Micropropagation of Populus trichocarpa ‘Nisqually-1’ and genetic engineering Populus with CaMV35s-AtWBC19, CaMV35s-AgNt84 and -60CaMV35s-GUS genes

In Chapter one, we described development of a highly efficient micropropagation protocol from greenhouse-grown shoot tips of ‘Nisqually-1’. The optimal micropropagation protocol involves growing in vitro shoots in plant growth regulator free Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 0.3% Gelrite and 5–10 g∙L^-1 of activated charcoal. Plants grown on this medium were significantly longer, and contained significantly higher concentrations of chlorophyll. In chapter II, we transferred the plant-originated Arabidopsis Atwbc19 gene encoding an ATP binding cassette transporter which confers resistance to aminoglycoside antibiotics. Transgenic plants were confirmed by polymerase chain reaction (PCR) with Atwbc19- specific primer pair. The expression was confirmed by the reverse transcription PCR. Transgenic plants were tested for aminoglycoside antibiotic resistance. The level of resistance conferred by CaMV35S-Atwbc19 is similar to that conferred by nptII gene. Therefore, plant-ubiquitous Atwbc19 gene can serve an alternative gene as a plant transformation selective marker gene to current bacterial antibiotic-resistance marker genes and alleviate the potential risk for horizontal transfer of bacterial resistance genes in transgenic plants. In Chapter III, we transformed a Populus clone with the enhancer trapping vector, pD991. All 250 transgenic lines were screened and 71 of them (28%) showed positive staining. They showed various patterns of the reporter gene expression, including expression in one tissue and simultaneously in two more tissues. These results confirmed the previously reports that enhancer trap lines can be produced in Populus, and these enhancer trap lines can be used for future gene cloning and studying gene expression in Populus. In Chapter IV, we transformed with the heavy metal binding protein agNt84 gene. Seven putative transgenic lines were confirmed by PCR with the agNt84 specific primers and two lines shoot tips of transgenic- and non-transgenic plants grown on cadmium (Cd) -containing rooting media to evaluate of Cd resistance. 33% of shoot tips from one line and 44% of those from another transgenic line survived on medium containing 250 mM Cd, respectively, but only 22% of the non-transgenic shoot tips survived on rooting medium with 150 mM Cd at week 8. Also, the Cd analysis by ICP-OES indicated that the transgenic plants which were grown on 100 mM Cd medium accumulated about 45% more Cd in the tissue than non-transgenic plants.