耐受偏二甲肼的芦苇变异株系的筛选

SELECTION OF UDMH TOLERANT VARIANT LINES OF AN AQUATIC REED

四氧化二氮/偏二甲肼(Unsymmetricaldimethyl_hybrazine,简称UDMH)为常用的航天器双组元液体推进剂。偏二甲肼沸点低,具有“三致”毒性,可在使用过程中释放到环境中,对污染地的生物多样性和人类健康构成威胁,因此迫切需要清除环境中的偏二甲肼。该文采用细胞工程的技术手段,以芦苇幼苗的下胚轴为材料诱导愈伤组织,继而通过逐步提高筛选压力选育出可耐受偏二甲肼的变异细胞系,再诱导变异细胞系分化,为可治理含偏二甲肼的废水的人工湿地处理系统的构建提供理想的工程植物。结果表明:芦苇的愈伤组织在含有0.5mg·L-12,4D的MS培养基上生长良好;偏二甲肼对该愈伤组织生长的半致死剂量为16.3mmol·L-1;在分别含有1.63、3.26和8.15mmol·L-1偏二甲肼的筛选培养基上进行3～6次继代培养后,可得到较稳定的抗性细胞系,培养23d后的相对生长量分别为对照的90.4%、84.3%和43.4%,培养43d后的相对生长量分别为对照的95.6%、91.7%和46.8%;但是只有前两类抗性细胞系可在含0.1g·L-1KT、0.01g·L-1NAA和相应浓度的偏二甲肼的MS培养基中诱导分化;将绿色再生苗转移到不含激素但含偏二甲肼的培养基上强化根的生长,再经过35d左右的适应性驯化,70%以上的再生苗可成功地转移至温室中培养,为日后人工湿地系统的构建奠定了基础。

Unsymmetrical dimethyl-hydrazine （UDMH） is a highly volatile and reactive compound widely used as a liquid rocket propellant fuel in manned and unmanned space and missile systems. Exposure to UDMH is an important occupational health issue, which will increase in significance as space applications increase. Several methods have been tested for removing UDMH from the environment, such as catalytic oxidation, active carbon absorption and ion exchange resins. However, little attention has been given to the application of construtted wetlands to remove UDMH from groundwater even though constructed wetlands are a promising measure for wastewater treatment. In expectation of constructing of an effective wetland by the variant reed lines to deal with UDMH polluted groundwater, attempts were made to screen for UDMH tolerant variant lines of reeds using cellular engineering technology in this study. Seeds of the reed （Phragmites communis ） collected from the river banks near by a satellite-launching basement were sterilized with 70% ethanol and 2% hypochlorous acid and then transferred to a MS medium for germination. After germination, hypocotyl seedling segments were used to induce callus on the MS medium supplemented with 1 mg· L^-1 2,4-dichlorophenoxyacetic acid. The UDMH-tolerant cell lines were obtained by continuous selection on the UDMH containing medium supplemented with 0.5mg· L^-1 2,4-dichlorophenoxyacetic acid. Three levels of UDMH tolerant cell lines were screened for that contained either 1.63, 3.26, or 8.15 mmol·L^-1 UDMH after 3 - 6 times of subculture. Relative growth, measured at the end of a 23-day incubation, was 90.4%, 84.3%, and 43.4% of the control in the three treatments, respectively, and, at the end of a 43-day incubation, was 95.6%, 91.7%, and 46.8% of the control, respectively. These results showed that UDMH could inhibit the growth of the callus at 8.15 mmol· L^-1. Regeneration of plantlets from the selected cell lines was conducted on UDMH containing MS medium supplemented with 0.1 g· L^-1 6-Benzylaminopurine and 0. 01 g· L- 1 1-naphthyl acetic acid. However, only the cell lines grown in the lower two treatments could be successfully differentiated, the 8.15 mmol·L^- 1 UDMH failed to differentiate. These resuits indicated that the suppressive effect of UDMH on callus differentiation was greater than on callus growth. Plantlets screened under UDMH stress were more tolerant to UDMH than the control, which were plantlets regenerated from callus without UDMH stress. Root growth of the differentiated plantlets was enhanced by transferring them to a hormone-free MS medium supplemented with UDMH for 35 days prior to successful transplantation of the plantlets into the soil. Finally, more than 70% of regenerated plantlets could be successfully transferred to the greenhouse. Further experiments estimating the growing status and remediation efficiency in UDMH polluted water of these variant seed lines were undertaken.