| Wheatgrass (Agropyron Gaertn.) is perennial sparse-fascicled grasses, which is also one of important Gramineae grazing grasses. Wheatgrass is planted widely in the arid and semiarid and desert grassland, with strong environment-stress resistance. It is also characterized as early turning green in spring, late withering and turning yellow in autumn.The stem and leaf of wheatgrass is tender and soft with plenty nutrition and favorable palatability. In northwest arid and semiarid area of China, wheatgrass is one kind of very important grasses used for establishing the made grassland and improving the ecological environment. In this paper, the wheatgrass tissue culture and regeneration system with the immature inflorescence as explant was established using four species as materials: A. mongolicum Keng, A.cristatum cv.'Fairway', A.desertorum cv.'Nordan', A.cristatum Ñ… A.desertorum cv.'Hycrest-Mengnong'. Based on the established regeneration system, the P5CS gene, which regulates the last step of proline synthesis in plant, was transformed into wheatgrass with phosphinothricin acetyltransferase (bar) conferring herbicide resistance as selecting gene. The transformation was conducted through microprojectile bombardment of callus derived from immature inflorescence. The obtained transgenic plants can provide the new material resource for wheatgrass genetically improvement.The transcription factors play important role in both plant growth and development and the response to the outside environment. CBF4 transcription activator is one kind of trans factors, which is induced by drought. It can specifically integrated with CRT/DREDNA complex regulator and then induce the expression of genes, whose promoters contain this regulator, and finally activate the various stress-resistance mechanisms in plant. But there are few reports about the function of CBF4 promoter. To study the expression pattern of CBF4 gene and its promoter in transgenic Agropyron and explore the application of CBF4 in the stress-resistance genetically engineering, the CBF4 gene and its promoter were isolated from Arabidopsis thaliana by PCR. The sequence analysis and function assay were conducted.The primary results and progress were summarized as following,1 The feasible medium for callus induction from immature inflorescence is improved MS + 2,4-D (2.0mg/L), the induction frequency is up to 83.5%. 2 The differentiation medium employed hormone free MS in 2002, the differentiation ratio is about 59.6%. In 2003, the differentiation medium was improved as MS+KT (0.2mg/L), the differentiation ratio is about 74.5%.3 The rooting medium was 1/2 MS. Rooting ratio was 100%.4 The size of the immature inflorescence was used as sampling standard for tissue culture. During the inflorescence developing stage, the out-striped inflorescence, which length is between 1.0 and 3.0 cm, is optimal to be used as explant to induce the callus.5 The results showed that callus initiation and plant regeneration could occur under induction in all four kinds of wheatgrass.6 The transgenic plants were obtained by microprojectile bombardment of callus induced from immature inflorescence. 7 The results of PCR and Southen analysis displayed that the exogenous P5CS gene had integrated into the genome of transgenic wheatgrass. 8 The assay of RT-PCR showed the transgenic P5CS had expressed at transcript level.9 The transgenic frequencies of P5CS gene were 0.09% in 2002 and 0.11% in 2003, respectively.10 The CBF4 gene and its promoter were isolated from Arabidopsis thaliana by PCR. 11 The analysis of CBF4 promoter sequence with bioinformatics software indicated that it maybe one of inducible promoters by outside environmental stresses. The GUS expression cassette driven by CBF4 promoter was constructed and transformed into tabacco(Wisconsin 38). The assay of the expression of GUS in the transgenic tobacco displayed that CBF4 promoter can be induced by drought stress, in accordance with the analysis with bioinformatic software.
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