Exogenous DNA Uptake via Cellular Permeabilization and Expression of Foreign Gene in Wheat Zygotic Embryos.

Mahalakshmi, Akella; Chugh, Archana; Khurana, Paramjit

doi:10.5511/plantbiotechnology.17.235

Cited by 8 publications

(8 citation statements)

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“…Peptide synthesis and fluorophore labelling Peptides (Table 1) [26,[50][51][52][53] were custom synthesised and fluoresceinated at the N-terminal amino group (Alberta Peptide Institute, Edmonton, Canada). FITC-dextran sulfate (4 kDa; Sigma Aldrich, Oakville, Canada) served as a nonpeptidic negative control in the initial experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorimetric analysis also indicated that transportan had relatively more penetration ability in the nonpermeabilized embryos than the Tat peptides and pVEC; nonetheless, an increase in uptake of labeled transportan (1.9-fold) was also observed in the embryos subjected to permeabilization treatment. Fluorescence uptake for negative controls [M-Tat and fluorescein isothiocyanate (FITC)-dextran sulfate] also doubled in permeabilized embryos but their relative fluorescence uptake level remained [53] significantly lower than the other CPPs investigated (Fig. 1B).…”

Section: Cellular Uptake Of Cpps Is Enhanced Remarkably By Permeabilimentioning

confidence: 98%

“…They also serve as a model tissue for genetic transformation studies owing to their amenability towards tissue culture procedures and a high efficacy for plant regeneration. We investigated the uptake of five fluorescently labeled CPPs [Tat (49)(50)(51)(52)(53)(54)(55)(56)(57), Tat 2 , mutated-Tat (M-Tat), pVEC, transportan] in wheat immature embryos. We demonstrate that permeabilization of immature embryos is a prerequisite to achieve efficient translocation of CPPs and their macromolecular cargoes.…”

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Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

Chugh¹,

Eudes²

2008

The FEBS Journal

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Cell-penetrating peptides (CPPs) comprise a fast growing class of short length peptides that differ in sequence, size and charge but share a common characteristic ability to translocate across the plasma membrane. It has been demonstrated that CPPs can act efficiently as nonviral delivery vehicles for macromolecules that are much larger in size than their own and lack the self-potential to enter living cells due to the The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat 2 , mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene ⁄ ethanol (1 : 20, v ⁄ v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cellpenetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active b-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat 2 -mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene ⁄ ethanol (1 : 40, v ⁄ v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat 2 -mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat 2 -GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat 2 -plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat 2 -plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent LipofectamineÔ 2000 to the Tat 2 -plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.Abbreviations AID, arginine-rich intracellular delivery; CPP, cell-penetrating peptide; EIPA, 5-(N-ethyl N-isopropyl) amirolide; b-GUS, b-glucuronidase; M-Tat, mutated-Tat; pVEC, peptide vascular endothelial-cadherin.

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Section: Methodsmentioning

confidence: 99%

Section: Cellular Uptake Of Cpps Is Enhanced Remarkably By Permeabilimentioning

confidence: 98%

mentioning

confidence: 99%

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Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

Chugh¹,

Eudes²

2008

The FEBS Journal

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“…Although most regeneration studies in wheat have been confined to immature embryos, scutellum and also to immature inflorescence tissue (Patnaik & Khurana, 2001), nonetheless, mature embryos have also been employed for callusing, regeneration and transformation studies (Mahalakshmi et al, 2000;Patnaik & Khurana, 2001;Khurana et al, 2002). Ozgen et al (1996a, b) appraised the callusing response of mature and immature embryos of seven genotypes of winter durum wheat cultured on MS medium supplemented with 2,4-D and found that mature embryos had low frequency of callus formation but a high regeneration capacity in comparison with immature embryos.…”

Section: Generation Of Pin2 Transgenic Plantsmentioning

confidence: 98%

Assessment of Nematode Resistance in Wheat Transgenic Plants Expressing Potato Proteinase Inhibitor (PIN2) Gene

et al. 2005

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Serine proteinase inhibitors (IP's) are proteins found naturally in a wide range of plants with a significant role in the natural defense system of plants against herbivores. The question addressed in the present study involves assessing the ability of the serine proteinase inhibitor in combating nematode infestation. The present study involves engineering a plant serine proteinase inhibitor (pin2) gene into T. durum PDW215 by Agrobacterium-mediated transformation to combat cereal cyst nematode (Heterodera avenae) infestation. Putative T(0) transformants were screened and positive segregating lines analysed further for the study of the stable integration, expression and segregation of the genes. PCR, Southern analysis along with bar gene expression studies corroborate the stable integration pattern of the respective genes. The transformation efficiency is 3%, while the frequency of escapes was 35.71%. chi(2) analysis reveals the stable integration and segregation of the genes in both the T(1) and T(2) progeny lines. The PIN2 systemic expression confers satisfactory nematode resistance. The correlation analysis suggests that at p < 0.05 level of significance the relative proteinase inhibitor (PI) values show a direct positive correlation vis-à-vis plant height, plant seed weight and also the seed number.

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“…Recently, Mahalakshmi et al (2000) exploited the altered membrane characteristics of dry tissues and coupled it with the use of membrane permeabilizing substances like saponin, toluene, triton, etc. ; and reported gus gene expression in mechanically isolated seed-derived dry embryos of wheat by incubating in a buffer containing plasmid DNA along with various membrane permeabilizing agents.…”

Section: Alternative Approachesmentioning

confidence: 99%

Wheat biotechnology: A minireview

Khurana¹,

Patnaik²

2001

Electron. J. Biotechnol.

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Exogenous DNA Uptake via Cellular Permeabilization and Expression of Foreign Gene in Wheat Zygotic Embryos.

Cited by 8 publications

References 41 publications

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

Assessment of Nematode Resistance in Wheat Transgenic Plants Expressing Potato Proteinase Inhibitor (PIN2) Gene

Wheat biotechnology: A minireview

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