L. Chinensis And Efficient Regeneration System For Transformation

Chinese Leymus is a perennial grass in Gramineae, which is widely distributed in Northern China and Mongolia. In recent years, being short of scientific management, overload on grassland and the "three low problems"(low setting percentage, low germination percentage, low tasseling percentage), the population of L chinensis has become more and more degenerated which restrict the artificial grassland construction, natural grassland improvement and control of deserted land. Thus, how to improve the quality of this forage by biotechnology to find a new germplasm, and how to improve the breeding process become the main subjects to be urgently solved. In this paper, initial research work have been done from three aspects as listed as below.First, High efficient germination in vitro shortens breeding cycle. The breeding cycle in Chinese Leymus is time-consuming because of its high level of seed dormancy. In order to improve the breeding process, we established a simple technique that shorten the breeding cycle by culturing the immature embryos in vitro to get germinating plantlet immediately. This technique showed a potential that may save one year in each sexual breeding cycle. Factors affecting immature embryos' germination were evaluated in this study. The immature embryos at different developmental stage were cultured on the MS basic medium. The immature embryos at 11-16 days after pollination expressed maximum germination rate (100%), but the maximum work efficiency (28 germinating plants/hour) was observed at 15-16 days after pollination. The germinating plantlets were 100% survival after field transplantation. Two generations were obtained in two years from the start of immature embryo culture, while it need at least two years for one generation by planting mature seeds.Second, established a high efficiency regeneration system and studied the factors affecting the plant regeneration. Concentration of different growth regulators and genotypes are the two main effecting factors to plant regeneration. Immature inflorescence segments of 3-5mm in length and immature embryos in different developing stages were cultured on N6 basal medium supplemented with 0-5.0 mg/1 2,4-D. The callus induction frequency of immature inflorescence ranged from 23.53 -100% depending on the medium used, and immature embryos' is from 71.43-100%.The shoots were differentiated from the calli on the basic medium containing N6 main elements and B5 microelements and supplemented with. Img/lKT and lmg/1 6-BA. Viable regenerants were developed on growth regulators free medium. Fertile plants were obtained after natural vernalization in the field. Meanwhile, the concentration of 2.4-D in the callus induction medium had strong effects on the successive plant regeneration. In addition, pH of the subculture medium L uenced the growth rate of calli.Third, the pilot study of transformation of Chinese leymus. The Pat gene had been transferred into the callus inducted from immature inflorescence by micro-projectile bombardement. The calli and shoots were selected respectively on the medium containing 20mg/l Basta, and obtained five Basta resistant plantlets. PCR eletrophoresis assay showed the foreign Pat gene had been transformed into the genomes of two Chinese leymus plantlets.