The Micropropagation Of Prunus Pseudocerasu ’Manaohong’ And Its Assessments Of Genetic Variation

To satisfy the increasing demand of intensive production, an efficient micropropagation system, genetic stability evaluation to the in vitro plantlets and shoot regeneration of ?Manaohong’ cultivars were carried out in this present study. The main results are as follow: 1. The establishtion and optimization of efficient propagationUsing dormant bud collected from the stock plants of orchard as explants, the dormant bud were soaked with 0.1% mercuric chloride and two drops of Tween-80 for sterilization, followed by rinse with sterile water for several tiumes. Subsequently, the dormant buds wree sterilized inoculated in Murashige and Skoog macro-elements(MS) medium containing the combination of 1.0 mg/L GA3, 1.0 mg/L 6-BA and 0.5 mg/L IBA(pH=6.2) for aseptic establishment to induced dormant buds germination. The pollution rate was 48%, the germination rate was 22%. Fifteen to twenty days later, dormant buds sprouted. Then turned them to the new medium MS+ 1.0 mg/L KT + 1.0 mg/L GA+ 0.5 mg/L 6-BA+ 0.5 mg/L IBA cultured 30 days. According to the orthogonal experiment screened the best multiplication culture medium is MS+ 1.0 mg/L KT + 1.5 mg/L GA+ 0.1 mg/L 6-BA+ 0.5 mg/L IBA, multiplication coefficient reached 11.5. Inoculated the multiplication seedlings into three different kinds of medium, MS+0.2 mg/L IBA+ 30 g/L Sucrose+ 7 g/L Argar,MS+0.3 mg/L IBA+ 30 g/L Sucrose + 7 g/L Argar, MS+0.2 mg/L IAA+ 30 g/L Sucrose + 7 g/L Argar,pH(6.0-6.2), rooting rates are 75%, growing well, taproots are stout and fibrous roots are rich. Experiments show that the GA3 has no significant impact on shoots rooting. 2. Genetic variation detection for in virtro material and ISSR molecular markerBy using 21 ISSR primers to investigate the seedlings of 1th to 8th leaves genome DNA through ISSR molecular maker. Further, ISSR markers amplified with 21 primers were also adopted to assess the somaclonal variation of 24 in vitro shoots randomly selected from the 8thcycle of transfer. No aberrant DNA marker was investigated, indaicating that, for the makers tested, no genetic variation was detected among in vitro shoots that had been multiplied as many as 8 cycle via organogenesis. 3. Epigenetic variation detection for in virtro material and MSAP molecular markerBy using 17 pairs selevtive-amplilfication primers to investigate the seedlings of 1th to 8th leaves genome DNA through MSAP molecular maker. In 8th cycles shoots has 5990 bands were created, has full-methylation bands 717, has hemi-methylation bands 395, obtained all cycle shoots full-methylation ration, hemi-methylation ration and total- methylation ration. From the result can infer that DNA methylation ration of ?Manaohong? increased with the times of cultivation. In the 8th cycle, hemi-methylation ration is 8.0%, full-methylation ration is 12.5%, total- methylation ration is 20.5%.