| Chinese leymus (Leymus chinensis (Trin.) Tzvel.), a member of Poaceae, is a dominate grass species distributed in the eastern grassland in Eurasia. As the king of forage, it is a strategicly biological resource with technological advantage in China and has an important role in stock raising and environmental protection in northern China. In recent years, due to scientific management, overgrazing and the "three-low-problem" (low seed setting, germination and tasseling),the population of L. chinensis has become more and more degenerated which restricts the artificial grassland construction, natural grassland improvement and control of deserted land. Therefore, improving Chinese leymus, fast evaluating and producing the new germplasm by biotechnology based on cell biology and molecular biology and improving the breeding process become the main subjects to be urgently solved. In this paper, initial works have been done from three aspects and obtained the results as listed as below. 1. Establishment of an in vitro regeneration system for L. chinensis and study of the factors affecting in vitro culture. Concentration of different growth regulators in the medium and genotypes of the explants were the main factors affecting plant regeneration. Immature inflorescence segments 3-5mm long were cultured on N6 basal medium supplemented with 0-5.0 mg/L of 2,4-D. The callus induction frequency of immature inflorescence varied depending on the medium used, and the maximal average induction frequency was 93.21% for genotype C6 and the minimal frequency was 33.35% for Jisheng1. Shoots were regenerated from the calli on the basic medium containing N6 macro-elements and B5 micro-elements and supplemented with 1.0 mg/L of KT and 1.0 mg/L of 6-BA. Viable regenerants developed roots on medium free of growth regulator. Meanwhile, the concentration of 2,4-D in the callus induction medium had strong effects on the subsequent plant regeneration. In addition, calli from different inflorescence from genotype W4 were cultured separately. The growth and regeneration frequency of calli from different inflorescence was investigated. These calli illustrated a little different referred to the growth and death. In the course of regeneration, the differentiation frequency was a bit different, the maximal frequency was 9.24%, and the minimal frequency was 5.26%. 2. The differential expressed genes of calli from different immature inflorescence of genotype W4 were studied. These calli displayed differences during regeneration. Differential cDNA fragments were isolated by means of DDRT-PCR analysis and silver-nitrate staining. The differential fragments were cloned and sequenced. Nucleotide sequence analysis of these two cDNA fragments showed that their homologous genes encode translation factor eEF-1 and glutathione S-transferase, respectively. 3. Establishment of genetic transformation techinique for Chinense leymus. The study on transformation of a gene for phosphinothricin acetyltransferase (PAT) through microprojectile bombardment was carried out in two genotypes of this species. Calli from immature inflorescence cultured on N6 medium supplemented with 2.0 mg/L of 2,4-D and 5.0 mg/L of glutamine were bombarded. In total, 1430 calli from genotype W4 and 1850 calli from C3 were bombarded. The bombarded calli survived on selection medium with 1.0 mg/L of phosphinothricin (PPT). Twenty-three plantlets were obtained from resistant calli on differentiation medium supplemented with 1.0 mg/l of 6-benzylaminopurine, 1.0 mg/L of kinetin, and 1.0 mg/L of PPT. Five of 23 regenerated plantlets survived on the rooting medium with 1.0 mg/L of PPT, and three of them were from genotype W4 and two from genotype C3. Polymerase chain reaction and Southern blotting analyses indicated that PAT gene had been integrated into the genomes of Chinese leymus plantlets and the gene was stably transferred to its clones. There were no other phenotypic effects associated with transgene expression during vegetative growth except tolerance to the herbicide Basta?.
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