Research Of Soybean Somatic Embryo Regeneration Optimized And Genetic Transformation

Soybean [Glycine max(L.)Merrill.] is one of the most important oilcrops and protein resource which is widely cultivated all over the worldas well. Soybean has been already regarded as a species which is difficultto be transformed. We can use all kinds of ways to operate genetictransformation, such as Agrobacterium-mediated, low pressure assistanceand particle bombardment. Effective soybean regeneration system isessential for proceeding transformation. We have optimized variousfactors needed by genetypes in order to perfect the whole system. Wecontinued to search the transient expression of Agrobacterium-mediated,particle bombardment and co-transformation while the biomass aquiredafter12wk screening process was valued by means of the three methodsin order to form a set of effective transformation system preliminary.With10genotypes of soybean sowed in different environmentconditions, we achieve somatic embryos by means of inducing theimmature cotyledons. The embryos belonging to4genotypes of soybeanconsidered as occupying potential capability to be induced have beenchosen for the next experiments according to situation which the somaticembryos were produced. The results show that various surroundings havean enormous impact on the same genotypes of soybean embryoinductivity. We will be informed the inductivity of somatic embryo ranking which is spring stage>greenhouse stage>autume stage>summerstage. When the various genotypes of soybean cultivated in the sameenvironment the abilities to produce embryos are different. GenotypesKenfeng23, North4217, Suinong28, Suinong30have a high levelinductivity in preference to another ones. Compared to living in solidculture, somatic embryos have a great advantage in proliferation during inliquid culture. The5mg/L concentration of2,4-D is ideal in reducing theaberration rate. The proliferation speed is quit fast while the clumpdiameter is3mm during solid cultivated. But while cultivated during inliquid the proliferation speed of Kenfeng23and North4217is faster thanothers left when their diameters are3mm and5mm. Suinong28showssimilar proliferation when the diameters are3mm,5mm and7mm.Suinong30is the same situation as Suinong28. The embryos of North4217, Suinong28, Suinong30will display a comparative high vitalitywhile they are subcultured during4-8days no matter which live in solidculture or in liquid culture. We use NFS (The number of single embryo toproliferate during4days) to measure the embryo vitality. But whenKenfeng23is sub-cultured during8-12days the vitality to producesecondary embryos is the highest and the NFS is0.88.To provide the best thesis guider about soybean transformation wehave researched the different transient expression of embryo using YFPreport gene with the cultivar North4217. In the study, we focused on the effect of co-transformation of particle bombardment and agrobacteriumon the transient expression of soybean somatic embryo. According to theobtained statistic data from agrobacterium-mediated transformation andparticle bombardment system, the quantity of transient expression is thebest with1350psi rupture disk,9cm distance as well as OD600=0.3. Thelightspots watched can reach16.8each embryo unit under fluorescencestereomicroscope. The result of PCR indicated that yfp has integrated thechoromosome of embryos. For the limitation of screening process,co-transformation did not maintain the original advantage. However thisoptimized genetic transformation has been certified to be applicable forrapid transient gene analysis and transgenic soybean.