The Colonization Of Azospirillum Brasilense Yu62 In The Wheat And Growth-Promoting Effect On It

Using of EGFP gene as a reporter gene of Azospirillum brasilenseYu62 tagged. First, the EGFP gene was amplified by using PCR amplification methods and cloned to the prokaryotic expression vector pVK-100 to construct the recombinant plasmid pVK-EGFP, the plasmid containing Azospirillum replicon can be copied stablity. The recombinant plasmid was transformed into Azospirillum brasilense Yu62 by electroporation. Detection of the expression of green fluorescent protein through polyacrylamide gel electrophoresis, the result showed that the EGFP gene can be expressed stablity in Azospirillum brasilenseYu62 .In restricted bacteria conditions, using of marker strains inoculated wheat (Xiaoyan 107) seedlings, after 1,3,5,7,30 days, disinfection from wheat seedlings with 1% sodium hypochlorite and 75% ethanol at the surface, and then, placed in YMA medium containing tetracycline to culture for the separation of Azospirillum and initial position in wheat. After cultured for 48 hours, observation under the UV-light of 365nm wavelength, the results showed that the fluorescent position from rootstalk growth point spread upward gradually until the stem and leaf with the change in inoculation time. Checking the wheat which inoculated the same number of days to make slices and observe the colonization law of Azospirillum in wheat by Fluorescence microscope. The results showed that marked strains mainly colonizing the root surface after inoculation the one day, After inoculation three days, marked strains into the cortex of wheat roots, mainly located in cell space. After inoculation five days, marker bacteria have entered the inner cortex, a small amount of strain into the vascular bundle. After inoculation seven days, a large number of strains into the vascular bundle, a small amount of strain into the catheter. After inoculation thirty days, marked bacteria colonization in the stems and leaves of wheat, The results showed that Azospirillum can migrate from the roots to the ground site.In open conditions, the big plump grains of wheat (Xiaoyan 107) were selected and then divided into two groups, as fertilizer, Azospirillum brasilenseYu62 was mixed with one group. The main method: when the Azospirillum brasilenseYu62 was cultured to OD value of approximately 0.6, mixed it with wheat seed soaking about 2h. After soaking, planting the inoculation and other wheat without any treatment in the fileds, with the groove width 0.2 m, uniform the plants per line, and Ensure that inoculation and the control is consistent in the quantity. In the fifth month after inoculation, the observation results show that the inoculation plants not only grow higher than the control plants, but also have more green leaves. In the seventh month after inoculation, the observation results show that the inoculation plants have large advandage in the height, tiller and the level of root developed compared to the control. All these lead us conclusion that as a grass endophytic diazotrophs, Azospirillum brasilenseYu62 has obvious growth-promoting effect on the plant.