectively

Conclusion: Thus, production of composite licorice plants was remarked due to it has low cost, fast and simple

Glycyrrhiza/genetics , Agrobacterium/genetics , Transformation, Genetic
Journal of Medicinal Plants. 2017; 16 (Supp. 10): 141-155
in Persian | IMEMR | ID: emr-185703


Background: Using Agrobacterium rhizogenes due to create hairy roots, is a useful method to increase secondary metabolites many plants

Objective: Purpose of this research is to transgenic hairy roots culture, in order to produce secondary metabolites in Datura innoxia

Methods: Explants leaf and cotyledon of Datura innoxia were inoculated for two months with A7, A4 and 15834 strains of Agrobacterium rhizogenes; Furthermore injection and Immersion methods were used in this scrutiny. The presence of T-DNA in transgenic hairy roots were confirmed by PCR. Transgenic hairy roots in liquid medium of 1/2MS were cultured. In order to induct elicitors, methyl jasmonate in tow densities of 50 micro M and 100micro M, and salicylic acid in three densities of 1mM, 0.1mM and 0.01 mM were used randomly three times. Atropine and scopolamine content of transgenic hairy roots were examined by HPLC

Results: The highest and lowest rate of transgenic hairy roots production was respectively related to the strains of A4 and 15834. Best explants for inoculation, leaf with A4 strain and cotyledon with A7 strain, were reported. With highest production rate of hairy roots, Simple deposit using a syringes method was recognized as the best method of inoculation. The effect of salicylic acid at a density of 0.1 mM increases the content of atropine concentrations. Also the results showed that usage of Methyl jasmonate at higher doses [100 micro M] reduces the content of atropine and scopolamine

Conclusion: A. rhizogenes as an appropriate method to produce hairy roots and elicitors the best treatment for increase alkaloids

Gene Transfer Techniques/trends , Agrobacterium/genetics , Transformation, Genetic , Salicylates , Oils, Volatile , Scopolamine Derivatives