The Biological Mechanism Of A Novel Hydro-electro Hybrid Priming To Recover Potential Vigor Of Onion Seeds

Onion?Allium cepa?is the most foreign-exchanging earner in Chinese exporting vegetables.The cultivars and seeds that are planted in China are mainly imported,so each seeds lot generally requires 4-5 years of storage for multi-season sales.However,onion seeds have a poor longevity that they lost their vigors faster than other vegetable seeds even in optimal storage causing retardation of germination and low seedling establishment.Technically,the lost vigor can be recovered by seeds priming.Hydro-priming is the most widely used in varieties of primings as it does not use chemicals and is long effective.However,traditional hydro-priming requires a sustained seed moisture,temperature and air flow precise control in priming chamber for more than 5 days.Its technological process is difficult to control.Electro-priming can instantly increase seed vigor through irradiating seeds with high voltage electrostatic field,though the effects are short-lived that it generally will disappear in 20 days.Here,we investigated the potential of combined hydro-electrostatic hybrid priming?HEHP?on the recovery of vigor in onion seeds.And I managed to stack the effects and advantages of the two single primings on onion seeds,that hybrid priming led to a shorter time and simpler processing than single hydro-priming,and longer lasting effects than single electro-priming,while seeds vigor was further improved without any chemicals.The biological mechanism of the hybrid priming were studied.Main processes and results are as follows:1 Single electro-priming recovers the vigor of onion seeds.One combination of parameters?10 kv/cm for 40 s?of single electro-priming with the most positive performance on onion seeds was acquired from massive preliminary experiments.Principal component analysis was applied to integrate all 8 indexes?germination percentage 2d,germination percentage 4d,germination rate,length of shoot,fresh weight,mean emergence time,germination index,vigor index?to one index to comprehensively evaluate the effects of priming and a mathematical model between the parameters?voltage of electristatics and irradiation duration?and the comprehensive index was established through quadratic general rotation combination experiment design to optimize the effect of electro-priming.The optimized parameters?8.7 kv/cm for 43 s?increased 14.0%of germination percentage?4d?,10.4%of germination rate,8.9 mm of length of radicle,0.123 mg of fresh weight,4.76 of germination index,184.06 of vigor index and decreased 0.7 day of mean germination time,comparing to control.The integrative index was further promoted.However,the effect waws totally disappeared after 21 days optimal storage.2 Hydro-electro hybrid priming recovers the vigor of onion seeds.Onion seeds were hybrid primed for 24h,48h and 96h with optimized electro-priming parameters.Seed moisture,temperature and air flow in priming chamber control were inaccurately controlled during priming.The result showed that the integrative index of HEHP for 24h was very closing to hydro-priming for 96h while HEHP for 48h and 96h was completely above that.After artificial aging,seeds with HEHP for 48h and 96h showed an improved aging-resistant comparing to hydro-priming for 96h.The technical aims of HEHP was archieved.3 Influence of different primings on the activity and structure of critical germination enzymes in onion seeds.The structure of enzymes were determined by circular dichroism spectrum after purification and their avtivities were also determined by assay kits.The results showed that the unit activity of superoxide dismutase was rapidly and significantly promoted as electrostatic field irradiation?8.7 kv/cm for 43 s?increased?-sheet and decreased random-coil of SOD secondary structure,while catalase and?-amylase were not influenced.With the electrostatic field irradiation's help,SOD activity could be promoted same to hydro-priming for 96h by HEHP for 24h.And it was not promoted through electrostatic field irradiation up-regulated SOD synthesis related gene to increase the quantity of SOD enzyme.SDS-PAGE showed that Cu/Zn-SOD was dominating the types of isoenzyme in onion seeds.4 Influence of different primings on the self-repairing of cell membrane and organelle of onion seeds embryo.The determination of EPR,EC and MDA increment showed that,with higher SOD activity,free radicals were scavenged by HEHP much quicker during priming.So that hybrid primed seeds had lower EPR signals,EC and MDA increment.The oxidizability of intracellular environment were further suppressed,so HEHP for 48h had the same suppression to hydro-priming for 96h,while HEHP for 96h was completely above that.TEM and SEM directly revealed that HEHP led to quicker and more complete self-repairing of cell membrane and tissues and less wounds could be observed on hybrid primed embryo.Single electro-priming had no substantial repairing or scavenging during priming.5 Influence of different primings on critical germination substances and their gene expression.The result of RNA-sequencing showed that 143 RNAs of onion seeds were directly quantitatively changed by electrostatic field irradiation,where SOD,GA systhesis and ABA degration related gene did not involved.GA systhesis and ABA degration related gene were activated and normally expressed during hydro-priming process,while HEHP up-regulated the expression of one GA₂₀ox and two8'-ABA hydroxylase synthesis to further increase GA and decrease ABA content.However,this three genes were not differently expressed through electricfield irradiation changed structure or sequence of DNA or RNA.Spearman correlation analysis demonstrated seeds vigor was positively correlated to GA content,and negatively correlated to ABA content and ABA/GA ratio.Hydro-priming and HEHP both down-regulated the expression of SOD synthesis related gene.6 Explanation and comparasion of biological mechanism of different primings.Based on the above results,the mechanism that HEHP led to higher seeds vigor,shorter time,simpler processing and longer effectiveness,and the difference of mechanism between different primings were concluded:1.The changing of SOD structure improved tissue recovery.Seed stayed in pre-germination after imbibition and enzymes related to germination were activated in this stage.SOD strated to scavenge free radicals,then self-repairing of embryo cell was initiated,and radicle finally went into full emergence.Single electro-priming instantly and temporarily increased?-sheet of SOD secondary structure and promote the unit activity of SOD to faster radical scavenging and tissue repairing,so germination time was shortened and germination rate was improved.Hydro-priming provided seeds with an extra pre-germination and part of repairing was completed before actual sowing,whose speed and level depended on current seeds vigor.HEHP further improved radical scavenging,tissue repair and seeds vigor through electricfield irradiation promoted SOD activity to improve the pre-germination.HEHP seeds had more complete tissues to be long effective,comparing to short-lived single electro-priming who had no substantial repairing or scavenging during priming.2.Hormone level regulation.Physiological processes of GA synthesis and ABA degration were activated during hydro-priming.Electricfield irradiation indirectly regulated the level of this two hormones to be positive germination,higher GA and lower ABA and ABA/GA ratio.So hybrid primed seeds had further improved vigor.In this paper,hydro-and electro-priming were firstly and innovatively combined to a novel hydro-electro hybrid priming,and it successfully stacked the effects and advantages of the two single primings on onion seeds.Technical aims of HEHP finally archieved that hybrid primed seeds had further improved vigor and longer effectiveness,while seed moisture,temperature and air flow in priming chamber control were inaccurately controlled during shorter priming time.A logical mechanism chain from the changes of SOD enzyme structure induced by electric field to the changes of SOD activity,oxidizability of intracellular environment,cell membrane repair speed and key hormone levels was constructed and scientifically explained.