Agrobacterium-mediated transformation of Arachis hypogaea L. utilizing the shoot apex method

Abstract

Regeneration of peanut (Arachis hypogaea L.) plants from the shoot apex of in vitro germinated seedlings was accomplished with initiation and rooting media. Explants were left on initiation medium containing MS salts and 0. I mg/L BAP for 5 or more weeks. After the shoot elongated to 2 mm, the explant was transferred to rooting medium containing MS and I mg/L NAA. Media having 250 mg/L antibiotic did not inhibit growth of the explant. Two Agrobacterium tumefaciens strains used for insertion of foreign genes into the peanut plant genome harbored different foreign gene construct. EHA105 had the N coat protein gene of tomato spotted wilt virus and two selectable marker genes. Bar gene harbored by EHA101 provided resistance to the herbicide bialaphos. Shoot apices were exposed to bacterial culture of one of the constructs, in some instances, induced by opines. Explants exposed to the bar gene were placed on shoot initiation medium containing 0.5 mg/L PPT in addition to clavamox. Explants that survived the co-cultivation were rooted, grown in soil and analyzed for transient expression of the foreign gene. All 250 putative transformants exposed to EHA105 were tested by ELISA for presence of the N coat protein, and one-half were tested by GUS histochemical assay for insertion of 0-glucuronidase. Neither test identified putative transformants. Six explants that were co-cultivated with EHA101 survived selection on PPT and rooted. These plants were tested for presence of the bar gene by the chlorophenol red and PAT spectrophotometric assays. One plant was analyzed by Southern hybridization. The blot showed a faint band in both undigested and digested putative transformant samples. Neither undigested nor digested control plant had any areas hybridized by the probe. Somatic chromosome preparations of root tips of Arachis hypogaea L. (2n=40) were accomplished by use of an enzyme hydrolysis technique which produced slides with hundreds of cells that had chromosomes amenable to counting. The chromosome numbers for 5 in vivo progeny and 5 in vitro micropropagated progeny seedlings were determined. One of the micropropagation derived progeny had abnormal chromosome counts in greater than 40 % of cells.