Establishment Of An Embryonic Tip Regeneration System Of Soybean And Gene IFS2, FAD3Genetic Transformation

Soybean (Glycine max (L.) Merr) is a major oil and economic crop. As thedevelopment of living standard, people made higher request on the quality and yield ofsoybean. Isoflavones synthetase (IFS2) is the key enzyme in flavonoids metabolic pathways.Isoflavones is the secondary metabolite of flavonoids metabolic pathways which have manyfunctions that defer senescence, prevent osteoporosis, anti-tumor, protect against heart andblood vessel disease, et al. Over expression of fatty acid desaturase gene (FAD3) caused theincreasing of linolenic acid. There was a positive correlation between the linolenic acid andplant cold resistance. Northeast is the main soybean producing areas in China which alwayssuffer to low temperature damage from seeding to mature period. So the research of fattyacid desaturase has important practical significance.We cloned two genes IFS2and FAD3from cDNA of Jilin32. Then, we constructedplant expression vectors: pCB35SR1R2GFP-IFS2and pCB35SR1R2GFP-FAD3.Agrobacterium strain of EHA105was transformed with the two plant expression vectorsrespectively for plant infections.Although Gm Soy has been planted all over the world, it is still difficult in the genetictransformation. However, the system of soybean tissue culture still exist some problems,such as difficult in regeneration, poor repeatability, dependent on genotype, complexoperation which limit the development of soybean tissue culture.In order to establish an effective and stable genetic transformation of soybean,embryonic tip as explants, we first screened ten varieties through inductivity and GUSactivity experiments. Then, we optimized the disinfectant methods, soak time, infect ways,infect time, the concentration of surfactant, concentration of the hormone, concentration ofthe antibiotics, browning, and rooting factors. The results show that Jilin35sensitive toagrobacterium tumefaciens most meanwhile has the high inductivity, so it is the best choiceas receptor materials; alcohol sterilization is the best way which has the smallest damage toseed;12h of soak time has the best result; the effect of add6-BA meanwhile with IBA isbest for regeneration; sucrose as carbon source,1/2MSB and1.0mg/L IBA is best forrooting; ultrasonication and vacuum infect15min with0.03%Silwet L-77gain thehighest effect;1.0mg/L proline and silver has the lowest browning; OD600=0.5～0.7is best;150mg/L cefotaxime is the lowest concentration which has no contamination;gradient screening has the highest efficiency with no selective agent in first week, then1.5mg/L glufosinate for two weeks and0.75mg/L glufosinate later.Through the engineering bacteria of gene IFS2and FAD3infect embryonic tip ofJilin35, we gained four T0transgenic plants of gene IFS2and two T0transgenic plants ofgene FAD3which confirmed by PCR molecular analysis and bar gene test strip. One T1transgenic plant of gene IFS2is confirmed by Glufosinate painting and PCR molecularanalysis.