Mediated Transformation Research Articles

Transformation of tetracycline by multipurpose Fe-Mn-Cu nano-composite oxide: Dual-synergies and dual-mechanisms

The interaction between tetracycline (TTC) and mixed metallic oxides remains unclear, and even complexation usually is ignored. This study firstly distinguished the triple functions of adsorption, transformation and complexation in presence of Fe-Mn-Cu nano-composite metallic oxide (FMC) on TTC. Rapid adsorption and faint complexation initiated the transformation that dominated the entire reactions at 180 min, which completed TTC removal (up to 99.04%) synergistically within 48 h. Environmental factors (dosage, pH and coexisting ions) had small influence on TTC removal, which primarily depended on the stable transformation characteristics of FMC. Kinetic models incorporating pseudo-second-order kinetics and transformation reaction kinetics demonstrated that the surface sites of FMC promoted electron transfer process through chemical adsorption and electrostatic attraction. ProtoFit program coupled with characterization methods concluded that Cu-OH was the main reaction site of FMC where the protonated surface favored to generate·O2–. Meanwhile, three metal ions developed simultaneous mediated transformation reactions on TTC in liquid phase, and·O2– induced the production of·OH. The transformed products were subjected to toxicity assessment, which had lost antimicrobial properties toward Escherichia coli. Insights gained from this study can refine the dual mechanisms of multipurpose FMC in solid and liquid phases underlying TTC transformation.

Improving Agrobacterium tumefaciens-Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.).

In the post-genomics era, Agrobacterium tumefaciens-mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot-regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co-cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot-regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot-regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non-transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2-1.7%. Screening among T1 OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T-DNA insertion as a potential source of mutagenesis. The Agrobacterium-mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.

Down-regulation of FBP1 in lung adenocarcinoma cells promotes proliferation and invasion through SLUG mediated epithelial mesenchymal transformation.

Metabolic reprogramming and epithelial-mesenchymal transformation (EMT) play an important role in lung cancer. In recent studies, metabolic enzymes such as Fructose-1,6-bisphosphatase 1 (FBP1) have shown potential functions beyond regulating metabolism. Western blot assay was performed to detect glycolysis-related and EMT-related protein expression levels. The glucose uptake kit and adenosine triphosphate (ATP) detection kit were used to detect glucose uptake rate and ATP content. Transwell assay was used to determine the invasiveness of lung adenocarcinoma cells. Wound healing assay was used to determine the metastatic ability of lung adenocarcinoma cells. Methyl thiazolyl tetrazolium (MTT) assay and EdU staining were performed to investigate the effect of FBP1 overexpression on lung adenocarcinoma proliferation. Overexpression of FBP1 down-regulated glycolysis-related protein levels and inhibited glucose uptake and ATP production, while knockdown of FBP1 had the opposite effect. Overexpression of FBP1 reversed EMT and inhibited Slug expression. Meanwhile, overexpression of FBP1 impaired the invasion, metastasis and proliferation ability of lung adenocarcinoma cells. In contrast, FBP1 knockdown promoted the EMT process, up-regulated Slug expression and enhanced the invasion, metastasis and proliferation of lung adenocarcinoma cells. Therefore, FBP1 can be used as one of the potential clinical targets through inhibiting glycolysis, cell invasion and proliferation by inhibiting Slug mediated EMT processes.

Impact of cefotaxime and kanamycin on in vitro regeneration via Agrobacterium mediated transformation in apple cv. Red Chief

Impact of cefotaxime and kanamycin on in vitro regeneration via Agrobacterium mediated transformation in apple cv. Red Chief

In planta transformation of Polianthes tuberosa for concomitant knockdown of flp-1, flp-12 and flp-18 genes induced root-knot nematode resistance

Polianthes tuberosa is a lucrative crop owing to its huge demand in floriculture, perfume and pharmaceutical industry. Root-knot nematodes, especially Meloidogyne incognita poses a major impediment on tuberose yield. Currently nematode management relies on the use of hazardous nematicides and other inefficacious means viz. crop rotation and soil solarization. The ongoing search for an ecologically sustainable and target specific approach, led to the progression of genetic modification based techniques to target genes critical for nematode survival. FMRFamide like peptide (FLPs) genes have drawn interest as prospective targets because of their diverse physiological roles in nematodes. Here, the effect of concomitant silencing of three vital nematode FLP genes (Mi-flp-1, Mi-flp-12 and Mi-flp-18) using host induced RNAi in tuberose was assessed against M. incognita. Firstly, the fusion (flp-1, flp-12 and flp-18) gene cassette was functionally validated using host-delivered RNAi in tobacco model system. Transgenic tobacco expressing dsRNA of fusion gene significantly reduced the nematode multiplication factor (MF) by 82.6%. Since, genetic transformation of tuberose has been neglected so far, and transgenic development is an inevitable step for host-generated RNAi, an Agrobacterium mediated transformation protocol was optimized in tuberose. Tuberose tubers were pricked at the apical meristem of the axis and at the tiny white meristematic buds emerging from them, followed by infection with Agrobacterium hosting pCAMBIA 3301 vector. Maximum transformation efficiency was achieved with 200 μM acetosyringone as observed from the GUS staining. For HIGS, fusion transgenic events were developed in tuberose and molecular analysis confirmed the presence and integration of fusion transgene using PCR, qRT-PCR and Southern blot analysis. Bioefficacy analysis of transgenic plants against M. incognita resulted in 73.75% reduction in nematode fecundity, thereby demonstrating the effectiveness of the three flp genes for nematode management in tuberose. This study established first ever report of in planta Agrobacterium-mediated transformation of tuberose for host delivered RNAi for M. incognita resistance.

Inorganic Ligand Triggered Transformation from Cs4PbBr6 Nanocrystals to Blue-Emitting CsPbBr3 Nanoplatelets

Blue-emitting perovskite nanoplates (NPLs) have been widely investigated for display application. However, the traditional synthetic techniques for high-quality perovskite NPLs are still limited due to their rapid crystallization rate. Here, we developed an inorganic ligand (NOBF4) mediated transformation strategy to obtain blue-emitting CsPbBr3 NPLs. The transformation process was based on the dissolution–recrystallization in a nonpolar environment, involving the dissociation of Cs4PbBr6 NCs to Pb ions, which then reformed PbBr64– octahedrons and finally formed two monolayer NPLs with pure blue-emission (line width ∼12 nm). The whole transformation process was clearly detected, and during the process the reorganization of organic ligands tuned by NO+ played a crucial role in the formation of NPLs. This work provides an effective strategy for obtaining pure blue-emitting NPLs and builds a platform for exploring the mechanism for the anisotropic growth of perovskite crystals.

Over-expression of Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) in transgenic pigeonpea confers resistance to the Helicoverpa armigera

Pigeonpea is an important food legume crop cultivated in tropical and sub-tropical regions around the world wherein the Indian subcontinent accounting for over 90 % of global production. It is a rich source of protein and is an important component of a well-balanced diet for the majority of Indians. Among the other insect pests, pigeonpea productivity is mostly affected by Helicoverpa armigera which is causing severe yield loss. Non-availability of resistant genes in germplasm and constraints with traditional breeding induce the application of a genetic engineering approach to generate insect resistance in pigeonpea. Expression of plant defensins in various crops provided enhanced resistance towards a variety of pests and pathogens. In the current study, two defensins Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) integrated by a linker peptide was transferred into pigeonpea as a fusion gene by Agrobacterium mediated transformation. Putative transgenic lines were confirmed through PCR and the promising lines were identified in the following generations based upon integration, expression and bioefficiency of the fusion gene. Leaf bioassay conducted against H. armigera larvae showed increased levels of insect resistance compared to the control, where six T2 plants were identified as superior lines showing less than 25 % of leaf damage. Our findings illustrates that Tfgd2–RsAFP2 fusion protein is efficient in imparting protection against the insect pest and the transgenic lines developed in this study could be used for further pigeonpea improvement projects.

Transgenic plants-immunomodulators for animal husbandry

In modern pharmacology, there is a tendency to move from low molecular weight drugs to protein drugs. Interferon is among them. Interferon is used in animal husbandry to prevent and treat viral and bacterial diseases, as a single drug and as an adjuvant for vaccines and antibiotics to increase their efficiency and reduce the amount of use. After interferon therapy, meat, milk and eggs can be consumed without restrictions. Currently, interferon for veterinary purposes is obtained using genetically modified organisms (mainly bacteria). The limiting factor is the high cost of the active substance isolating and purifying, which is up to 8090% of the product cost. Edible plants-producers can solve this problem. Plants have the lowest price per unit of biomass, are not infected by mammalian pathogens, and have an eukaryotic system of protein synthesis.
 Transgenic tobacco plants (Nicotiana tabacum L.) for the production of bovine gamma-interferon were obtained by agrobacterium mediated transformation at the Department of Genetics and Biotechnology, Saint Petersburg State University. Testing of this model showed that interferon is successfully produced in plants and retains biological activity, including oral administration. Problems were also identified: tobacco toxicity and low levels of protein accumulation. At the next stage of the experiment, it was decided to transform edible carrot (Daucus carota) plants, modify the target protein (shorten) to increase its resistance to proteolytic degradation, and use the pSRD1 root-specific promoter. At present, the corresponding transgenic constructs have been obtained, the effectiveness of which will be determined first on the carrot hairy roots, and then on whole carrot plants.
 The research was supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2020-922 date 16.11.2020 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center Future Agrotechnologies.

Composite plants of cucumber and buckwheat as a tool to study auxin distribution and transport in the root system

Genetic transformation of most dicotyledonous plants by Rhizobium rhizogenes (also known as Agrobacterium rhizogenes) results in production of composite plants plants consisting of wild-type shoot and transgenic root system. Composite plants are the suitable model for investigation of hormonal mechanisms related to development of the root system as regulatory links between the root system and the shoot maintains in such plants.
 In most plants initiation of lateral root primordia occurs above the elongation zone [1]. However, in cucurbits and some other species, including important cereal crop buckwheat (Fagopyrum esculentum Moench), lateral root primordia initiation and development occurs in the apical meristem of the parental root [2, 3].
 The phytohormone auxin is a key regulator of lateral root development. Fusions of auxin-responsive promoters and reporter genes can be used to study the role of auxin in the development of root system of non-model plants such as cucumber (Cucumis sativus L.) and buckwheat [4].
 The agrobacterium mediated transformation technique of cucurbits [5] has been adapted for buckwheat. R. rhizogenes strain R1000 was used in all transformations. Set of binary vectors based on pKGW-RR-MGW or pKGW-MGW was developed to study auxin response maxima (DR5::mNeonGreen) or auxin transport (fusions of genes encoding auxin efflux proteins PIN and mNeonGreen).
 Pattern of auxin response maxima was similar in both species and included quiescent center and initial cells, columella, xylem cell files and lateral root primordia on all stages of development. Members of CsPIN1 (CsPINb and CsSoPIN1) group contributed unequally in generation of auxin maximum required for lateral root primordium initiation.
 The research was supported by the RFBR grant 20-016-00233-a.

AtGCS promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by Agrobacterium rhizogenes-mediated transformation.

Agrobacterium rhizogenes-mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive Cas9 expression. Here, we identified a highly efficient Arabidopsis thaliana gamma-glutamylcysteine synthetase promoter, termed AtGCSpro, with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. AtGCSpro achieved higher homozygous/biallelic mutation efficiency than the most widely used CaMV 35S promoter in driving Cas9 expression in soybean, Lotus japonicus, and tomato roots. Using the pAtGCSpro-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p2 × 35Spro-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the pAtGCSpro-Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing. AtGCSpro is conservatively active in various eudicots species, suggesting that AtGCSpro might be applied in a wide range of dicots species.

In vitro</em></strong><strong> direct regeneration and <em>Agrobacterium tumefaciens</em> mediated <em>in planta</em> transformation of <em>Ocimum sanctum L.

An in vitro regeneration system for propagation has been successfully developed for a valuable medicinal and aromatic plant ‘ Ocimum sanctum L’. In the present study, petiole explants, from in-vitro grown cultures of O. sanctum, was used for direct regeneration. The developed protocol employed 98% of regeneration frequency in addition to 9.6 shoots per explant when cultured on Murashige and Skoog (MS) medium fortified with 3 mg/L benzylamino purine (BAP) and 1 mg/L Naphthalene acetic acid (NAA). Furthermore, Agrobacterium tumefaciens Mediated genetic Transformation (ATMT) protocol (transient and stable) was also developed using LBA4404 strain harboring pBI121 with uid-A and neomycin phosphotransferase genes. The regenerated transformants were shifted on MS with kanamycin (50 mg/L) and afterwards placed on the half-strength MS medium. The validation was done through polymerase chain reaction (PCR) with neomycin phosphotransferase-II ( npt-II) & β-glucoronidase ( uid-A) gene primers. The maximum stable transformation frequency of 70% ± 0.35 was achieved. Hence, it is apparent that the established protocols i.e. in vitro direct regeneration and ATMT are appropriate for integrating novel enzymes/genes through high throughput techniques such as gene tagging, and targeted gene replacement to modulate the primary as well as secondary metabolic flux towards desired agronomic product or trait in planta.

Dihydromyricetin alleviates methotrexate-induced hepatotoxicity via suppressing the ‎TLR4/NF-κB pathway and NLRP3 inflammasome/caspase 1 axis

The anticancer drug methotrexate (MTX) is known to cause hepatotoxicity as a possibly fatal adverse effect that hinders its clinical application. Although the natural flavonoid, dihydromyricetin (DHM), has antioxidant and anti-inflammatory effects; its role against MTX-induced hepatotoxicity has not been explored yet. For this, rats were administrated DHM orally for two weeks at a dose of 300 mg/kg per day, with or without a single i.p. injection of 40 mg/kg MTX on the 9th day of the experiment. MTX caused deterioration in liver structure and function, depicted by an increase in liver enzymes; ALT and AST. Moreover, MTX induced oxidative stress, shown by increasing malondialdehyde and decreasing reduced glutathione and total antioxidant capacity, initiated the inflammatory response via upregulated expression of Toll-like receptor 4 (TLR4) and its downstream transcription factor, nuclear factor-kappa B (NF-κB p65). Consequent to TLR4 signaling cascade, Nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammosome was activated and caused caspase 1 mediated transformation of proinflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18) into their active forms. Interestingly, administering DHM with MTX improved liver structure and function, as well as significantly decreased all oxidative stress and inflammatory signaling. Collectively, DHM possesses antioxidant and anti-inflammatory properties that can ameliorate MTX-induced hepatotoxicity, through down-regulation of liver TLR4/NF-κB and therefore prohibit activation of NLRP3/caspase 1 pathway.

An Attempt to Establish an Agrobacterium-mediated Transient Expression in Euphorbia tirucalli (L.) an Important Medicinal Plant

Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Euphorbia tirucalli evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Optimized parameters resulted in high transformation efficiency 3 fold increase at transient GUS expression .The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (100 µM) and cocultivation temperature of 25°C. Methods: Nodal explants were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56, 0.8 and 1.2 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of NAA 2 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400, 600 and 800 µM. Histochemical analysis of gus gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 72% when the infection process was carried out with acetosyringone in co-cultivation medium (100 µM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.

Silencing P25, HC-Pro and Brp1 of Potato Virus (Viroid) Using Artificial microRNA Confers Resistance to PVX, PVY and PSTVd in Transgenic Potato.

Virus infection is the key constraint to potato cultivation worldwide. Especially, coinfection by multiple viruses could exacerbate the yield loss. Transgenic plants expressing artificial microRNAs (amiRNAs) have been shown to confer specific resistance to viruses. In this study, three amiRNAs containing Arabidopsis miR159 as a backbone, expressing genes targeting P25, HC-Pro and Brp1 of potato virus X (PVX), potato virus Y (PVY) and potato spindle tuber viroid (PSTVd), were constructed. amiR-159P25, amiR-159HCPro and amiR-159Brp1 were cloned into the plant expression vector pCAMBIA1301 with a CaMV35S promoter, producing the p1301-pre-amiRP25−HCPro−Brp1 vector. Twenty-three transgenic plants (Solanum tuberosum cv. ‘Youjin’) were obtained by Agrobacterium tumefaciens–mediated transformation, and ten PCR-positive transplants were chosen for further analysis. Quantitative real-time PCR results indicated that 10 transgenic plants could express amiRNAs successfully. Southern blotting hybridization proved that amiR-159P25−HCPro−Brp1 had integrated into potato genome in transgenic lines. Viral (viroid) challenge assays revealed that these transgenic plants demonstrated resistance against PVX, PVY and PSTVd coinfection simultaneously, whereas the untransformed controls developed severe symptoms. This study demonstrates a novel amiRNA-based mechanism that may have the potential to develop multiple viral resistance strategies in potato.

Optimization of Agrobacterium tumefaciens-mediated Transformation in Oxalis corniculata (L.)

Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Oxalis corniculata evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Five fold transformation efficiency is achieved in transient gus gene expression after optimizing various parameters. The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (400 μM) and cocultivation temperature of 22°C. Methods: Oxalis corniculata (L.) were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56,0.80 and 1.34 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of BAP 2 mg L-1 and NAA 0.5 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400,600 and 800 μM. Histochemical analysis of gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 64% when the infection process was carried out with acetosyringone in co-cultivation medium (400 μM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.

Molecular level insights into HO• and Cl2•−-Mediated transformation of dissolved organic matter in landfill leachate concentrates during the Fenton process

Cl2•− can be the dominant radical species when high salinity wastewater is treated by advanced oxidation processes (AOPs). In this study, the steady-state concentrations of HO•, Cl• and Cl2•− were determined during the Fenton treatment of two landfill leachate concentrates with significantly different Cl− concentrations (XPNF: 1406 mg/L; SNNF: 8036 mg/L). Based on the steady-state concentrations of the radical species and measured second-order rate constants with dissolved organic matters (DOM), HO• accounted for 76.2% of DOM transformation in XPNF, while Cl2•− accounted for 96.5% in SNNF. The DOM transformation dominated by HO• or Cl2•− was then thoroughly compared to investigate the reactivities of HO• and Cl2•− with DOM at molecular level using high-resolution mass spectrometry. The results showed that formulas with low oxidation degree (O/C < 0.5) were preferentially removed by HO• and HO• was more reactive towards compounds with high saturation degree (H/C > 1.5) compared with Cl2•−. In contrast, Cl2•− was more reactive to the compounds with lower saturation level (H/C < 1.5) and the reaction of Cl2•− with DOM was less dependent on DOM oxidation degree. In addition, HO• preferentially reacted with the CHON compounds with low N content, while Cl2•− was more reactive to CHON compounds with higher N content compared with HO•. To our knowledge, this is the first study to distinguish the difference in the reactivity of HO• and Cl2•− towards DOM at molecular level.

Efficient synthesis of terminal-diazirine-based histone peptide probes

Histone post-translational modifications (hPTMs) regulate series fundamental cellular processes through the specific recognition of “reader” or “effector” proteins. The chemical probes containing photo-cross-linking groups, especially diazirine group has been developed to capture proteins recognized by hPTMs. However, the construction of diazirine-based photo-reactive amino acids requires multiple synthetic steps. Herein, we report the efficient synthesis of a new diazirine-based histone peptide probes through the construction of terminal diazirine via phenyliodonium diacetate (PIDA) mediated transformation. The key building block tert-butyl 3-(3H-diazirin-3-yl)propanoate can be obtained in one step with 90% yield. The synthetic H3K4Me3 photo-cross-linking probe was demonstrated to label H3K4Me3 specific reader in vitro and in cell lysates.

Highly efficient site-specific integration of DNA fragments into the honeybee genome using CRISPR/Cas9.

Functional genetic studies in honeybees have been limited to transposon mediated transformation and site directed mutagenesis tools. However, site- and sequence-specific manipulations that insert DNA fragments or replace sequences at specific target sites are lacking. Such tools would enable the tagging of proteins, the expression of reporters and site-specific amino acid changes, which are all gold standard manipulations for physiological, organismal, and genetic studies. However, such manipulations must be very efficient in honeybees since screening and crossing procedures are laborious due to their social organization. Here, we report an accurate and remarkably efficient site-specific integration of DNA-sequences into the honeybee genome using clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9-mediated homology-directed repair. We employed early embryonic injections and selected a highly efficient sgRNA in order to insert 294 and 729 bp long DNA sequences into a specific locus at the dsx gene. These sequences were locus-specifically integrated in 57% and 59% of injected bees. Most importantly, 21% and 25% of the individuals lacked the wildtype sequence demonstrating that we generated homozygous mutants in which all cells are affected (no mosaicism). The highly efficient, locus-specific insertions of nucleotide sequences generating homozygous mutants demonstrate that systematic molecular studies for honeybees are in hand that allow somatic mutation approaches via workers or studies in the next generation using queens with their worker progeny. The employment of early embryonic injections and screenings of highly efficient sgRNAs may offer the prospect of highly successful sequence- and locus-specific mutations also in other organisms.

Agrobacterium mediated transformation of ABA biosynthetic pathway coding genes for enhanced drought tolerance in Nicotiana tabacum

Abscisic acid (ABA) is an important hormone that regulates stomata closure, developmental stages and drought tolerance in plants. NCED (EC 1.13.11.51) is a key enzyme involved in the biosynthesis of abscisic acid. Transgenic tobacco plants with the integration of nced and rpk genes under the control of stress inducible promoters viz., sal T and lea P were developed through Agrobacterium mediated transformation. Over expression of integrated genes were confirmed by leaf disc assay method by imposing artificial stress using methyl Viologen (5 μMmethyl Viologen for 36 hrs).On exposure of stress, transgenic plants showed high content of chlorophyll pigments and low level of electrolyte leakage and MDA were observed than the wild type plants. Over expression of nced and rpk enhanced the abscisic acid content and increased the tolerance to drought stress.

Overexpression of Choline Oxidase Gene in Three Filial Generations of Rice Transgenic Lines.

Glycinebetaine (GB) accumulation in many halophytic plants, animals, and microorganisms confers abiotic stress tolerance to salinity, drought, and extreme temperatures. Although there are a few genetic and biochemical pathways to synthesize GB, but isolation of a single gene Choline Oxidase (codA) from Arthrobacter spp. have opened a new hatch to engineer the susceptible plants. The effects of overexpressed codA gene, through multiple copy insertion and GB accumulation on salinity tolerance in rice were studied. Seed-derived embryogenic calli of 'Tarom Molaie' cultivar were targeted with two plasmids pChlCOD and pCytCOD both harboring the codA gene using the biolistic mediated transformation. The regenerated T0 plants were screened by PCR analysis. A line containing three copies of codA gene and harboring pChlCOD and pCytCOD was identified by Southern blot analysis. The expression of codA gene in this transgenic line was then confirmed by RT-PCR. The Mendelian segregation pattern of the inserted sequences was accomplished by the progeny test using PCR. The effects of overexpression of codA on salinity tolerance were evaluated at germination and seedling stage using T2-pChl transgenic line and control seeds in the presence of 0, 100, 200, and 300 mM NaCl. Finally, leaf growth dynamics of T2-pChlCOD transgenic line and control line under hydroponic conditions in the presence of 0, 40, 80, and 120 mM NaCl were assessed. The seed germination experiment results showed that the transformed seeds had a higher germination rate than the controls under all salinity treatments. But also, the leaf growth dynamics showed that the control plants had a more favorable leaf growth dynamic in all of the treatments. Although, the transgenic lines (T0, T1 and T2) exhibited lower performance than the wild type, the transgenic line varied for GB and choline contents and increasing codA gene copy number led to increased GB content. In a salinity sensitive crop such as rice, GB may not significantly contribute to the plant protection against salt stress. Also, insufficiency of choline resources as GB precursor might have affected the overall growth ability of the transgenic line and resulted in decreased leaf growth dynamics.