Plant Regeneration and Microprojectile-Mediated Gene Transfer in Embryonic Leaflets of Peanut (Arachis hypogaea L.)

Abstract

Excised leaflets from mature embryos of peanut cultivars Robut, Gajah, McCubbin and NC-7 produced multiple shoot primordia via organogenesis on Murashige and Skoog medium supplemented with 3 mg L-1 BAP and 1 mg L-1 NAA. Plants were regenerated by elongation of shoot primordia on transfer to MS medium supplemented with 5 mg L-1 BAP. The regeneration frequency of five plants per embryonic leaflet was 10-40-fold higher than previously reported from seedling leaflets. Microprojectile bombardment parameters were optimised for high transient expression rates of the β-glucuronidase (uidA or gus) reporter gene in peanut embryos (> 2000 cells per bombardment) and in excised embryonic leaflets (> 1200 cells per bombardment). The firefly luciferase reporter gene (luc) allowed repeated non-toxic assays, revealing the transition from hundreds of transiently expressing cells soon after bombardment, to a few stably expressing regions in callus grown without selection for 8 weeks after bombardment of embryonic leaflets.