Plant regeneration and genetic transformation in Kentucky bluegrass (Poa pratensis L.)

Four different types of callus were produced when the coleoptile, leaf, and stem tissues of dark-germinated 'Touchdown' Kentucky bluegrass (Poa pratensis L.) seedlings were cultured on Murashige and Skoog (MS) medium. Both the compact friable callus (type 3) and the moderately-compact friable callus (type 2) tissues regenerated plants, while the non-structured watery callus (type 4) and the brown nodular callus (type 1) produced only roots. The efficiency of shoot regeneration was highest from coleoptile tissues (32%) as compared to stem sections (12%) or young leaf tissues (2%) when cultured on MS medium supplemented with 0.2 mg L{dollar}sp{lcub}-1{rcub}{dollar} picloram and 0.01 mg L{dollar}sp{lcub}-1{rcub} alpha{dollar}-naphthaleacetic acid (NAA). Some callus lines produced green plants, while others produced albino plants or plants with green and albino tissues.; Coleoptile tissues were transformed with the rolC gene by either particle-wounded Agrobacterium infection or biolistic method. The coleoptile tissues were treated with the pGA-GUSGF plasmid carrying the npt II, gus, and rolC genes and then cultured on MS medium containing 0.2 mg L{dollar}sp{lcub}-1{rcub}{dollar} picloram, 0.01 mg L{dollar}sp{lcub}-1{rcub}{dollar}NAA, and 250 mg L{dollar}sp{lcub}-1{rcub}{dollar} kanamycin for two weeks. The regenerated plants from these calli were analyzed for the presence of the gus and rolC genes by histochemical GUS assay, PCR, and Southern hybridization. The frequency of the rolC gene transformation from either method ranged from 3.7% to 4.2% when determined by GUS assay. Most of the putative transformants were either albinos or varigated plants composed of white and green tissue sectors. The albino transgenic plants had little or no 56-kDa and 14-kDa subunits in Rubisco proteins in the stroma. They also lacked 110-kDa, 43-47 kDa, and 57-58-kDa polypeptides in photosystem I (PS I), photosystem II (PS II), and coupling factors (Cf{dollar}alphabeta{dollar}), respectively, in the thylakoid membranes. No light-harvesting complex (LHC) proteins were present in these albino clones. Our data suggest that both the nucleus and plastid gene products were impaired in the rolC transgenic plants.