Mechanism Of Hyperhydricity Mediated By Reactive Oxygen Species Of Garlic Plantlets In Vitro

Hyperhydricity is a common physiological disorder during plant in vitro culture.Plantlets in vitro of garlic AAllium sativum L.)is easy to be hyperhydric and difficult to recover.Hyperhydricity affected the application of elimination of virus,micropropagation,innovation of germplasm and genetic improvement severely.The mechanism of hyperhydricity was still unclear.Lacking theoretical basis led to the limit effects of prevention and control of hyperhydricity.The previous study found the metabolic disorder of reactive oxygen species(ROS)in hyperhydric plantlets in vitro,but the relationship between them needs to be studied systematically.Garlic variety 'ershuizao' was selected as the material to study the mechanism of hyperhydricity from the aspect of endogenous ROS by physiological and biochemical analysis,qPCR,high-throughput sequencing,and RACE.ROS metabolism characteristics of plantlets under hyperhydricity-aggravated and hyperhydricity-alleviated treatments were investigated.The alleviation mechanisms of exogenous H2O2 on hyperhydricity were discussed.Expression stability of reference genes for qPCR of garlic was validated.The critical genes of hyperhydricity were identified.The main results were as follows:1.On the day two of high concentration of H2O2 and low media pH and the day eight of low concentration of agar and high concentration of BA treatments,endogenous ROS of plantlets in vitro increased rapidly along with the increasing of hyperhydric rate.Under four hyperhydricity-aggravated treatments,the expression levels and enzyme activities of CAT,SOD,and POD increased significantly.The content of GSH and AsA,the DPPH-radical scavenging activity,and FRAP value decreased;the MDA content and the relative membrane permeability increased;the integrity of cell membrane declined;the ROS metabolism was in disorder.Under 100 ?M SA and 500 ?M AsA treatments,the ROS increased slowly;the expression level and enzyme activity of APX,GR,GPX increased;the content of AsA and GSH increased;the metabolism of ROS was in a stable status.The results showed that maintain the stability of the ROS metabolism was the key to alleviate hyperhydricity.2.Plantlet pretreated with the low concentration of exogenous H2O2(50?M)showed a high O2.-generation rate and suitable endogenous H2O2 content by improving the expression level and enzyme activity of GPX,APX,and GR and by declining the expression level and enzyme activity of CAT.The content of AsA and GSH and the ratio of AsA/DHA and GSH/GSSG increased.During the following hyperhydricity-aggravated treatments,the plantlets pretreated with low concentration H2O2 maintained the balance of endogenous ROS metabolism and showed less damage to the cell membrane and lower hyperhydric rate.On the contrary,the high concentration H2O2(250?M)pretreatment induced oxidative stress in plantlets in vitro.During the following stress treatments,the ROS accumulation,the damage of cell membrane and the hyperhydric rate increased.The result proved that the exogenous ROS played a dual role in hyperhydricity based on the concentration.Low concentration of exogenous H2O2 played as a signal molecular in regulating the relative stability of ROS metabolism and then alleviated the hyperhydricity;high concentration of H2O2 played as the damage substance and induced the oxidative stress and keep the hyperhydricity at a high level,3.Gene expression stability was identified base the qPCR results.TUA,GAPDH,UBQ,CYP,and ACT were the most suitable of the eight reference genes for different garlic genotypes,organs,developmental stages,abiotic stresses and media treatments,respectively.4.Garlic variety 'ershuizao' under control and oxidative stress treatments were selected as the material to construct libraries of transcriptomic sequencing.The sequencing result showed that 735 related genes were involved in the occurrence of hyperhydricity.Besides the ROS production and scavenging gene of NADPH oxidase,CAT and POD,the plant hormone metabolism and aquaporin were involved in the occurrence of hyperhydricity.Three key candidate genes(AsPIP1;1,AsP1P1;2 and AsPIP2;1)were cloned to analyze the gene sequence and expression profile The results found that the variety 'ershuizao' and inflorescence which were easier to be hyperhydric had a lower expression level of AsPIPs.AsPIPs participated in the occurrence and recovery of hyperhydricity.The hyperhydricity-aggravated treatments(low and high concentration H2O2,low media pH value and high concentration of BA,and low concentration of agar)induced the specific expression of AsPIPs during hyperhydricity.