Nanopriming With Green Synthesis Of Silver Effects On The Germination And Physiological Characteristics Of Alfalfa(Medicago Sativa L.)

Nanoparticles（NPs）have become a hot topic in many fields,especially in agriculture,due to exceptional physicochemical properties.With the development of nanotechnology,NPs synthesized using plant extracts have proven to be an alternative to agrochemicals and traditional NPs in the field of sustainable.Due to the lack of application of NPs prepared by this method on alfalfa and the fact that research is mostly limited to toxicology,it is necessary to apply NPs to alfalfa in a suitable way.In this study,we investigated the effect of Ag NPs priming treatment on seed germination and seedling growth of alfalfa（Medicago sativa L.）based on the green synthesis of Ag NPs from alfalfa extracts at different concentrations and priming time treatments.The main findings are as follows:1.Green synthesis of Ag NPs based on alfalfa extracts.Active substances in the above-ground parts of alfalfa were extracted by ultrasonic alcohol extraction.The reaction conditions for the preparation of two Ag NPs suspensions were investigated:room temperature,450 rpm,1:2 volume ratio of reaction material（Ag NO₃:alfalfa extracts）,2 d in the dark or 3 d in the light.A series of characterization techniques were analyzed and the prepared Ag NPs were spherical with good crystalline structure and the outer surface was wrapped by chemical substances such as esters,phenols,carboxylic acids,amino acids and sugars.Both Ag NPs suspensions had the same composition as the alfalfa extracts,with very low free Ag⁺in the concentration range of 0-200 mg/L.The free Ag⁺in the pink Ag NPs suspension under light exposure is lower,only about 2%of the original proportion of the Ag NPs suspension.2.The effects of green synthetic Ag NPs on the germination characteristics of seeds were studied.Alfalfa seeds were treated with different concentrations（0,12.5,25,50,100and 200 mg/L）and different priming times（1 h,2 h and 3 h）of pink Ag NPs suspensions for priming.It was shown that low and medium concentrations of Ag NPs（12.5 mg/L,25mg/L and 50 mg/L）tended to significantly increase（P<0.05）the germination energy,germination index and vigor index of alfalfa seeds,while high concentrations of Ag NPs treatments（100 mg/L and 200 mg/L）decreased.The priming concentration of Ag NPs significantly affected germination energy,germination index,vigor index and germination rate（P<0.05）,while the priming time of Ag NPs significantly affected vigor index and germination index（P>0.05）.There was no significant difference（P>0.05）inα-amylase activity of all concentrations of Ag NPs-treated and hydro-primed seeds compared to hydro-priming.However,the surface cellular interstices of the treated seeds increase and pores were formed,thus increasing the water absorption of the seeds.Furthermore,the distribution of silver was detected in the cross-sectional dicotyledon of the treated seeds,indicating that Ag NPs could enter the seeds and affect seed germination of alfalfa.3.The effects of green synthetic Ag NPs on the physiological properties of seedlings were studied.Results of seedling growth and physiological characteristics after different priming treatments showed that the distribution of silver corresponded to physiological indicators of alfalfa:at all priming times and at low to medium concentrations of Ag NPs（12.5 mg/L,25 mg/L and 50 mg/L）treatments,silver accumulated in the leaves and seedling length decreased significantly（P<0.05）,but total leaf chlorophyll increased significantly（P<0.05）and no leaf cell death was found by Evans Blue Stain.SOD and POD enzyme activities were significantly enhanced in leaf and root cells（P<0.05）,CAT enzyme activity was not significantly increased,and the plasma membrane was not damaged.In contrast,silver accumulated in the roots under high concentrations of Ag NPs（100 mg/L and 200 mg/L）treatment,which had a positive effect on root cell division and significantly increased root activity and elongation（P<0.05）.SOD and POD enzyme activities were significantly enhanced in leaf and root cells（P<0.05）,CAT enzyme activity was not increased,and the plasma membrane was damaged.Both the priming concentration and time of Ag NPs affected seedling vigor indicators and antioxidant enzyme systems（P<0.05）,while the priming concentration of Ag NPs affected significantly more content of chlorophyll,leaf vigor,root activity,biomass,proline and MDA than the priming time（P<0.05）.Besides,biomass of seedlings reached a maximum when the distribution ratio of silver in the leaves to roots of seedlings was about 1:1.In conclusion,appropriate light induction,active substance coverage and nanopriming can minimize the toxicity of Ag NPs to alfalfa,and at suitable concentrations and times,the green synthesized Ag NPs promotes alfalfa seed germination and enhances the antioxidant enzyme system of seedling growth.This study can provide a theoretical reference for the toxicology of nanomaterials affecting alfalfa and provide technical support for the further promotion and application of green nanomaterials.