The particle gun, cocultivation with Agrobacterium tumefaciens, and imbibition in DNA solutions were compared as methods to transfer DNA into mature and immature pollen of Nicotiana tabacum. Bombardment of mature pollen with the β-glucuronidase gene cloned behind the pollen-specific PA2 promoter of the chalcone isomerase gene of Petunia hybrida resulted in the expression of the β-glucuronidase gene in 0.025% of the pollen grains. Bombardment of younger stages followed by in vitro maturation also resulted in the formation of mature pollen that expressed β-glucuronidase, although at a lower frequency. Cocultivation of pollen during in vitro maturation or in vitro germination with Agrobacterium tumefaciens did not yeild β-glucuronidase-expressing pollen. In these cases, an intron-containing β-glucuronidase gene was used which effectively prevented β-glucuronidase expression in the bacteria. Imbibition of mature, dry pollen in various DNA solutions of the same constructs also did not lead to the formation of β-glucuronidase expressing pollen.