Genetic Transformation Of Oxalate Oxidase Gene (OxO) Into Soybean(Glycine Max(L.) Merr.)

Soybean, which is one of the most important oil crops in China and around the world, is a food of the most rich in nutrition and easy to digest in the all of Leguminous plants, and also is the most abundant and cheapest source of protein. Sclerotinia is a seriously harmful disease for soybean. It can harm soybean during the whole growth period of soybean, and cause a series of symptoms, such as leaves rot, seedling blight, pod rot. Sclerotinia can cause the reduction of soybean. And the reduction of soybean can reach20%to30%, and50%to90%when Sclerotinia is seriously for soybean, and even more have no harvests. For agricultural production, people take measures such as chemical control, cropping system to prevent Sclerotinia. But it is not obvious for the effect of prevention. So it is necessary for us to cultivate new soybean varieties of high-quality. However, the traditional breeding methods is time-consuming and uncertainty of the phenotype of offspring. With the development of the technology of genetic engineering, transgenic breeding has become an important method for the present stage to obtain excellent crop varieties.Many researches indicate that there is an important relationship between Sclerotinia and oxalic acid which Sclerotinia scleroterum secrete. Oxalate oxidase (oxalate oxidase, OxO， E.C.1.2.3.4) is one of the most primary oxalic acid degradation enzyme. Oxalate oxidase mainly exists in the few cereal plants. It can degrade oxalic acid into H2O2and CO2to weaken the toxicity of oxalic acid, so that delay infection of pathogenic fungi. So we use oxalate oxidase gene (OxO) to transfect soybean plants, in order to get soybean plants which have high resistance to Sclerotinia.In this study, we use OxO gene to transfect embryonic tips of mature soybeans mediated by Agrobacterium tumefaciens. First, we established a regeneration system for embryonic tips of soybean. Second, we discussed the influence factors of genetic transformation of soybean which are the time of transformation mediated by Agrobacterium tumefaciens, the time of co-culture and recovery-culture and so on. As a result, we established a transformation system that adapts embryonic tips of soybean. The main results were as follows:1. Establishment of regeneration system for embryonic tips of soybeanSelected mature soybean seeds which are smooth and no lesion on surfaceâ†'washed the soybean seeds with70%ethanol for50s-1minâ†'soaked seeds in2%NaCIO for20min, we need shake irregularlyâ†'washed seeds with sterile water4～5timesâ†'soaked in sterile water for24hâ†'stripped embryo tip as explants, and cultured on pre-incubation medium for2daysâ†'cultured the explants on shoot induction medium which contains3mg/L6-BA and0.05mg/L IBAâ†'when the shoots were about2-3cm, cut the shoots and transferred them to rooting medium which contains0.3mg/L IBAâ†'when the roots of shoots were strong, transferred them to nutritive soil.2. Establishment of Agrobacterium-mediated transformation system for embryonic tips of soybeanSterilized soybean seeds and soaked in sterile water for the whole nightâ†'stripped embryo tip as explants, and cultured on pre-incubation medium for2daysâ†'activated and cultured A. tumefaciens LBA4404/3304-OxOâ†'infected embryo tips with infection solution whose OD600=0.6-0.8ABS for20h, then co-culture for5days in the darknessâ†'washed explants2times, and transferred them to recovery-culture medium for7daysâ†'transferred explants to selection-culture medium for30days, and changed another fresh selection-culture medium for10daysâ†'selected the survival shoots to rooting mediumâ†'when the roots of shoots were strong, transferred them to nutritive soil.3. Histochemical detection and identification of transgenic soybean plantsCut fresh leaves of soybean plants which were survival from the selection-culture medium, to detection with GUS staining, observation of gfp fluorescence and PCR amplification. The results showed, there were6strains which were dyed blue, and can observe green fluorescence. And PCR amplification also showed that there were same band with positive control. All the results show that the foreign gene OxO has been successfully integrated into the soybean genome.