| Garlic(Allium sativum L.)is an important medicinal and edible vegetable,widely cultivated and edible around the world,China is the major garlic production,consumption and exporting countries.But the garlic mainly to bulb asexual reproduction,not only low reproductive coefficient,high production costs,and easy to accumulate the virus,resulting in seed degradation,production decreased,quality decline.The use of tissue culture technology can be carried out garlic virus and rapid propagation and purification of rejuvenation,but in this process there is a serious phenomenon of hyperhydricity,limiting tissue culture technology in the rapid development of detoxification.hyperhydrticity of plantlets in vitro abnormalities,metabolic disorders,differentiation ability is low,proliferation,budding and rooting difficult,after transplanting is difficult to survive,transplanting is difficult to survive,or into seedlings weak,low production potential,resulting in serious economic losses.The causes of the hyperhydrticity of plantlets in vitro were studied extensively at home and abroad,and the role of endogenous hormones in plants was also paid attention to.Abscisic acid as one of the five major hormones,not only in the plant to adapt to the external environment play an important role,but also regulate the plant growth and development of the various stages.Although abscisic acid as an exogenous regulated substances has been used in plant tissue culture,but the study of the occurrence of hyperhydrticity is rarely reported.The preliminary study found that high concentrations of H2O2 can significantly improve the hyperhydrticity rate of plantlets in vitro.Therefore,from the point of view of abscisic acid and hydrogen peroxide,the mechanism of the hyperhydrticity of plantlets in vitro and the control technology are discussed,which has important theoretical and practical value.In this study,garlic variety ’Ershuizao’ was used as the material.The changes of endogenous hormones such as zein(ZR),abscisic acid(IAA),abscisic acid(ABA)and gibberellin(GA3)were measured and analysis of its relationship with hyperhydrticity.Further analysis the effects of exogenous ABA and H2O2 on the hyperhydrticity and antioxidant system of garlic plantlets.The main researches were as follows:1.The changes of endogenous hormones in normal plantlets in vitro and the hyperhydrticity of plantlets in vitro were determined.The study found that for different degrees of hyperhydrticity(mild,moderate and severe)of plantlets in vitro,in the subculture process,the content of ABA of the hyperhydrticity of plantlets in vitro was significantly lower than that in the normal plantlets in vitro on the 16th day;the ZR in medium and severe hyperhydrticity plantlets was significantly higher than that in normal plantlets,and the content of ZR increased with the degree of hyperhydrticity;while the 1AA and GA3 contents were not the same as those of normal plantlets in vitro.During the subculture(0-16 d),the content of ABA of the hyperhydrticity of plantlets in vitro was significantly lower than that in the normal plantlets in vitro at the later stage of culture(8-16 d).The ZR content of the hyperhydrticity of plantlets in vitro was higher than that of the normal.The IAA/ZR of hyperhydrticity of plantlets in vitro was higher than that of normal plantlets in vitro,while ZR/ABA was lower than that of normal.It was presumed that the increase of ZR content and the decrease of ABA content are the important factors leading to the hyperhydrticity of plantlets in vitro.2.The effects of exogenous ABA on the hyperhydrticity of plantlets in vitro and the optimum treatment concentration were studied.Studies have shown that the addition of suitable concentration exogenous ABA in the culture medium could effectively inhibit the hyperhydrticity of plantlets in vitro.In different concentrations,ABA with 0.1-0.5 mg-L-1 had the best effect,the lowest hyperhydrticity rate.Among them,0.5 mg-L-1 ABA could significantly increase the activities of superoxide dismutase(SOD),catalase(CAT),peroxidase(POD)and glutathione reductase(GR).3.The effects of exogenous H2O2,ABA and H2O2+ABA on the hyperhydrticity of garlic plantlets in vitro were studied.The results showed that the ABA could significantly decrease the hyperhydrticity rate of the plantlets in vitro compared with the control.The H2O2 treatment significantly increased the hyperhydrticity rate of the garlic plantlets in vitro,and ABA could significantly reduce the hyperhydrticity rate of the plantlets caused by H2O2,ABA treatment could significantly reduce the rate of O2·-production rate,H2O2 content,malondialdehyde(MDA)content and plasma membrane permeability,which may be the reason for the decrease of hyperhydrticity rate.Compared with the control,exogenous ABA could significantly increase the activities of SOD,CAT,POD,APX and GR,and increased the contents of AsA and GSH.Compared with H2O2 alone,H2O2+ABA treatment was significant increased the activities of SOD,CAT,POD,APX and AsA and GSH at 8-16 days.In summary,exogenous ABA can improve the antioxidant capacity of plantlets in vitro,reduce the accumulation of reactive oxygen species,thereby inhibiting the occurrence of hyperhydrticity.4.The changes of antioxidant-related genes in vitro after H2O2,ABA and H2O2+ABA treatment were studied.Research shows that the relative expression of POD and GR reached the highest level at 0.5 d after treatment with ABA.The relative expression level of SOD and APX gene reached the highest level at 8 days after treatment,and the expression level of RBOH was 0.5 d reached the highest value,and then decreased,in the treatment 1-16 d were lower than the control;The relative expression of SOD reached the maximum at 0.5 d after treatment,and the relative expression of CAT and APX reached the maximum at 12 d after H2O2 treatment.The expression of RBOH was significantly higher than that of control at 8-12 d after treatment,and reached the highest value on the 8th day;While H2O2 and ABA were added in the medium,the relative expression level of CAT reached the highest value at 1 d after treatment.The relative expression level of SOD and GR gene reached the peak at 12 d after treatment,RBOH expression was inhibited,were lower than the control.In summary,the results showed that exogenous ABA could inhibit the expression of RBOH and induced the expression of SOD,CAT,POD,APX and GR,to enhance the antioxidant capacity of plantlets in vitro and reduce the hyperhydrticity rate of plantlets in vitro. |
