Author
|
Submitted to: In Vitro Cellular And Developmental Biology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 5/14/2003 Publication Date: 11/1/2003 Citation: Shatters, R.G. 2003. Electroporation of embryogenic protoplasts of sweet orange (citrus sinensis (l.) osbeck) and regeneration of transformed plants. In Vitro Cellular And Developmental Biology. Interpretive Summary: When a plant is genetically engineered it is necessary to first insert the piece of foreign DNA into the plants cells in some manner. The first part of this paper explains how to use a strong electrical pulse to form holes in single cells from sweet orange cells grown in tissue culture. This is called electroporation (i.e., the formation of pores using electricity). When the cells are mixed with DNA and the cells are electroporated the DNA flows through the holes and into the cell. The second part of this paper utilizes the electroporation method developed in part one to insert a gene for the green fluorescent protein into sweet orange cells. Cells that glowed green under UV light were picked out of the culture dish and regenerated into trees that also glowed green. This demonstrates a method to insert foreign genes into citrus. Genes for traits of agricultural value such as fruit quality or disease resistance can now be inserted into sweet orange cells and trees produced that have these valuable characteristics. Technical Abstract: Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet orange [Citrus sinensis (L.) Osbeck cv. Hamlin]. Electric field strength (375-450 V cm-1), vector DNA concentration (100 ?g ml-1), carrier DNA concentration (100 ?g ml-1), electroporation buffer (pH 8), and pre-electroporation heat shock of protoplasts (5 min @ 45o C) were optimized using the plasmid vector pBI221 containing the GUS coding sequence under the control of the CaMV 35S promoter and measuring GUS activity 24 h after electroporation. All variables significantly affected transfection efficiency and when optimal conditions for each were combined, GUS activity was 7,714 pmol 4-MU min-1 mg protein-2. Protoplasts were then electroporated in the presence of GFP expression vectors pARS101 or pARS108. Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted excitation peak, and optimized for human codon¿usage. pARS101 was constructed by ligating EGFP as a PCR-amplified NcoI-BamHI fragment from pEGFP-1 into pBI524. This placed the EGFP coding sequence under the control of the 35S-35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly except EGFP was modified with the addition of a 5' terminal signal sequence from an Arabidopsis vacuolar basic chitinase and a C-terminal HDEL sequence for retention in the lumen of the endoplasmic reticulum. Embryogenic calluses transgenic for the ER-targeted EGFP were significantly brighter than those transformed with nontargeted EGFP. |
