Mechanism Of Carbon Nano Materials On The Cell Viability Of Agapanthus Praecox Callus During Cryopreservation

Cryopreservation has been proved to be the best way for long-term preservation of plant germplasm resources.How to improve the survival rate of plant cryopreservation and reveal its regulating mechanism are important scientific issues in cryobiology.As a new-type nanomaterial,carbon nanomaterials（CNMs）have been widely applied in biological research because of their excellent biocompatibility,however no report has been seen in plants cryopreservation yet.The present research focused on the effects of four different carbon nanomaterials on improving survival rate of Agapanthus praecox callus（NEC）during cryopreservation,and reveal the mechanism based on interdisciplinary methods,which provides a theoretical basis for the application of carbon nanomaterials in cryopreservation field,and further enriches the theory of plants cryopreservation.The results were as follows:（1）In four kinds of carbon nanomaterials optimized cryopreservation procedure（CNMs-PVS2）,single-walled carbon nanotubes（SWCNT）,fullerenes(C₆₀),graphene（GR）and graphene quantum dots（GQDs）were added to plant plant vitrification solution 2（PVS2）at concentrations of 0.1g/L,0.3 g/L and 0.5g/L.According to the surivial rates,the best treatment was 0.3g/L C₆₀–PVS2,which increased the survival rate of A.praecox callus from 23.4%to 60.9%,compared with the control（PVS2）.For the different concentrations of SWCNT–PVS2,the highest survival rate（48.5%）was obtained after 0.1g/L SWCNTs-PVS2 treated procedure.Besides,0.3g/L Graphene and 0.1g/L GDQs-PVS2 had the better effect respectively.Synthetically considering the optimization effect and cost of the CNMs,0.1 g/L SWCNT-PVS2 and 0.3 g/L C₆₀-PVS2 are recommended to be used in the cryopreservation.（2）To find out the effect of CNMs on the physicochemical property of PVS2,the thermal properties of four kinds of CNMs-PVS2 and PVS2were analyzed by differential scanning calorimetry（DSC）.The results suggested that there was no significantly difference of the glass-transition temperature（T_g）between CNMs-PVS2 and PVS2,which indicated that T_g of CNMs-PVS2 was not the key factor that affects the survival,but SWCNT reduced the stability of PVS2,which influenced the trans-vitrification behavior,and resulting in the recrystallization during the rewarming step.（3）In order to analyze the subcellular location of CNMs and estimate its effect on the cell structure,the Raman spectroscopy results showed that the particles of SWCNT,C₆₀0 and GQDs entered into the callus cells at the step of dehydration.And the transmission electron microscopy（TEM）image showed that SWCNTs just located the cell membrane and the cell wall,while C₆₀0 particles were only accumulated in the mitochondria,and the structure of callus cell with C₆₀0 and SWCNT was mostly maintained intact,and the structural damages reduced.However,most of the CNMs were removed from the cell in the dilution step.（4）The difference of oxidative stress response between the PVS2（CK）and the optimal CNMs-PVS2 system(0.3 g/L C₆₀)was compared.The results suggested that antioxidant enzymes,POD and CAT remove the excess ROS fractions in the optimized CNMs-PVS2 system.During the steps of dehydration,freezing and washing,the activities of antioxidant enzymes increased with the expression of the antioxidant related genes.As a result,the levels of ROS content,such as,H₂O₂ and the production activity of OH?were decreased effectively.Moreover,lipid peroxidation of callus was decreased.It effectively maintained the cell membrane structure and protected the it from oxidative stresses induced damage.