Application of polymeric dimethylaminoethyl methacrylate based carriers of plasmid DNA for genetic transformation of Ceratodon purpureus moss

Introduction. Genetic engineering in plants is of great importance for agriculture, biotechnology and medicine, and nanomaterials are widely used for genetic engineering. The aim of present study was to evaluate the potential of poly(2-dimethylamino)ethyl methacrylate (DMAEMA)-based comb-like polymers as gene delivery systems in moss Ceratodon purpureus (Hedw.) Brid. protoplasts and determine the level of phytotoxicity of these polymers. Materials and Methods. In order to confirm the formation of complex of poly-DMAEMA carrier with plasmid DNA pSF3, gel retardation assay was used. The PEG-mediated transformation protocol was adapted to transform the protoplasts of C. purpureus moss with poly-DMAEMA carriers. Light microscopy was used to study a toxicity of polymers for moss protoplasts. The level of the polymers toxicity was estimated as IC50 value. Results and Discussion. The formation of pDNA complex with DMAEMA-based carriers took place at 0.03% concentration of the polymers BGA-21, BGA-22(2ph), BG-24, BG-25, BG-26 or 0.1% concentration of the BGA-22 polymer. Poly-DMAEMA carriers were able to deliver plasmid DNA pSF3 into protoplasts of C. purpureus moss. Three stable transformants of C. purpureus were obtained at using BGA-22 polymer, 2 clones – at using BGA-21 carrier, and 1 clone – at using BGA-22(2ph), BG-24, BG-25, BG-26 polymers. The poly-DMAEMA carriers at the working 0.0025% dose were relatively non-toxic for protoplasts of C. purpureus moss. 83.1-88.4% of viable protoplasts of C. purpureus moss were detected after treatment with studied carriers at 0.0025% dose. A survival ratio of protoplasts reached 66.7-72.9% under the effect of these polymers at 0.025% dose, which is 10 times higher than their working concentration. The IC50 value of poly-DMAEMA carriers was in the range of 0.113-0.164%, that was approximately 10 times higher than that of the PEG-6000 used for gene delivery in plants. Conclusion. Novel synthetic poly-DMAEMA carriers delivered the gene of interest into moss C. purpureus protoplasts and possessed a low phytotoxicity. Thus, these carriers can be useful for gene delivery into plant cells.

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Polyglycerol-functionalized nanodiamond as a platform for gene delivery: Derivatization, characterization, and hybridization with DNA

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.10.64 ◽

2014 ◽

Vol 10 ◽

pp. 707-713 ◽

Cited By ~ 25

Author(s):

Li Zhao ◽

Yuki Nakae ◽

Hongmei Qin ◽

Tadamasa Ito ◽

Takahide Kimura ◽

...

Keyword(s):

Gene Delivery ◽

Zeta Potential ◽

Plasmid Dna ◽

Ring Opening ◽

Ring Opening Polymerization ◽

Gene Vector ◽

Organic Transformations ◽

Gel Retardation Assay ◽

Basic Polypeptides ◽

Basic Polypeptide

A gene vector consisting of nanodiamond, polyglycerol, and basic polypeptide (ND-PG-BPP) has been designed, synthesized, and characterized. The ND-PG-BPP was synthesized by PG functionalization of ND through ring-opening polymerization of glycidol on the ND surface, multistep organic transformations (–OH → –OTs (tosylate) → –N3) in the PG layer, and click conjugation of the basic polypeptides (Arg8, Lys8 or His8) terminated with propargyl glycine. The ND-PG-BPP exhibited good dispersibility in water (>1.0 mg/mL) and positive zeta potential ranging from +14.2 mV to +44.1 mV at neutral pH in Milli-Q water. It was confirmed by gel retardation assay that ND-PG-Arg8 and ND-PG-Lys8 with higher zeta potential hybridized with plasmid DNA (pDNA) through electrostatic attraction, making them promising as nonviral vectors for gene delivery.

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Gene delivery into cultured plant cells by DNA-coated gold particles accelerated by a pneumatic particle gun

Theoretical and Applied Genetics ◽

10.1007/bf00224198 ◽

1990 ◽

Vol 80 (6) ◽

pp. 813-816 ◽

Cited By ~ 59

Author(s):

A. Iida ◽

M. Seki ◽

M. Kamada ◽

Y. Yamada ◽

H. Morikawa

Keyword(s):

Gene Delivery ◽

Plant Cells ◽

Particle Gun ◽

Gold Particles

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Plasmid DNA microgels for cancer treatment through combination of chemotherapy and gene delivery

Journal of Biotechnology ◽

10.1016/j.jbiotec.2014.07.118 ◽

2014 ◽

Vol 185 ◽

pp. S35

Author(s):

Diana Barata Costa ◽

Artur Monteiro Valente ◽

João Sampaio Queiroz

Keyword(s):

Gene Delivery ◽

Cancer Treatment ◽

Plasmid Dna

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Efficient Genetic Transformation of Rice for CRISPR/Cas9 Mediated Genome-Editing and Stable Overexpression Studies: A Case Study on Rice Lipase 1 and Galactinol Synthase Encoding Genes

Agronomy ◽

10.3390/agronomy12010179 ◽

2022 ◽

Vol 12 (1) ◽

pp. 179

Author(s):

Tanika Thakur ◽

Kshitija Sinha ◽

Tushpinder Kaur ◽

Ritu Kapoor ◽

Gulshan Kumar ◽

...

Keyword(s):

Genetic Transformation ◽

Crop Improvement ◽

Rice Cultivar ◽

Climatic Conditions ◽

Rice Transformation ◽

Transformation Protocol ◽

Galactinol Synthase ◽

Biotic Stresses ◽

Putative Transformants ◽

Efficient Genetic Transformation

Rice is a staple food crop for almost half of the world’s population, especially in the developing countries of Asia and Africa. It is widely grown in different climatic conditions, depending on the quality of the water, soil, and genetic makeup of the rice cultivar. Many (a)biotic stresses severely curtail rice growth and development, with an eventual reduction in crop yield. However, for molecular functional analysis, the availability of an efficient genetic transformation protocol is essential. To ensure food security and safety for the continuously increasing global population, the development of climate-resilient crops is crucial. Here, in this study, the rice transformation protocol has been effectively optimized for the efficient and rapid generation of rice transgenic plants. We also highlighted the critical steps and precautionary measures to be taken while performing the rice transformation. We further assess the efficacy of this protocol by transforming rice with two different transformation constructs for generating galactinol synthase (GolS) overexpression lines and CRISPR/Cas9-mediated edited lines of lipase (Lip) encoding the OsLip1 gene. The putative transformants were subjected to molecular analysis to confirm gene integration/editing, respectively. Collectively, the easy, efficient, and rapid rice transformation protocol used in this present study can be applied as a potential tool for gene(s) function studies in rice and eventually to the rice crop improvement.

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Efficiency of Genetic Transformation via Pollen-Tube Pathway of Jatropha (Jatropha curcas L.) Based on Histochemical and Molecular Analysis

Jurnal Agronomi Indonesia (Indonesian Journal of Agronomy) ◽

10.24831/jai.v45i3.12925 ◽

2018 ◽

Vol 45 (3) ◽

pp. 316

Author(s):

Agus Zainudin ◽

Bambang Sapta Purwoko ◽

Tri Joko Santoso ◽

Sintho Wahyuning Ardie ◽

And Trikoesoemaningtyas

Keyword(s):

Pollen Tube ◽

Genetic Transformation ◽

Molecular Analysis ◽

Plasmid Dna ◽

Jatropha Curcas ◽

Marker Gene ◽

Pcr Analysis ◽

Jatropha Curcas L ◽

Gus Reporter ◽

Combination Treatments

The genetic transformation via pollen-tube pathway is an alternative method to overcome the constraints imposed by genotype specificity in transformation and regeneration in jatropha (Jatropha curcas L.) tissue culture. Therefore, it is necessary to establish important parameters for efficient genetic transformation of jatropha via pollen-tube pathway. The objective of the research was to study the efficiency of direct transformation of jatropha via pollen-tube pathway based on histochemical and molecular analysis. Solution of purified pCAMBIA1301 DNA plasmid carrying a hptII marker gene and a gus reporter gene with concentration level of 0.05, 0.25, 0.50 µg µl-1 were applied to stigma of flowers at 1, 2, 4, 7, 10 h after pollination. Seedling of IP3A, IP3P and JcUMM18 jatropha’s genotypes derived from 15 combination treatments of plasmid DNA concentration and application time, also wild type was subjected to histochemical and molecular analyses. Based on those analyses, the efficiency of transformation via pollen-tube pathway of three jatropha genotypes ranged from 1.5-16.7%. PCR analysis showed that a number of positive plants were identified by using specific primers hptII and gus, i.e. 1-3 and 3-7 plants of the 15 combined treatments, respectively. It indicated that the transformation efficiency via the pollen-tube pathway varied in each jatropha genotype.<br /><br />Keywords: Jatropha curcas L., pCAMBIA1301, plasmid DNA, stigma-drip<br /><br />

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Block Copolymer/Plasmid DNA Micelles Postmodified with Functional Peptides via Thiol–Maleimide Conjugation for Efficient Gene Delivery into Plants

Biomacromolecules ◽

10.1021/acs.biomac.8b01304 ◽

2018 ◽

Vol 20 (2) ◽

pp. 653-661 ◽

Cited By ~ 9

Author(s):

Takaaki Miyamoto ◽

Kousuke Tsuchiya ◽

Keiji Numata

Keyword(s):

Block Copolymer ◽

Gene Delivery ◽

Plasmid Dna ◽

Efficient Gene ◽

Functional Peptides

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The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae

Metabolites ◽

10.3390/metabo9030049 ◽

2019 ◽

Vol 9 (3) ◽

pp. 49 ◽

Cited By ~ 1

Author(s):

Ana Molina-Márquez ◽

Marta Vila ◽

Javier Vigara ◽

Ana Borrero ◽

Rosa León

Keyword(s):

Genetic Transformation ◽

Selectable Marker ◽

Genetic Manipulation ◽

Plant Cells ◽

Phytoene Desaturase ◽

Carotenoid Content ◽

Great Promise ◽

Encoding Gene ◽

Average Transformation ◽

Selection Of

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

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