An optimized particle bombardment protocol to introduce DNA into Arundo donax L. (giant reed) embryogenic callus cells was developed. The physical and biological parameters tested for optimal transient expression of ß-glucuronidase (GUS) and green fluorescent protein (GFP) genes were: helium pressure, distance from stopping screen to target tissue and vacuum pressure together with other factors such as gold microparticle size, DNA concentration and the number of bombardments. The highest transient GUS and GFP expression was obtained when cells were bombarded twice at 1100 psi, with 9 cm target distance, 24 mm Hg vacuum pressure, 1 mm gold particle size, 1.5 mg DNA per bombardment, three days pre-culture prior to bombardment and six days post bombardment culture. This is the first report of optimization of particle bombardment parameters for high-efficiency DNA delivery combined with minimum damage to target giant reed tissues.
Key words: Arundo donax, particle bombardment, ß-glucuronidase (GUS), green fluorescent protein (GFP), transient gene expression, genetic transformation.
GUS, ß-Glucuronidase; DNA, deoxyribonucleic acid; GFP, green fluorescent protein; MS, Murashige and Skoog’s medium; 2,4-D, 2,4-dichlorophenoxy acetic acid; EDTA, ethylene diamine tetraacetic acid; TE, tris- EDTA buffer; RNase, ribonuclease.
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