| Soil salinization is a very serious issue impact on crop yields around the world, theexcessive amount of salt in the soil may cause many adverse effects on plant growth, iontoxicity t mainly reflected in the penetration of the stress, it inhibits plant’s growth,photosynthetic rate, causes the acceleration of plant’s senescence, even the death of plant.Severely stress will make serious injury to the growth and development of plants , and havevery serious influence to complete their life-history of biological, lead to the decline of cropyield, quality of crops, agricultural production.Limonium sinense ,which is a kind of recretohalophyte ,is from plumbagenaceae. Salt ionscan be absorbed from the external environment into its body and excreted out of its body via astructure called salt glands. The tolerance to salt stress is much higher than the average plant,and can grow very well in soil salinization nvironment. The salt gland of Limonium can secretsalt from the habitat out of the body, which causes desalination of the saline soil. This processcan reduce the degree of soil salinization, having a important role in the improvement of soilstructure.The process of understanding to salt excretion of the Limonium is still stucked on a veryshallow level, the molecular structure of the salt excretion is not enough, the key factors stillneed to be explored. Along with the development of the powerful techniques of geneticengineering. Such as gene-knockout,a stable and efficient genetic transformation system ofLimonium sinense is desperately needed. Limonium ,which has a very wide range ofcultivation, is a common floral materials, with high ornamental value, can be found all overthe world. It is called“Do not forget me”in chinese. In horticulture, seeds of the Limoniumsinense are few and do not germination well; its growth cycle is too long; only has few colors.these can all be genetically engineered to improved varieties of Limonium. This also requiresefficient transformation systemThis article is optimization of Agrobacterium-mediated genetic transformation system ofLimonium sinense ,which was established before. GUS and GFP are used as a reporter genein the system to make the detection more convenient, intuitive, higher resolution, whilemaking further comparison and research to Agrobacterium-mediated Limonium leaf disctransformation system:1) The plasmid GUS and GFP is resistanced to hygromycin and bar, first of all identifiedthe sensitivion to the concentration of these selective factors in Limonium sinense leaves. thesensitive to hygromycin and Bar concentration is 20mg / L and 2 mg / L,.2) Inoculating with different strains, the conversion rate of Limonium Limonium is 45%,slightly higher than inoculating with EHA105 and, and much higher than LBA4404 andGV3101 strains.3) After the disinfection, the mature leaves of Limonium sinense are cuted into small pieces1cm2 by size, the concentration of resuspended bacterium liquid, OD600, is 1.0-1.5. It’s themost significant effect concentration, the conversion rate reachs to 24%.4) Co-cultivation days of process: the conversion rate of Limonium have a certain impact onthe efficiency of 3 -4 days is significantly higher than 1-2 days only; 3days’conversion rate isslightly better, longer time does not make the improvement of conversion efficiency, andAgrobacterium’s excessive growth means the reduce of the conversion efficiency. 5) pre- cultivation days: the difference between 2 to 3 days is slightly. However,the mortalitywhen explants without pre-cultured is too high; the efficiency of two days is increased slightlythan one days; with time prolonging ,efficient does not improve significantly, and the callusbegins to emergence, the chimerism rate is greatly increased. This shows that the pre-culturedfor 2 days is enough.The identification of transformed plants with the PCR amplification, transgenic plants, andthe fluorescence under the microscope, GUS staining effect. Plasmid carrying the exogenousgene was not found been expressed in Limonium.In this experiment, the transcriptome of Limonium sinense is been secquenced, and comments grasp of the genetic information of Limonium.these sequences were assembled intounigene and been annotated through many database. we found many tolerance mechanism-related genes in these annotation. many genes that may be related with mechanism oflimonium stress-tolerance were found：ion transport、osmotic protection-ralated gene .suchas, NHX family、P5CS gene. The preliminary analysis of these genes was done. The structureof unigene and SSR were preliminary analysised. These work provide a solid foundation forthe subsequent cloning of salt tolerance gene. |
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