Effects Of AgNO₃ On Shoot Regeneration In In Vitro Leaves Of Pear

Two varieties of pear mainly cultured in sichuan such as Pyrus brestchneideri Rehd. cv.Jinshui and Pyrus bretschneideri Rehd.cv.Jinhua were used as materials to set up the non-bacillus propagation system,then chose the leaves from it to culture.Use different kinds of basic medium and hormones to decide which one was the best combination for leaves shoot regeneration by uniform design.Add different concentrations of AgNO₃ into the chose combination and observe the process of calluses induction and tissue's morphology changes.Analyze the changes of antioxidant enzyme,free radical,protein and endogenous polyamines when leaves deal with AgNO₃.Discuss the mutual relation among the cell differentiation, organogenesis and the physiologically biochemical basis in the process of pear's tissue culture.The results were as follows:First,uniform design was carried to find the best culture condition for shoot regeneration of 'jinhua' and 'jinshui'.From regeneration rate,shoot per leaf and adventitious shoot regeneration efficiency,the optimal initiation medium for shoot regeneration of each cultivar was respectively as follows:For 'jinhua',shoots regenerated best on NN69 supplemented with 1.0 mg/L BA and 0.1mg/LIBA with regeneration rate of 40.49%.For'jinshui',shoots regenerated best on NN69 supplemented with 1.0 mg/L TDZ and 0.6mg/L NAA with regeneration rate of 58.07%.Second,according to the observation of the callus's modality,the process of regeneration including four stages:firstly,the edge of leaves became swollen and nervations expanded.Some white calluses emerged at the tip of leafstalk and the edge of leaf blocks.In the second stage,all the leaf blocks produced calluses and a few of calluses emerged single yellow green bud spot which can be seen with eyes.The third stage is the period which was the pinnacle of shoot regeneration.In the forth stage, the numbers of leaves which produce shoot became less and less.In the last days, only the buds grew form the shoot.Third,the most effective concentration of AgNO₃ was 0.1mg/L for 'jinshui'. However,for 'jinhua',AgNO₃ enhanced the regeneration efficiency of leaves when its concentration varied from 0.1 mg/L to 0.5mg/L.Fourth,according to the study on the relationship between different concentration of AgNO₃ and the change of antioxidant enzyme and free radical,the conclusions were as follows:During the period of dark treatment,CAT and POD activities in leaves were changed heavily,especially the changes of POD activity in 'jinhua' leaves and CAT activity in 'jinshui' leaves were higher than the other three treatments when they dealt with 0.1 mg/L AgNO₃.At the same time,O₂^- production rates and H₂O₂ content would increase but the ascensive degree was lower than the control.In the stage of light treatment,the pinnacle of SOD would enhance when adding apt concentration of AgNO₃,while H₂O₂ content and O₂^- production rates would decrease.On the contrary,higher concentration of AgNO₃ would cause H₂O₂ content and O₂^- production rates rising again.Though the changes of antioxidant enzymes in different varieties of pears were not the same,it could get the conclusion that 0.1 mg/L AgNO₃ was best to increase the activity of antioxidant enzymes in 'jinshui' and 'jinhua' leaves among the four treatments.Fifth,the study on the changes of protein content when leaves deal with different concentration of AgNO₃ showed that protein content rise with shoot regenerated. During the dark cultured period,there was no difference among all levels.However, when they went into the period of shoot germination,protein content of 'jinshui' and 'jinhua' which deal with 0.1mg/L AgNO₃ always showed markedly difference to the check and reached the highest on 35th day.Sixth,the research of endogenous polyamines content during the process of shoot regeneration showed that the content of endogenous polyamine would enrich with cells divided and differentiated.Put content and Spm content formed pinnacles in 5th day and 20th.day respectively.However,Spm content enhanced in both 10th and 20th day.It could draw the conclusion that Put,Spm and Spd regulated the leaves cells divided and differentiated alone or in whole.Leaves deal with AgNO₃ whose content of endogenous polyamine was much higher than the control,especially the treatment with 0.1 mg/LAgNO₃.