Construction Of Genetic Transformation System And Optimization Of ISSR-PCR System Of ’Tunisia Soft-seed’ Pomegranate

ABSTRACT:For the genetic transformation study, leaves of’Tunisia soft-seed’ pomegranate were used as experimental materials, the sensitivity tests of Kan, Glu and Cef on callus of pomegranate were carried out, and the effects of different agrobacterium concentrations and different immerged time on leaves and callus were discussed, and the optimal explant for genetic transformation was selected; the optimal methods of leaves DNA extraction and optimization of ISSR-PCR system were studied. The main results were as follows:1. The sensitivity tests of Kan, Glu and Cef. on callus of’Tunisia soft-seed’ pomegranate. Callus was inoculated the MS medium of containing different concentrations Kan, Glu and Cef respectively, and observed, after cultivated40d. The results showed that callus was significantly inhibited when the concentration of Kan was50mg·L-1or the concentration of Glu was20mg·L-1, the genetically engineered materials could be sifted; the strains growing could be effectively restrained when the concentration of Cef was500mg·L-1.2. Effects of different bacterium concentrations and immerged time on leaves and callus. The precultured leaves and callus were immerged under different conditions of bacterium concentrations and immerged time, after cocultured3d, then cultured14d in the selection culture medium. The results showed that leaves and callus of’Tunisia soft-seed’ pomegranate could grow well when the bacterium concentration and immerged time were OD600=0.5and10min respectively.3. Selection of optimal immerged materials for genetic transformation. Earlier observations suggested, there was no significant difference between leaves and callus. Leaves and callus were immerged under certain conditions of the bacterium concentration OD600=0.5and immerged10time, then were transferred into selection medium, and observed, after cultured for30d. The results showed that leaves browned seriously, but the callus did not appear pollution phenomenon, and the browning rates were lowed than leaves, grew well, was the optimal transformation material.4. The extraction of genomic DNA. Using young leaves of ’Tunisia soft-seed’ pomegranate as materials, conventional CTAB, improved CTAB, conventional SDS and SDS-CTAB binding assay were used to extract the DNA of leaves respectively, the extraction effects of the four DNA extraction methods were compared by NanoDrop and agarose gel electrophoresis analysis. The results showed that improved CTAB made high DNA concentrations, and the clarity and brightness of DNA bands were significantly higher than other methods. So it was suitable for leaves DNA extraction of ’Tunisia soft-seed’ pomegranate.5. The optimization of ISSR-PCR system. The orthogonal experiment of five factors which would be affected the results of ISSR was designed to seek the most optimal reaction system of ISSR-PCR. The results showed that the most optimal system was:25mmol-L-1Mg2+2.0μl,2.5mmol·L-1dNTPs1.2μl,1U·μl-1Tag DNA polymerases1.0μl,30ng·μ-1DNA template2.0μl,10μmol·L-1primer1.2μl.