Transient Expression of Green Fluorescent Protein in Rice Calluses : Optimization of Parameters for Helios Gene Gun Device

Carsono, Nono; Yoshida, Tomohiko

doi:10.1626/pps.11.88

Cited by 12 publications

(13 citation statements)

References 37 publications

(43 reference statements)

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“…Evenly-sliced leaf sheath tissue, a clear cell structure with little auto-fluorescence, was used for the biolistic bombardment assay. Expression efficiencies of nearly 30%, which were similar to levels in previously-published Arabidopsis and rice callus systems 6,11 , were observed using a mCherry reporter gene expressed under the maize ubiquitin promoter. Our protocol provides a rapid and efficient way to analyze expression and subcellular localization of proteins of interest in rice cells.…”

Section: Introductionsupporting

confidence: 81%

A Transient Gene Expression Protocol for Subcellular Protein Localization and Protein Secretion Analyses in Rice

Wang¹,

Wu²,

Kim³

et al. 2013

Protocol Exchange

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We developed a protocol for in planta gene expression analyses in sliced rice sheath cells by modifying and optimizing biolistic particle bombardment techniques. By obtaining thin sections (400 um) of rice sheath cells, auto-fluorescence tissues from chlorophyll were eliminated. This system was validated through the localization of marker genes specifically expressed in nuclei, plasma membranes, and vacuolar membranes. Additionally, high transformation efficiencies of approximately 30% were achieved. Therefore, this protocol provides a new and rapid method for transient gene expression assays in rice. Protein secretion was examined in rice sheath cells using predicted secretory proteins from rice blast fungus, indicating that this method is practical for host plantmicrobe interaction studies. The transient expression protocol established here is well suited for protein localization, secretion, and host-pathogen protein interaction studies in rice. A typical experiment can be completed in 3 days.

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Section: Introductionsupporting

confidence: 81%

A Transient Gene Expression Protocol for Subcellular Protein Localization and Protein Secretion Analyses in Rice

Wang¹,

Wu²,

Kim³

et al. 2013

Protocol Exchange

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“…Biolistic mediated transformation is preferred over Agrobacterium-mediated transformation as it does not require construction of a specialized vector. Also, the advantage of biolistic technology for direct gene transfer into plant cell/tissues has facilitated plant transformation on monocots such as maize (12), wheat (13), rice (14), barley (15), palm date (16) and oil palm (17). The efficiency of biolistic transformation depends on certain physical and biological parameters (18).…”

Section: Introductionmentioning

confidence: 99%

Sugarcane (NCo310) Transient Transformation Using uidA Reporter Gene

et al. 2013

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Background: Sugarcane is a monocotyledonous crop that is cultivated in the tropical and subtropical regions of the world. One of the most important criteria, influencing the efficiency of the sugarcane transformation is known to be related to physical and biological factors during the transformation procedure. Objectives: The objective of this research was to study the response of callus induction and embryogenic callus production and to identify the major parameters controlling DNA delivery by particle bombardment into sugarcane (Saccharum officinarum L.) cv. NCo310. Materials and Methods: For callus induction and embryogenic callus production, leaf base segments were subjected to in vitro culture medium supplemented with two plant growth regulators (2,4-D and Dicamba). Results showed that 1 mg.L -1 2,4-D was significantly influential in callus induction and embryogenic callus production. Considering both physical and biological factors, the efficiency of DNA (uidA gene) delivery was assessed by scoring transient GUS (gene (β-glucuronidase) ) expression in bombarded tissues. Results: The highest transient GUS expression was obtained when callus was bombarded with the construct harboring rice Act1 promoter, and having 9 cm target distance, 25 inHg vacuum pressure, 1 µm gold particles, 12.5 µg DNA per bombardment and one day pre-culture prior to the bombardment. Conclusions: A bombardment procedure suitable for elite sugarcane varieties was developed, which allowed high-efficiency DNA delivery combined with reduced damage to target tissues.

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“…Helenius et al [28] and Carsono and Yoshida [24] suggested that pressure of between 200 and 250 psi is the best to use with the distance of about 2-3 cm to intact plant cells. The helium pressure and the distance used in this sago palm embryonic callus transformation were slightly higher due to the different type of explants used.…”

Section: Resultsmentioning

confidence: 99%

Genetic Transformation ofMetroxylon sagu(Rottb.) Cultures viaAgrobacterium-Mediated and Particle Bombardment

Ibrahim

Hossain

Roslan

2014

BioMed Research International

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Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance.

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Transient Expression of Green Fluorescent Protein in Rice Calluses : Optimization of Parameters for Helios Gene Gun Device

Cited by 12 publications

References 37 publications

A Transient Gene Expression Protocol for Subcellular Protein Localization and Protein Secretion Analyses in Rice

A Transient Gene Expression Protocol for Subcellular Protein Localization and Protein Secretion Analyses in Rice

Sugarcane (NCo310) Transient Transformation Using uidA Reporter Gene

Genetic Transformation ofMetroxylon sagu(Rottb.) Cultures viaAgrobacterium-Mediated and Particle Bombardment

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