scholarly journals Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

2021 ◽  
Vol 12 ◽  
Author(s):  
Gabriela Conti ◽  
Beatriz Xoconostle-Cázares ◽  
Gabriel Marcelino-Pérez ◽  
Horacio Esteban Hopp ◽  
Carina A. Reyes
Keyword(s):  
Genetic Transformation ◽  
New Technologies ◽  
Negative Impact ◽  
Protoplast Isolation ◽  
Future Perspective ◽  
Juvenile Period ◽  
Breeding Programs ◽  
Regulatory Processes ◽  
Marker Free

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.

scholarly journals Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

2022 ◽  
Vol 23 (2) ◽  
pp. 837
Author(s):  
Sudip Biswas ◽  
Nancy J. Wahl ◽  
Michael J. Thomson ◽  
John M. Cason ◽  
Bill F. McCutchen ◽  
...  
Keyword(s):  
Genome Editing ◽  
New Technologies ◽  
Fluorescent Protein ◽  
Peanut Butter ◽  
Processing System ◽  
Protoplast Isolation ◽  
Sequencing Analysis ◽  
Ara H 2

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

scholarly journals Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments

2003 ◽  
Vol 60 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Weliton Antonio Bastos de Almeida ◽  
Francisco de Assis Alves Mourão Filho ◽  
Beatriz Madalena Januzzi Mendes ◽  
Alexandra Pavan ◽  
Adriana Pinheiro Martinelli Rodriguez
Keyword(s):  
Genetic Transformation ◽  
Citrus Sinensis ◽  
Transformation Efficiency ◽  
Petri Dish ◽  
Cultivation Medium ◽  
Breeding Programs ◽  
Rangpur Lime ◽  
Citrus Breeding ◽  
In Vitro Plantlets

Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck). Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod) were used as explants. These were co-cultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluate the transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days; explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation medium was supplemented with acetosyringone at 0, 100 or 200 µmol L-1; Explants ends had a longitudinal terminal incision (2-3 mm); co-cultivation temperatures of 19, 23 or 27°C were imposed. The experimental design was completely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm) with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in the explant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmed from explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27°C, in the absence of acetosyringone.

scholarly journals Olive (Olea europaea L.) Genetic Transformation: Current Status and Future Prospects

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 386
Author(s):  
Elena Palomo-Ríos ◽  
Isabel Narváez ◽  
Fernando Pliego-Alfaro ◽  
José A. Mercado
Keyword(s):  
Genetic Transformation ◽  
Olea Europaea ◽  
Fungal Resistance ◽  
Current Status ◽  
Biotic And Abiotic Stress ◽  
Juvenile Period ◽  
Factors Affecting ◽  
Olea Europaea L ◽  
Olea Europaéa

Olive (Olea europaea L.) is the most characteristic and important oil crop of the Mediterranean region. Traditional olive cultivation is based on few tens cultivars of ancient origin. To improve this crop, novel selections with higher tolerance to biotic and abiotic stress, adaptable to high-density planting systems and resilient to climate change are needed; however, breeding programs are hindered by the long juvenile period of this species and few improved genotypes have been released so far. Genetic transformation could be of great value, in the near future, to develop new varieties or rootstocks in a shorter time; in addition, it has currently become an essential tool for functional genomic studies. The recalcitrance of olive tissues to their in vitro manipulation has been the main bottleneck in the development of genetic transformation procedures in this species; however, some important traits such as fungal resistance, flowering or lipid composition have successfully been manipulated through the genetic transformation of somatic embryos of juvenile or adult origin, providing a proof of the potential role that this technology could have in olive improvement. However, the optimization of these protocols for explants of adult origin is a prerequisite to obtain useful materials for the olive industry. In this review, initially, factors affecting plant regeneration via somatic embryogenesis are discussed. Subsequently, the different transformation approaches explored in olive are reviewed. Finally, transgenic experiments with genes of interest undertaken to manipulate selected traits are discussed.

scholarly journals Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange

2002 ◽  
Vol 37 (7) ◽  
pp. 955-961 ◽  
Author(s):  
Beatriz Madalena Januzzi Mendes ◽  
Raquel Luciana Boscariol ◽  
Francisco de Assis Alves Mourão Filho ◽  
Weliton Antonio Bastos de Almeida
Keyword(s):  
Genetic Transformation ◽  
Culture Medium ◽  
Transformation Efficiency ◽  
Sweet Orange ◽  
Pcr Amplification ◽  
Culture Conditions ◽  
Direct Organogenesis ◽  
Breeding Programs ◽  
Citrus Breeding

The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 muM of acetosyringone. Genetic transformation was confirmed by performing beta-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes.

Ecological Innovations in the Automotive Sector. Part I: Construction Innovations

2018 ◽  
Vol 19 (11) ◽  
pp. 30-35
Author(s):  
Marta Wójcik
Keyword(s):  
Automotive Industry ◽  
Diesel Engines ◽  
New Technologies ◽  
Negative Impact ◽  
The Other ◽  
Automotive Sector ◽  
Environmental Aspects ◽  
Car Industry ◽  
Human Transport ◽  
Sector Part

The automotive sector is one of the fastest growing sectors of economy. The increasing amount of cars both in Polish and world roads results in the immeasurable benefits associated with the goods and human transport. On the other hand, this phenomenon caused the contamination of the environment. During the fuel combustion in petrol or diesel engines, the harmful gases, for example CO2, NOx and SOx are emitted. Apart from the negative impact on the environment, the emission of the aforementioned gases results in the deterioration of human conditions, as well as, the development of civilization diseases. In order to minimalize the harmful influence of an automotive industry on the environment, new technologies which can reduce the consumption of fuel or limit the fumes emission are developed. The first part of paper presents new solutions in an automotive sector which influence on the decline of the negative impact of automobiles on the environment. Additionally, proposed solutions affect the development of a car industry, taking into consideration environmental aspects.

Introduction of Eucommia ulmoides Oliv. to in vitro Culture

2020 ◽  
pp. 38-50
Author(s):  
A.V. Zhigunov ◽  
◽  
Q.T. Nguyen
Keyword(s):  
Herbal Medicines ◽  
Naphthaleneacetic Acid ◽  
Eucommia Ulmoides ◽  
Juvenile Period ◽  
Dioecious Species ◽  
Relict Species ◽  
Mass Reproduction ◽  
Special Value ◽  
The Republic

The increasing need for herbal medicines requires the study of not only biological resources of medical plants, but also methods for their reproduction. Of special value are the medicinal plants that have a long history of success in traditional medicine. One of such plants is Eucommia ulmoides Oliv., which belongs to a rare relict species growing in natural conditions, for the most part, in the undergrowth of humid subtropical forests in China, mainly in the middle course of the Yangtze river. E. ulmoides compares favorably with most subtropical plants owing to its significant frost resistance, which makes it possible to cultivate it outside the humid subtropics. It has been widely introduced in Krasnodar Krai and in the Republic of Adygea (Russia) since the mid-20th century and successfully adapted to various environmental conditions in the Northwest Caucasus. The increasing demand for E. ulmoides bark can only be satisfied by laying out industrial plantations. However, the difficulties encountered in the traditional seed reproduction of E. ulmoides (dioecious species, pollen low quality, parthenocarpy, prolonged seed dormancy, irregular fruiting, long juvenile period, etc.) make scientists turn to modern biotechnological methods of plant propagation. While considering cultivation of planting material, we should focus on highly efficient methods that ensure stable and mass reproduction of the plants under study. An important role is played here by in vitro plant regeneration. The effectiveness of biotechnology methods is due to a reduction in timing of obtaining a large number of vegetative progeny of plants difficult for propagation, as well saving of the area required for their cultivation. The conditions for producing an aseptic culture of E. ulmoides were chosen based on the results of the studies. The highest degree of sterilization of E. ulmoides shoot segments was achieved when the explants were sequentially immersed first in 70 % ethanol (30 s) and then in 0.1 % mercuric chloride solution (5 min). With such a sterilization procedure, 63.3 % of the studied cuttings were made sterile, and 56.7 % of them proved to be viable. The optimal composition of the nutrient medium for regeneration of E. ulmoides microshoots has been determined: MS medium complemented with 1 mg/L 6-Benzylaminopurine (BAP) + 0.2 mg/L 1-Naphthaleneacetic acid (NAA). The best media for explant rooting are the following: 2/3 MS + 1.5 mg/L NAA + 30 g sucrose + 7 g agar; 2/3 MS + 1 mg/L NAA + 0.4 mg/L IBA + 30 g sucrose + 7 g agar.

Current Status and Future Perspective for Research on Medicinal Plants with Anticancerous Activity and Minimum Cytotoxic Value

2019 ◽  
Vol 20 (12) ◽  
pp. 1227-1243
Author(s):  
Hina Qamar ◽  
Sumbul Rehman ◽  
D.K. Chauhan
Keyword(s):  
Side Effects ◽  
Medicinal Plants ◽  
Anticancer Agents ◽  
Drug Targets ◽  
Psidium Guajava ◽  
Current Status ◽  
Future Perspective ◽  
Wide Range

Cancer is the second leading cause of morbidity and mortality worldwide. Although chemotherapy and radiotherapy enhance the survival rate of cancerous patients but they have several acute toxic effects. Therefore, there is a need to search for new anticancer agents having better efficacy and lesser side effects. In this regard, herbal treatment is found to be a safe method for treating and preventing cancer. Here, an attempt has been made to screen some less explored medicinal plants like Ammania baccifera, Asclepias curassavica, Azadarichta indica, Butea monosperma, Croton tiglium, Hedera nepalensis, Jatropha curcas, Momordica charantia, Moringa oleifera, Psidium guajava, etc. having potent anticancer activity with minimum cytotoxic value (IC50 >3μM) and lesser or negligible toxicity. They are rich in active phytochemicals with a wide range of drug targets. In this study, these medicinal plants were evaluated for dose-dependent cytotoxicological studies via in vitro MTT assay and in vivo tumor models along with some more plants which are reported to have IC50 value in the range of 0.019-0.528 mg/ml. The findings indicate that these plants inhibit tumor growth by their antiproliferative, pro-apoptotic, anti-metastatic and anti-angiogenic molecular targets. They are widely used because of their easy availability, affordable price and having no or sometimes minimal side effects. This review provides a baseline for the discovery of anticancer drugs from medicinal plants having minimum cytotoxic value with minimal side effects and establishment of their analogues for the welfare of mankind.

scholarly journals Evaluation of the Impact of Different Pain Medication and Proton Pump Inhibitors on the Osteogenic Differentiation Potential of hMSCs Using 99mTc-HDP Labelling

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 339
Author(s):  
Tobias Grossner ◽  
Uwe Haberkorn ◽  
Tobias Gotterbarm
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Bone Metabolism ◽  
Negative Impact ◽  
Pain Medication ◽  
Group 3 ◽  
Group 5 ◽  
The Impact

First-line analgetic medication used in the field of musculoskeletal degenerative diseases, like Nonsteroidal anti-inflammatory drugs (NSAIDs), reduces pain and prostaglandin synthesis, whereby peptic ulcers are a severe adverse effect. Therefore, proton pump inhibitors (PPI) are frequently used as a concomitant medication to reduce this risk. However, the impact of NSAIDs or metamizole, in combination with PPIs, on bone metabolism is still unclear. Therefore, human mesenchymal stem cells (hMSCs) were cultured in monolayer cultures in 10 different groups for 21 days. New bone formation was induced as follows: Group 1 negative control group, group 2 osteogenic differentiation media (OSM), group 3 OSM with pantoprazole (PAN), group 4 OSM with ibuprofen (IBU), group 5 OSM with diclofenac (DIC), group 6 OSM with metamizole (MET), group 7 OSM with ibuprofen and pantoprazole (IBU + PAN), group 8 OSM with diclofenac and pantoprazole (DIC + PAN), group 9 OSM with metamizole and pantoprazole (MET + PAN) and group 10 OSM with diclofenac, metamizole and pantoprazole (DIC + MET + PAN). Hydroxyapatite content was evaluated using high-sensitive radioactive 99mTc-HDP labeling. Within this study, no evidence was found that the common analgetic medication, using NSAIDs alone or in combination with pantoprazole and/or metamizole, has any negative impact on the osteogenic differentiation of mesenchymal stem cells in vitro. To the contrary, the statistical results indicate that pantoprazole alone (group 3 (PAN) (p = 0.016)) or diclofenac alone (group 5 (DIC) (p = 0.008)) enhances the deposition of minerals by hMSCS in vitro. There is an ongoing discussion between clinicians in the field of orthopaedics and traumatology as to whether post-surgical (pain) medication has a negative impact on bone healing. This is the first hMSC in vitro study that investigates the effects of pain medication in combination with PPIs on bone metabolism. Our in vitro data indicates that the assumed negative impact on bone metabolism is subsidiary. These findings substantiate the thesis that, in clinical medicine, the patient can receive every pain medication needed, whether or not in combination with PPIs, without any negative effects for the osteo-regenerative potential.

scholarly journals Genetic Perturbation of Pyruvate Dehydrogenase Kinase 1 Modulates Growth, Angiogenesis and Metabolic Pathways in Ovarian Cancer Xenografts

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 325
Author(s):  
Carolina Venturoli ◽  
Ilaria Piga ◽  
Matteo Curtarello ◽  
Martina Verza ◽  
Giovanni Esposito ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Pyruvate Dehydrogenase ◽  
Metabolic Pathways ◽  
Negative Impact ◽  
Digital Pathology ◽  
Pyruvate Dehydrogenase Kinase ◽  
Ovarian Cancer Cells ◽  
Pyruvate Dehydrogenase Kinase 1

Pyruvate dehydrogenase kinase 1 (PDK1) blockade triggers are well characterized in vitro metabolic alterations in cancer cells, including reduced glycolysis and increased glucose oxidation. Here, by gene expression profiling and digital pathology-mediated quantification of in situ markers in tumors, we investigated effects of PDK1 silencing on growth, angiogenesis and metabolic features of tumor xenografts formed by highly glycolytic OC316 and OVCAR3 ovarian cancer cells. Notably, at variance with the moderate antiproliferative effects observed in vitro, we found a dramatic negative impact of PDK1 silencing on tumor growth. These findings were associated with reduced angiogenesis and increased necrosis in the OC316 and OVCAR3 tumor models, respectively. Analysis of viable tumor areas uncovered increased proliferation as well as increased apoptosis in PDK1-silenced OVCAR3 tumors. Moreover, RNA profiling disclosed increased glucose catabolic pathways—comprising both oxidative phosphorylation and glycolysis—in PDK1-silenced OVCAR3 tumors, in line with the high mitotic activity detected in the viable rim of these tumors. Altogether, our findings add new evidence in support of a link between tumor metabolism and angiogenesis and remark on the importance of investigating net effects of modulations of metabolic pathways in the context of the tumor microenvironment.

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