Studies On The Mechanism And Recovery Measures Of Hyperhydricity Of Pyrus Calleryana Decne In Vitro

On the basis of establishing the low hyperhydricity and efficient and rapid propagation of Pyrus calleryana Decne, analyzes the mechanism of hyperhydricity at the physiological and biochemical and molecular level, and studies the recovery technology for hyperhydric shoots. The main results are as follows:1. Establishing the low hyperhydricity and efficient and rapid propagation of Pyrus calleryana Decne. Taking the stem of Pyrus calleryana Decne as explants, the author has studied the influence of factors such as 6-BA, NAA, sucrose, agar, activated carbon in tissue culture of Pyrus calleryana Decne, discussed the effect of the four basic mediums (MS, AS, NN69, WPM),6-BA and NAA in different concentrations and two different cultrue types on the proliferation of Pyrus calleryana Decne, and conducted research on the effect of NAA and 6-BA on the rooting of Pyrus calleryana Decne. The results have shown that during in tissue culture of Pyrus calleryana Decne, higher concentration of 6-BA plays a significant role in promoting the multiplication factor and hyperhydric rate of Pyrus calleryana Decne in vitro, activated carbon had an inhibitory effect on the multiplication factor, hyperhydric rate and the shoot height, higher concentration of NAA promotes the growth of stem. Moreover, while using MS as the basic medium, the suitable proliferation medium is MS+6-BA0.6mg/L+NAA0.2mg/L, the appropriate culture type is alternate inter-generational culture between the above-mentioned proliferation medium and the MS blank medium, and the appropriate rooting medium is 1/2 MS+1.5 mg/L IBA.2. Studying the recovery measures of hyperhydric shoots.Taking the hyperhydric shoots (light and heavy) produced in normal proliferation as the material,the author has adopted differents measures to recover hyperhydric shoots and made comparison among the hyperhydric shoots (light and heavy), recovered shoots and normal shoots in terms of anatomy anatomy, physiological and biochemical, and chlorophyll fuorescence.The results have shown the hyperhydric shoots (light and heavy) have different degrees of recovery if being provided with proper moisture control and exposure toultraviolet light. And if the material is put in a sealed strilized empty flask for three days for reducing its water content, and then was transferred into MS blank medium for thirty days, the maximum recovery rate reaches up to 100%.3.Comparing the differences in leaf ultrastructure, chlorophyll fluorescence and antioxidant enzyme activities between hyperhydric shoots and normal shoots. The results have shown that:(1) the hyperhydric shoots'stomata are closed, the organellae are damaged, the number of chloroplast was lower than in normal shoots, and the thylakoid stacked irregularly in hyperhydric shoots.(2) the Fv/Fm, Fv/FoqP and PhiPS2 in hyperhydric shoots were lower than those in normal shoots. More ROS would be produced by excess energy accumulating in PSⅡreaction cente.(3)compared with the normal shoots, the hyperhydric shoots have lower SOD and CAT activity, but higher POD activity.4. Analyzing the the differences in methylation patterns and levels of CCGG sequences between normal shoots and hyperhydric shoots by use of MSAP.The results have shown that the methylation ratio of the tolal genome DNA in hyperhydric shoots is 83.3%, lower than that in the normal shoots which is 87.6%. In addition, demethylation sequences of hyperhydric shoots are associated with decline of tissues, such as leaves, and at the DNA level, it is possible that the changes the of methylation status in the declining tissues has a direct impact on plant development.