Preparation Of Azafullerenol And Its Promotional Effect On Seed Germination And Seedling Growth Of Pepper

In recent years,plant nanoscience as a new interdisciplinary subject,has attracted the attention of many researchers.Much research about the application of carbon nanomaterials（CNMs）in agriculture,especially fullerenes and their derivatives have excellent performance in promoting crop seed germination,growth and regulating plant metabolism,and can be used as potential nano plant regulators in agriculture.Due to the strong hydrophobicity of fullerenes,it is necessary to modify the fullerenes with water solution to improve their hydrophilicity and biocompatibility,and then improve the convenience of application.In recent years,plant nanoscience as a new interdisciplinary subject,has attracted the attention of many researchers.Much research about the application of carbon nanomaterials（CNMs）in agriculture,especially fullerenes and their derivatives have excellent performance in promoting seed germination,growth and regulating plant metabolism,which can be used as potential nano plant regulators in agriculture.Due to the strong hydrophobicity of fullerenes,it is necessary to modify as the water-soluble derivatives of fullerenes for improving their hydrophilicity,biocompatibility,and convenience of application.In this paper,hydroxyl-functionalized fullerenes,azafullerenol,were prepared by onestep nitrene reaction.The method can be extended to prepare hydroxyl functionalized oneand two-dimensional CNMs derivatives.Moreover,pepper seeds were treated with azafullerenol,to explore its promotional effects on seed germination,seedling growth and to analyze its accumulation and transport pathway in pepper.The detailed research contents of this paper are as follows:（1）zero-dimensional C₆₀ were dispersed by 2-chloropropanol,and then reacted with azidoethanol.The dialysis was carried out after washing with ethyl acetate,methylene chloride and ethanol to produce purified hydroxyl-functionalized and water-soluble zerodimensional product,C₆₀（NCH₂CH₂OH）n.The structure of C₆₀（NCH₂CH₂OH）n was characterized by solid nuclear magnetic resonance spectroscopy,infrared spectroscopy,and X-ray photoelectron spectroscopy,and thermogravimetric analysis,and its average addition number was 14,which was marked as C₆₀（NCH₂CH₂OH）₁₄·9H₂O.In addition,hydroxyl functionalized one-and two-dimensional CNMs were prepared by nitrene reaction of one-dimensional multi-walled carbon nanotubes（MWCNTs）,singlewalled carbon nanotubes（SWCNTs）,and two-dimensional graphene（G）,which the average addition numbers of MWCNTs-OH,SWCNTs-OH and G-OH per thousand carbon atoms were 138,258,and 191,which were marked as MWCNTs（NCH₂CH₂OH）138,SWCNTs（NCH₂CH₂OH）258,and G（NCH₂CH₂OH）191,respectively.（2）It was carried out to measure the aggregation and surface charge properties and free radical scavenging efficiency for C₆₀（NCH₂CH₂OH）₁₄·9H₂O.The hydration particle size of C₆₀（NCH₂CH₂OH）₁₄·9H₂O in aqueous solution was 78.8±7.1 nm,showing a good watersolubility.The zeta potential is 20.7 ± 0.3 eV,showing the surface positive charge characteristics.The inhibition rate of ABTS⁺· radical in vitro was 50% under C₆₀（NCH₂CH₂OH）₁₄·9H₂O treatment at the concentration of 45.7 mg/L.（3）the promotional effects of C₆₀（NCH₂CH₂OH）₁₄·9H₂O on seed germination and seedling growth of pepper was analyzed.Pepper seeds were treated by C₆₀（NCH₂CH₂OH）₁₄·9H₂O with petri dish hydroponic method and shaking table hydroponic method,respectively.Statistical analysis was conducted by SPSS.At the early growth stage of pepper seeds,the hormesis effect of C₆₀（NCH₂CH₂OH）₁₄·9H₂O on pepper seeds was verified.The root elongation was promoted at lower concentrations of 5,10 and 20 mg/L,while the treatment at higher concentration of 50 mg/L inhibited root growth.There was no obvious effect on buds.Then,the enzymes and metabolites of roots were measured.The pepper seedlings were cultured by shaking table hydroponic method with a series of concentrations of C₆₀（NCH₂CH₂OH）₁₄·9H₂O.The contents of Hydrogen peroxide（H₂O₂）,malondialdehyde（MDA）and the activities of catalase（CAT）and superoxide dismutase（SOD）were determined,and CAT activity increased after treatment with 5,10,and 20 mg/L of C₆₀（NCH₂CH₂OH）₁₄·9H₂O.H₂O₂ content is decreased;MDA content did not change significantly;SOD activity is slightly increased.The results showed that C₆₀（NCH₂CH₂OH）₁₄·9H₂O treatment can regulate the intracellular oxidation balance and promote H₂O₂ metabolic cycle to stimulate the growth of root cells by affecting oxidation related enzyme activities in the roots of pepper seeds.The potted experiments was carried out to explore promoting effect on pepper seedling growth treatment with various C₆₀（NCH₂CH₂OH）₁₄·9H₂O by the methods of irrigation,sprayed casting processing,respectively.The irrigation treatment under the concentration of 5 mg/L can promote root and seedling growth.The plant was significantly higher than that of the control group.However,under 50 mg/L C₆₀（NCH₂CH₂OH）₁₄·9H₂O treatment,the root and seedling growth was significantly inhibited.（4）The accumulation and transport of C₆₀（NCH₂CH₂OH）₁₄·9H₂O in pepper was analyzed.The pepper seeds were observed with a microscope in different growth stages.At the seed germination stage,C₆₀（NCH₂CH₂OH）₁₄·9H₂O was absorbed and accumulated on the surface of seed coat.At the stage of seed-root growth,the accumulation of C₆₀（NCH₂CH₂OH）₁₄·9H₂O was observed on the root crown surface of the root tip and formed a coating substance.The root-hair growth was not inhibited at low concentrations of5 and 10 mg/L,and a few amount of C₆₀（NCH₂CH₂OH）₁₄·9H₂O was adsorbed on the surface of root hair.Under 50 mg/L C₆₀（NCH₂CH₂OH）₁₄·9H₂O treatment,the growth of root hair was inhibited to show a phenomenon of shrinkage,and a large amount of azafullerenol was accumulated on the surface of root hair.It was speculated that azafullerenol had broken through the surface cells of root hair,resulting in the loss of water and inactivation of cells,thus inhibiting the development of root hair.At the concentration of 5 mg/L,azafullerenol was observed in the elongation zone of pepper root,and the color of azafullerenol was clearly visible through microscopic observation of the transverse section of the root.According to the above results,it can be inferred that azafullerenol can accumulate on the seed epidermis,attach to the root hair surface,and enter the internal root column during the seedling growth stage for transport in xylem.