scholarly journals Functional Improvement of Human Cardiotrophin 1 Produced in Tobacco Chloroplasts by Co-Expression with Plastid Thioredoxin m

Plants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 183 ◽  
Author(s):  
María Ancín ◽  
Ruth Sanz-Barrio ◽  
Eva Santamaría ◽  
Alicia Fernández-San Millán ◽  
Luis Larraya ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Therapeutic Potential ◽  
Plastid Transformation ◽  
Growth Conditions ◽  
Functional Improvement ◽  
Fusion Strategy ◽  
Expression Levels ◽  
Young Leaves ◽  
New Strategies

Human cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant protein’s overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts.

scholarly journals Therapeutic Potential of Thrombomodulin in Renal Fibrosis of Nephrotoxic Serum Nephritis in Wistar-Kyoto Rats

2020 ◽  
Vol 45 (3) ◽  
pp. 391-406
Author(s):  
Nobuhiro Kanazawa ◽  
Masayuki Iyoda ◽  
Shohei Tachibana ◽  
Kei Matsumoto ◽  
Yukihiro Wada ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Renal Fibrosis ◽  
Therapeutic Potential ◽  
The Body ◽  
Expression Levels ◽  
Nephrotoxic Serum Nephritis ◽  
Collagen Iii ◽  
Wistar Kyoto ◽  
Mrna Expression Levels

Background: Recombinant human soluble thrombomodulin (rhTM) was approved in 2008 and has been used for treatment of disseminated intravascular coagulation in Japan. The antifibrotic effects of rhTM in acute exacerbation of idiopathic pulmonary fibrosis are well established, but the therapeutic potential of rhTM in renal fibrosis remains poorly understood. Methods: Nephrotoxic serum nephritis (NTS-N) was induced in 22 female Wistar-Kyoto (WKY) rats on day 0. Rats were administered either rhTM or vehicle intraperitoneally, every day from day 4 to day 55. Rats were sacrificed on day 56 when renal fibrosis was established and renal morphological investigations were performed. In vitro, rat renal fibroblasts (NRK-49F) were pretreated with rhTM or saline, and expression levels of profibrogenic gene induced by thrombin were analyzed by real-time reverse transcription polymerase chain reaction. Results: Compared to WKY-GN-vehicle rats, the body weights of WKY-GN-rhTM rats were significantly greater on day 55. By day 56, rhTM had significantly reduced serum creatinine levels in NTS-N. On the other hand, urinary protein excretion was comparable between the two treatment groups throughout the study. The percentage of Masson trichrome-positive areas in WKY-GN-rhTM rats was significantly lower compared to that in WKY-GN-vehicle rats. Glomerular fibrin deposition was significantly reduced in WKY-GN-rhTM rats. In addition, rhTM significantly reduced the renal cortical mRNA expression levels of TNF-α, Toll-like receptor 4, MYD88, TGF-β, αSMA, collagen I, collagen III, fibronectin, and protease-activated receptor 1 (PAR1), a thrombin receptor. In vitro, thrombin stimulation of NRK-49F cells significantly enhanced the mRNA expression levels of αSMA and PAR1, and these upregulations were significantly reduced by pretreatment with rhTM. Conclusions: Administration of rhTM after establishment of crescentic glomerulonephritis (GN) attenuated the subsequent development of renal fibrosis in NTS-N, possibly in part by inhibiting thrombin-mediated fibrogenesis. Our results suggest that rhTM may offer a therapeutic option for limiting the progression of chronic kidney disease in crescentic GN.

scholarly journals Introduction to in vitro culture of Ginkgo biloba (Linnaeus, 1771)

2020 ◽  
Vol 203 (12) ◽  
pp. 43-49
Author(s):  
Varvara Bessonova ◽  
Ol'ga Cherepanova
Keyword(s):  
Ginkgo Biloba ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Biologically Active ◽  
Future Research ◽  
Planting Material ◽  
Murashige Skoog ◽  
Young Leaves ◽  
High Proliferative Activity

Abstract. The purpose of this research was to introduce Ginkgo biloba into culture, to study the composition and properties of its biologically active compounds. Methods. We researched the optimal growth conditions for obtaining a viable tissue culture, such as: concentration of phytohormones and other organic and nonorganic substances in Murashige – Skoog medium and light hours. The effectiveness of the standard method of sodium hypochloride sterilization of young leaves and vegetative buds also was verified. As a result, of conducting the experiment we were able to grow a living callus from leaves of G. biloba. Based on this result we can conclude that these conditions are acceptable for high proliferative activity of the plant. We were studied the effect of phytohormones NAA, at a concentration of 0.5 ml and 6-BAP, at a concentration of 2.5 ml. Also, was selected the ideal planting material for callus production – young leaves that were more sensitive to treatment with hypochloride. This research serves as the foundation for future research not only for our laboratory, but also for other research groups. The callus can be used to clone specimens of G. bilobain greenhouses. It will be use to extract and study unique chemical compounds, such as ginkgolides, bilobalides and various terpenes, contained in the extract of plants of this group.

scholarly journals Subacute Transplantation of Native and Genetically Engineered Neural Progenitors Seeded on Microsphere Scaffolds Promote Repair and Functional Recovery After Traumatic Brain Injury

ASN NEURO ◽  
2019 ◽  
Vol 11 ◽  
pp. 175909141983018 ◽  
Author(s):  
Nolan B. Skop ◽  
Sweta Singh ◽  
Henri Antikainen ◽  
Chaitali Saqcena ◽  
Frances Calderon ◽  
...  
Keyword(s):  
Stem Cell ◽  
Functional Recovery ◽  
Subventricular Zone ◽  
Brain Injuries ◽  
Radial Glia ◽  
Therapeutic Potential ◽  
Ventricular Zone ◽  
Neural Progenitors ◽  
Functional Improvement

There is intense interest and effort toward regenerating the brain after severe injury. Stem cell transplantation after insult to the central nervous system has been regarded as the most promising approach for repair; however, engrafting cells alone might not be sufficient for effective regeneration. In this study, we have compared neural progenitors (NPs) from the fetal ventricular zone (VZ), the postnatal subventricular zone, and an immortalized radial glia (RG) cell line engineered to conditionally secrete the trophic factor insulin-like growth factor 1 (IGF-1). Upon differentiation in vitro, the VZ cells were able to generate a greater number of neurons than subventricular zone cells. Furthermore, differentiated VZ cells generated pyramidal neurons . In vitro, doxycycline-driven secretion of IGF-1 strongly promoted neuronal differentiation of cells with hippocampal, interneuron and cortical specificity. Accordingly, VZ and engineered RG-IGF-1-hemagglutinin (HA) cells were selected for subsequent in vivo experiments. To increase cell survival, we delivered the NPs attached to a multifunctional chitosan-based scaffold. The microspheres containing adherent NPs were injected subacutely into the lesion cavity of adult rat brains that had sustained controlled cortical impact injury. At 2 weeks posttransplantation, the exogenously introduced cells showed a reduction in stem cell or progenitor markers and acquired mature neuronal and glial markers. In beam walking tests assessing sensorimotor recovery, transplanted RG cells secreting IGF-1 contributed significantly to functional improvement while native VZ or RG cells did not promote significant recovery. Altogether, these results support the therapeutic potential of chitosan-based multifunctional microsphere scaffolds seeded with genetically modified NPs expressing IGF-1 to promote repair and functional recovery after traumatic brain injuries.

scholarly journals Role of protein arginine methyltransferase 5 in group 3 (MYC-driven) Medulloblastoma

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Nagendra K. Chaturvedi ◽  
Sidharth Mahapatra ◽  
Varun Kesherwani ◽  
Matthew J. Kling ◽  
Mamta Shukla ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Physical Interaction ◽  
Methyl Transferase ◽  
Primary Tumors ◽  
Expression Levels ◽  
Group 3

Abstract Background MYC amplification or overexpression is common in Group 3 medulloblastoma and is associated with the worst prognosis. Recently, protein arginine methyl transferase (PRMT) 5 expression has been closely associated with aberrant MYC function in various cancers, including brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Here, we report the role of PRMT5 as a novel regulator of MYC and implicate PRMT5 as a potential therapeutic target in MYC-driven medulloblastoma. Methods Expression and association between PRMT5 and MYC in primary medulloblastoma tumors were investigated using publicly available databases. Expression levels of PRMT5 protein were also examined using medulloblastoma cell lines and primary tumors by western blotting and immunohistochemistry, respectively. Using MYC-driven medulloblastoma cells, we examined the physical interaction between PRMT5 and MYC by co-immunoprecipitation and co-localization experiments. To determine the functional role of PRMT5 in MYC-driven medulloblastoma, PRMT5 was knocked-down in MYC-amplified cells using siRNA and the consequences of knockdown on cell growth and MYC expression/stability were investigated. In vitro therapeutic potential of PRMT5 in medulloblastoma was also evaluated using a small molecule inhibitor, EPZ015666. Results We observed overexpression of PRMT5 in MYC-driven primary medulloblastoma tumors and cell lines compared to non-MYC medulloblastoma tumors and adjacent normal tissues. We also found that high expression of PRMT5 is inversely correlated with patient survival. Knockdown of PRMT5 using siRNA in MYC-driven medulloblastoma cells significantly decreased cell growth and MYC expression. Mechanistically, we found that PRMT5 physically associated with MYC by direct protein-protein interaction. In addition, a cycloheximide chase experiment showed that PRMT5 post-translationally regulated MYC stability. In the context of therapeutics, we observed dose-dependent efficacy of PRMT5 inhibitor EPZ015666 in suppressing cell growth and inducing apoptosis in MYC-driven medulloblastoma cells. Further, the expression levels of PRMT5 and MYC protein were downregulated upon EPZ015666 treatment. We also observed a superior efficacy of this inhibitor against MYC-amplified medulloblastoma cells compared to non-MYC-amplified medulloblastoma cells, indicating specificity. Conclusion Our results reveal the regulation of MYC oncoprotein by PRMT5 and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven medulloblastoma.

scholarly journals Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3

2016 ◽  
Vol 60 (9) ◽  
pp. 5198-5207 ◽  
Author(s):  
Wei Li ◽  
Andrés Obregón-Henao ◽  
Joshua B. Wallach ◽  
E. Jeffrey North ◽  
Richard E. Lee ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Therapeutic Potential ◽  
Bactericidal Effect ◽  
Growth Conditions ◽  
Mycolic Acid ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Acid Transporter ◽  
Multiple Chemical

ABSTRACTIn recent years, whole-cell-based screens for novel small molecule inhibitors active againstMycobacterium tuberculosisin culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, thatmmpL3is required for the replication and viability ofM. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencingmmpL3had a rapid bactericidal effect on actively replicating cellsin vitroand reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further renderedM. tuberculosishypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies.

scholarly journals POS0394 NAD+ BOOSTERS REESTABLISH THE ALTERED NAD+ METABOLISM OF LEUKOCYTES FROM RHEUMATOID ARTHRITIS PATIENTS IMPROVING THEIR OXIDATIVE, APOPTOTIC AND INFLAMMATORY STATUS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 426.2-426
Author(s):  
C. Perez-Sanchez ◽  
L. M. Sánchez-Mendoza ◽  
M. D. C. Abalos-Aguilera ◽  
N. Barbarroja Puerto ◽  
M. Luque-Tévar ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Disease Activity ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Therapeutic Effects ◽  
Rt Pcr ◽  
Nad Metabolism ◽  
Expression Levels ◽  
Inflammatory Status

Background:NAD+ is an important cofactor and second messenger for multiple cellular processes that exhibits antioxidant, anti-apoptotic and anti-inflammatory properties. Pre-clinical studies in animal models of Rheumatoid Arthritis (RA) have demonstrated the therapeutic potential of NAD+ boosters in the control of the disease activity. However, to date no studies have been set up to evaluate the NAD+ metabolism and the therapeutic effects of NAD+ boosters in RA patients.Objectives:1- To study the NAD+ metabolism in RA patients and its association with key clinical features. 2- To evaluate the effect of anti-TNF therapy in the NAD+ metabolism.3- To analyze the beneficial effects of NAD+ boosters in leukocytes from active RA patients.Methods:Plasma and PBMCs were purified from 100 RA patients and 50 healthy donors (HDs). Moreover, an additional cohort of 50 RA patients treated with Anti-TNF therapy was analyzed before and after 6 months of treatment. NAD+ levels were determined by using the NAD/NADH-Glo Assay. NAD+-consuming genes expression were analyzed by RT-PCR. In parallel, PBMCs from eight HDs and eight active RA patients were treated ex vivo with 1 mM of NAD+ boosters including nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN). After 24 hours, intracellular reactive oxygen species (ROS) levels (DFCHDA) and the percentage of apoptotic PBMCs (annnexin V/PI) were assessed by flow cytometry. Lastly, a panel of key pro-inflammatory genes were evaluated by RT-PCR.Results:NAD+ and NADH levels were significantly reduced in plasma and PBMCs of RA patients compared with HDs and directly related to disease activity (DAS28, CDAI, SDAI). Accordingly, the expression levels of genes involved in the consumption of NAD+ such as SIRT-1, CD38 and PARP-1 were found up-regulated in PBMCs from RA patients. Anti-TNF therapy for 6 months restored the altered NAD+ levels towards those showed by HDs. Furthermore, the clinical response promoted by Anti-TNF therapy (changes in DAS28) correlated with changes in NAD+ levels. The in vitro treatments of PBMCs isolated from active RA patients with NAD+ boosters significantly increased the NAD+ levels and promoted a deep reduction of intracellular ROS levels, the percentage of apoptotic cells and the expression levels of key inflammatory mediators, such as IL-6, IL-8, IL-1ß, TNF-α, CCL2, IL-23, and STAT-3.Conclusion:1. NAD+ metabolism is altered and associated with the disease activity of RA patients, involving both, reduced NAD+ levels and increased expression of NAD+-consuming genes. 2. Anti-TNF therapy restored NAD+ levels, which were directly linked to the clinical effectiveness. 3. NAD+ boosters reduced the oxidative, apoptotic and inflammatory profiles of RA leukocytes through the parallel increase of intracellular NAD+ levels. Thus, NAD+ boosters might be considered novel therapeutic tools for RA patients.Acknowledgements:Supported by PI18/00837, RIER RD16/0012/0015, RTI2018-100695-B-I00, P18-RT-4264 and CVI276, co-funded with FEDER.Disclosure of Interests:None declared

Cytotoxic Effect of the Combination of Gemcitabine and Atorvastatin Loaded in Microemulsion on the HCT116 Colon Cancer Cells

2017 ◽  
Vol 9 (2) ◽  
Author(s):  
Mayson H. Alkhatib ◽  
Dalal Al-Saedi ◽  
Wadiah S. Backer
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Anticancer Drugs ◽  
Therapeutic Potential ◽  
Morphological Changes ◽  
In Vitro Study ◽  
Colon Cancer Cells ◽  
Apoptosis Detection ◽  
Hct116 Cells

The combination of anticancer drugs in nanoparticles has great potential as a promising strategy to maximize efficacies by eradicating resistant, reduce the dosage of the drug and minimize toxicities on the normal cells. Gemcitabine (GEM), a nucleoside analogue, and atorvastatin (ATV), a cholesterol lowering agent, have shown anticancer effect with some limitations. The objective of this in vitro study was to evaluate the antitumor activity of the combination therapy of GEM and ATVencapsulated in a microemulsion (ME) formulation in the HCT116 colon cancer cells. The cytotoxicity and efficacy of the formulation were assessed by the 3- (4,5dimethylthiazole-2-yl)-2,5-diphyneltetrazolium bromide (MTT) assay. The mechanism of cell death was examined by observing the morphological changes of treated cells under light microscope, identifying apoptosis by using the ApopNexin apoptosis detection kit, and viewing the morphological changes in the chromatin structure stained with 4′,6-diamidino-2-phenylindole (DAPI) under the inverted fluorescence microscope. It has been found that reducing the concentration of GEM loaded on ME (GEM-ME) from 5μM to 1.67μM by combining it with 3.33μM of ATV in a ME formulation (GEM/2ATV-ME) has preserved the strong cytotoxicity of GEM-ME against HCT116 cells. The current study proved that formulating GEM with ATV in ME has improved the therapeutic potential of GEM and ATV as anticancer drugs.

Polyphenols of leaves of Apium graveolens inhibit in vitro protein glycation and protect RINm5F cells against methylglyoxal-induced cytotoxicity

2018 ◽  
Vol 8 (3) ◽  
pp. 193 ◽  
Author(s):  
Rosa Martha Perez Gutierrez ◽  
Alethia Muñiz-Ramirez ◽  
Abraham Heriberto Garcia Campoy ◽  
Jose Maria Mota Flores ◽  
Sergio Odin Flores
Keyword(s):  
Therapeutic Potential ◽  
Health Benefits ◽  
Protein Glycation ◽  
Apium Graveolens ◽  
Ionization Mass ◽  
Intensity Level ◽  
Edible Plants ◽  
Rinm5f Cells ◽  
Protein Carbonyl Content

Background: The health benefits of edible plants have been widely investigated and disseminated. However, only polyphenols have been found to have sufficient therapeutic potential to be considered in clinical trials. Fewer manuscripts have other applications such as prospective health benefits and disease treatment. Other components of edible plants are responsible for a range of other benefits including antimalarial, burns, flu, cancer, inflammation, diabetes, glycation, antimicrobial, prevention of neurodegeneration, analgesic, antimigraine activity, sedative activities, etc. Accordingly, the public needs to be informed of the potential edible plants have to act on different targets and maintain better control over diabetes compared to commercial drugs which can be toxic, have side effects, do not have the capacity to maintain blood glucose at normal levels, and do not protect the patient from the complications of diabetes over time. Consequently, edible plants, such as Apium graveolen, which have therapeutic targets on AGEs formation, are potentially a better alternative treatment for diabetes.Methods: The leaves of celery were extracted with methanol (CM). Polyphenols contents in CM were investigated by liquid chromatography-electrospray ionization mass. The ability of the compounds to inhibit formation of AGEs was evaluated in vitro models using formation of AGE fluorescence intensity, level of fructosamine, Nε-(carboxymethyl)lysine (CML), methylglyoxal (MG)-derived protein, and formation of amyloid cross β structure. Protein-oxidation was determined by thiol group and protein carbonyl content. Inhibition of MG-derived AGEs and MG-trapping ability were also measured. Additionally, insulin production was determined in methylglyoxal-treated pancreatic RINm5F cells assay. Results: Apigenin, kaempferol, apiin, rutin, caffeic acid, ferulic acid, chlorogenic acid, coumaroylquinic acid, and p-coumaric acid were the major polyphenols contained in CM. In all the model tests CM displayed potent AGE inhibitory activity, suggesting that CM delayed the three stages of glycation. Accordingly, the mechanisms of action of celery involving dicarbonyl trapping and breaking the crosslink structure in the AGEs formed may contribute to the protection of pancreatic RINm5F cells against MG conditions.Conclusion: These findings indicate that CM have an excellent anti-glycation effect which may be beneficial for future development of antiglycating agents for the treatment of diabetes.Keywords: Apium graveolens, anti-glycation, polyphenols methylglyoxal, insulin, pancreatic cells

Formulation of Modified Release Atorvastatin Nanoparticles by Emulsion Interfacial Reaction Method

2015 ◽  
Vol 8 (3) ◽  
pp. 2937-2940
Author(s):  
Sudhakar Sekar ◽  
Shee Sim May
Keyword(s):  
Interfacial Reaction ◽  
Therapeutic Potential ◽  
Chitosan Nanoparticles ◽  
In Vitro Release ◽  
Morphological Characteristics ◽  
Preparation Technique ◽  
Modified Release ◽  
Reaction Method ◽  
Vitro Release

The aim of the study is to formulate a modified release chitosan nanoparticles for the oral delivery of atorvastatin and to study the in vitro release of atorvastatin from chitosan nanoparticles. Atorvastatin-loaded chitosan nanoparticles were prepared with different concentration of cross-linking agent (glutaraldehyde) by emulsion interfacial reaction method. The formed nanoparticles were characterized in terms of size and morphological characteristics by scanning electron microscopy (SEM) and transmission electron microscope (TEM). Spherical and regular nanoparticles with the size range of 100-250nm were formed. Atorvastatin encapsulation efficiency of nanoparticles was found to be highest in ANP3, followed by ANP2 and ANP1. The in vitro release of atorvastatin was studied by membrane diffusion technique. The resulted cumulative percentage of drug released for ANP1, ANP2 and ANP3 were 60.08%, 34.81% and 20.39% respectively. Through this study, the nanoparticles preparation technique has shown to be a promising approach for enhancing the dissolution of hydrophobic drugs like atorvastatin calcium. The application of this novel delivery system offers good therapeutic potential in the management of hypercholesterolemia and dyslipidemia.

Development of an Agrobacterium-mediated Transformation System for `Beurre Bosc' Pear

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 461d-461
Author(s):  
Richard L. Bell ◽  
Ralph Scorza ◽  
Chinnathambi Srinivasan
Keyword(s):  
Shoot Proliferation ◽  
Induction Medium ◽  
Shoot Induction ◽  
Transformation System ◽  
Gus Histochemical Assay ◽  
Young Leaves ◽  
Leaf Tissues ◽  
Shoot Induction Medium ◽  
Basal Salts

An efficient regeneration/transformation system was developed for `Beurre Bosc' pear. Young leaves were harvested from in vitro shoots proliferated on a medium containing MS basal salts and 5 BAP, 0.5 μM IBA, and 0.6M3. Shoot regeneration was optimized using a modification of the medium of Chevreau and Leblay (1993). Explants were cultured on shoot induction medium contained 10 μM TDZ and 1 μM IBA for 4 weeks in the dark, and then transfered to a similar, but auxinless, regeneration medium until shoots developed, usually after an additional 4 to 8 weeks. Leaf tissues were transformed by co-cultivation for 3 days with Agrobacterium tumefaciens EHA101 carrying a pGA482 plasmid containing NPTII, GUS, and rolC genes, followed by cultivation on SIM containing 300 mg/L timentin. Putative transgenic plants were selected on shoot induction medium containing 80mg/L kanamycin, and multiplied on shoot proliferation medium. Four clones were confirmed as transgenic using the GUS histochemical assay and Southern blots for the NPTII and rolC genes. Plants of each clone have been rooted and successfully transfered to the greenhouse for further analysis of gene expression.

Sign in / Sign up

Export Citation Format

Share Document