| Soybean (Glycine max L.) is a very important food crop in China and it is also a source of both oil and plant protein. The China’s soybean import and export situation has been fundamentaly reversed since1996and become a net importer country, the vast majority of which is genetically modified soybean products. It is very important to develop GMO soybean production in order to alleviate the cultivated land tension and the food security problem. But at present the soybean transformation efficiency is still very low and poor repeatability in China. How to improve the soybean transformation efficiency is an urgent problem to be solved. In this study, we optimized the Agrobacterium-tumefaciens mediated soybean embryo meristerm transformation sytem by genotypes, Agrobacterium concentration, inoculation methods and plant hormones in shoot induction medium. At the same time, the identification and segregation had been studied in the progeny of transformed Sporamin and Chitinase KDEL soybean. The agronomic and yield characteristics were also analyzed in these transgenic progeny.The Agrobacterium-tumefactions mediated transformation system of embryo meristerm explants was optimized using PTF102vector which contains GUS reporter gene and bar selection markers gene as binary vector. The different genotype (HN37, ZD32, ZH10, ZH30, YC03-3and YC04-5) influenced on the GUS transient expression percentage and the shoot induction percentage was studied. The result showed that the best genotype for GUS transient expression percentage and the shoot induction percentage was YC03-3. OD6500.6-0.8and infected3d showed the highest GUS transient expression percentage and the shoot induction percentage later among different Agrobacterium oncentration (OD6500.4,0.6,0.8,1.0and1.2). Comparing conventional infection with ultrasonic assisted infection at the inoculation stage, the latter is help to improve the embryo tips of GUS transient expression percentage. Different concentration combination of plant growth regulation (6-BA and IBA) was closely related to shoot induction, the result revealed the combination of0.4mg/l6-BA and0.04mg/1IBA with higher shoot induction, up to72.33%. In addition, we studied the influence of shoot induction inliquid culture and solid culture, liquid culture was help to shorten the transgenic seedlings regeneration cycle than solid culture. In conclusion, The optimized Agrobacterium-mediated transformation system for soybean embryo tip was using the germinated1d embryo tip of YC03-3cultivars as explants, the concentration of Agrobacterium0.6-0.8(OD650) and with sonication assisted Agrobacterium-mediated transformationco-infected30min, the3d lighting co-cultivation on the co-cultivation medium. Then shoots induced on shoot induction medium (SI), which contains6-BA0.4mg/1and IBA0.04mg/1, and elongated the shoots on WPM then rooted on RM. Finally, the putative transgenic plants were got.This study also identified Sporamin and ChitinaseKDEL transgenic progeny using PCR method, bar quick dip stick detection method and leaf painting method using135mg·L-1glufosinate. The results suggested that leaf painting using135mg/1glufosinate could be an effective and costless method to detect genetically modified soybeans progeny using bar gene as a selective agent. There were four independent lines of foreign genes in T1generation genetic from45plants were TO positive transgenic plants.With two lines of3:1ratio on genetic separation according to Mendelian genetics separation ratio.There were165plants in T2generation is positive plant, accounting for47.97%of the total number. Furthermore, we studied the influence of transformation progeny on growth period and agronomic characters. The results showed there were no agronomic and growth stage difference difference between the transgenic plants and wildtype. |
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