Study On The Role Of Ethylene In Programmed Cell Death Of Dendrobium Nobile PLBs During Cryopreservation And Its Interaction With H₂O₂

The cryopreservation technology is currently one of the most effective and long-term preservation methods for plant germplasm resources,but the death or the low survival rate of some plant materials after preservation has been an unsolved problem.Programmed cell death（PCD）is one of the main reasons for the decrease in survival rate of some animal materials after cryopreservation.Regulating the occurrence of PCD may be one of the ways to reduce mortality after cryopreservation.However,the study of PCD in cryopreservation has just started,and the endogenous signal is still unclear.As important signal molecules,ethylene and H₂O₂ participate in PCD of plants under a variety of stress,and their role in cryopreservation is still unclear.In this paper,the protocorm-like stems（PLBs）of Dendrobium nobile Lindl.’Hamana Lake Dream’,which has a high reproduction rate but low regeneration rate after cryopreservation,are used as materials to explore the role and mechanism of ethylene and H₂O₂ as signal molecules in the occurrence of PCD in cryopreservation,and explore the interaction between it and hydrogen peroxide（H₂O₂）in this process,in order to further reveal the cryopreservation The mechanism provides the basis.The result is as follows:1.Adding 400mg·L^-1 exogenous ethephon in the preculture and loading stages,the endogenous ACC content and ACO and ACS activities of PLBs stored during cryopreservation were significantly increased,which increased by 20.64%、21.80%and 29.47%respectively compared with the control group.While,the expression of ACO and ACS genes was significantly up-regulated;at the same time,the survival rate of PLBs after cryopreservation in liquid nitrogen increased by 7.97%compared with the control.It shows that exogenous ethylene increases the survival rate of spring Dendrobium protocorms preserved in vitrified ultra-low temperature by increasing the content of endogenous ethylene.2.After adding an exogenous ethephon in the preculture and loading stages,the Caspse-3-like activity was reduced by 44.17%and 26.80%respectively compared with the control group.At the same time,the PCD defense genes were up-regulated and the promoting genes were down-regulated.It shows that ethylene inhibits the occurrence of PCD by regulating genes and proteases.Both ethephon and cobalt nitrate up-regulated the expression of four genes MAPK,MAPK1,MAPK3,and MAPK4 that have signal transmission effects in PCD.It shows that ethylene is involved in the signal transmission of PCD.3.The addition of exogenous ethylene in the preculture stage or the Loading stage significantly reduced the content of endogenous H₂O₂,which was 12.91%-42.90%lower than that of the control group,and significantly increased the activities of SOD,CAT,APX,GR and other enzymes in PLBs and the two non-enzymatic substances of As A and GSH.Content,while reducing NADPH enzyme activity.It shows that ethylene reduces the content of endogenous H₂O₂ and the oxidative stress mediated by it by regulating catalase and non-enzymes,and participates in the response of PLBs to the stress of cryopreservation.4.At the same time,exogenous H₂O₂ significantly reduces the content and production of endogenous ethylene in PLBs.With the addition of H₂O₂ in the preculture stage,the ACC content,ACO,and ACS enzyme activities were greatly reduced,all of which were reduced by more than 90%compared with the control group.It shows that the pre-cultivation link is the main period when ethylene plays a role.This study shows that ethylene affects the survival rate of PLBs after cryopreservation;it is clear that ethylene signal is involved in the occurrence of PCD in PLBs cryopreservation,and has a negative regulation on PCD;it clarifies the interaction between ethylene signal and H₂O₂ signal in cryopreservation relationship.It is confirmed that the H₂O₂ and As A-GSH cycle may be one of the important regulatory pathways for ethylene to participate in PCD during cryopreservation.