Poster: Vegetative Development

Abs # 448: Genetic background and tissue age affect GUS reporter gene expression levels in creeping bentgrass

Presenter:	Chandlee, Joel M., joel_chandlee@uri.edu
Authors	Chandlee, Joel M. (A)   Basu, Chhandak  (A)   Martin, Jonathan  (A)   Luo, Hong  (B)   Kausch, Albert  (B)
Affiliations:	(A): University of Rhode Island, Department of Cell and Molecular Biology, Kingston, RI (B): Hybrigene, LLC, West Kingston, RI

Embryogenic callus was generated from seeds of turfgrass (creeping bentgrass, cv. Penn A4 and Providence) on appropriate tissue culture medium for purposes of genetic modification of turfgrass via gene transfer strategies. The ubiquitin rice promoter-GUS reporter gene construct produced a high number of spots in callus and in both young and mature leaf tissue following microprojectile bombardment. The number of GUS spots in mature leaves was higher relative to young leaves. High nuclease activity levels in young leaves compared to mature leaves may be responsible for this result. Nuclease activity in leaf and callus tissue of Penn A4 was sufficiently high enough to degrade exogenously added plasmid DNA within one minute. Four different nuclease inhibitors (heparin, spermidine, aurintricarboxylic acid and polyethylene glycol) were analyzed for their efficacy in protecting exogenously added plasmid DNA. Heparin and spermidine were observed to be effective in vitro nuclease inhibitors protecting exogenously added plasmid DNA from degradation by turfgrass callus and leaf extracts. 14-month-old callus tissue of Penn A4 was incubated for 24 hours in the presence of heparin salt solution and bombarded with the ubiquitin rice-GUS construct. We observed a 1000-fold increase in GUS spots in heparin-treated callus compared to equivalent age, untreated Penn A4 callus. However, we did not observe similar enhancement from heparin pretreatment (0.6% or 1.2%) either in 6-month-old callus of Penn A4 and Providence or 12-month callus of Providence. This result is likely the outcome of varying nuclease activity levels between cultivars and the age differences of the callus used.