Particularly, transformation efficiency of perennial ryegrass was improved extensively by reducing defense responses of calluses in Agrobacterium-mediated transformation, which made a solid foundation for functional-gene research and genetic improvement of perennial ryegrass 15. Thereafter, high-efficiency Agrobacterium-mediated transformation systems of perennial ryegrass were established 13, 14, 15. In 2005, Agrobacterium-mediated transformation of perennial ryegrass was reported 12. Transgenic ryegrass plants were first obtained in 1999 by using silicon carbide fiber-mediated transformation 11. Later, mature embryos, immature inflorescence, leaf, and meristem cells of perennial ryegrass were used as explants for perennial ryegrass regeneration 6, 7, 8, 9, 10.
In 1977, fertile perennial ryegrass regeneration plants were obtained by using the shoot-tip meristem as an explant 5. The application of biotechnology to ryegrass was initiated early. Breeding new varieties of perennial ryegrass with enhanced salt tolerance through genetic engineering is expected 3, 4. In recent years, genetic engineering has been widely used in plant genetic improvement and has showed obvious advantages. However, perennial ryegrass is a cross-pollinated, self-infertile plant, resulting in slow progress in breeding new varieties with conventional strategies. Therefore, it is necessary to improve the salt tolerance of perennial ryegrass. However, the growth of perennial ryegrass as turfgrass is hampered by the aggravation of soil salinization and the shortage of water resources. It is widely cultivated as a turfgrass and forage with favorable agronomic traits, including rapid establishment rate, strong tiller ability, strong trample resistance, as well as high yield 3. Perennial ryegrass ( Lolium perenne L.) is an important cool-season grass in temperate regions worldwide. Salinity stress has become one of the major abiotic factors that severely affects plant growth. The area of saline land worldwide is nearly 1 billion hectares, and accounts for 10 percent of the total land area 1, 2. To our best knowledge this study is the first report of utilizing Chimeric Repressor gene-Silencing Technology (CRES-T) in turfgrass and forage species for salt-tolerance improvement. Physiological analyses including relative leaf water content, electrolyte leakage, proline content, malondialdehyde (MDA) content, H 2O 2 content and sodium and potassium accumulation indicated that the OsDST-SRDX fusion gene enhanced salt tolerance in transgenic perennial ryegrass by altering a wide range of physiological responses.
Transgenic lines overexpressing the OsDST-SRDX fusion gene showed obvious phenotypic differences and clear resistance to salt-shock and to continuous salt stresses compared to non-transgenic plants.
Integration and expression of the OsDST-SRDX in transgenic plants were tested by PCR and RT-PCR, respectively. Here, the rice DST gene was linked to an SRDX domain for gene expression repression based on the Chimeric REpressor gene-Silencing Technology (CRES-T) to make a chimeric gene ( OsDST-SRDX) construct and introduced into perennial ryegrass by Agrobacterium-mediated transformation. Phylogenetic analysis of six homologues of DST genes in different plant species revealed that DST genes were conserved evolutionarily. The Drought and Salt Tolerance gene ( DST) encodes a C 2H 2 zinc finger transcription factor, which negatively regulates salt tolerance in rice ( Oryza sativa).
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