Toshinori Komatsu

Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene

We introduced the rice chitinase (Cht-2; RCC2) gene into calli of Italian ryegrass (Lolium multiflorum Lam.), with a hygromycin phosphotransferase (HPT) gene as a selectable marker, by particle bombardment. Hygromycin-resistant calli were selected and transferred to regeneration medium for shoot formation. Polymerase chain reaction (PCR) analysis revealed regenerants containing the HPT gene. The RCC2 gene was detected in 65.5% of those regenerants. Southern hybridization detected both HPT and RCC2 genes and indicated that the transgenic plants were independently transformed. Expression of the RCC2 gene in the transgenic plants was confirmed by Northern hybridization, reverse transcription–PCR and Western blotting. Bioassay of detached leaves indicated increased resistance to crown rust (Puccinia coronata) in transgenic plants, which exhibited higher chitinase activity than a nontransgenic plant.

Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.)

The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of β(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for β(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against β(2,1)-linked fructans, but considerably less than against β(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy.

Screening of Regenerable Genotypes of Italian Ryegrass (Lolium multiflorum Lam.)

Abstract We screened regenerable genotypes from 12 cultivars of Italian ryegrass (Lolium multiflorum Lam.) through tissue culture of mature seeds, shoot tips and root tips. Although three morphological types of calli, friable, watery, and compact with fine root hair were observed, green shoots regenerated only from the friable calli. The highest frequency of the formation of regenerable callus from mature seeds was 1.4% in the diploid cultivar Waseaoba and 1.1% in the tetraploid cultivar Meritra. The plants regenerated from mature seed-derived calli were maintained and propagated aseptically in vitro, and the method of regenerating plantlets via callus culture was established by using their shoot tips. Sixty-three seeds of Waseaoba and 44 seeds of Meritra were aseptically sown, each seedling (genotype) was propagated in vitro, and the shoot tip from each genotype was subjected to tissue culture. More than 22% of these genotypes formed regenerable calli. From these results, we conclude that the shoot tip of the in vitro-preserved plantlet is useful for producing the regenerable calli routinely. The information obtained in the present study is useful for establishing a reliable transformation system for Italian ryegrass.