Research On Transformation Of Maize With Marker-free Expression Vector Of BADH And CMO Genes

Maize (Zea mays L.) is a very important crop used as food and feedstuff. And it also can be used asraw material in modern medicine and industry fields. Maize is a crop which middle sensitive to salinity.The salinization of the field is a chiefly environmental reason for the decline of productivity. So it is verymeaningful to breed high-resisted maize. It is extremely slow to select and breed species which bear salt bytraditional methods. With the development of molecular biology, the employment of gene engineeringtechnique has been an important trend in plant stress-tolerance breeding in 21 st century. In present, all thetransgenic plants are almost use the selectable marker genes such as antibiotic and herbicide resistance toselect transplants. Recent years, more and more people worry about the safety of transgenic plant. Althoughthere is no report shows that selectable marker genes can affect the human health and the environmentsecurity. Marker-free is a rapidly developed method which can eliminate the safety concern of transgenicplant. In this experiment, we have been done on generic transformation of maize with marker-free genesBADH and CMO, which make a good foundation for breeding species that good ability and safety toendure saline-alkaline stress.In this experiment, we used the immature callis of general maize inbred lines as receptor materials.Studies have been done on genetic transformation of BADH and CMO., The sensitive characteristic onsaline-alkaline of maize calli and maize seedling was studied. We select calli and seedling which gene havetransferred by the limit concentration of saline-alkaline liquor. Testing the physiological index of maizecalli and seedling and evaluate the ability of resisting saline-alkaline of maize which transferred CMO andBADH gene. The results of study were acquired as follows:1. Further optimized the corn bruise condition of Agrobacterium Turnefaciens-mediated transformationThis experiment mainly used general maize inbred lines H99 and P2 as materials and did sensitivityexperiments by using salt to confirm the suitable selection pressure. We transferred the salt tolerance geneinto maize inbred lines. The results indicated that we could get better result by using the value of OD0.5-0.6, the time of incursion 25min. The transformation rate was high.2. Transform the gene BADH and CMO into corn via Agrobacteriun Turnefaciens-mediatedTransformation,then obtain transferred gene adult plantwe examined the transformed calli after they were differentiated. That indicated that the genes hadbeen transferred into the genome of maize.3.Transform the gene BADH and CMO into corn via Pollen Tube Pathway Method, then obtain transferred gene adult plantWe also applied Pollen Tube Pathway Method to transform gene into maize inbred lines. In thisexperiment, we transformed salt tolerance gene into maize inbred lines Jinong201 and 030 by using PollenTube Pathway Method. and discussed the effects of different entry time, different DNA concentration andentry content on transformation rate in order to get the best transformation conditions. The results indicatedthat the best condition was that we selected them with salt liquor 1.0%, dissolved plasmid with SSC and thebest time was 6:00-8:00 am and 4:00-7:00 pm. The best interval was 8-20 hours. And plasmidconcentration was 500ug/L and entry content was 200ul. Then we analysed the transformed plant by usingPCR and Southern Blot. The result indicated that the salt tolerance gene had been transferred into thegenome of maize and expressed in the descendant4.Make sure the critical concent of the corn bruise organization is 32mmol/L,the critical concent ofseedling is 1.05.Has carried on the appraisal to the seedling salt endurance associated physical targetThe indexes consist of membrane permeability, content of MDA, activity of SOD and activity ofPOD. To contrast the indexes of transformed to untransformed maize in the concentration of 1.0%, wediscovered that the membrane permeability and the content of MDA of transformed maize are apparentlylower and in the opposite that the activity of SOD and POD of transformed maize are higher than theuntransformed maize.