Study On Stress-resistance Transgenic Plant Without Selective Marker

Abiotic stresses such as drought,salting,and low tempetature cause adverse effects on the growth of plants and the yield of crops.To improve stress-tolerance of crops, the employment of gene engineering technique has been an important trend in plant stress- tolerance breeding in 21st century.The use of selectable marker genes encoding antibiotic or herbicide resistance for transgenic crops has potential negative effects.It maily causes safety of environment and human health,which brings increasing public concern and their acceptanc to transgenic products, precludes the use of the same marker gene for the selection of multi-transformed plants, delays the market process of transgenic crops. Presently many plant transformation systems have been developed in order to remove selectable marker genes such as co-transformation system,site-specific recombination system,transposable element system and MAT vector system etc. Double T-DNA vector system of co-transformation system was a simple and efficient strategy to obliterate marker genes.The two double T-DNA plant expression vectors, pCDMARpWDT-Hyg and pCDMAR-USOD-Hyg including target gene DREB and SOD respectively, were constructed in our research.They contained plant selectable marker hygromycin phosphotransferase gene (hpt) and target gene in two independent T-DNA region.The flanking of DREB and SOD gene is MAR sequence from pea which could increase gene expression level.A stress-induced promoter was used to drive the expression of DREB gene. These two vectors were introduced into maize(Zea mays L.) and rice by Agrobacterium-mediated transformation, then transgenic maize and rice plants were obtained through about two months of selection on N6 medium containing HygB, a month of differentiation and half a month of rootage. The transgenic maize result of PCR assay showed that frequency of co-transformation with hpt gene and DREB gene was 23.8% in transgenic DREB maize plants. By sexual propagation the two genes will be separated in transgenic plant descendants, and we will obtain marker-free transgenic plants.Low transformation efficiency is still a critical factor which affects plant gene engineering development. In order to optimize the transformation system and improve transformation efficiency, we analyzed certain influencing factors of maize by Agrobacterium- mediated transformation by gus gene transient expression. We studied theinfluencing factors of gene transformation efficiency by setting high-osmotic treatments to calli, different pH infectious suspension and dicotyledon sensitive tissue in calli. According to statistics analysis, low pH infectious suspension had the most evident effect on transient expression efficiency of gus, and the infectious suspension of pH5.2 represented the highest transient expression efficiency of gus; the treatment of dicotyledon sensitive tissue in calli had higher transient expression efficiency of gus, Besides, high-osmotic treatment with 10g/L mannitol to calli in co-culture medium had also an effect on improving gene transformation efficiency.