Factors affecting transient gene expression in electroporated Glycine max protoplasts

Dhir, Sarwan K.; Hepburn, Angus G.; Widholm, Jack M.

doi:10.1007/bf00236468

Cited by 17 publications

(8 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…2, 3), as reported for other species (42)(43)(44)(45). This result highlights an advantage of FCM over biochemical methods, namely, the ability to quantify gene expression on a per-cell basis in a small (Ͻ6%; Fig.…”

Section: Discussionsupporting

confidence: 80%

Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts

Hagenbeek

Rock

2001

Cytometry

View full text Add to dashboard Cite

Background: Quantifying plant gene expression by flow cytometry (FCM) would allow multidimensional cell-parameter analysis on a per-cell basis, thereby providing insight into the cellular mechanisms of plant gene regulation. Here we sought to establish quantitation by FCM of plant hormone (abscisic acid, ABA)-inducible green fluorescent protein (GFP) expression and to compare the method directly with traditional reporter enzyme assays. Materials and Methods: GFP, ␤-glucuronidase, and luciferase reporter genes driven by ABA-inducible or constitutive promoter constructs were expressed in transiently cotransformed rice protoplasts and reporter activities quantified by FCM (for GFP) or traditional enzyme assays. Treatments included cotransformations with specific ABA signaling effector cDNA constructs (encoding VIVIPAROUS-1, an ABA transcription factor, and ABA-INSENSITIVE1-1, a dominant-negative protein phosphatase regulator) and the ABA agonist lanthanum chloride. Dual-color FCM was also performed on GFP-expressing cells immunodecorated with an mAb recognizing a rice cell surface epitope. Results: Quantitative analysis of ABA-inducible gene expression by FCM using GFP as reporter gave comparable results to traditional reporter enzyme assays, although the signal-to-noise ratio was less for FCM, which can be a limitation of the method at low promoter strengths. Multiparameter-correlated analysis of ABA-inducible GFP expression with a plasma membrane marker showed no apparent correlation between ABA sensitivity, marked by GFP, and presence of a cell surface arabinogalactan glycoprotein.Conclusions: Quantitative FCM of GFP-expressing plant cells is a rapid, robust, reproducible, and value-added method relative to traditional enzymatic reporter gene assays. Cytometry 45:170 -179, 2001.

show abstract

Section: Discussionsupporting

confidence: 80%

Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts

Hagenbeek

Rock

2001

Cytometry

View full text Add to dashboard Cite

show abstract

“…2a) . Similar frequencies were observed by other groups in different species (Zhang & Wu, 1988 ;Dhir et al ., 1991 ;Diaz & Carbonero, 1992) .…”

Section: Transient Gus Expression (Tge) In Electroporated Protoplastssupporting

confidence: 91%

Transient gene expression in transformed banana (Musa cv. Bluggoe) protoplasts and embryogenic cell suspensions

et al. 1995

View full text Add to dashboard Cite

“…One ml of protoplast suspension was mixed with 20 mg of the plasmid DNA to which was added 200 ml of 15% (w/v) PEG 4000. Electroporation was performed by delivering an electric pulse at 25 mF from the capacitor charged at 150 V (350 V cm 21 ) as described previously (Dhir et al, 1991;Mitchell et al, 1998). Electroporated protoplasts were diluted immediately after electroporation with KM8P (Kao and Michayluk, 1975) medium to a density of 10 5 ml 21 , and incubated in the dark for 1 h before embedding in 1.2% SeaPlaque (FMC) agarose.…”

Section: Methodsmentioning

confidence: 99%

Transformation of sweet potato tissues with green-fluorescent protein gene

Winfield

Lawton

Daniell

et al. 2001

In Vitro Cell.Dev.Biol.-Plant

View full text Add to dashboard Cite

The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) cv. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embryogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1 Â 10 6 protoplasts ml 21 was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm 21 . Approximately 25±30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidified with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro calluses out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600±700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic callus. In bombarded cultures, several stable GFP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful screenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.

show abstract

Factors affecting transient gene expression in electroporated Glycine max protoplasts

Cited by 17 publications

References 14 publications

Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts

Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts

Transient gene expression in transformed banana (Musa cv. Bluggoe) protoplasts and embryogenic cell suspensions

Transformation of sweet potato tissues with green-fluorescent protein gene

Contact Info

Product

Resources

About