TRANSIENT CHIMERIC GENE-EXPRESSION IN POLLEN OF 5 CONIFER SPECIES FOLLOWING MICROPARTICLE BOMBARDMENT

Mature pollen of lodgepole pine (Pinus concorta Dougl.), yellow cypres s (Chamaecyparis nootkatensis (D. Don) Spach), western hemlock (Tsuga heterophylla (Raf.) Sarg.), jack pine (Pinus banksiana Lamb.), and bla ck spruce (Picea mariana (Mill.) B.S.P.) was bombarded with gold parti cles coated with four different plasmid constructions, pRT99GUS, pBM11 3Kp, pAct1-D, and pGA984, using the biolistic PDS-1000/He device. A pr otocol was devised for efficient gene transfer and gene expression ass ay in pollen. False positive results for expression of the beta-glucur onidase (GUS) gene assayed with the substrate X-glucuronide were obser ved with pollen of yellow cypress, western hemlock, and lodgepole pine . The highest levels of transient GUS gene expression were obtained wi th plasmid pBM113Kp, which carried the GUS gene under the control of t he wheat abscisic acid inducible early methionine promoter. The plasmi ds pRT99GUS (35S promoter) and pAct1-D (rice actin promoter) yielded s imilar intermediate levels of transient GUS gene expression. The polle n-specific promoter of the alpha-tubulin gene from Arabidopsis thalian a (pGA984) yielded the lowest levels of gene expression in pollen. Of the four species, yellow cypress showed the lowest levels of transient GUS gene expression and black spruce yielded the highest levels. The neomycin phosphotransferase II (NPT II) gene was also tested as a repo rter gene for pollen transformation and was easily assayed via ELISA. The fusion gene between NPT II and GUS genes was detected at a lower l evel than the nonfused NPT II gene when under the control of the same 35S promoter. The method devised here could be used for the study of t issue-specific gene expression in conifer pollen.