Regeneration of transgenic peanut plants from stably transformed embryogenic callus

Embryogenic tissue cultures of Arachis hypogaea L. (peanut or groundnut), have been transformed via microprojectile bombardment. We introduced a gene (hph) conferring resistance to the antibiotic hygromycin under the control of the CaMV 35S promoter. Selection for resistant callus was initiated 4–5 weeks post-bombardment on medium containing 10–20 mg/l hygromycin. Twelve percent of the bombardments resulted in recovery of a transgenic cell line. An average of two transgenic embryogenic cell lines was isolated per bombardment experiment over 4–6 months of continuous selection. Each bombardment experiment consisted of 11–19 plates, and each plate contained approximately fourteen 25 mm2 embryogenic callus pieces. Thus, nearly 1% of the bombarded callus pieces produced a stably transformed cell line. Over 100 plants have been regenerated collectively from all of the transformed cell lines. The presence and integration of foreign DNA in hygromycin-resistant callus lines and regenerated plants has been confirmed by polymerase chain reaction amplification of a defined portion of the chimeric gene and by Southern hybridization analysis. Hygromycin resistance was expressed in leaflets from transformed plants which remained green when cultured on basal medium containing hygromycin. Leaflets from control, non-transformed plants turned brown within 3 weeks on the hygromycin-containing medium.