Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germp lasm base and lacks sources of resistance to several major diseases. The sp ecies is considered recalcitrant to transformation, with few confirmed tran sgenic plants upon particle bombardment or Agrobacterium treatment. Reporte d transformation methods are limited by low efficiency, cultivar specificit y, chimeric or infertile transformants, or availability of explants. Here w e present a method to efficiently transform cultivars in both botanical typ es of peanut, by (1) particle bombardment into embryogenic callus derived f rom mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubatio n on charcoal and cytokinin-containing media; resulting in efficient conver sion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bomb ardment of 10 cm(2) embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bomb ardment. Transgene copy number ranged from one to 20 with multiple integrat ion sites. There was ca. 50% coexpression of hph and luc or uidA genes copr ecipitated on separate plasmids. Reporter gene (luc) expression was confirm ed in T-1 progeny from each of six tested independent transformants. Insuff icient seeds were produced under containment conditions to determine segreg ation ratios. The practicality of the technique for efficient cotransformat ion with selected and unselected genes is demonstrated using major commerci al peanut varieties in Australia (cv. NC-7, a virginia market type) and Ind onesia (cv. Gajah, a spanish market type).