Studies On Transformation Of CMO-BADH Gene Into Maize

China is the second largest maize producer in the world. And it is an important food crop or feed crops. But maize is a crop which middle sensitive to salinity. Drought, high salinity, low temperature and other environmental factors limit its growth and yield. So it is very meaningful to breed high-resisted maize. Salt tolerance mechanism of plants is very complex, osmotic adjustment is one of the important physiological mechanism of higher plants to adapt to salt stress, which is considered the most hope osmotic adjustment substances is betaine. Closely related to plant salt tolerance and drought resistance by its accumulation in the cells. Betaine synthesis pathway in plants is based on choline as substrate two-step enzymatic oxidation, these two enzymes are present in the chloroplast stroma. Its enzymatic activity induced by salt. Under salt stress conditions, plant cells can be rapidly accumulated betaine to improve the salt tolerance of plants.It is extremely slow to select and breed species which bear salt by traditional breeding methods. In this study, through genetic engineering techniques to make betaine synthesis related to choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) gene into corn, so that the accumulation of betaine will help to enhance the purpose of the plant salt tolerance. At present, the selectable marker gene used by most plant genetic transformation, such as antibiotic or herbicide resistance gene to select transplants. Although there is no direct study results show that the selectable marker gene can affect human health or environmental safety, but in order to eliminate concerns about the safety of genetically modified crops.In this study, we have been done on generic transformation of maize with marker-free genes CMO+BADH,At the same time,we achieve the corn which have double gene and analyzed betaine content and salt tolerance of transformed plants.The results of study were acquired asfollows:firstly, the conditions of Agrobacterium tumefaciens transforming system were studied. The experiment mainly used general maize inbred lines H99and Dan988embryogenic callus as materials. Anti-sense expression vector of pCAMBIA-1301-CMO-BADH gene was transformed into maize inbred callus by Agrobacterium tumefaciens. In the case of the same time of infection, the infection results of EH A105is better than EHA101and the infection time was25min, EHA105have the highest transformation ability. So, EHA105is so more suitable for genetic transformation of corn monocotyledons. EHA105strain vibrates20h concentration of fermenting liquor was OD0.5～0.6is most conducive to the transformation of maize embryogenic callus. By NaCl sensitivity experiments which determine the callus suitable selection pressure and suitable for the transformation of callus salt concentration is200mmol/L. Secondly, we also applied the Pollen Tube Pathway Method to transform gene into maize inbred lines HW-3and D6199. And plasmid DNA concentration was200～500μg/mL and entry content was100～200μl can achieve a higher conversion efficiency. The study use agrobacterium-mediated and pollen tube channel two methods to make the CMO+BADH double marker-free gene into maize inbred line, detect the To transgenic plants and prove that DNA had been integrated into maize genome.The test of physiological index of CMO+BADH double marker-free gene maize seedlings.The indexes consist of membrane permeability, content of malondialdehyde (MDA), activity of peroxidase (POD) and activity of superoxide dismutase (SOD). Transformation of the seedlings at NaCl concentration of0%,1.5%,2%, each treatment three replicates, pouring NaCl solution for two days. After handling6days under NaCl solution, taking the functional leaves and measurement of physiological index. In order to test and compare the salt tolerance of all lines and the functions of CMO-BADH double gene and BADH gene and provide theoretical evidence for salt-tolerant new variety breeding, the effect of NaCl stresses was studied in this paper on the tissue culture of transgenic plants with CMO-BADH double gene and BADH gene. The relative electrical conductivity, MDA content, SOD activity and POD activity were measured. The results show that the relative electrical conductivity and MDA content of all lines were increased along with increased salt stress and the order of all lines in those two physiological traits was CK>transgenic plants with BADH gene>transgenic plants with CMO-BADH double gene. The SOD activity and POD activity of all lines were also increased under the salt stress and the order of all lines in those two physiological traits was transgenic plants with CMO+BADH double gene>transgenic plants with BADH Gene>CK. In summary of all the physiological traits, the salt tolerance order of all tested lines was transgenic plants with CMO-BADH double gene>transgenic plants with BADH gene>CK.