Over-expression Of MsCu/Zn-SOD Gene In Petunia Hybrida And The Cold Tolerance Analysis Of Transgenic Plants

As a common perennial herbaceous plant in northeast China, Petunia has some features like short life cycle, easy to survive and rich in colors and is extensively applied in the urban gardens. Moreover, Petunia is used as explants due to its good regeneration capacity and easy to be transformed by Agrobacterium. However, Petunia has poor cold tolerance and can’t grow in the cold seasons. If the cold tolerance of Petunia could be improved by engineering technology, the more colorful Petunia will be available for the plant landscape in Northeastern China during cold seasons. In this study, the suitable explant of Petunia was firstly selected among the four cultivars depending on the frequency of regeneration and then the Cu/Zn-SOD gene, which isolated from alfalfa cultivar "long Mu 803”, was transferred into the genome of this explant by Agrobacterium-mediated transformation. The cold tolerance of transgenic Petunia was improved due to the over-expression of the target gene. The results are showed as following:1. Four Petunia hybrids cultivars with different colors, Pink, Red, Pink vein and Peppermint, were used to investigate the frequency of adventitious bud induction and subsequently provide explants for transformation. The MS medium was served as basic medium supplemented with different concentrations of 6-BA, NAA and IBA, respectively, with the addition of bialaphos sodium. According to DPS multiple comparisons among cultivars and hormonal factors, Pink cultivar was finally determined to be the most suitable explant, and the components of induction medium of the Pink cultivar were listed: MS+1.0 mg·L-16-BA+0.5 mg·L-1NAA + 3 mg·L-1 bar.2. The total RNAs were isolated from alfalfa cultivar “long Mu 803”, which has better cold tolerance, and transcripted into cDNA. The Cu/Zn-SOD gene was cloned using a pair of specific-designed primers with cDNA as template. The expression vector p3301-SOD was constructed for nuclear transformation and then transferred into the genome of Pink.3. Cu/Zn-SOD gene was transformed into three putative transgenic plants by PCR and integrated into the genomes of two trangenic plants with single copy according to the Southern blot analysis. No signal band for another one plant and its bar test strip test suggested Agrobacterium stainedit.4. Both transgenic and wild-type plants were put into the incubator under 4 °C for six days. The growth potential of transgenic plants was better than the wild-type plants. Secondly, the relative conductivity of transgenic plants showed less rising trend compared with wild-type plants. Thirdly, the decline of chlorophyll content in the leaves of transgenic plants was less than that of the wild-type ones. Taken all above together, over-expression of Cu/Zn-SOD gene in Petunia promoted its cold tolerance.