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 germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm(2) embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T-1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).

Differentiation of peanut seedborne potyviruses and curcumoviruses by RT-PCR

Seedborne peanut viruses pose important constraints to peanut production and safe movement of germ plasm. They also pose a risk of accidental introduction into previously disease-free regions. We have developed reverse transcription-polymerase chain reaction (RT-PCR) assays based on identical cycling parameters which identified peanut stripe, Peanut mottle, Peanut stunt, and Cucumber mosaic viruses through production of specific DNA fragments of 234 bp, 327 bp, 390 bp, and 133 bp, respectively. Assay sensitivity in the picogram range was achieved. The two potyviruses and two cucumoviruses could be differentiated using duplex RT-PCR assays. These assays should be useful for testing peanut leaves or seeds for virus identification in epidemiological studies, seed testing or in post-entry quarantine.