Description

Abstract: An efficient protocol for protoplast-mediated transformation of peanut (Arachis hypogaea) using ration has been successfully developed. Co-cultivation of protoplasts isolated from immature cotyledons of a variety of peanut genotypes with peanut nurse cells resulted in the formation of callus colonies. Up to 80% of protoplast-derived colonies differentiated after transfer to a regeneration medium. Multiple shoots developed from differentiating colonies within a period of two months. Protoplast-derived plants of cultivated peanut, grown to maturity in the greenhouse, were fertile. A poration medium containing biodegradable glycine or glycylglycine was developed to improve direct gene transfer into peanut protoplasts using electroporation. Transformation efficiency, as determined by a transient GUS expression analysis, was increased by 8 to 430-fold using the newly-devised potation medium, when compared to other commonly-used media containing stable, non-metabolizable buffers. Further transient gene expression analysis indicated that both protoplast viability and gene transfer efficiency were enhanced by using the new poration medium. Kanamycin-resistant colonies have been obtained Mu electroporation with the plasmid pBI426 containing the GUS gene and the NYM kanainycin-resistance gene. DNA analysis using PCR and Southern blot hybridization confirmed the integration of multiple copies of intact tmsgenes in the peanut genome. Multiple shoots have been regenerated from transgenic callus colonies and are being rooted for further evaluation. Using this protoplast-mediated transformation system, a number of virus coat protein genes are being introduced into protoplasts of several major US peanut cultivars for the induction of virus resistance.