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Study on Transformation of Cut Chrysanthmemum with DdICE1 Gene

Author: ChenLin
Tutor: ChenFaZuo
School: Nanjing Agricultural College
Course: Ornamental Plants and Horticulture
Keywords: cut chrysanthemum DdICE1 gene genetic transformation molecular analysis stress tolerance assays
CLC: S682.11
Type: Master's thesis
Year: 2011
Downloads: 4
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Abstract


Chrysanthemum as one of ten traditional famous flowers in China and main four cut flowers in the world plays an important position on gardens and greening. In the study, using leaf disks of cut-flower chrysanthemum’Jinba’, the sense and anti-sense directions of DdICE1 gene from Dendranthema dichrumwas were introduced into’Jinba’using the Agrobacterium-mediated transformation method.Transgenic chrysanthemum was identified with PCR and RT-PCR. In addition, studies on the tolerance of transgenic plants under cold, drought and salt conditions and the stress related-gene expression level in transgenic chrysanthemum were also conducted, the detailed results were listed as follows:1. The transformation of DdICE1 gene to cut chrysanthemum and the descendants of transgenic chrysanthemum.DdICEl gene was inserted in the pCAMBIA1301 vector in the sense or anti-sense directions driven by the CaMV35S promoter. The construction vectors were introduced successfully into’Jinba’using the Agrobacterium-mediated transformation. A gradual-reducing bacterium controlling method was carried with choosing 12 mg·L-1 as the optimal concentration of Hyg in the early stage, then decreasing to 10 mg·L-1 for leaf disc regeneration and 10 mg-L-1 for root selection, and using 500 mg·L-1 of Carb concentration in the early stage, then declined to 250 mg·L-1 (He et al,2010). PCR and RT-PCR were conducted to confirm the Hyg screening plants. The results showed that the DdICEl gene have been integrated into the genome of transformed chrysanthemum and expressed successfully. Totally, ten overexpresson (Ti) and three anti-sense (TAi) transgenic lines were obtained.2. Analysis of stress tolerance of the To transgenic plants.Studies on the tolerance of transgenic plants under cold, drought and salt conditions, and the gene expression level and mechanisms about stress in transgenic chrysanthemum were conducted. Analysis of cold tolerance indicated that the LT50 of the TAi2 was lower and the LT50 of over-expression lines (Ti2、Ti3、Ti4) were higher than that of the WT. After the recovery at the normal temperature 23±2℃for two weeks, the survival rates of the WT was 81.7%at-6℃and 46.7% at-9℃; the lines TAi2 was 55% at-6℃and 23.3%at-9℃, while that of the Ti2 was 93.3%at-6℃and 70%at-9℃. These results showed the Ti2 plants were highly tolerant, while the TAi2 plants were sensitive to the freezing stress than the wild type plants. When the plants exposed to cold, the transcript levels of ICE1 accumulated quickly and reached the highest at 9 h (hours) in the Ti2 while at 6h in the WT plants, but always kept lower in the TAi2 line. Ti2 plants had the highest expression level of CgDREBa comparing with WT and TAi2 plants corresponding to the ICE1 expression level. Under low temperature, activity of SOD、POD and proline content were all remarkable increased in Ti2 plant, while MDA content remarkable lower than WT and TRNAi2 plants. These results showed the Ti2 plants were highly anti-oxidant and anti-osmosis than WT and TAi2 plants.For the drought stress treatment, the survival rates of Ti2 (88.3%) was higher than WT (43.3%) and TAi2 (11.7%) plants, indicating that the over-expression DdICE1 in Ti2 plant improved the tolerance to drought stress. When withholding water of the plants for 9 d, the expression level of DdICE1 in the WT and transgenic plants all displayed a tendency of accumulating quickly and reaching the highest at 3 d then decreasing, and the ICE1 expression level in the Ti2 was obviously higher than the WT and TAi2 plants all the time. The expression level of CgDREBa indicated a similar result comparing with the ICE1 expression level. Under drought stress, activity of SOD、POD and proline content were all remarkable increased in Ti2 plant, while MDA content remarkable lower than WT and TAi2 plants. These results showed the Ti2 plants were highly anti-oxidant and anti-osmosis than WT and TAi2 plants, and drought tolerance of Ti2 plants were enhanced.For the salt stress treatment, the survival rates of the WT was 61.7%, the line TAi2 was 41.7%, while that of the Ti2 was 96.7%, indicating that the over-expression DdICE1in Ti2 plant improved tolerance under salt stress. Under salt stress the gene expression level in plants showed a similar result to the cold and drought stress, but the highest level was at 10 d. The activity of SOD、POD and proline content were all increased in Ti2 plants, while MDA content remarkable lower than in WT and TAi2 plants.Our data suggest that the multiple stress tolerance of Ti2 plants was all increased comparing to WT and TAi2 plants under cold, drought and salt conditions.

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CLC: > Agricultural Sciences > Gardening > Ornamental Horticulture ( flowers and ornamental trees) > Perennial Flowers > Perennial Flowers Class > Chrysanthemum
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