Improvement of peanut (Arachis hypogaea L.) transformation efficiency and determination of transgene copy number by relative quantitative real-time PCR

Abstract

The biolistic method is reliable for delivering genes of interest into various species, but low transformation efficiency can be a limiting factor in its application. To test various conditions that could improve peanut transformation via particle bombardment, embryogenic tissues of the peanut cultivar Georgia Green were co-bombarded with two plasmids: one containing a green fluorescent protein gene and one containing a gene of interest plus a selectable marker. The fluorescence in bombarded embryogenic tissues was measured to evaluate transformation efficiency. 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 plasmid DNA and 50 μg/shot gold. Unexpectedly, the reduction of plasmid DNA from 700 to 70 ng/shot produced transgenic lines with significantly increased numbers of transgene copies. To determine the transgene copy number during plantlet regeneration, relative quantitative real-time polymerase chain reaction (qPCR) was established using fluorescently labeled universal library probes. A correlation of 95% was found for estimation of copy number between Southern blot and qPCR 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 number events as early as the shoot formation stage of regeneration.