TRANSIENT GENE-EXPRESSION IN VEGETATIVE SHOOT APICAL MERISTEMS OF WHEAT AFTER BALLISTIC MICROTARGETING

A method has been established that allows the targeted delivery of DNA-carrying gold particles to vegetative shoot apical meristems of cereal species. Meristems of 8- to 10-day-old seedlings of wheat (Triticum aestivum), rice (Oryza sativa) and sorghum (Sorghum bicolor) were manually exposed by removal of the coleoptile and the first three to four leaves. Using ballistic microtargeting, equal-sized gold particles of different diameters ranging from 1.0 to 2.0 mu m were propelled to meristems by pulses of compressed nitrogen ranging from 9 to 13 MPa. When accelerated by 13 MPa, particles of 1.4 mu m or larger penetrated to cells of L2 in 80% of the bombarded wheat meristems. Expression vectors containing either the gene for beta-glucuronidase (gusA) or genes regulating the anthocyanin biosynthesis (Bperu and C1 from maize) were delivered to wheat meristems. The level of transient gene expression was dependent on the osmotic pressure of the MS-based medium that was used to mount the explants for shooting: an increase of the sucrose concentration from 2 to 10% in the medium resulted in an increase of transient gene expression from 5 to 25% of the bombarded meristems. Although particles of 1.4 mu m were efficiently delivered to L1 and L2, transient gene expression occurred predominantly in the L1 layer. In contrast, up to 10% of the bombarded meristems had expressing cells in L2 when particles of 2.0 mu m were used. Ten days after bombardment, GUS-positive sectors in meristems and in leaf primordia were observed. When destructive GUS detection was omitted, between 80 and 90% of the bombarded explants recovered in vitro on basal MS medium and then regenerated to fertile plants in the greenhouse.