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United States Department of Agriculture

Agricultural Research Service

Research Project: Genetic Improvement of Maize and Pearl Millet for Resistance to Insects and Aflatoxin

Location: Crop Genetics and Breeding Research

Title: Improvement of peanut (Arachis hypogaea) transformation efficiency and determination of transgene copy number of relative quantitative realtime-PCR

Authors
item Chu, Ye -
item Bhattacharya, Anjanabha -
item Wu, Congling -
item Knoll, Joseph
item Ozias-Akins, Peggy -

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 1, 2013
Publication Date: April 30, 2013
Citation: Chu, Y., Bhattacharya, A., Wu, C., Knoll, J.E., Ozias-Akins, P. 2013. Improvement of peanut (Arachis hypogaea) transformation efficiency and determination of transgene copy number of relative quantitative realtime-PCR. In Vitro Cellular and Developmental Biology - Plants. 49:266-275.

Interpretive Summary: The biolistic method is reliable for delivering genes of interest into various species. This method utilizes an apparatus known as a gene gun to bombard plant tissue with tiny DNA-coated gold particles. However, a low rate of success (low transformation efficiency) has been a limiting factor for this technique, particularly in peanut. The DNA coating agent protamine has been shown to improve transformation efficiency in rice, while a reduction of DNA in the bombardment mixture has been reported to elevate the chance of recovering single-copy integration events in corn. Plants with only one copy of the introduced gene are usually more desirable than those with multiple copies. To test various conditions that could improve peanut transformation, peanut cultivar Georgia Green was co-bombarded with two different circular DNA molecules, called plasmids. One contained a green fluorescent protein (GFP) gene, while the other contained a gene of interest, which silences a major allergen of peanut (Ara h 1). After bombardment under various conditions the green fluorescent signal in embryogenic tissues was measured to evaluate transformation efficiency for each bombardment condition. A 4.6-fold improvement of transformation efficiency was achieved in stably transformed peanut lines by introducing protamine instead of conventional spermidine in a bombardment mixture with 70 ng/shot of plasmid DNA and 50 µg/shot of gold. Unexpectedly, the reduction of plasmid DNA from 700 ng/shot to 70 ng/shot produced transgenic lines with significantly increased number of transgene copies. Southern blotting is the usual way of estimating transgene copy number, but it is labor-intensive and requires a large amount of DNA. Because a large amount of DNA is needed, bombarded tissue must be regenerated for a long time (several months or more in peanut) before its copy number can be determined. PCR-based techniques are much faster and only require minute quantities of DNA. In order to determine the transgene copy number during the early stages of plantlet regeneration, relative quantitative realtime-PCR (qPCR) was established using fluorescently labeled universal library probes. A correlation of 95% was found for estimation of copy number between Southern blot and q-PCR data. Given its speed and high-throughput nature, qPCR can be employed as an effective screening tool to separate high copy number events from low copy events as early as the shoot formation stage of regeneration.

Technical Abstract: The biolistic method is reliable for delivering genes of interest into various species. Low transformation efficiency has been a limiting factor for its application. The DNA coating agent protamine was shown to improve transformation efficiency in rice, while a reduction of plasmid DNA in the bombardment mixture was reported to elevate the chance of recovering single copy integration events. To test various conditions that could improve peanut transformation, peanut cultivar Georgia Green was co-bombarded with two plasmids, one containing a green fluorescent protein (GFP) gene and a second containing a gene of interest plus selectable marker. Fluorescent signal in bombarded embryogenic tissues was measured to evaluate transformation efficiency for each bombardment condition. A 4.6-fold improvement of transformation efficiency was achieved in stably transformed peanut lines by introducing protamine instead of conventional spermidine in a bombardment mixture with 70 ng/shot of plasmid DNA and 50 µg/shot of gold. Unexpectedly, the reduction of plasmid DNA from 700 ng/shot to 70 ng/shot produced transgenic lines with significantly increased number of transgene copies. In order to determine the transgene copy number during plantlet regeneration, relative quantitative realtime-PCR (qPCR) was established using fluorescently labeled universal library probes. A correlation of 95% was found for estimation of copy number between Southern blot and q-PCR data. Given its speed and high-throughput nature, qPCR can be employed as an effective screening tool to separate high copy number events from low copy events as early as the shoot formation stage of regeneration.

Last Modified: 10/1/2014
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