We created transgenic bentgrass with a bacterial gene (ipt) encoding the enzyme adenine isopentenyl phosphotransferase. The ipt gene was ligated to a stress- activated promoter from Arabidopsis, SAG12, which is activated at the start of leaf senescence (Psag12-ipt). Each plasmid contains the hygromycin resistance gene (hyg) for transgenic plant tissue selection, and the GUS reporter gene (uid) for easy identification of transgenic plants. Creeping bentgrass callus derived from stolons was transformed with the plasmids using the Agrobacterium tumefaciens (agro) infection technique.
A total of 142 senescence-activated plants were confirmed as transgenic using PCR. Over 40 Psag12-ipt clones have been confirmed positive with Northern blotting. Psag12-ipt plants were screened for chlorophyll content and maintenance of green color after the activation of senescence using an excised leaf bioassay. Leaves from some Psag12-ipt plants remained green longer in the dark and had higher chlorophyll content than controls one and three weeks after excision and after 19 d of growth in the dark (growth chamber without light). Some Psag12-ipt plants had increased number of tillers and roots, and higher root dry weight than non-transgenic plants. Some transgenic plants had higher cytokinin in the leaves and the roots after stress treatments, suggesting a role for this hormone in delaying leaf senescence and regulating tiller and root production in creeping bentgrass.