Factors affecting adventitious shoot formation and expression of microprojectile-introduced DNA in Douglas-fir cotyledons

Graduation date: 1991Our goal was to develop knowledge that will enable insertion of foreign genes into Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] trees. This required improvement of a tissue culture system for producing adventitious shoots from cotyledons, and study of factors affecting gene delivery, expression, and analysis. Administering a short-duration, concentrated liquid pulse of\ud cytokinin induced more shoots than published protocols that incorporate cytokinins in the growth medium. Cotyledons from the youngest seedlings formed the most shoots, and medium solidified with agarose was less inhibitory than media with gellan gum or either of\ud two agars tested. Factors affecting transient expression of microprojectile-introduced DNA were investigated in Douglas-fir cotyledons. Physical factors---the number of bombardments, particle loading density, and particle velocity---did not significantly affect the number of cells expressing the B-glucuronidase (GUS) gene over the ranges tested. Biological factors were more important; preculturing cotyledons with\ud cytokinins, or auxins plus cytokinins, enhanced expression after approximately seven days of pretreatment. A plasmid containing the GUS, Bacillus thuringiensis 6-endotoxin (Bt), and neomycin phosphotransferase II (NPT-II) genes was introduced into cotyledons on microprojectiles. Callus was grown from the\ud cotyledons on media with and without kanamycin, but no resistant cell lines developed. Eight months after DNA introduction, callus lines on kanamycin-free medium were selected on the basis of GUS expression for further molecular analysis. Six callus lines consistently yielded\ud appropriately-sized PCR (polymerase chain reaction) fragments in DNA amplification reactions using primers complementary to the GUS gene, but no correctly-sized fragments were seen when Bt primers were used. No GUS activity was observed in these callus lines despite improvements to the sensitivity of the fluorometric assay. Callus fed to larvae of the Douglas-fir tussock moth failed to inhibit growth. Southern analysis demonstrated the absence of foreign genes in DNA of the callus lines, and showed that the comigrating PCR fragment was not homologous to the GUS gene