Improved electroporation buffer enhances transient gene expression in Arachis hypogaea protoplasts

Genome ◽  
1995 ◽  
Vol 38 (5) ◽  
pp. 858-863 ◽  
Author(s):  
Zhijian Li ◽  
Ming Cheng ◽  
James W. Demski ◽  
Robert L. Jarret
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Arachis Hypogaea ◽  
Transient Gene Expression ◽  
Gus Expression ◽  
Histochemical Staining ◽  
Media Analysis ◽  
Gus Activity ◽  
Arachis Hypogaea L ◽  
Protoplast Viability

An electroporation medium containing 50 mM glycine or 10 mM glycylglycine (glygly), 70 mM potassium glutamate, and 0.4 M mannitol was evaluated for its ability to improve transient β-glucuronidase (GUS) expression in immature cotyledonary protoplasts of Arachis hypogaea L. GUS activity in electroporated protoplasts was 8- to 430-fold greater than that obtained using any of other four commonly employed poration media. Analysis of viability and histochemical staining of protoplasts indicated that electroporation using the glycine- or glygly-based poration medium resulted in increased protoplast viability and GUS expression when compared with other poration media. Replacement of glygly with MES or HEPES buffers significantly reduced the level of GUS expression in electroporated protoplasts.Key words: transient expression, electroporation, Arachis, protoplasts, GUS.

scholarly journals Transient Expression of β-Glucuronidase (GUS) Activity in Embryogenic Callus Tissue of Alkaligrass (Puccinellia distans) using Particle Bombardment

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 913A-913
Author(s):  
Aaron Brown ◽  
Harrison G. Hughes
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Particle Bombardment ◽  
Callus Induction ◽  
Embryogenic Callus ◽  
Callus Tissue ◽  
Transient Gene Expression ◽  
Plant Cells ◽  
Gus Activity ◽  
Puccinellia Distans

Callus induction and regeneration of alkaligrass (Puccinellia distans) was developed in our laboratory for use in transformation studies of turfgrass. Particle bombardment of the embryogenic callus is being evaluated using a helium particle inflow gun constructed at Colorado State Univ., according to the design of Philippe et al. (Ohio State Univ., 1993). Its utility in delivering DNA to plant cells is being tested by measuring the frequency of transient gene expression of a reporter gene (GUS pBI121) in embryogenic callus of alkaligrass. Varying pressure of helium and the distance of the calli in the chamber are also being evaluated for efficiency in transformation.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Overexpression of a Redox-Regulated Cutinase Gene, MfCUT1, Increases Virulence of the Brown Rot Pathogen Monilinia fructicola on Prunus spp.

2010 ◽  
Vol 23 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Miin-Huey Lee ◽  
Chiu-Min Chiu ◽  
Tatiana Roubtsova ◽  
Chien-Ming Chou ◽  
Richard M. Bostock
Keyword(s):  
Gene Expression ◽  
Redox Regulation ◽  
Brown Rot ◽  
Gus Expression ◽  
Apple Fruit ◽  
Gus Activity ◽  
Monilinia Fructicola ◽  
Gus Reporter ◽  
Flanking Regions ◽  
Prunus Spp

A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUT1, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUT1 carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUT1 expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUT1 gene expression in cutin-induced cultures, and in the present study, where H2O2 enhanced MfCUT1 gene expression. A β-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUT1 under the control of the MfCUT1 promoter, and this construct was then used to generate an MfCUT1-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUT1-GUS fusion protein was expressed correctly under the control of the MfCUT1 promoter. MfCUT1-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUT1 expression within 5 h of inoculation in both almond and peach petals. MfCUT1-GUS transformants expressed MfCUT1 transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUT1 contributing to the virulence of M. fructicola.

scholarly journals Biolistic-mediated transient gene expression in shoot apical meristems of the prickly-pear (Opuntia ficus-indica)

1999 ◽  
Vol 42 (3) ◽  
pp. 299-302 ◽  
Author(s):  
Romulo Marino Llamoca-Zárate ◽  
Luiz Ferreira Aguiar Ponte ◽  
Joerg Landsmann ◽  
Francisco de Assis Paiva Campos
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Transient Gene Expression ◽  
Dna Delivery ◽  
Prickly Pear ◽  
Target Tissue ◽  
Gus Gene ◽  
Transformation System ◽  
Opuntia Ficus Indica ◽  
Apical Meristems

We have demonstrated the transient expression of the GUS gene in cells of the meristematic apical dome of Opuntia ficus-indica. DNA delivery into the cells was achieved using a biolistic PDS-1000He instrument from Bio-Rad Laboratories. The transforming DNA was coated in tungsten particles with diameter of 1.3 m m and the distance between the flying disk and the target tissue was 7.5cm and the shooting pressure was adjusted to 1200 psi. This is the first demonstration that the biolistic transformation system can be used to express a transgene in a member of the Cactaceae.

scholarly journals Transient gene expression of beta-glucuronidase in citrus thin epicotyl transversal sections using particle bombardment

2003 ◽  
Vol 46 (1) ◽  
pp. 1-6 ◽  
Author(s):  
João C. Bespalhok Filho ◽  
Adilson K. Kobayashi ◽  
Luiz F. P. Pereira ◽  
Rafaelo M. Galvão ◽  
Luiz G. E. Vieira
Keyword(s):  
Gene Expression ◽  
Particle Bombardment ◽  
Citrus Sinensis ◽  
Sweet Orange ◽  
Transient Gene Expression ◽  
Gus Expression ◽  
Poncirus Trifoliata ◽  
Helium Pressure ◽  
High Osmolarity ◽  
Carrizo Citrange

Studies were carried out to optimize the conditions for transient gene expression through particle bombardment on Carrizo citrange (Citrus sinensis x Poncirus trifoliata) thin epicotyl sections. The best conditions for transient GUS expression were: M-25 tungsten particles, 1550 psi helium pressure, 9 cm distance between specimen and DNA/particle holder and culture of explants in a high osmolarity medium (0.2 M mannitol + 0.2 M sorbitol) 4 h prior and 20 h after bombardment. Under these conditions, an average of 102 blue spots per bombardment (20 explants/plate) were achieved. This protocol is currently being used for transformation of Carrizo citrange and sweet orange (Citrus sinensis).

scholarly journals Transient Gene Expression is an Effective Experimental Tool for the Research into the Fine Mechanisms of Plant Gene Function: Advantages, Limitations, and Solutions

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1187
Author(s):  
Alexander A. Tyurin ◽  
Alexandra V. Suhorukova ◽  
Ksenia V. Kabardaeva ◽  
Irina V. Goldenkova-Pavlova
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Gene Function ◽  
Transient Gene Expression ◽  
Physiological Role ◽  
Gene Products ◽  
Plant Gene ◽  
Gene Functions ◽  
Insight Into

A large data array on plant gene expression accumulated thanks to comparative omic studies directs the efforts of researchers to the specific or fine effects of the target gene functions and, as a consequence, elaboration of relatively simple and concurrently effective approaches allowing for the insight into the physiological role of gene products. Numerous studies have convincingly demonstrated the efficacy of transient expression strategy for characterization of the plant gene functions. The review goals are (i) to consider the advantages and limitations of different plant systems and methods of transient expression used to find out the role of gene products; (ii) to summarize the current data on the use of the transient expression approaches for the insight into fine mechanisms underlying the gene function; and (iii) to outline the accomplishments in efficient transient expression of plant genes. In general, the review discusses the main and critical steps in each of the methods of transient gene expression in plants; areas of their application; main results obtained using plant objects; their contribution to our knowledge about the fine mechanisms of the plant gene functions underlying plant growth and development; and clarification of the mechanisms regulating complex metabolic pathways.

scholarly journals Activation of Latent Transgenes in Arabidopsis Using a Hybrid Transcription Factor

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 633-639 ◽  
Author(s):  
Dave Guyer ◽  
Ann Tuttle ◽  
Sabrina Rouse ◽  
Sandra Volrath ◽  
Marie Johnson ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Activation ◽  
Gus Expression ◽  
Histochemical Staining ◽  
Gus Activity ◽  
Synthetic Promoter ◽  
Cis Acting ◽  
Transcription Activation Domain ◽  
Adenylosuccinate Synthetase ◽  
Transgenic Lines

Abstract A hybrid transcription factor comprising a fusion of the DNA-binding domain of Saccharomyces cerevisiae GAL4 and the transcription activation domain of maize C1 was expressed in stably transformed Arabidopsis. Additional transgenic lines were created containing test genes controlled by a synthetic promoter consisting of concatemeric copies of the cis-acting site recognized by GAL4 (UASG) fused to a minimal promoter. The GAL4/C1 effector line was crossed to two lines containing a synthetic promoter/GUS fusion. Both histochemical staining and GUS activity assays indicate strong activation of GUS expression was achieved only after crossing. The GAL4/C1 effector line was also crossed to 15 lines containing a synthetic promoter/antisense adenylosuccinate synthetase gene. Severely retarded growth, and in some cases lethality, was observed in 40% of the F1 lines. This system of activation by crossing is generally useful for activating expression of test transgenes.

scholarly journals Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Guoliang Yuan ◽  
Haiwei Lu ◽  
Dan Tang ◽  
Md Mahmudul Hassan ◽  
Yi Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Plants ◽  
Transient Expression ◽  
Plant Species ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Research Work ◽  
Transient Gene Expression ◽  
Stable Transformation ◽  
Herbaceous Plant

AbstractGreen fluorescent protein (GFP) has been widely used for monitoring gene expression and protein localization in diverse organisms. However, highly sensitive imaging equipment, like fluorescence microscope, is usually required for the visualization of GFP, limitings its application to fixed locations in samples. A reporter that can be visualized in real-time regardless the shape, size and location of the target samples will increase the flexibility and efficiency of research work. Here, we report the application of a GFP-like protein, called eYGFPuv, in both transient expression and stable transformation, in two herbaceous plant species (Arabidopsis and tobacco) and two woody plant species (poplar and citrus). We observed bright fluorescence under UV light in all of the four plant species without any effects on plant growth or development. eYGFPuv was shown to be effective for imaging transient expression in leaf and root tissues. With a focus on in vitro transformation, we demonstrated that the transgenic events expressing 1x eYGFPuv could be easily identified visually during the callus stage and the shoot stage, enabling early and efficient selection of transformants. Furthermore, whole-plant level visualization of eYGFPuv revealed its ubiquitous stability in transgenic plants. In addition, our transformation experiments showed that eYGFPuv can also be used to select transgenic plants without antibiotics. This work demonstrates the feasibility of utilizing 1x eYGFPuv in studies of gene expression and plant transformation in diverse plants.

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