| Switchgrass(Panicum virgatum L.) is a kind of native North America C4 perennial herb. In recent years, it has been widely used as an ideal bio-energy plants internationally because of its strong resistance, high biological yields and in-depth research has been started on swichgrass. We use switchgrass spike and young internode as explants. After in vitro propagation and further proliferation, we can get a large number of buds or spike buds, which is of positive significance on speeding up the switchgrass breeding process and save switchgrass germplasm. Study the process of buds occurrance mechanism allows us to have a good understanding of the physiological mechanism of adventitious buds development, and thus can provide some technical guidance in the production. Therefore, the study of inducing switchgrass spike buds physiological mechanisms can improve the efficiency of switchgrass tissue culture and optimize and improve its organizational culture condition.Switchgrass is also a kind of strict self-incompatibility crops with high sexual reproduction traits separation ratio, resulting in the limit of modern biotechnology utilization. A clone group has the characteristic that its material has the same genotype background, so exploring the use of clones in the same genetic background to created different mutants of switchgrass will likely promote breeding and molecular biology biotechnology research which has important significance.In this experiment,we use the different treatment, the frequency of significant differences switchgrass artificial spike buds as materials, its physiology difference formation process were studied and analyzed;with switchgrass artificial spike buds clones as materials mutant library were created and conducted a preliminary study of its variation types. the following results were achieved:1. We used two genotypes of switchgrass cultivars ’Alamo’, during the process that spike buds induced, taking E4(elongation phase 4)-E6 end of tillering as stem explants, which were inoculated in MS medium and SIM medium(Shoot Induction Medium, spike buds induction medium, MS basic medium supplemented with 3mg.L-1 6- Bian amino adenine). Spike buds on 30 days, 60 days and 90 days(30 days subculture once) were subjects, and physiological and biochemical parameters and endogenous hormone content were measured. The results showed that at the same cultured time, explants on SIM medium induced more spike buds and spike buds on SIM medium has higher cell proliferation and differentiation vitality; on SIM medium switchgrass spike buds antioxidant enzymes and polyphenol oxidase activity in general is higher than the material MS medium, it was a further proof that the physiological activity of switchgrass spike buds on SIM medium is stronger; materials from SIM medium has a higher N use efficiency and its spike buds chlorophyll content is generally lower than the control group, but the results failed to obtain energy restriction spike buds, soluble sugar content is always consistent with the control group; applying a 6-BA on switchgrass spike buds in vivo source no much impact on the synthetic of zeatin type cytokinins, but the synthesis of IAA can play a certain extent; GA content on SIM medium spike buds are always significantly higher than that from MS medium spike buds, indicating that 6-BA to play a role in promoting the synthesis of GA3, GA3 may be involved in cytokinin spike buds formation, and play a positive role; ABA level to some extent affected by the exogenous 6-BA. But in the two genotypes were not consistent in performance, indicating that ABA is not a key factor in the process of spike bud formation. Therefore, switchgrass artificial spike buds formation may be related enzymes and polyphenol oxidase activity increased and antioxidant, N use efficiency, certain inhibition of the synthesis of IAA, GA3 content increases and other physiological and biochemical factors, exogenous 6-BA promoted the synthesis of GA3, while prohibited the synthesis of IAA.2. Switchgrass cultivars ’Alamo’ spike buds were obtained by tissue culture propagation as explants, treat the materiel with ethyl methane sulfonate(EMS), three time gradient(4h、6h&8h) and concentration gradient(0.2%、0.5%&0.8%)were setted, we analyzed the best combination of concentration and time of EMS treatment. The results showed that the best mutagenic treatment for switchgrass was 0.5% EMS concentration with 4 hours, 110 regenerated plants were gotten. Field testing found the slow development mutants and precocious mutants, proving that creation of mutants with shoot clones in switchgrass was feasible. |
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