Transgenic Plants of Tall Fescue (Festuca arundinacea Schreb.) Obtained by Direct Gene Transfer to Protoplasts

Abstract

Chimeric hygromycin phosphotransferase (hph) and phosphinothricin acetyltransferase (bar) genes were introduced, using polyethylene glycol treatment, into protoplasts isolated from embryogenic cell suspension cultures of tall fescue (Festuca arundinacea Schreb.), a graminaceous plant that is an important forage crop in temperate pastures. Colonies resistant to either 200 mg/l hygromycin or 100 mg/l phosphinothricin, respectively, were recovered upon selection using bead-type culture systems. Stable integration of the transgenes in the genomes of plants regenerated from resistant callus clones was shown by Southern hybridization analysis. In situ hybridization of a labeled transgene-probe to metaphase chromosomes is shown for one transgenic primary regenerant. Expression of the transgenes in mature plants was demonstrated by HPH enzyme assay or by phosphinothricin-herbicide spraying.

Cool‐Season Forage Grasses

“…Transgenic cool-season grass plants were first obtained by direct gene transfer to protoplasts (Horn et al, 1988;Wang et al, 1992). Because protoplast culture and transformation are very delicate systems to work with, in recent years biolistic transformation and Agrobacteriummediated transformation have become the main methods for producing transgenic grasses.…”

“…As shown in Table 1, most of the protoplast works were done in the 1990s. Recovery of transgenic plants from protoplasts was reported for orchardgrass (Horn et al, 1988), tall fescue (Ha et al, 1992;Wang et al, 1992;Dalton et al, 1995;Kuai et al, 1999), Italian ryegrass , and perennial ryegrass .…”

Genetic Transformation in Buffel Grass (Cenchrus ciliaris L.)

“…The resistance of pasture grasses to antibiotics or herbicides has been investigated in cell suspensions, but not in callus. Panicum maximum cells are reported to be killed by methotrexate at 1 mg/l; Dactylis glomerata by hygromycin at 20 mgll; and Festuca arundinacea by hygromycin at 200mg/1 and phosphinothricin at 100mg/1 (Hauptmann et al 1988;Horn et al 1988;Wang et al 1992). We have determined the dose responses for postbombardment selection of buffel grass callus for resistance to G418 (geneticin), hygromycin, chlorsulfuron, and phosphinothricin.…”

Genetic transformation ofLotus corniculatus withAgrobacterium tumefaciens and the analysis of the inheritance of transgenes in the T1 generation

“…The PCR reactions were performed in 30/xl volumes containing 10 mM Tris-HC1, pH 8.3, 50 mM KC1, 1.25 mM MgCI> 200 ~xM dATP, dCTP, dGTP, dTTP, 0.05UJx1-1 of Taq polymerase (Perkin Ehner Cetus), 0.5 Nvl oligonucleotide primers and 100ng of plant DNA. Amplification of the aph IV gene was performed with the primers described by Wang et al (1992): 5 '-GCTGGGGCGTCGGTTTCCACTATCCG-3 ' and 5'-CGCATAACAGCGGTCATTGACTGGAGC-3'. These were designed to amplify a 375bp internal aphIV fragment.…”