RECOVERY OF TRANSGENIC ASPARAGUS PLANTS BY PARTICLE GUN BOMBARDMENT OF SOMATIC-CELLS

Transgenic asparagus (Asparagus officinalis L.) plants were obtained t hrough particle gun bombardment of suspension cells using the pKGUS pl asmid containing both neomycin phosphotransferase II (nptII) and P-glu curonidase (uidA) genes. In experiments to optimize bombardment parame ters, the combination of 1.1 mu m tungsten particles and 1000 psi bomb ardment pressure was best; 65 cells or cell clumps per plate of 10(5)- 10(6) suspension culture derived cells expressed uidA in transient ass ays. No significant genotypic effects were detected for the number of cells or cell clumps expressing uidA among four genotypes. A two-step selection procedure was established for the recovery of transformed pl ants. The bombarded cells were first placed on embryo induction medium with 25 mg l(-1) kanamycin for 4 weeks, and the resistant calli were then transferred to the same medium with 50 mg l(-1) kanamycin for emb ryo development. A total of 10 plants from six kanamycin resistant cal li were recovered. The expression of uidA was detected in the stems an d cladodes of these 10 plants. Southern analysis confirmed the integra tion of nptII and uidA into the asparagus genome, and revealed that th e 10 transgenic plants from six resistant calli were from one transfor mation event. In addition to the detection of junction fragments where the pKGUS plasmid integrated into the genome, Southern analysis also provided evidence that most of the pKGUS plasmid DNA was integrated in tactly and organized as head-to-tail concatemers in these plants. uidA assays of the hybrid seeds from the crosses between one transgenic ma le asparagus T-0 plant and nontransformed female asparagus suggested t hat uidA activity was inherited as a single locus with Mendelian segre gation. (C) 1997 Elsevier Science Ireland Ltd.