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EoE Sub Articles

1. Rice genetic transformation and plant tissue culture
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	<li>Callus induction and regeneration 
	NOTE: Use fresh young rice inflorescences as the starting material to induce callus (Figure 1).
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		<li>Collect the young inflorescences from the paddy field or green house at meiosis stage, determined by floret length of 1.6-4.8 mm (Figure 1A). Ensure that the rice inflorescences are covered with leaf sheath. Wipe each inflorescence with a 70% alcohol swab an...

agrobacterium-mediated genetic transformation: a method to genetically transform the rice genome via genetically engineered agrobacterium tumefaciens

Source: Xu, D., et al. <a href="https://www.jove.com/v/61665">Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice.</a> J. Vis. Exp. (2020).
In this video, we demonstrate genetic transformation of the rice genome using an Agrobacterium tumefaciens binary vector. This bacterium facilitates the transfer of a foreign DNA plasmid using Agrobacterium virulence proteins.

<img alt="Figure 1" src="/files/ftp_upload/20991/20991_Figure1.jpg" /> 
Figure 1: Starting materials for inducing callus. (A) Young rice inflorescences at meiosis stage. (B) Dissected young inflorescences growing on the NBD2 medium. Bar = 2 cm in (A).
<img alt="Figure 2" src="/files/ftp_upload/20991/20991_Figure2.jpg" /> 
Figure 2: Procedure for producing transgenic lines by Agroba...

Agrobacterium-Mediated Genetic Transformation: A Method to Genetically Transform the Rice Genome via Genetically Engineered Agrobacterium tumefaciens

Source: Xu, D., et al. Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive ...

Agrobacterium tumefaciens, a plant pathogenic bacterium, can be genetically engineered by introducing an artificially prepared Ti plasmid, carrying a gene of interest in its transfer DNA or T-DNA region, and a helper plasmid carrying the virulence or Vir genes for transferring the T-DNA into plant cells.
For Agrobacterium-mediated genetic transformation, begin with a young rice inflorescence and cut it into small pieces. Transfer the cuttings to a growth medium and incubate to facilitate the growth of the cuttings into an undifferentiated cell mass called a callus.
Now, transfer the callus to an Agrobacterium infiltration medium containing acetosyringone - a chemoattractant for Agrobacterium. Next, add a culture of genetically engineered Agrobacterium to the medium and incubate briefly.
The acetosyringone molecules activate the cell receptor protein - VirA - on the Agrobacterium membrane, which further mediates the phosphorylation of a transcriptional activator protein - VirG. Phosphorylated VirG moves toward the helper plasmid and activates the transcription of a cascade of virulence genes, including channel proteins and endonucleases - VirD1 and VirD2.
The channel proteins form a transport channel connecting the bacterial and plant cell, whereas the endonucleases bind to the border sequences in the Ti plasmid and cleave the T-DNA with VirD2, bound at one end. Thereafter, VirD2 directs the entry of T-DNA via the transport channel into the callus cells and facilitates its integration into the rice genome.

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Research

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Biological Techniques

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In this video, we demonstrate genetic transformation of the rice genome using an Agrobacterium tumefaciens binary vector. This bacterium facilitates the transfer of a foreign DNA plasmid using Agrobacterium virulence proteins.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/20991/20991_Figure1.jpg" /> 
Figure 1: Starting materials for inducing callus. (A) Young rice inflorescences at meiosis stage. (B) Dissected young inflorescences growing on the NBD2 medium. Bar = 2 cm in (A).
<img title="figure-representative results-393" alt="figure-representative results-393" src="/files/ftp_upload/20991/20991_Figure2.jpg" /> 
Figure 2: Procedure for producing transgenic lines by Agroba...

Agrobacterium-Mediated Genetic Transformation: A Method to Genetically Transform the Rice Genome via Genetically Engineered Agrobacterium tumefaciens

In This Article

Overview

Protocol

Representative Results

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