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).

Gene replacement by homologous recombination in the multicellular green alga Volvox carteri

With only two different cell types, the haploid green alga Volvox represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter/arylsulfatase gene). After bombardment of Volvox reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in Volvox. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (nitA) with a single G → A nucleotide exchange in a 5′-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in Volvox to apply the powerful tool of gene disruption for functional studies of novel genes.

Gas and particle dynamics of a contoured shock tube for pre-clinical microparticle drug delivery

We investigate the gas-particle dynamics of a device designed for biological pre-clinical experiments. The device uses transonic/supersonic gas flow to accelerate microparticles such that they penetrate the outer skin layers. By using a shock tube coupled to a correctly expanded nozzle, a quasi-one-dimensional, quasi-steady flow (QSF) is produced to uniformly accelerate the microparticles. The system utilises a microparticle cassette (a diaphragm sealed container) that incorporates a jet mixing mechanism to stir the particles prior to diaphragm rupture. Pressure measurements reveal that a QSF exit period - suitable for uniformly accelerating microparticles - exists between 155 and 220 mus after diaphragm rupture. Immediately preceding the QSF period, a starting process secondary shock was shown to form with its (x,t) trajectory comparing well to theoretical estimates. To characterise the microparticle, flow particle image velocimetry experiments were conducted at the nozzle exit, using particle payloads with varying diameter (2.7-48 mu m), density (600-16,800 kg/m(3)) and mass (0.25-10 mg). The resultant microparticle velocities were temporally uniform. The experiments also show that the starting process does not significantly influence the microparticle nozzle exit velocities. The velocity distribution across the nozzle exit was also uniform for the majority of microparticle types tested. For payload masses typically used in pre-clinical drug and vaccine applications (