Comparative Study On Normal And Vitrificated Shoot Of ’huang-guan’Pear In Vitro

Tissue culture and rapid propagation were base of shoot-tip detoxification,seed multiplication and genetic transformation. A large number of studies have reported on tissue culture of pear.But different genotype stuff have big differences in the degree of difficulty.In recent years,the acreage of’Huang-guan’ pear is growing,because it has the advantage of strong resistance, high yield and excellent quality. On the other hand, it is not conducive to storage and easy to aging. After several subculture, serious vitrification is discoved in the process of improvement with biological technology. It caused much inconvenience to the research.. We want to reveal the occurrence and mechanism by studying the possibility of vitrification with Rehd.cv.Huang-guan. Experimental content and research results are as follows:Firstly, influence on tissue culture proliferation of ’Huangguan’ pear with the dosage of three kinds of basic medium (MS, AS,1/2MS),6-BA, NAA, GA3is studied by ’Huangguan’ pear stems. The influence on tissue culture proliferation frequence is followed by6-BA> NAA> GA3> basic medium. The results showed:The fittest medium for the proliferation is MS+6-BA1.0mg/L+NAA0.2mg/L+GA30.1mg/L, its proliferation coefficient can reach to5.1or above.Secondly, influence on’Huangguan’ pear regeneration of different concentrations TDZ respectively with IAA, NAA combination,6-BA and IAA combination, and different ways to place’Huangguan’ pear leaf. The results showed:TDZ and IAA combination is fittest in the effect of inducing adventitious bud regeneration frequence. TDZ and NAA combination is better than6-BA and IAA combination. There was no significant influence of the different on ways to place’Huangguan’ pear leaf. On’Huangguan’ regeneration,By dark incubation for21days in the NN69+TDZ5.0mg/L+IAA0.3mg/L, the highest regeneration frequence and average number of axillary buds explants were found to be77%and3.7.Thirdly, comparison of rapid organizational propagation, leaf regeneration, physiology and biochemistry and organizational structure was studied on normal and vitrificated shoot. The results showed:compared with the normal shoot, proliferation and regeneration ability of vitrificated shoot was significantly lower,The proliferated and regenerated plant of vitrificated shoot was still vitrificated and it’s vitrification degree increased;compared with the normal shoot,the dry matter accumulation of vitrificated shoot reduced by31.3%and the soluble sugar content of vitrificated shoot increased by13.92%,while the starch content reduced by29.57%。3. Compared with the normal shoot, Chlorophyll a, chlorophyll b and total chlorophyll of vitrificated shoot reduced by40.16%,43.24%and39.41%respectively;Compared with the normal shoot, the content of ZR,IAA and GA of vitrificated shoot increased and the hormone ratio of IAA/GA, ZR/GA, IAA/ZR were significantly lower; Compared with the normal shoot,the chloroplast number of vitrificated shoot reduced, the chloroplast volume decreased. The thylakoid stacked irregularly.and the nucleus was deformed or missing. After vitrification, the photosynthetic structure was destroyed, the capacity of the photosynthetic product accumulation was weakened and the endogenous hormone was disordered.and thus the degree of the vitrification was further aggravated. The reduction of nuclear material resulted in lower capacity of its proliferatin and division.Fourthly, the change of chlorophyll fluorescence parameters was studied on ’Huang-guan’pear tissue culture vitrificated shoot. Compare with the normal shoot,the value of Fv/Fo was significantly higher and no significant difference was found in Fv/Fm. The value of qN increased significantly in both the high-light and low-light intensity. The value of qP, PhiPS2and Fv’/Fm’ was significantly lower and photosynthetic capacity decreased in the high-light intensity. Ps Ⅱ reaction center had not been damaged.Fifthly, the cDNA sequence of pseudoresponse regulator gene was cloned from ’Huang-guan’ pear by RT-PCR. The fragment was902bp, containing four exons and3introns, the length of the reconnected RNA was610bp,with an open reading frame of182bp. The predicted amino acids sequence included a REC domain which showed hig identity to the bean, poplar, and arabidopsis.The results indicted that the DNA fragment was pseudoresponse regulator gene.