FACTORS ENHANCING AGROBACTERIUM-TUMEFACIENS-MEDIATED GENE-TRANSFER INPEANUT (ARACHIS-HYPOGAEA L.)

Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) falls were investigated. Bn intron-con taining P-glucuronidase uidA (gusA) gene under the transcriptional con trol of CaMV 35S promoter served as a reporter. Transformation frequen cy was evaluated by scoring the number of sectors expressing GUS activ ity on leaf and epicotyl explants. The 'Valencia Select' market type c v. New Mexico was more amenable to Agrobacterium transformation than t he 'runner' market type cultivars tested (Florunner, Georgia Runner, S unrunner, or South Runner). The disarmed Agrobacterium tumefaciens str ain EHA101 was superior in facilitating the transfer of uidA gene to p eanut cells compared to the disarmed strain C58. Rinsing of explants i n half-strength Murashige-Skoog (MS) media prior to infection by Agrob acterium significantly increased the transformation efficiency. The us e of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 mu M or 11.31 mu M) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 mu M or 2.0 mu M) BA] promoted higher transformation than either hormone -free or thidiazuron-containing medium. The polarity of the epicotyl d uring cocultivation was important; explants incubated in an inverted ( vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Precult ure of explants in MS basal medium or with 2.5 mg thidiazuron per 1 pr ior to infection drastically decreased the number of transformed zones , The optimized protocol was used to obtain transient transformation f requencies ranging from 12% to 36% for leaf explants, 15% to 42% for e picotyls. Initial evidence of transformation was obtained by polymeras e chain reaction and subsequently confirmed by Southern analysis of re generated plants.