Genetic Transformation System Of Sinningia Speciosa And Regeneration Of Transgenic Plants Containing LEA Gene

sinningia speciosa is a kind of ornamental flower in greenhouse, and originated in Brazil. It likes wet and warm cultivation environment,but does not tolerance drought. Late embryo genesis abundant proteins(LEA) is a series of proteins that are accumulated during late embryogenesis in higher plant cell. Under the drought stress,salt stress and cold stress,LEA gene is induced in nutritional organ in all kinds of plants. LEA proteins are hyper hydrophilicness,which are involved in protecting membrane system and Biomacromolecules from damage in drought: In this study ,in order to establish drought resistance, some biophysical chemical indexes of sinningia speciosa under different drought stress conditions were determined. a transformation system of sinningia speciosa was firstly established , and we want to obtain transenic sinningia speciosa with LEA gene which can resist drought by using genetic engineering technology .The results are as following:.1. In order to establish drought resistance,some biophysical chemical indexes of sinningia speciosa under different drought stress conditions were determinedThe experiment was carried out under5%,15%,25%,35% PEG analogue drought condition . researched on drought resistance from physiology,determination was made on the content of chlorophyll, permeability of protoplasm etc. The results are as following: the concent of chlorophyll decreased with the increasing PEG concentration;the concent of MDA increased with the increasing PEG concentration; the membrane penetrability increased with the increasing PEG concentration; POD activity increased first and decreased afterwards with the increasing PEG concentration. 2. Establishment of the high efficient regeneration systems of leavesThe small and tender leaves of sinningia speciosa were cultured on MS2(MS+1.0mg/LBA+0.1mg/LNAA) to induce bud initation,the regeneration frequency amounted 83.3%.3. Leaf dish transformation system of sinningia speciosaThe optimized transformation condition was that: The leaves were cut into l*1cm^2,, and precultured on MS2(MS+1.0mg/L BA+0.1mg/L NAA) for 2d .The Agrobacterium tumefaceins which OD600 comes up to o.5 or so were collected by centrifugation and resuspended in same volume of liquid AAM . The leaves segments were infection by A .tumefaceins suspension as described aboved for 10 min . Dry them to MS2 medium and co-cultured for 4d in darkness .After co-cultivation , all the explants were washed in liquid MS medium containing 500mg/L Cef for 1 hour or so ,Then washed with sterile water for 4 or 5 times and dries on the filter paper . Subsequently , the explants were transferred to MS2 with 200mg/L Cef to tall the overgrown Agrobacterium for delay culture .After 6 days , The explants were transferred to MS4(MS+1.0 mg/L BA+0.1 mg/L NAA+50 mg/LKm + 200 mg/L Cef ). Regenerated shoots were inoculated into MS5(l/2 MS +10 mg/L Km +200 mg/L Cef )for rooting selection .4. Detection of transgenic plantsUsing the optimized transformation system , LEA gene were introduced into sinningia speciosa , 16 Km-resistant plantlets were obtained . The PCR assay showed that the target gene had been integrated into three of them . resistance to drought is observing in transgenic plants when cultivated in the greenhouse .