Optimisation of procedures for microprojectile bombardment of microspore-derived embryos in wheat

Using the PDS-1000/He Biolistic(R) Particle Delivery System, the microprojectile travel distance, rupture disk pressure and DNA/gold particle concentrations were assessed in order to optimise short and longer-term beta-glucuronidase reporter gene expression in microspore-derived embryos of wheat. The effects were also evaluated of using sterile filter paper to support explants and treatment with a high osmoticum medium (0.2 M mannitol/0.2 M sorbitol or 0.4 M maltose). In the optimised procedure, wheat microspore-derived embryos (MDEs), were placed on filter paper and incubated on medium containing 0.4 M maltose, for 4 h pre- and 45 h post-bombardment. Five mu l pAHC25 (0.75 mg ml(-1) in TE buffer) was precipitated onto 25 mu l gold particles (60 mg ml(-1) in sterile water), using 20 mu l spermidine (0.1 M) and 50 mu l CaCl2 (2.5 M). The particles were centrifuged and resuspended in 75 mu l absolute ethanol prior to the preparation of 6 macrocarriers. A microprojectile travel distance of 70 mm, a rupture pressure of 1300 p.s.i., and a vacuum of 29 " Hg were employed. Maltose at 0.4 M in the support medium was the most important factor influencing GUS activity in bombarded tissues. GUS activity, 1 day post-bombardment, reached 52 +/- 17 GUS-positive foci/MDE (mean +/-s.e.m, n=3), with 17 +/- 4 foci/MDE at 15 days, giving a 3.0-fold increase (p<0.05) compared to expression in MDEs bombarded on medium without a high osmoticum treatment.