Agrobacterium-Mediated Transformation Of Ponkan Mandarin With CG1-400-RNase And GFP Gene

Obtaining high quality and seedless varieties is one of the main objectives of citrus breeding. However, the conventional breeding technique has been constrained by the complex biological and/or genetic characteristics of citrus, such as nucellar polyembryony, pollen/ovule sterility, long juvenility, and high genetic heterozygosity etc. The application of transgenic technique and alternative genes related to seed development provided an efficient method for citrus seedless breeding.In this study, chimeric ribonuclease gene (Barnase) driven by seed-specific promoter (CG1-400)for reducing seed numbers, was introduced into seedy citrus variety Ponkan mandarin, using etiolated epicotyl segment and the trimmed etiolated shoot/root region seedling as explants, to create’less seedy’or seedless transgenic lines. Meanwhile, the Agrobacterium-mediated transformation of green fluorescent protein (GFP) gene with etiolated epicotyl segment and the trimmed etiolated shoot/root region seedling of Ponkan mandarin was conducted. The main results are as follows:1. Transformation of Ponkan mandarin with CG1-400-RNase gene. Regeneration efficiency of etiolated epicotyl segment and the trimmed etiolated shoot/root region seedling were 17.1% and 11.4%, respectively.40 transgenic buds were achieved by PCR analysis, the positive rate of regenerated buds was 12.86%, and the transformation efficiency was 0.55%.15 of them were well-developed by grafting in vitro, but none of the 15 seedlings were confirmed as transformants by further PCR analysis. The result suggested that Ponkan mandarin was recalcitrant for genetic transformation.2. Transformation of Ponkan mandarin with GFP gene. Regeneration efficiency of etiolated epicotyl segment and the trimmed etiolated shoot/root region seedling were 20.6% and 21.4%, respectively, which were both higher than that of the transformation of gene CG1-400-RNase. The results indicated that regeneration efficiency was different after transformation of different vectors in the same citrus genotype. Observed by fluorescence microscopy in vivo,24 buds with stable expression of GFP were obtained, and 7 of them were chimeric shoots. Transformation efficiency of etiolated epicotyl segment and the trimmed etiolated shoot/root region seedling were 4.1% and 1.3%, respectively, and chimerism efficiency was 15.4%.