Optimizing parameters for efficient introduction of transgenes into turfgrass
Embryogenic callus was generated from seeds of turfgrass species (creeping bentgrass, cv. Penn A4 and Providence) on tissue culture medium. After 12 weeks, embryogenic calli were identified and used for bombardment with chimeric gene constructs that confer phosphinothricin resistance (bar) as a selectable marker and β-glucuronidase (GUS) as a reporter gene. After bombardment, the calli were transferred to selection medium and then to regeneration medium. Putative transgenic turfgrass plants were generated and molecular analysis (PCR) was performed to confirm presence of the transgene (bar, GUS). We analyzed reporter gene (GUS) expression in turfgrass cells over a developmental time period of 15 days following microprojectile bombardment with a plasmid containing a GUS reporter gene driven by the ubiquitin rice promoter. Varietal differences for tissue developmental stage specific patterns of reporter gene expression have been observed. We observed transient expression patterns of reporter genes (GUS) driven by four different promoters (ubiquitin rice, ubiquitin corn, ubiquitin-3 potato and CaMV 35S) in calli, root and leaf tissue of turfgrass. The ubiquitin rice promoter driving GUS produced the highest number of GUS spots compared to three other promoters both in young and mature leaves. We analyzed GUS expression at the two different developmental stages of the leaves, young leaf vs. fully mature leaf. Dramatically higher GUS expression was observed in mature leaves compared to young leaves following microprojectile bombardment. From these observations we can say that transient expression of a reporter gene (GUS in this case) does change considerably with the developmental stage of the creeping bentgrass plant. Fully expanded mature leaves were found to be much more efficient in GUS expression than young leaves. We have successfully inhibited in vitro endogenous nucleolytic activity in turfgrass cells by different nuclease inhibitors (0.6% heparin, aurintricarboxylic acid, polyethylene glycol and spermidine). We have observed more than a 1000-fold enhancement of GUS expression in 0.6% heparin treated 14-month-old Penn A4 callus compared to equivalent aged untreated callus. These results will lead to development of efficient strategies of use of biotechnology for trait enhancement and modification of turfgrass. ^
Biology, Molecular|Biology, Plant Physiology
"Optimizing parameters for efficient introduction of transgenes into turfgrass"
Dissertations and Master's Theses (Campus Access).