Study On The Mechanism Of Programmed Cell Death During Vitrification-cryopreservation In Dendrobium Protocorm-like Bodies

Because of its safety and stability,cryopreservation has become an ideal means for the long-term storage of plant genetic resources.However,this preservation technology cannot be applied to all plant species.Although some biomaterials can be cryopreserved.the low regrowth rate after cryopreservation has been a bottleneck problem hindering their successful preservation.Over the past two decades,many studies in hunman and animal tissues have shown that programmed cell death(PCD)plays a key role in cell death following cryopreservation.However,there are few studies on plant PCD induced by cryopreservation.Therefore,it is of great significance to reveal the damage mechanism of plant cryopreservation from the PCD,thus providing an new idea for the optimization of cryopreservation technology.Protocorm-like bodies(PLBs)arc suitable and reliable materials for Dendrobium germplasm conservation,since they have the potential to develop into healthy plants and propagate easily in vitro.However,PLBs after cryopreservation always become bleached and show delayed cell death during the re-culture period.Using PLBs of D.nobile Lindl.'Hamana Lake Dream as materials,this study aims to investigate the effects of vitrification-cryopreservation on PCD and find the key stages of PCD occurrence,on this basis,to further study the possible role of nitric oxide(NO)and hydrogen peroxide(H2O2)as signal molecules in PCD.The main results and conclusions arc as follows:(1)Plant vitrification solution 2(PVS2)dehydration and liquid nitrogen(LN)exposure induced PCD in PLBs,which was evidenced by the following morphological and biochemical characteristics:?The main changes in cell ultrastructure were noted at the PVS2 stage with severe plasmolvsis,nuclei irregular,vesicle formation,nucleolus disintegration and chromatin condensation.LN exposure increased autophagy activity with the appearance of many autophagic vesicles and broken organelles.? Caspase-3-like activity,an executor of PCD,was increased significantly at the PVS2 dehydration and LN cooling-rewarming stage.Expressions of the autophagy-related protein 8C gene(Atg 8C),reticulon-like protein B8 gene(Rtnl B8),bax inhibitor 1-like gene(BIL I)and heat shock protein 70 gene(Hsp 70)were increased at the LN cooling-rewanning stage compared with expression levels in fresh PLBs(CK).(2)Preculture may be a signal initiation stage of PCD.PLBs did not show obvious PCD morphological characteristics and caspase-3-like activity increase,but showed significantly up-regulated PCD-related regulating genes at the preculturc stage.LN exposure during cryoprescrvation accelerated PCD.PCD-related regulating genes were highly expressed within 3 hours of recovery culture after cryopreservation.Cell structure showed serious damage after 3 hours of recovery culture,and DNA fragmentation began to appear at this moment.This may be one of the main reason why D.nobile PLBs can not regenerate after cryopreservation.(3)H2O2 produced at the preculture and loading stage during vitrification-cryopreservation of PLBs played a signaling role in PCD.H2O2 scavenger added at the preculture and loading stage significantly decreased the caspase-3-like activity of cryopreserved PLBs,alleviated PCD,and improved the survival rate of cryopreserved PLBs.While hydroxyl radical(·OH)mainly played a harmful role in cryopreservation.(4)NO also played a signaling role in PCD during vitrification-cryopreservation of PLBs,and there were difference in PCD regulation between NO produced at the preculture and loading stage.Exogenous NO inhibitor(50 ?M cPTIO)added at the preculture stage inhibited the expression of some pro-PCD regulating genes,decreased the caspase-3-like activity,alleviated PCD and increased the survival of cryopreserved PLBs by 17.78%.Exogenous NO donor(100 ?M SNP)added at the preculture stage aggravated PCD and decreased survival of cryopreserved PLBs by 20.28%.While.exogenous NO donor(1 mM SNP)added at the loading stage increased expression of Hsp 70,BIL1 and signal transduction genes,decreased aspase-3-like activity after cryopreservation.alleviated PCD and increased the survival of cryopreserved PLBs by 18.44%.But there were no obvious effect on PCD and survival of cryopreserved PLBs when exogenous NO inhibitor cPTIO was added at the loading stage.(5)NO produced iii the preculture and loading stage performed its signaling function in PCD and cell death by affecting the activity of CAT and ascorbate-glutathione(AsA-GSH)recycle and interacting with H2O2.Moreover,large accumulation of NO in unloading stage is disadvantageous to the success of cryopreservation.(6)Photoinhibition in PLBs occurred at the preculture stage,photosynthetic apparatus began to be damaged at the loading stage,and photosynthetic activity declined severely at the unloading stage during the vitrification-cn opreservation of PLBs.The impaired photosynthetic apparatus were not been improved during the recovery culture after cropreservation.The loss of photosynthetic function may occur at 1 hour after cryopreservation.which occured earlier than severe damage in morphology structure.The main contributions of this study:the survival rate of PLBs after cryopreservation was verified to be related to PCD;PCD occurred during the vitrification-cryopreservation in PLBs was confirmed to be related to PVS2 dehydration and LN exposure;NO and H2O2 produced in the preculture and loading stage was found to be involved in PCD as a signal molecule,and the interaction with H2O2 and ASA-GSH cycle may be one of possible regulatory pathways of NO.