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In Vitro Rapid Propagation System of Peach Yu Dwarfing Rootstock No.1 and Optimization of Its SSR-PCR System

Author: XiongMingGuo
Tutor: LiJing
School: Henan Agricultural University
Course: Pomology
Keywords: peach dwarfing rootstock in vitro propagation genomic DNA SSR-PCR
CLC: S662.1
Type: Master's thesis
Year: 2011
Downloads: 3
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Abstract


The research was carried out from 2008 to 2011 in Henan Agricultural University. The stem segments with bud and leaf of the peach Yu dwarfing rootstock No. 1 were used as experimental materials. This study was researched on the selction of medium of inducing bud, disinfection method of leaf, inducing callus and sub-culture, the DNA extraction and optimization of SSR-PCR system on peach Yu dwarfing rootstock No. 1. The main results were as follows:1. The selection of medium on inducing buds from stem segments. The stem segments with buds were innoculated in the primary culture medium. The results showed that MS medium with 1.4 mg/L BA plus 0.3 mg/L IBA was the most effective medium for promoting bud.2. Establishment of the disinfection system with leaf. The disinfection effect of treatment 75% ethanol and disinfection piece was better than 75% ethanol with NaClO and HgCl2. The most effective method was used 75% ethanol 30s then 5.0 g/L disinfection piece 8 min.3. The induction of callus from leaf. The effect on inducing callus from leaf were analyzed by comparing the concentration of 2,4-D and light condition. The results indicated that the inductivity of callus become higher with the concentration of 2,4-D rangeed from 0 mg/L to 2.0 mg/L; the highest induction efficiency (83.3%) was obtained when the concentration of 2,4-D was 2.0 mg/L and it become lower when the concentration was over 2.0 mg/L.The highest induction efficiency was obtained when the leaves were cultured on the MS with 2.0 mg/L 2,4-D in darkness.4. The sub-culture of callus. Compare to the different medium and the period of sub-culture, the TN2 and TN3 types of callus could be maintained for a long time and kept exuberant growth when they were alternately cultured on the MS medium containing 1.0 mg/L 2,4-D and the wpm medium containing 1.0 mg/L 2,4-D.5. The DNA extraction of young leaves. The DNA extraction effect of conventional CTAB, improved CTAB, conventional SDS and SDS-CTAB binding assay was compared by UV scanning,agarose gel electrophoresis analysis and EcoRI analysis by using the young leaves of Yu dwarfing rootstock No. 1 as the experimental materials. The results showed the DNA extraction of conventional SDS and SDS-CTAB binding assay which salt, protein, carbohydrates, phenols and other impurities had relatively cleaned and high concentrations. Meanwhile, the clarity and brightness of DNA bands were significantly higher than the other two methods, and the DNA was digested completely by restriction enzyme. In summary, conventional SDS and SDS-CTAB binding assay were suitable for the DNA extraction of Yu dwarfing rootstock No. 1.6. The optimization of SSR-PCR system. The orthogonal experiment of six factors which would be affected the results of SSR was designed to seek the most optimal reaction system of SSR-PCR. The result showed that the most optimal system as follows: Mg2+ (25 mmol/L) 2.0μl,2.0 mmol/L dNTPs 1.2μl,10×Buffer 3.0μl,5U Taq polymerase 0.2μl,50 ng/μl DNA 0.6μl,primer (10μmol/L) 1.5μl.

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CLC: > Agricultural Sciences > Gardening > Fruit trees gardening > Drupe > Peach
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