Sky Full Of Stars Vitro Vitrification And Normal Seedlings Of A Comparative Study

Hypeahydric shoot in tissue culture is refers that the outward appearance oftissue shoot is semitransparent and abnormal phenomena. Once forms theHypeahydric shoot, the rapidity of propagateon namely drops obviously, cannotgenerate roots and multiplication. After transplants survival rate extremely low.Therefore, the vitrification is a question which urgently awaits to be solved. Normaland vitrification shoots was used in this experiment. Through physiological andbiochemical characteristic, EST isozyme activity, and RAPD amplification. The mainfindings are as follows:Water content of vitrification shoot (95.8%) was higher than normal shoot(91.7%). For chlorophyll average content, solubility and reductive sugar content,vitrification shoot was 14.0%, 63.8%and 86.9% of normal shoot, respectively, butPOD activity of vitrification shoot increased, was 2.6 folds of normal shoot. Theresult of EST isozyme analysis using PAGE method, electrophoresis of EST isozymeappeared abnormity of lacking band, moreover, band color was slight. RAPD analysiswas performed in this experiment also, a specific band was found at 1500 bp in primerof OPG-17, this indicated that variation of the vitrification shoots was existed inmolecular level. The result showed that the high-quality genomie DNA was obtainedby the CTAB method. The optimal PCR system for RAPD analysis was as follows:14.17μl ddH₂O, 150μmol/L dNTP, 1.5 mmol/L Mg²⁺, 2.5μl Buffer, 0.3μmol/Lrandom primer, 10 ng template, 1 U Taq polymerase in 25μl reaction system. Theprogram of amplifying reaction was as follows: After pre-denaturing at 94℃for 5min, under the condition of denaturing at 94℃for 1 min, annealing at 36℃for 1 min,extension at 72℃for 1.5 min, amplifying for 45cycles, and extension at 72℃for 7min at last. applying RAPD (Random Amplified Polymorphic DNA) fingerprintingmethod, PCR of polymorphic DNA analysis was performed on the normal andvitrificat(?)m shoots in vitro of Gypsophila paniculata. One hundred random primersbased on the method of bulked segregation analysis (BSA) were used, and sevenpolymorphic p(?)mers were screened among them, The seven random primers wereused on seven no(?)mal shoots and seven vitrification shoots for PCR amplification, RAPD data indicated that OPG17 was capable of repeatedly amplifying a specificband, The RAPD marker (primer OPG17) and SCAR marker from OPG17 primer bycloning, sequenceing and designing primer were established.