Location: Crop Genetics and Breeding ResearchTitle: Progress on genotyping and phenotyping recombinant inbred line populations of peanut) Author
Submitted to: American Peanut Research and Education Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 5/15/2013
Publication Date: 10/1/2013
Citation: Chu, Y., Holbrook Jr, C.C., Isleib, T., Tillman, B., Burow, M., Brenneman, T., Culbreath, A., Ozias-Akins, P. 2013. Progress on genotyping and phenotyping recombinant inbred line populations of peanut. Proc. Amer. Peanut Res. and Educ. Soc. 45:31. Interpretive Summary: not required
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.