scholarly journals Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein

2021 ◽  
Vol 22 (4) ◽  
pp. 1757
Author(s):  
Vanesa Cano ◽  
Mᵃ Teresa Martínez ◽  
José Luis Couselo ◽  
Elena Varas ◽  
Francisco Javier Vieitez ◽  
...  
Keyword(s):  
Somatic Embryos ◽  
Transformation Efficiency ◽  
Phytophthora Cinnamomi ◽  
Lethal Dose ◽  
Cork Oak ◽  
Pcr Analysis ◽  
Qpcr Analysis ◽  
Bacteriostatic Agent ◽  
Transgenic Lines

We present a reproducible procedure for transforming somatic embryos of cork oak with the CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the CsTL1 gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.

scholarly journals Vegetative Propagation of Phytophthora cinnamomi-Tolerant Holm Oak Genotypes by Axillary Budding and Somatic Embryogenesis

Forests ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 841
Author(s):  
Maria Teresa Martínez ◽  
Francisco Javier Vieitez ◽  
Alejandro Solla ◽  
Raúl Tapias ◽  
Noelia Ramírez-Martín ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
Somatic Embryos ◽  
Phytophthora Cinnamomi ◽  
Shoot Proliferation ◽  
Holm Oak ◽  
Induction Medium ◽  
Axillary Shoot ◽  
Oak Decline ◽  
Shoot Cultures

Holm oak (Quercus ilex) is one of the most widely distributed tree species in the Mediterranean basin. High mortality rates have been observed in holm oak populations in the southwest of the Iberian Peninsula as a result of oak decline syndrome. Selection and propagation of genotypes tolerant to this syndrome could aid the restoration of affected areas. In this article, we report micropropagation and conservation procedures based on axillary budding and somatic embryogenesis (SE) of holm oak plants, selected for their tolerance to Phytophthora cinnamomi—the main biotic factor responsible for oak decline. Forced shoots were obtained from potted plants of eight different genotypes, and used as stock material to establish in vitro shoot proliferation cultures. Reliable shoot proliferation was obtained in seven out the eight genotypes established in vitro, whereas multiplication rates were genotype-dependent. The highest rooting rates were obtained by culturing shoots for 24 h or 48 h on rooting induction medium containing 25 mg L−1 indole-3-butyric acid, followed by transfer to medium supplemented with 20 µM silver thiosulphate. Axillary shoot cultures can be successful conserved by cold storage for 12 months at 4 °C under dim lighting. Shoot tips, excised from axillary shoot cultures established from tolerant plants, were used as initial explants to induce SE. Somatic embryos and/or nodular embryogenic structures were obtained on induction medium with or without indole-acetic acid 4 mg L−1, in two out the three genotypes evaluated, and induction rates ranged between 2 and 4%. Plantlet recovery was 45% after two months cold stratification of somatic embryos and eight weeks of culture on germination medium. Vegetative propagation of P. cinnamomi-tolerant Q. ilex trees is a valuable milestone towards the restoration of disease-affected areas.

scholarly journals An efficient in vitro regeneration protocol to generate stable transgenic lines of Black gram (Vigna mungo)

2018 ◽  
Vol 78 (3) ◽  
Author(s):  
Bidyut Kumar Sarmah ◽  
Trishna Konwar ◽  
Borsha Borah ◽  
Arun Kumar Handique ◽  
Sumita Acharjee
Keyword(s):  
Transformation Efficiency ◽  
In Vitro Regeneration ◽  
Multiple Shoots ◽  
Vigna Mungo ◽  
Ms Medium ◽  
Black Gram ◽  
Transgenic Lines ◽  
Half Strength ◽  
The Individual

An efficient and quick in vitro regeneration protocol was developed for black gram (Vigna mungo) using wounded embryonic axis with cotyledon as explant. Murashige and Skoog (MS) medium supplemented with 4.44 μM BAP and 2.32 μM Kinetin was found to be effective in producing maximum number (mean 7.80) of multiple shoots. The individual shoots elongated to 4.5 cm when MS medium was supplemented with 2.89 μM GA3 along with 0.44 μM BAP and 0.46 μM KIN. A novel in vitro rooting technique was also optimized for black gram using half-strength liquid MS medium supplemented with 1.34 μM NAA. The shoots in this medium produced the highest number (mean 7.50) of roots with root length of 6.02 cm. The plantlets were transferred to soil mixture and placed in greenhouse where more than 80% successfully grew to maturity. The same protocol was successfully used to generate transgenic black gram lines carrying Bt-Cry2Aa gene through Agrobacteriummediated transformation with a transformation efficiency of 0.42%. The rooted T0 plants grew to maturity and produced T1 seeds with the presence and expression of transgene in T1 plants. Thus, we have standardized an in vitro regeneration protocol suitable for generation of stable transgenic black gram plants.

scholarly journals Efficient Biolistic Transformation of Immature Citrus Rootstocks Using Phosphomannose-isomerase Selection

Plants ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 390 ◽  
Author(s):  
Wu ◽  
Acanda ◽  
Canton ◽  
Zale
Keyword(s):  
Fluorescent Protein ◽  
Transformation Efficiency ◽  
Biolistic Transformation ◽  
Phosphomannose Isomerase ◽  
Gene Encoding ◽  
Chlorophenol Red ◽  
Citrus Rootstock ◽  
Transgenic Lines ◽  
Green Fluorescent

This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.

scholarly journals In Vitro Selection of a Salt-tolerant Embryogenic Line in Habanero Pepper (Capsicum chinense Jacq.)

HortScience ◽  
2011 ◽  
Vol 46 (12) ◽  
pp. 1666-1671 ◽  
Author(s):  
Daniela Solís-Marroquín ◽  
Carlos Alberto Lecona-Guzmán ◽  
Adriana Canto-Flick ◽  
Jericó Jabín Bello-Bello ◽  
Nancy Santana-Buzzy ◽  
...  
Keyword(s):  
Salt Stress ◽  
Somatic Embryos ◽  
Growth And Development ◽  
In Vitro Selection ◽  
Lethal Dose ◽  
Intermediate Stage ◽  
Cellular Mechanisms ◽  
Stage Of Development ◽  
Habanero Pepper

The effect of NaCI salinity on growth and development of somatic embryos of Habanero pepper was examined. Addition of 75 and 100 mm NaCI into the medium greatly increased the growth and development of somatic embryos and both of these concentrations favored the proliferation of somatic embryos. However, supplementation of 200 and 300 mm NaCI to the medium showed a negative effect on the growth and development of somatic embryos. Concentration increases of NaCl provoked a significant reduction of the embryos survival rate with the average lethal dose (46%) being registered in the treatment of 100 mm. Furthermore, a lower tolerance to salt stress (NaCl) was observed in deformed somatic embryos. Concentrations of 200 and 300 mm NaCl significantly delayed development in the surviving embryos in both treatments. These embryos remained at the globular stage throughout culture time. At 75 mm NaCl, most of the embryos were observed in the torpedo stage. However, the embryos exposed to 100 mm NaCl were observed mainly in globular and cotiledonar stages. It is quite likely that the transition from one intermediate stage of development to another occurs rapidly. With the exception of the concentration at 300 mm NaCl, salt stress stimulated embryonic germination, particularly at 100 mm NaCl. The content of proline in somatic embryos increased substantially in response to salinization. The results suggest that somatic embryos of C. chinense can tolerate concentrations of NaCl up to 100 mm without their development being affected. Moreover, they have sufficient cellular mechanisms to tolerate salinity at relatively higher levels.

scholarly journals TheIn VitroEffect of Ivermectin on the Activity of Trehalose Synthesis Pathway Enzymes and Their mRNA Expression in the Muscle of Adult FemaleAscaris suum(Nematoda)

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Małgorzata Dmitryjuk ◽  
Elżbieta Łopieńska-Biernat ◽  
Ewa Anna Zaobidna
Keyword(s):  
Mrna Expression ◽  
Prolonged Exposure ◽  
Lethal Dose ◽  
Weak Correlation ◽  
Control Groups ◽  
Qpcr Analysis ◽  
And Control ◽  
Trehalose Synthesis ◽  
Real Time Qpcr

Thein vitroeffect of ivermectin lethal dose on the activity of trehalose-6-phosphate synthase (TPS) and phosphatase (TPP) and the expression of their mRNA (tps1, tps2, andtppgenes) in the muscle of adult femaleAscaris suumwas investigated. The presence of ivermectin in the medium caused a decrease in TPS and TPP activities during the experiment compared with the start and control groups. The exception was the group of worms grown for 8 hours in a IVM solution, in which there was a little higher TPS activity than in the control. Real-time qPCR analysis showed reduced expression oftps1andtps2, and unchangedtppexpression after 20 hours of incubation relative to the expression at time zero. Relative to the appropriate control groups, the expression oftps2gene was slight increased but the other two genes were reduced after 8-hours of IVM-treatment. Then the expression of all three genes was lower at the end of cultivation. The level of gene expression was positively correlated with the activity of specific enzymes. In the case oftppgene there was only a weak correlation. Prolonged exposure to ivermectin was effective in lowering TPS and TPP activity and their mRNA expression. However, the drug did not block the pathway.

Somatic Embryogenesis and In vitro Plant Regeneration in Vigna trilobata (L.) Verd. - A Potential Pasture Legume

1970 ◽  
Vol 19 (1) ◽  
pp. 89-99
Author(s):  
K. Choudhary ◽  
M. Singh ◽  
M. S. Rathore ◽  
N. S. Shekhawat
Keyword(s):  
Somatic Embryogenesis ◽  
Growth Regulators ◽  
Somatic Embryos ◽  
Basal Medium ◽  
Long Term Study ◽  
Continuous Application ◽  
First Time ◽  
Pasture Legume

This long term study demonstrates for the first time that it is possible to propagate embryogenic Vigna trilobata and to subsequently initiate the differentiation of embryos into complete plantlets. Initiation of callus was possible on 2,4-D. Somatic embryos differentiated on modified MS basal nutrient medium with 1.0 mg/l  of 2,4-D and 0.5 mg/l  of Kn. Sustained cell division resulted in globular and heart shape stages of somatic embryos. Transfer of embryos on to a fresh modified MS basal medium with 0.5 mg/l of Kn and 0.5 mg/l of GA3 helped them to attain maturation and germination. However, the propagation of cells, as well as the differentiation of embryos, were inhibited by a continuous application of these growth regulators. For this reason, a long period on medium lacking these growth regulators was necessary before the differentiation of embryos occurred again. The consequences for improving the propagation of embryogenic cultures in Vigna species are discussed. Key words: Pasture  legume, Vigna trilobata, Globular, Heart shape, somatic embryogenesis D.O.I. 10.3329/ptcb.v19i1.4990 Plant Tissue Cult. & Biotech. 19(1): 89-99, 2009 (June)

Hepato- and Nephroprotective Effects of the Polyphenol Concentrate From Cabernet Sauvignon Grape Varieties Cultivated in Kazakhstan in the Experimental Model Of CCL4-Induced Toxicity

2017 ◽  
Vol 9 (03) ◽  
Author(s):  
Nurgozhin T. ◽  
Sergazy S. H. ◽  
Adilgozhina G. ◽  
Gulyayev A. ◽  
Shulgau Z. ◽  
...  
Keyword(s):  
Carbon Tetrachloride ◽  
Experimental Model ◽  
Lethal Dose ◽  
Radical Scavenging ◽  
Cabernet Sauvignon ◽  
Intragastric Administration ◽  
Liver And Kidney ◽  
Antioxidant Stress

Objective:This study investigates the hepatoprotective effect and the antioxidant role of polyphenol concentrate in the experimental model of carbon tetrachloride (CCl4) induced toxicity. Methods: Antioxidant activity of Cabernet Sauvignon grape polyphenol were evaluated by radical scavenging of 1,1-diphenyl-2-picryl hydrazyl radical (DPPH), 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS.+). In addition, the effects of polyphenol concentrate on the survival of Wistar rats in the toxicity model, was also investigated. The polyphenol concentrate was administered for 5 five days prior to injection of carbon tetrachloride in a sub-lethal dose of 300 mg/kg of animal body weight in order to perform histological examinations of the liver and kidney, and detect the levels of AST, ALT and bilirubin. Results: Administration of polyphenol concentrate increased animal survival in the experimental model. Moreover, the intragastric administration of polyphenol concentrate prior to the initiation of the experimental model of toxicity, which was caused by a sub-lethal CCl4 dose, reduced morphological injuries in the liver and kidney, decreased the AST and ALT levels of the blood serum. Discussion and conclusion: Our data demonstrate that polyphenol concentrate possesses an antioxidant potential both in vitro and in vivo by reducing antioxidant stress that was caused by CCl4 administration into rats.

Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

2020 ◽  
Vol 18 ◽  
Author(s):  
Zirui Zhang ◽  
Shangcong Han ◽  
Panpan Liu ◽  
Xu Yang ◽  
Jing Han ◽  
...  
Keyword(s):  
Wound Healing ◽  
Mrna Expression ◽  
Tissue Injury ◽  
Inflammatory Cells ◽  
Pcr Analysis ◽  
Protective Effects ◽  
Deep Tissue ◽  
Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

In Vitro and In Vivo Neutralizing Activity of Uvaria chamae Leaves Fractions on the Venom of Naja nigricollis in Albino Rat and Bovine Blood

2020 ◽  
Vol 14 (4) ◽  
pp. 295-311
Author(s):  
Ada Gabriel ◽  
Mamman Mohammed ◽  
Mohammed G. Magaji ◽  
Yusuf P. Ofemile ◽  
Ameh P. Matthew ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Neglected Tropical Diseases ◽  
Lethal Dose ◽  
Positive Control ◽  
Haemolytic Activity ◽  
Cytotoxic Activities ◽  
Albino Rat ◽  
Aqueous Residue

Background: Snakebite envenomation is a global priority ranked top among other neglected tropical diseases. There is a folkloric claim that Uvaria chamae is beneficial for the management of snakebite and wounds in African ethnobotanical surveys. Besides, there are many registered patents asserting the health benefits of U. chamae. Objective: This study aimed to investigate U. chamae’s potentials and identify candidates for the development of tools for the treatment and management of N. nigricollis envenomation. Methods: Freshly collected U. chamae leaves were air-dried, powdered, and extracted in methanol. The median lethal dose of the extract was determined and further fractionated with n-hexane, n-butanol and ethyl acetate. Each fraction was tested for neutralizing effect against venom-induced haemolytic, fibrinolytic, hemorrhagic, and cytotoxic activities. Results: U. chamae fractions significantly (p<0.05) neutralized the haemolytic activity of N. nigricollis venom in n-butanol; 31.40%, n-hexane; 33%, aqueous residue; 39.60% and ethyl acetate; 40.70% at the concentration of 100mg/ml of each fraction against 10mg/ml of the snake venom when compared to the positive control. The fibrinolytic activity of N. nigricollis venom was significantly (p<0.05) neutralized in n-hexane at 73.88%, n-butanol; 72.22% and aqueous residue; 72.22% by the fractions of U. chamae. In addition, haemorrhagic activity of N. nigricollis venom was significantly (p<0.05) neutralized by U. chamae fractions at the concentrations of 100mg/ml, 200mg/ml and 400mg/ml except for n-butanol and aqueous residues at 400 mg/ml. Conclusion: U. chamae leaves fractions possess a high level of protection against N. nigricollis venoms-induced lethality and thus validate the pharmacological rationale for its usage in the management of N. nigricollis envenomation.

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